Engineered adenylate kinase variants

Abstract

The present disclosure provides engineered adenylate kinases, as well as recombinant polynucleotides encoding the same. The present disclosure also provides uses of the engineered adenylate kinases for converting NMPs to NDPs.

Description

Cross-references to related applications

[0001] This application claims the benefits of U.S. Provisional Application No. 63 / 589,828, filed October 12, 2023, and U.S. Provisional Application No. 63 / 661,366, filed June 18, 2024, each of which is incorporated herein by reference in its entirety for all purposes.

[0002] References to sequence lists, tables, or computer programs The sequence list, created on October 10, 2024, with a file size of 5,616,375 bytes and named CX10-258WO3_ST26.xml, which was filed concurrently with this application, is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to engineered adenosine kinase variants, polynucleotides encoding engineered adenosine kinase variants, and the use of engineered enzymes. Background Technology

[0004] Adenosine kinase (AK or AdyK) is an ATP:AMP phosphotransferase, an enzyme belonging to the nucleoside monophosphate (NMP) kinase family. This enzyme mediates the interconversion of adenine nucleotides (ATP + AMP). 2. ADP). This enzyme is ubiquitous in nature, found in Archaea, Bacteria, and Eukaryotes (excluding sulfur bacteria), and is a key enzyme in energy metabolism and adenine nucleotide balance in the body. Although ATP is the substrate for most AK enzymes, human AK isoenzyme 3 uses GTP as a substrate (GTP: AMP phosphotransferase).

[0005] Adenosine kinase can act as an ATP regeneration system in the presence of excess ADP, converting two ADP molecules (one as a phosphate acceptor and the other as a phosphate donor) into ATP + AMP. Adenosine kinase is also suitable for enzymatic cascade reactions that convert AMP into ADP and other NDPs. However, adenosine kinase can exhibit substrate-specific preferences and different activities for certain NDP substrates. Summary of the Invention

[0006] This disclosure provides engineered adenosine kinase, a polynucleotide encoding said engineered adenosine kinase, and a method of using said adenosine kinase. In some embodiments, the engineered adenosine kinase has improved properties compared to a reference adenosine kinase.

[0007] In one aspect, this disclosure provides an engineered adenosine kinase comprising a reference sequence corresponding to amino acid residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 2-1302, 1356-2652, and 2676-3192, or 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 higher sequence identity with respect to SEQ ID NOs: 2-1302, 1356-2652, and 2676-3192, wherein the amino acid sequence comprises an amino acid sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 83%, 96%, 97%, 98%, 99% or higher sequence identity with respect to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0008] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104. The reference sequences corresponding to these sequences have 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 higher sequence identity, wherein the amino acid sequences contain amino acid sequences relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0009] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence that corresponds to a reference sequence of amino acid residues 12-231 of SEQ ID NO: 2, or has 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 higher sequence identity with respect to the reference sequence of amino acid residues 12-231 of SEQ ID NO: 2, or one or more substitutions thereof.

[0010] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 4. An amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or one or more substitutions to the reference sequence corresponding to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2.

[0011] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to amino acid residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, or 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 higher sequence identity with the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2.

[0012] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions at at least the following amino acid positions: 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions at at least the following amino acid positions: 13, 18, 29, 30, 31, 32, 34, 35, 48, 50, 53, 55, 59, 60, 61, 62, 66, 68, 77, 78, 80, 83, 87, 100, 104, 105, 109, 118, 119, 127, 128, 133, 136, 139, 142, 143, 150, 151, 155, 157, 168, 170, 175, 184, 186, 190, 201, 207, 212, 213, 214, 216, 217, 218, 224, or 226, or combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0014] In some embodiments, the engineered adenosine kinase amino acid sequence contains substitutions or sets of substitutions at at least the following amino acid positions: 29 / 30 / 128 / 142 / 186 / 212 / 213, 18 / 142 / 212 / 213, 29 / 30 / 128 / 142 / 186, 40 / 135 / 214, 18 / 29 / 30 / 127 / 150, 82, 69, 118, 29 / 30, 206, 32, 16, 104, 111 / 135 / 136 / 214, 29 / 128 / 175, 178, 18 / 128 / 142, 32 / 108 / 111 / 156 / 191, 18 / 29 / 128 / 129 / 142 / 186, 32 / 133, 18 / 29 / 30 / 212 / 213 / 222 29 / 65 / 127, 32 / 40 / 43 / 52 / 155 / 156 / 217, 18 / 29 / 128 / 129, 80, 32 / 43 / 138 / 152 / 191 / 214 / 215, 183, 18 / 175 / 186 / 212 / 213, 129, 18 / 29 / 30 / 65 / 129 / 142 / 150 / 186, 127 / 142 / 212 / 213, 30 / 65 / 142 / 222, 40 / 43 / 215, 136, 29 / 150 / 211 / 212 / 213 / 221, 43 / 111 / 136 / 219, 226, 88, 140 / 215 / 216, 32 / 133 / 134 or 18 / 29 / 128 / 186, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0015] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes at least one substitution shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0016] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the substitutions or sets of substitutions of the engineered adenosine kinase variants shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0017] In some implementations, the engineered adenosine kinase comprises substances included in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, and 42. The reference amino acid sequence of the engineered adenosine kinase variant shown in .2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2 and 49.2 has 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 higher sequence identity, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to residues 12-231 of SEQ ID NO: 4. The reference sequences corresponding to 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104 have amino acid sequences with 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 higher sequence identity.

[0019] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, or 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 higher sequence identity to the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192.

[0020] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to residues 12-231 of SEQ ID NO: 4. An amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0021] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, or 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%, or 99% or higher sequence identity with the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, wherein the amino acid sequence comprises an amino acid sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 83%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to SEQ ID NO: 4. The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0022] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions at at least the following amino acid positions: 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231, or combinations thereof, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

[0023] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions at at least the following amino acid positions: 13, 18, 29, 30, 31, 32, 34, 35, 48, 50, 53, 55, 59, 60, 61, 62, 66, 68, 77, 78, 80, 83, 87, 100, 104, 105, 109, 118, 119, 127, 128, 133, 136, 139, 142, 143, 150, 151, 155, 157, 168, 170, 175, 184, 186, 190, 201, 207, 212, 213, 214, 216, 217, 218, 224, or 226, or combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 4.

[0025] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 86-372, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0026] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 100 / 101, 94, 179, 198, 57, 78, 68, 93, 77, 62, 14, 51, 118, 91, 163, 65, 173, 224, 66, 109, 60, 40 / 88, 191, 63, 68 / 170, 100, 108, 58, 180, 178, 199, 40, 80 / 203, 40 / 43, 39, 55, 110, 51 / 59, 155 / 226, 80, 219, 166, 202, 59, 38, 90, 80 / 184 / 203, 35, 222. 54, 111 / 153 / 155, 112, 175, 74, 105, 170, 117, 128, 61, 119, 155, 79, 220, 226, 108 / 155, 108 / 111, 111, 69 / 88, 43, 43 / 69 / 138, 40 / 43 / 88 / 134 / 178, 214, 80 / 82 / 184 / 219, 88 / 136, 16 / 40 / 43 / 88, 40 / 134, 88, 111 / 155, 223, 115, 190, 201, 126, 56, 216, 76, 69, 120, 116, 203, 194 or 138, wherein the amino acid sequence comprises relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 4.

[0027] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or one or more substitutions relative to the reference sequence corresponding to the reference sequence corresponding to SEQ ID NO: 172.

[0028] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 374-444, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or relative to the reference sequence corresponding to SEQ ID NO: 172.

[0029] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 66 / 78, 78 / 80 / 224, 78, 65 / 66, 78 / 224, 78 / 80 / 109, 40 / 60 / 94 / 226, 66 / 68, 78 / 80, 94 / 117 / 118 / 226, 68 / 109, 68, 94 / 179, 51 / 66 / 68 / 224, 94 / 117, 51 / 66 / 68 / 78 / 224, 66, 118, 93 / 94 / 180, 109, 68 / 224, 40 / 62 / 93, 93, 51 / 66, 94 / 117 / 118, 198 / 220, 93 / 117, 117 / 118, 93 / 198, 68 / 173, 40 / 117, 224, 40 / 118 / 226, 93 / 94, or 40 / 62 / 118, wherein the amino acid sequence comprises a reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or one or more substitutions of the reference sequence corresponding to SEQ ID NO: 172.

[0030] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376.

[0031] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 446-510, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO: 376.

[0032] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 13, 60, 126, 104, 201, 59, 55, 133, 61, 53, 190, 57, 62, 181, 168, 54, 173, 170, 34, 183, 14, 60 / 62 / 124 / 170, 60 / 170, or 60 / 62, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO: 376.

[0033] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 508.

[0034] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 512-580, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or relative to the reference sequence corresponding to SEQ ID NO: 508.

[0035] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 15, 15 / 68, 15 / 181, 13 / 15, 13 / 15 / 201, 54 / 55, 55 / 133, 55 / 133 / 197, 54 / 55 / 133 / 197, 68, 54 / 133 / 197, 54 / 197, 13 / 15 / 68, 34, 54, 133, 120, 34 / 133, 13 / 15 / 181, 55, 15 / 59, 68 / 181, 201, 181, 66, 15 / 59 / 181, 197, 13 / 15 / 59, 61, 34 / 61, 13, 34 / 69, or 163, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of 508, or relative to the reference sequence corresponding to SEQ ID NO: 508.

[0036] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 524.

[0037] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 582-608, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO: 524.

[0038] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions or sets of substitutions at at least the following amino acid positions: 120, 61 / 201, 34, 36, 105, 112, 31, or 146, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO: 524.

[0039] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 584.

[0040] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 610-666, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or relative to the reference sequence corresponding to SEQ ID NO: 584.

[0041] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 13 / 30 / 31 / 53 / 109 / 119 / 168, 36 / 126 / 190, 126, 36 / 39 / 126, 126 / 190, 36 / 126, 36 / 190, 39 / 126, 13 / 109 / 119, 109, 36 / 120 / 190, 126 / 148, 190, 108 / 126, 30 / 31, 13 / 118 / 182, 31 / 53, 39, 119, 13 / 119 / 168, 53 / 168, 34, 34 / 65 / 146, 36 / 39, 119 / 168, 34 / 112, or 173, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of 584, or relative to the reference sequence corresponding to SEQ ID NO: 584.

[0042] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610.

[0043] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 668-780, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO: 610.

[0044] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 190, 115 / 126 / 190, 126 / 190, 34 / 35 / 105, 126, 59 / 126, 34 / 105 / 116 / 146, 48, 66, 59, 148, 82, 100, 27, 135, 77, 138, 52, 68, 139, 184, 81, 65, 153, 50, 181, 214, 172, 69, or 154, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO: 610.

[0045] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 674.

[0046] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 782-868, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO: 674.

[0047] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 81 / 148 / 219, 68 / 148 / 219, 68 / 81 / 219, 148 / 219, 81 / 219, 136 / 138 / 139, 68 / 219, 66 / 68 / 219, 66 / 148 / 219, 66 / 68 / 148, 59, 59 / 135, 219, 59 / 139, 138, 126 / 148 / 219, 59 / 181, 81, 66 / 81 / 219, 126 / 219, 66 / 68 / 81 / 148 / 219, 181, 27 / 148, 135 / 136 / 139, 148, 59 / 135 / 138 / 181, 59 / 135 / 136 / 138 / 139, 59 / 135 / 136 / 138 / 181, 27, 27 / 126 / 148, 135 / 136 / 138, 68 / 81 / 126 / 148 / 219, 136, 68, 126 / 148 or 138 / 139, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO: 674.

[0048] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 816.

[0049] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 870-1014, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO: 816.

[0050] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 48 / 100 / 148, 48 / 66 / 148 / 219, 48 / 100, 48 / 100 / 135 / 136, 48 / 66 / 77 / 100 / 135 / 136 / 148, 48 / 100 / 136, 48 / 135 / 148, 48 / 148, 48 / 136 / 148, 48 / 135, 48 / 135 / 136, 66 / 100, 48 / 66 / 219, 48 / 135 / 219, 100 / 148, 48 / 136 / 219, 48 / 219, 48 / 135 / 148 / 219, 100 / 135 / 136, 100 / 136, 66 / 77 / 100 / 136 / 219, 100 / 135, 48 / 77 / 100 / 135 / 136, 100, 77 / 100 / 135, 48 / 66 / 100 / 135 / 136 / 148 / 219, 52 / 184 66, 52 / 68, 136 / 148, 126 / 184, 148, 184, 52, 135 / 136 / 148, 126 / 138 / 184, 27 / 184, 27 / 126, 27 / 126 / 148, 27, 27 / 126 / 184, 66 / 100 / 135 / 219, 27 / 68, 52 / 68 / 81 / 126, 48 / 66 / 136 / 148, 66 / 136 / 148, 66 / 135, 66 / 136, 66 / 13 5 / 219, 136, 135 / 148 / 219, 68 / 126, 66 / 148 / 219, 27 / 68 / 126, 27 / 68 / 184, 126, 68 / 81 / 126, 77 / 135, 135 / 136, 77 / 136, 77 / 148, 82, 138 / 184, 77 / 135 / 136, 27 / 138, 81, 81 / 126, 27 / 126 / 138, 68 or 81 / 138, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO: 816.

[0051] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874.

[0052] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 1016-1302, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO: 874.

[0053] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 32 / 60 / 135 / 175 / 225, 32 / 81 / 127 / 128 / 135 / 175 / 225, 83 / 175 / 225, 135 / 175 / 225, 54 / 84 / 190, 55 / 190 / 212, 32 / 60 / 81 / 83 / 127 / 128 / 135 / 225, 175 / 225, 32 / 127 / 175, 84 / 190 / 212, 32 / 128 / 175, 81 / 175, 21 / 54, 54 / 55 / 212, 32 / 175 / 225, 128 / 175 / 225, 6 2 / 84 / 190, 81 / 135 / 225, 21 / 54 / 62 / 190 / 212, 60 / 81 / 83 / 175, 55 / 84 / 190, 32 / 81 / 83 / 135 / 225, 175, 62 / 212, 32 / 81 / 83, 32 / 81 / 225, 54 / 55 / 84 / 212, 55 / 19 0, 62 / 179, 54 / 84 / 179 / 212, 54 / 55 / 62 / 84, 81 / 135, 32 / 81 / 135, 179 / 190 / 212, 55 / 62, 100, 21 / 54 / 62 / 190, 190 / 212, 128 / 175, 21 / 62 / 190, 54 / 179 / 190, 5 4 / 190, 21 / 55 / 190, 21 / 54 / 62, 21 / 84, 21 / 55, 21 / 62, 21 / 179, 55 / 62 / 190 / 212, 54 / 62 / 84, 127 / 128 / 175 / 225, 54 / 62, 100 / 104, 54 / 55, 21 / 55 / 212, 62, 55 / 84, 55 / 62 / 212, 21 / 84 / 190, 225, 21 / 54 / 179 / 190, 83 / 135, 32 / 175, 21 / 54 / 78 / 190, 55 / 62 / 179 / 190 / 212, 21 / 54 / 190, 21 / 55 / 84 / 190, 62 / 84, 21 / 55 / 62, 2 1 / 62 / 84, 135, 32 / 60 / 81 / 127, 21 / 54 / 55 / 62 / 78, 54 / 84, 55 / 62 / 179 / 190, 54 / 55 / 62 / 84 / 179 / 190 / 212, 190, 83, 78, 54 / 62 / 190, 21 / 55 / 62 / 179 / 190, 32 / 6 0 / 127, 54 / 62 / 84 / 190 / 212, 21 / 62 / 179, 127 / 128 / 225, 32 / 225, 32 / 60 / 81, 81, 54, 21, 60 / 81 / 128 / 175, 84, 21 / 190 / 212, 60 / 128 / 225, 179, 21 / 55 / 62 / 190,128, 60, 212, 55, 127, 32, 80, 220, 62 / 78 / 179, 55 / 62 / 84, 21 / 55 / 190 / 212, 32 / 127 / 128 / 225, 21 / 54 / 55 / 84 / 179, 21 / 190, 32 / 60 / 81 / 83 / 128 / 135, 127 / 128, or 21 / 55 / 179, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO: 874.

[0054] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032.

[0055] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 1356-1552, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0056] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following amino acid positions: 54 / 80 / 225, 55 / 80 / 180, 54 / 55 / 80 / 208 / 225, 84 / 128 / 155 / 190 / 212, 62 / 155 / 190, 83 / 84 / 155 / 190, 62 / 128 / 135 / 190, 225, 84 / 155 / 190, 128 / 190, 190 / 212, 83 / 84 / 155, 54 / 208 / 225, 84 / 155 / 190, 84 / 212, 84 / 123 / 155 / 212, 62 / 128 / 190, 55 / 80 / 208 / 22 5, 155 / 190, 83 / 84 / 190 / 212, 83 / 128 / 155 / 190 / 212, 55 / 180 / 225, 84 / 190 / 212, 54 / 80 / 180 / 208 / 225, 80 / 208, 135 / 155 / 190, 212, 80 / 180 / 208, 128 / 135 / 190 / 212, 128 / 155 / 212, 80 / 208 / 225, 83 / 212, 62 / 84 / 155 / 190, 54 / 179 / 225, 84, 83 / 84 / 190, 128 / 212, 54 / 55 / 80 / 208, 55, 62 / 128, 54 / 80 / 208 / 225, 62 / 83 / 84 / 190, 179 / 225, 190, 80 / 169 / 208, 62 / 128 / 212, 80, 62, 54 / 55 / 80 / 225, 54 / 80 / 180 / 208, 83 / 84 / 128 / 190 / 212, 62 / 83 / 190, 62 / 190, 80 / 179, 80 / 18 0, 54 / 80, 62 / 83 / 84 / 128 / 190 / 212, 83 / 84 / 135 / 212, 54 / 80 / 179 / 208, 62 / 84 / 128, 11 / 62 / 84 / 128 / 190 / 212, 80 / 179 / 208, 80 / 225, 62 / 84 / 128 / 190 / 212, 6 2 / 84 / 212, 80 / 179 / 180 / 208 / 225, 62 / 135, 55 / 208, 62 / 84, 128 / 135 / 190, 55 / 208 / 225, 84 / 128 / 190, 62 / 84 / 128 / 190, 62 / 135 / 212, 54 / 179 / 180 / 225, 62 / 84 / 135 / 212, 135, 54 / 55 / 225, 55 / 80 / 179 / 180, 84 / 135 / 190, 84 / 128, 54, 84 / 128 / 135, 54 / 179 / 208 / 225, 55 / 179 / 225, 128, 62 / 83 / 84 / 155 / 190, 84 / 13584 / 128 / 155, 55 / 179 / 180, 83 / 84 / 128 / 190, or 54 / 55 / 179 / 225, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0057] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388.

[0058] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 1554-1786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388 or relative to the reference sequence corresponding to SEQ ID NO: 1388.

[0059] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 143, 228, 136, 82, 156, 205, 226, 150, 219, 217, 218, 68, 142, 212, 213, 79, 230, 207, 155, 227, 129, 210, 133, 151, 43, 204, 182, 82 / 136 / 150 / 217, 136 / 150, 79 / 81 / 143 / 156 / 212 / 228, 82 / 136, 7 9 / 81 / 143 / 212, 79 / 133 / 228, 81 / 143 / 156 / 212, 136 / 219, 82 / 136 / 226, 136 / 150 / 217, 133 / 156, 133 / 143 / 228, 150 / 217, 82 / 136 / 150, 81, 81 / 143, 81 / 133 / 228, 143 / 228, 212 / 228, 79 / 81, 133 / 228, 133 / 212, 136 / 217, 216, 215 or 127, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or relative to the reference sequence corresponding to SEQ ID NO: 1388.

[0060] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to SEQ ID NO: 1708.

[0061] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 1788-1968, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708 or relative to the reference sequence corresponding to SEQ ID NO: 1708.

[0062] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 127, 143 / 155 / 219 / 228, 219, 127 / 142, 142 / 151 / 207 / 218, 143 / 213, 151 / 218, 127 / 142 / 218, 142 / 151, 143 / 155 / 205 / 213 / 228, 127 / 142 / 207, 127 / 216 / 218, 142 / 151 / 207, 143 / 219, 127 / 207, 127 / 218. 143 / 219 / 228, 127 / 216, 68 / 127 / 207, 143 / 155 / 212 / 228, 151, 143 / 213 / 228, 143 / 155 / 205, 151 / 207 / 216, 143 / 228, 68 / 127 / 207 / 218, 143 / 155, 143, 205 / 213 / 219, 155 / 212 / 219 / 228, 205 / 219 / 228, 143 / 205, 142 / 207, 143 / 205 / 219, 143 / 212 / 213, 127 / 207 / 218, 142, 127 / 142 / 151 / 207 / 218, 143 / 205 / 219 / 228, 205 / 213 / 228, 68 / 151 / 207, 68 / 127 / 142 / 207, 68 / 127, 218, 127 / 142 / 196, 142 / 207 / 218, 68 / 142 / 216, 68 / 127 / 216 / 218, 68 / 127 / 218, 151 / 216 / 218, 143 / 212 / 228, 127 / 142 / 151, 151 / 207, 127 / 142 / 216, 129 / 207 / 218, 68 / 142 / 151 / 207, 68 / 142 / 151, 151 / 207 / 218, 228, 155 / 213, 127 / 142 / 151 / 218, 127 / 151, 155 / 228, 68 / 142 / 207, 80 / 151 / 207, 205 / 228, 68 / 142 / 207 / 216 / 218, 68 / 218, 68 / 207, 216 / 218, 68 / 142, 212, 205 or 142 / 207 / 216 / 218, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or relative to the reference sequence corresponding to SEQ ID NO: 1708.

[0063] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952.

[0064] 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%, 96%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 1970-2032, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952 or relative to the reference sequence corresponding to SEQ ID NO: 1952.

[0065] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 18 / 118, 87 / 119 / 192, 119 / 173 / 192, 118, 87 / 119, 18 / 118 / 170, 18, 119, 192, 87, 89, 59, 170, 64, 112, 93, 184, 146, 51, 125, 192, 191, 117, 172, or 163, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or relative to the reference sequence corresponding to SEQ ID NO: 1952.

[0066] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980.

[0067] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2034-2134, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

[0068] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 87 / 119 / 192, 87 / 192, 87 / 119, 119 / 192, 192, 119, 146 / 192, 87, 59 / 87, 59 / 87 / 112 / 146, 146, 87 / 112 / 119, 74, 75, 66, 181, 81, 73, 77, 188, 216, 82, 136, 214, 21, 217, 154, 133, 71, 212, or 228, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

[0069] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072.

[0070] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2136-2264, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

[0071] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions or sets of substitutions at at least the following amino acid positions: 87, 194, 197, 93, 202, 38, 91, 183, 39, 146, 203, 37, 92, 36, 89, 131, 132, 90, 148, 35 / 197, 231, or 56, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

[0072] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138.

[0073] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2266-2366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

[0074] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 161, 162, 153, 216, 212, 156, 214, 229, 77 / 143 / 214 / 216, 143 / 156 / 216, 77 / 143, 77 / 143 / 216, 77 / 214 / 216, 77 / 216, 143 / 156, 156 / 216, 143, 77, 77 / 214, 77 / 143 / 161 / 162 / 212 / 214, 77 / 162 / 214 / 216, 77 / 143 / 156 / 214 / 216, 77 / 143 / 156 / 162 / 212 / 216, 77 / 143 / 156 / 161 / 214 / 216, 77 / 156 / 162 / 216, 156 / 161 / 214 / 216, 77 / 143 / 162 / 214 / 216, 143 / 216, 214 / 216, 143 / 156 / 214 / 216, 143 / 214 / 216, 77 / 143 / 212 / 216, 143 / 156 / 161 / 162 / 216, 156 / 162 / 214, 77 / 143 / 156 / 161 / 162, 143 / 161 / 214 / 216, 77 / 161 / 216 or 143 / 212 / 214 / 216, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

[0075] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294.

[0076] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2368-2594, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

[0077] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions or sets of substitutions at least at the following amino acid positions: 80, 105, 228, 50, 175, 170, 82, 173, 65, 53, 184, 122, 195, 68, 89, 34, 118, 119, 87, 179, 117, 113, 54, 190, 216, 88, 36, 166, 38, 169, 66, 142, 136, 131, 187, 40, 74, 127, 55, 148, 215, 64, 155, 116, 90, 132, 60, 94, 112, 120, 37, 108, 35, 51, 61, 212, 172, 59, or 56, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

[0078] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368.

[0079] 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%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2596-2652 or 2676-2714, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368 or relative to the reference sequence corresponding to SEQ ID NO: 2368.

[0080] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 20, 21, 22, 43, 44, 46, 48, 138, 181, 182, 136 / 228, 82 / 142 / 173 / 184 / 216, 184 / 216, 50 / 184 / 216, 127 / 173 / 184, 105 / 136 / 170 / 175 / 228, 105 / 175, 50 / 184, 105 / 170 / 175 / 228, 50 / 127 / 173 / 184, 5 0 / 142 / 184 / 216, 50 / 82 / 127 / 216, 50, 50 / 82 / 173 / 216, 50 / 127 / 142 / 184 / 216, 50 / 82 / 127, 105 / 136, 105 / 228, 105 / 136 / 175 / 228, 173 / 216, 50 / 173, 228, 127 / 173, 105 / 136 / 170 / 195, 50 / 82 / 216, 175 / 228 or 50 / 82 / 127 / 142 / 184 / 216, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

[0081] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to SEQ ID NO: 2602.

[0082] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2716-2988, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0083] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 18, 18 / 56, 20, 21, 26, 27, 29, 30, 32, 42, 43, 50, 66, 67, 69, 70, 80, 98, 99, 102, 135, 137, 139, 141, 142, 143, 148, 149, 152, 153, 156, 173, 184, or 216, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0084] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions or substitution groups at at least the following amino acid positions: 65, 65 / 104, 65 / 104 / 105 / 116 / 127, 65 / 104 / 105 / 127 / 136, 65 / 104 / 136, 65 / 104 / 136 / 170, 65 / 105 / 116 / 136 / 170, 65 / 105 / 127 / 136, 65 / 105 / 127 / 136 / 175, 65 / 105 / 127 / 170, 65 / 105 / 127 / 175, 65 / 105 / 136, 65 / 105 / 136 / 170, 65 / 105 / 170, 65 / 116, 65 / 127 / 136, 65 / 1 27 / 136 / 170, 65 / 127 / 170, 94 / 173, 94 / 187, 104 / 105, 104 / 105 / 127, 104 / 105 / 127 / 136 / 175 / 195, 104 / 105 / 136 / 170, 104 / 127, 104 / 127 / 136 / 170, 104 / 127 / 136 / 175, 105, 105 / 116 / 127 / 136 / 170 / 175, 105 / 127 / 136, 105 / 127 / 136 / 195, 105 / 136, 105 / 136 / 195, 116 / 127 / 136 / 175, 127 / 195, 132, 136 or 170, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0085] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions at at least the following amino acid positions: 36, 37, 40, 50, 59, 89, 117, 120, 128, 168, or 203, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0086] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832.

[0087] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2990-3032, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or relative to the reference sequence corresponding to SEQ ID NO: 2832.

[0088] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions or sets of substitutions at at least the following amino acid positions: 36, 36 / 50, 36 / 50 / 89 / 93 / 110, 36 / 50 / 89 / 93 / 139, 36 / 50 / 89 / 139 / 170, 36 / 50 / 89 / 172, 36 / 89, 36 / 170, 50, 50 / 89 / 93, 50 / 89 / 93 / 139, 50 / 93, 50 / 170, 89 / 170 / 172, 90 / 104, 90 / 151 / 157, 104 / 151 / 154, 104 / 151 / 154 / 157, 104 / 154 / 157 / 216, 151 / 157, 151 / 216 or 216, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or relative to the reference sequence corresponding to SEQ ID NO: 2832.

[0089] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994.

[0090] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 3034-3150, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

[0091] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions or sets of substitutions at at least the following amino acid positions: 50, 58, 59, 68, 74, 76, 79, 80, 83, 90, 112, 113, 119, 157, 170, 172, 182, 184, 217, 224, 226, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

[0092] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions or sets of substitutions at at least the following amino acid positions: 79 / 104 / 157 / 228, 79 / 157 / 228 / 229, 79 / 157 / 229, 79 / 228, 83, 83 / 104, 83 / 104 / 151 / 168 / 173 / 190, 83 / 113, 83 / 173. / 190, 83 / 173 / 190 / 201, 83 / 173 / 201, 83 / 190, 83 / 190 / 201 / 216, 83 / 216, 104 / 157, 157, 157 / 173 / 190 / 216, 157 / 183, 157 / 190, 157 / 190 / 216, 157 / 228, 173 / 216, 201 / 216 or 229, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

[0093] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 3104, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to SEQ ID NO: 3104.

[0094] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NOs in SEQ ID NO: 3152-3192, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 3104, or relative to the reference sequence corresponding to SEQ ID NO: 3104.

[0095] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions or sets of substitutions at at least the following amino acid positions: 104, 104 / 157, 104 / 157 / 190 / 228, 104 / 170 / 228, 104 / 157 / 228 / 229, 104 / 157 / 229, 104 / 170 / 190, 104 / 170 / 190 / 228, 104 / 190, 104 / 190 / 201, 104 / 190 / 228, 104 / 201 / 228, 104 / 228 / 229, 157 / 228, 157 / 228 / 229, 170 / 190 / 228, 170 / 190 / 228 / 229, 190, 190 / 229, 201 / 228 or 228, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 3104, or relative to the reference sequence corresponding to SEQ ID NO: 3104.

[0096] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes at least one substitution shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to SEQ ID NO: The reference sequences corresponding to 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

[0097] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the substitutions or sets of substitutions of the engineered adenosine kinase variants shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to SEQ ID NO: The reference sequences corresponding to 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

[0098] In some implementations, the engineered adenosine kinase comprises substances included in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, and 42. The reference amino acid sequence of the engineered adenosine kinase variant shown in SEQ ID NO: 2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2 has 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 higher sequence identity, wherein the amino acid position relative to SEQ ID NO: 2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 84%, 85%, 86%, 97%, 98%, 99% or higher sequence identity, wherein the amino acid position relative to SEQ ID NO: 2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 87%, 98%, 99%, or higher sequence identity, wherein the amino acid position relative to SEQ ID NO: 2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49. ... For the reference sequences corresponding to ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0099] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence containing residues 12-231 of SEQ ID NO. with even numbers in SEQ ID NO: 4-1302, 1356-2652 and 2676-3192, or contains SEQ ID NO. with even numbers in SEQ ID NO: 4-1302, 1356-2652 and 2676-3192, optionally wherein the engineered adenosine kinase has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions in the amino acid sequence.

[0100] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence of residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or said amino acid sequence comprises SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, optionally wherein the engineered adenosine kinase has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions in the amino acid sequence.

[0101] In some embodiments, the engineered adenosine kinase has adenosine kinase activity and at least one improved property compared to a reference adenosine kinase. In some embodiments, the engineered adenosine kinase has improved properties selected from the following compared to a reference adenosine kinase: i) increased activity to unmodified nucleoside monophosphates (NMPs), ii) increased stability, iii) increased thermal stability, iv) increased activity to 2'-fluoromodified nucleoside monophosphates, and v) increased activity to 2'-O-methylmodified nucleoside monophosphates, or any combination of i), ii), iii), iv), and v). In some embodiments, the reference adenosine kinase has the amino acid sequence corresponding to residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or the amino acid sequence corresponding to residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104. In some embodiments, the reference adenosine kinase has the amino acid sequence corresponding to residues 12-231 of SEQ ID NO: 2, or the amino acid sequence corresponding to SEQ ID NO: 2.

[0102] In some embodiments, the engineered adenosine kinase is purified. In some embodiments, the engineered adenosine kinase is immobilized on a supporting medium.

[0103] In another aspect, this disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered adenosine kinase as described herein.

[0104] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of SEQ ID NO: 1, 3, 171, 375, 507, 523, 583, 609, 673, 815, 873, 1031, 1387, 1707, 1951, 1979, 2071, 2137, 2293, 2367, 2601, 2831, 2993, or 3103, or a sequence corresponding to SEQ ID NO: The reference polynucleotide sequence corresponding to 1, 3, 171, 375, 507, 523, 583, 609, 673, 815, 873, 1031, 1387, 1707, 1951, 1979, 2071, 2137, 2293, 2367, 2601, 2831, 2993, or 3103 has a sequence identity of 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 higher, wherein the recombinant polynucleotide encodes adenosine kinase.

[0105] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of the odd-numbered SEQ ID NOs in SEQ ID NOs: 3-1301, 1355-2651, and 2675-3191, or a reference 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 higher sequence identity to the reference polynucleotide sequence corresponding to the odd-numbered SEQ ID NOs in SEQ ID NOs: 3-1301, 1355-2651, and 2675-3191, wherein the recombinant polynucleotide encodes an engineered adenosine kinase.

[0106] In some embodiments, the polynucleotide sequence encoding engineered adenosine kinase has preferred codons, for example, the polynucleotide sequence is codon-optimized. In some embodiments, the polynucleotide sequence is codon-optimized for expression in bacterial cells, fungal cells, or mammalian cells.

[0107] In some embodiments, the polynucleotide sequence comprises nucleotide residues 34-693 of the odd-numbered SEQ ID NOs in SEQ ID NOs: 3-1301, 1355-2651 and 2675-3191, or comprises the odd-numbered SEQ ID NOs in SEQ ID NOs: 3-1301, 1355-2651 and 2675-3191.

[0108] In some embodiments, the polynucleotide sequence comprises nucleotide residues 34-693 of SEQ ID NO: 1, 3, 171, 375, 507, 523, 583, 609, 673, 815, or 873, 1031, 1387, 1707, 1951, 1979, 2071, 2137, 2293, 2367, 2601, 2831, 2993, or 3103, or comprises nucleotide residues 34-693 of SEQ ID NO: 1, 3, 171, 375, 507, 523, 583, 609, 673, 815, or 873, 1031, 1387, 1707, 1951, 1979, 2071, 2137, 2293, 2367, 2601, 2831, 2993, or 3103.

[0109] In another aspect, this disclosure provides an expression vector comprising a recombinant polynucleotide encoding an engineered adenosine kinase as described herein. In some embodiments, the recombinant polynucleotide of the expression vector is operatively linked to a control sequence.

[0110] In another aspect, this disclosure also provides a host cell comprising an expression vector containing a recombinant polynucleotide encoding an engineered adenosine kinase. In some embodiments, the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

[0111] In another aspect, host cells are used to generate engineered adenosine kinase. In some embodiments, a method for generating an engineered adenosine kinase peptide includes culturing host cells containing the expression vector described herein under suitable culture conditions, thereby generating the engineered adenosine kinase peptide. In some embodiments, the method further includes recovering or isolating the adenosine kinase peptide from the culture and / or host cells. In some embodiments, the method further includes purifying the adenosine kinase peptide.

[0112] In another aspect, engineered adenosine kinase is provided as a composition. In some embodiments, the composition further comprises a nucleoside monophosphate substrate. In some embodiments, the nucleoside in the composition is a modified nucleoside monophosphate.

[0113] In another aspect, engineered adenosine kinases are used to convert nucleoside monophosphates (NMPs) to their corresponding nucleoside diphosphates (NDPs). In some embodiments, the method for converting a nucleoside monophosphate (NMP) to a nucleoside diphosphate (NDP) includes contacting the nucleoside monophosphate with the engineered adenosine kinase described herein under suitable reaction conditions in the presence of a substrate NTP to convert the nucleoside monophosphate to its corresponding nucleoside diphosphate.

[0114] In some embodiments of the method, the substrate nucleoside monophosphate is an unmodified nucleoside monophosphate. In some embodiments of the method, the substrate nucleoside monophosphate is a modified nucleoside monophosphate. In some embodiments, the modified nucleoside monophosphate comprises a modified sugar moiety, a modified nucleobase, or a modified phosphate, or any combination thereof. Detailed Implementation

[0115] This disclosure provides an engineered adenosine kinase for the interconversion of ATP:AMP, and a recombinant polynucleotide encoding the engineered adenosine kinase. In some embodiments, the engineered adenosine kinase has adenosine kinase activity and one or more improved properties compared to the parental wild-type enzyme, including increased activity for NDPs (including NDPs other than ADP) and increased activity for modified NDPs.

[0116] Abbreviations and Definitions For the purposes of this invention, unless otherwise expressly defined, the technical and scientific terms used in the description herein will have the meanings commonly understood by one of ordinary skill in the art. Therefore, the following terms are intended to have the following meanings.

[0117] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, references to “a polypeptide” include more than one polypeptide.

[0118] Similarly, “comprise” and “include” are interchangeable and are not intended to be restrictive. Therefore, as used herein, the term “comprise” and its cognates are used in their inclusive sense (i.e., equivalent to the term “include” and its corresponding cognates).

[0119] It should also be understood that, in the context of the use of the term "comprising" when describing various embodiments, those skilled in the art will understand that, in certain specific cases, the language "substantially consisting of" or "consisting of" may be used to alternatively describe the embodiment.

[0120] "Approximately" refers to the acceptable error for a specific value. In some cases, "approximately" means within 0.05%, 0.5%, 1.0%, or 2.0% of the given value. In other cases, "approximately" means within 1, 2, 3, or 4 standard deviations of the given value.

[0121] "EC" refers to the enzyme nomenclature system of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system of enzymes based on the chemical reactions they catalyze.

[0122] "ATCC" refers to the American Type Culture Collection, whose biobank collection includes genes and strains.

[0123] "NCBI" refers to the National Center for Biotechnology Information and the sequence database it provides.

[0124] "Adenosine kinase," "AdyK," or "AK" refers to an enzyme that catalyzes the interconversion of ATP, ADP, and AMP by transferring a phosphoryl group. In some embodiments, adenosine kinase also includes an enzyme capable of catalyzing the interconversion of NTP, NDP, and NMP. In some embodiments, adenosine kinase is an enzyme classified as EC 2.7.3.4.

[0125] Creatine kinase is an enzyme that catalyzes the reversible interconversion of creatine:ATP and phosphocreatine:ADP.

[0126] "Polyphosate kinase" refers to an enzyme that catalyzes the transfer of a phosphate group from a high-energy, phosphate donor molecule (such as polyphosphate (PolyPn)) to a specific substrate / molecule. Two polyphosphokinase families are known: PPKK1 and PPK2. PPK1 preferentially synthesizes polyphosphates from NTPs and undergoes the corresponding reverse reaction, while PPK2 preferentially consumes polyphosphates to phosphorylate nucleoside monophosphates or diphosphates and undergoes the corresponding reverse reaction. In some embodiments, polyphosphokinases include enzymes classified as EC 2.7.4.1.

[0127] Acetyl kinase (“AcK”) is an enzyme capable of catalyzing the phosphorylation of nucleoside diphosphate or its analogues to nucleoside triphosphate or its equivalent using acetyl phosphate or another phosphoryl donor. As used herein, acetate kinase includes naturally occurring wild-type enzymes or engineered enzymes. In some embodiments, acetate kinase is a naturally occurring wild-type alkaline metabolic enzyme primarily found in prokaryotes that catalyzes the following reaction: acetic acid + ATP Acetyl phosphate + ADP. Acetyl phosphate is an intermediate in the formation of acetyl-CoA. In some implementations, acetate kinase is derived from a naturally occurring wild-type enzyme.

[0128] “Adenosine kinase,” “AdoK,” or “Adk” refers to an enzyme that catalyzes the phosphorylation of adenosine (A or ADO) to produce adenosine-5'-monophosphate (AMP). In some embodiments, adenosine kinase is classified as EC 2.7.1.20. Although the primary substrate is adenosine, as used herein, adenosine kinase includes enzymes that act on other nucleosides.

[0129] "Pyruvate oxidase" or "Pox" refers to an enzyme that catalyzes the reaction between pyruvate, inorganic phosphate, and oxygen to produce acetyl phosphate and H₂O₂. In some embodiments, pyruvate oxidase includes enzymes classified as EC 1.2.3.3.

[0130] "Catase" refers to an enzyme that converts hydrogen peroxide (H2O2) into H2O and O2. In applications where hydrogen peroxide is present or is a product of the process, catalase can be used to remove residual hydrogen peroxide. In some embodiments, catalase includes enzymes classified as EC 1.11.1.6.

[0131] The terms “protein,” “polypeptide,” and “peptide” may be used interchangeably herein to refer to polymers of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). This definition includes D-amino acids and L-amino acids, mixtures of D-amino acids and L-amino acids, and polymers comprising D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids.

[0132] "Fusion protein," "chimeric protein," and "chimera" refer to hybrid proteins produced by the conjugation of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are produced using recombinant technologies (e.g., molecular biology techniques known in the art).

[0133] Amino acids are referred to in this document by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides are referred to by their generally accepted single-letter codes. The abbreviations used for gene-encoding amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When using three-letter abbreviations, amino acids can be L- or D-configurations with respect to the α-carbon (Cα) unless specifically preceded by "L" or "D" or clearly indicated from the context of the abbreviation. For example, "Ala" represents alanine without specifying the α-carbon configuration, "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When using single-letter abbreviations, uppercase letters indicate amino acids with the L-configuration with respect to the α-carbon, and lowercase letters indicate amino acids with the D-configuration with respect to the α-carbon. For example, "A" represents L-alanine, and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or mixtures thereof), the sequence is conventionally presented in the order of amino (N) to carboxyl (C).

[0134] "Polynucleotide" and "nucleic acid" refer to two or more nucleotides covalently linked together. A polynucleotide can consist entirely of ribonucleotides (i.e., RNA), entirely of 2'-deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2'-deoxyribonucleotides. While nucleosides are typically linked together by standard phosphodiester bonds, polynucleotides can contain one or more non-standard bonds. Polynucleotides can be single-stranded or double-stranded, or can include both single-stranded and double-stranded regions. Furthermore, while polynucleotides are typically composed of naturally occurring nucleobases encoding nucleotides (i.e., adenine, guanine, uracil, thymine, and cytosine), they can contain one or more modified and / or synthetic nucleotides, such as inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleotides are nucleotides encoding amino acid sequences.

[0135] The abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise specified, the abbreviation for nucleosides can be either ribonucleoside or 2'-deoxyribonucleoside. Nucleosides can be designated as ribonucleoside or 2'-deoxyribonucleoside on a single basis or on an aggregate basis. When a nucleic acid sequence is represented by a string of single-letter abbreviations, the sequence is presented in a 5' to 3' orientation according to common convention, and phosphate groups are not indicated.

[0136] "Nucleobase" or "base" refers to a naturally occurring or synthetic heterocyclic moiety known to humankind, which is used in the production of polymers capable of hybridizing with polynucleotides in a sequence-specific manner using nucleic acid or polynucleotide technologies. Non-limiting examples of nucleobases include, in particular, adenine, cytosine, guanine, thymine, uracil, 5-propynyluracil, 2-thio-5-propynyluracil, 5-methylcytosine, pseudoisocytosine, pseudoisouridine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deazoguanine), N9-(7-deazo-8-azaguanine), and N8-(7-deazo-8-azaaadenine).

[0137] "Nucleoside" refers to a glucosylamine comprising a nucleobase and a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. Conversely, the term "nucleotide" refers to a glycosamine comprising a nucleobase, a 5-carbon sugar, and one or more phosphate groups, as further described herein. In some embodiments, nucleosides may be phosphorylated by a kinase to produce nucleotides.

[0138] "Nucleoside monophosphate" or "NMP" refers to a glucosylamine 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. In some contexts, "nucleoside" and "nucleotide" are used interchangeably.

[0139] "Nucleoside diphosphate" or "NDP" refers to a glucosylamine comprising a nucleobase, a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose), and a diphosphate (i.e., pyrophosphate) moiety at the 5'-position. In some embodiments herein, "nucleoside diphosphate" is abbreviated as "NDP". Non-limiting 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. In some contexts, "nucleoside" and "nucleotide" are used interchangeably.

[0140] "Nucleoside triphosphate" or "NTP" refers to a glycosylamine comprising a nucleobase, a 5-carbon sugar (e.g., ribose, deoxyribose, or arabinose), 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. In some contexts, "nucleoside" and "nucleotide" are used interchangeably.

[0141] In the context of nucleosides or nucleotides, "modified" or "non-natural" refers to a nucleoside or nucleotide that has been altered to be a non-naturally occurring nucleoside or nucleotide. In some embodiments, modifications to the nucleoside or nucleotide include modifications to the nucleobase, sugar moiety, and / or phosphate group. In some embodiments, the common modification of the 2'-position of the sugar residue with fluorine (F) or -O-CH3 is indicated by "fN" and "mN," respectively, where N represents a nucleobase on the nucleoside or nucleotide. In some embodiments, the presence of 5'-thiophosphate is indicated by "*N."

[0142] "Locked nucleoside" or "locked nucleotide" refers to a nucleoside or nucleotide whose ribose moiety is modified by a bridge connecting the 2' oxygen and 4' carbon (see, for example, Obika et al., Tetrahedron Letters, 1997, 38(50):8735–8738; Orum et al., Current Pharmaceutical Design, 2008, 14(11):1138–1142). In some embodiments, the bridge is a methylene bridge or an ethylene bridge. In some embodiments, the ribose moiety of the locked nucleoside or locked nucleotide is in a C3'-inner (β-D) or C2'-inner (α-L) conformation.

[0143] "Biocatalysis," "biocatalytic," "biotransformation," and "biosynthesis" refer to the use of enzymes to carry out chemical reactions of organic compounds.

[0144] "Wild-type" and "naturally occurring" refer to forms found in nature. For example, wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms, can be isolated from natural sources, and have not been intentionally modified by human manipulation.

[0145] When used with respect to cells, nucleic acids, or peptides, "recombinant," "engineered," "variant," "non-natural," and "not naturally occurring" refer to materials or materials corresponding to the natural or native form of such materials that have been modified in a manner not found in nature. In some embodiments, the cells, nucleic acids, or peptides are identical to naturally occurring cells, nucleic acids, or peptides, but are produced or derived from synthetic materials and / or through manipulation using recombinant technologies. Non-limiting examples include, in particular, recombinant cells that express genes not present in the natural (non-recombinant) form of the cell or that express natural genes that would otherwise be expressed at different levels.

[0146] The term "sequence identity percentage (%)" is used herein to refer to comparisons between polynucleotides or peptides, and is determined by comparing two optimally aligned sequences within a comparison window. For optimal alignment, a portion of the polynucleotide or peptide sequence within the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence. The percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears, or by aligning nucleic acid bases or amino acid residues with vacancies to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Those skilled in the art will understand that many established algorithms exist for aligning two sequences. As is known in the art, optimal alignment of sequences for comparison can be performed by any suitable method, including but not limited to Smith and Waterman’s local homology algorithm (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), Needleman and Wunsch’s homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), Pearson and Lipman’s similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or visual inspection. Examples of algorithms suitable for determining sequence identity and sequence similarity percentages include, but are not limited to, the BLAST and BLAST 2.0 algorithms 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 analysis is publicly available from the website of the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. When compared with words of the same length in the database sequence, the short word matches or satisfies a certain positive threshold score “T”. T is called the neighborhood word score threshold (see Altschul et al., ibid.).These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended as far as possible in both directions along each sequence, as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Word hit extension in each direction stops when: the cumulative alignment score decreases by an amount "X" from its maximum realized value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residues; 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) defaults to a word length of 11 (W), an expected value of 10 (E), M=5, N=-4, and a comparison of two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3 (W), an expected value of 10 (E), and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and sequence identity percentage can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) with the provided default parameters.

[0147] "Substantial identity" refers to a polynucleotide or polypeptide sequence having at least 80%, at least 85%, at least 89% to 95%, or more commonly, at least 99% sequence identity with a reference sequence over a comparison window of at least 20 residue positions, typically at least 30 to 50 residue positions. The percentage of sequence identity is calculated by comparing the reference sequence with sequences containing deletions or additions that together constitute 20% or less of the reference sequence over the comparison window. In some specific embodiments applied to polypeptides, the term "substantial identity" means that, when optimally aligned, for example, using the GAP or BESTFIT procedure with default vacancy weights, the two polypeptide sequences share at least 80% sequence identity, preferably at least 89%, at least 95%, or higher (e.g., 99%). In some embodiments, the different residue positions in the compared sequences differ due to conserved amino acid substitutions.

[0148] A “reference sequence” is a defined sequence used as the basis for sequence and / or activity comparisons. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) contain sequences similar to each other (i.e., a portion of the complete sequence) and (2) may also contain sequences different from each other, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides on 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, wherein the reference sequence is a sequence that may have one or more variations in the primary sequence.

[0149] A “comparison window” refers to a conceptual segment of adjacent nucleotide positions or amino acid residues, where the sequence can be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 consecutive nucleotides or amino acids, and wherein, for optimal alignment of the two sequences, the sequence portion within the comparison window may contain 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (which does not contain additions or deletions). In some embodiments, the comparison window may be longer than 15-20 consecutive residues, and optionally include windows of 30, 40, 50, 100, or longer.

[0150] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference," and "relative to" refer to specifying the number of residues in the reference sequence when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbering or position of a given polymer is specified relative to the reference sequence, not by the actual numerical position of residues within the given amino acid or polynucleotide sequence. For example, by introducing vacancies to optimize residue matching between two sequences, a given amino acid sequence (such as the amino acid sequence of an engineered adenosine kinase) can be aligned to a reference sequence. In these cases, despite the presence of vacancies, the numbering of residues in the given amino acid or polynucleotide sequence is relative to the reference sequence with which it is aligned.

[0151] "Amino acid difference," "residue difference," and "substitution" refer to the difference of an amino acid residue at a position in a polypeptide sequence relative to the corresponding amino acid residue at a position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X13 compared to SEQ ID NO: 2" refers to the difference of an amino acid residue at the polypeptide position corresponding to position 13 of SEQ ID NO: 2. Therefore, if the reference polypeptide of SEQ ID NO: 2 has methionine at position 13, then "residue difference at position X13 compared to SEQ ID NO: 2" refers to an amino acid substitution of any residue other than methionine at the polypeptide position corresponding to position 13 of SEQ ID NO: 2. In most cases herein, a specific amino acid residue difference at a position is denoted as "XnY," where "Xn" indicates the corresponding position as described above, and "Y" is a single-letter identifier of the amino acid present in the engineered polypeptide (i.e., ...). ， (Residues different from those in the reference polypeptide). In some cases (e.g.) ， In the tables provided in the embodiments, the present invention also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a single-letter identifier of a residue in the reference sequence, "n" is a residue position number in the reference sequence, and B is a single-letter identifier of a residue substitution in the engineered polypeptide sequence. In some cases, amino acid residue differences or substitutions may be missing and may be represented by "-". In some cases, the polypeptide of the present invention may contain one or more amino acid residue differences relative to the reference sequence, which are represented by a list of specified positions where residue differences exist relative to the reference sequence. In some embodiments, amino acid differences (e.g., substitutions) are represented by the abbreviation "nB" without an identifier of a residue in the reference sequence. In some embodiments, the phrase "amino acid residue nB" indicates the presence of an amino residue in the engineered polypeptide, the presence of which may or may not be a substitution in the context of the reference polypeptide or amino acid sequence.

[0152] In some cases, the polypeptide of this disclosure may include one or more amino acid residue differences relative to a reference sequence, indicated by a list of designated positions where residue differences exist relative to the reference sequence. In some embodiments, where more than one amino acid may be used at a particular residue position of the polypeptide, the various amino acid residues that may be used are separated by " / " (e.g., X13G / X13S, X13G / S, or 13G / S).

[0153] A “set of amino acid substitutions” or “substitution set” refers to a group of amino acid substitutions in a polypeptide sequence compared to a reference sequence. A substitution set may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions present in any of the variant adenosine kinases listed in the table provided in the examples. In these substitution sets, each substitution is separated by a semicolon (“;”; for example, Y29Q;A30S) or a forward slash (“ / ”; for example, Y29Q / A30S or 29Q / 30S).

[0154] "Conservative amino acid substitution" refers to the substitution of residues with different residues having similar side chains, and therefore generally involves the substitution of amino acids in a polypeptide with amino acids within the same or similar defined amino acid classes. 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 a hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is substituted with another amino acid with 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 basic 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 substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0155] "Non-conservative substitution" refers to the substitution of amino acids in a polypeptide with amino acids having significantly different side chain properties. Non-conservative substitutions can use amino acids between rather than within groups and affect (a) the structure of the peptide backbone in the substitution region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions can be acidic amino acids substituted with basic or aliphatic amino acids; aromatic amino acids substituted with small amino acids; and hydrophilic amino acids substituted with hydrophobic amino acids.

[0156] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, representing up to 10% or up to 20% of the total number of amino acids constituting the reference enzyme, while retaining the enzymatic activity of the engineered adenosine kinase and / or retaining the improved properties of the engineered adenosine kinase. Deletion may target internal and / or terminal portions of the peptide. In various embodiments, deletion may include continuous segments or may be discontinuous. Deletion is typically indicated by a "-" in the amino acid sequence.

[0157] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. Insertion can occur within the polypeptide itself, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions can be continuous segments of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0158] The terms “functional fragment” and “bioactive fragment” are used interchangeably herein and refer to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding position in the sequence being compared (e.g., the full-length engineered adenosine kinase of the present invention) and substantially retains all the activities of the full-length polypeptide.

[0159] "Isolated polypeptides" refers to polypeptides that are essentially associated with other naturally occurring contaminants (e.g., ， Polypeptides (proteins, lipids, and polynucleotides) isolated from their natural environment or expression systems (e.g., proteins, lipids, and polynucleotides). This term includes peptides that have been isolated from their natural environment or expression systems. ， A polypeptide that has been removed or purified (either within host cells or through in vitro synthesis). Recombinant adenosine kinase polypeptides can be present in cells, in cell culture media, or prepared in various forms (such as lysates or isolated preparations). Therefore, in some embodiments, the recombinant adenosine kinase polypeptide may be an isolated polypeptide.

[0160] "Essentially pure polypeptides" or "purified proteins" means that polypeptides are the main component of a protein (i.e.,...). ，A composition that is more abundant (based on molar or weight) than any other single macromolecule in the composition, and is typically substantially purified when the target substance contains at least about 50% of the macromolecule present in molar or weight %. However, in some embodiments, compositions containing adenosine kinase contain less than 50% (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%) of adenosine kinase. Typically, substantially pure adenosine kinase compositions contain 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 macromolecules present in the composition in molar or weight %. In some embodiments, the target substance is purified to substantially homogeneity (i.e., ， (Contaminants that cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecule. Solvents, small molecules (<500 Daltons), and elemental ions are not considered macromolecules. In some embodiments, the isolated recombinant adenosine kinase peptide is an essentially pure peptide composition.

[0161] "Improved properties of the enzyme" refers to at least one improved property of the enzyme. In some embodiments, the present invention provides engineered adenosine kinase peptides that exhibit any improvement in enzymatic properties compared to a reference adenosine kinase peptide and / or a wild-type adenosine kinase peptide and / or another engineered adenosine kinase peptide. Therefore, the level of "improvement" can be determined and compared among various adenosine kinase peptides (including wild-type adenosine kinase peptides and engineered adenosine kinase peptides). Improved properties include, but are not limited to, properties such as increased protein expression, increased thermal activity, increased thermal stability, increased pH activity, increased stability, increased enzyme activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or final 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 signature. In other embodiments, the term is used to refer to at least one improved property of adenosine kinase. In some embodiments, the present invention provides an adenosine kinase polypeptide that exhibits any improvement in enzymatic properties compared to a reference adenosine kinase polypeptide and / or a wild-type adenosine kinase polypeptide and / or another engineered adenosine kinase polypeptide. Therefore, the level of “improvement” can be determined and compared among various adenosine kinase polypeptides, including wild-type adenosine kinase polypeptides and engineered adenosine kinase polypeptides.

[0162] "Increased enzyme activity" and "enhanced catalytic activity" refer to the improved properties of engineered peptides, which can be assessed by comparing their specific activities (e.g., compared to a reference enzyme). ， An increase in the product / time / protein weight or the percentage of substrate converted to product (e.g., ， The term is expressed as an increase in the percentage of substrate converted to product using a specified amount of enzyme and starting material over a specified time period. In some embodiments, this term refers to the improved properties of the engineered adenosine kinase peptide provided herein, which can be expressed by comparing its specific activity (e.g., compared to a reference adenosine kinase) with improved properties. ， An increase in the product / time / protein weight or the percentage of substrate converted to product (e.g., ， The term "adenosine kinase" is expressed as the percentage increase in the conversion of a given amount of substrate to the product using a specified amount of adenosine kinase over a specified time period. In some embodiments, this term is used to refer to the improved adenosine kinase provided herein. Exemplary methods for determining the enzymatic activity of the engineered adenosine kinase of the present invention are provided in the embodiments. Any properties related to enzyme activity may be affected, including... K m , V max or k cat The classic enzyme properties, changes of which can lead to increased enzyme activity. For example, the improvement in enzyme activity can be from about 1.1 times the enzyme activity of the corresponding wild-type enzyme to 2, 5, 10, 20, 25, 50, 75, 100, 150, 200 or more times the enzyme activity of naturally occurring adenosine kinase or another engineered adenosine kinase derived from an adenosine kinase polypeptide.

[0163] "Conversion" refers to the enzymatic (or biotransformation) of a substrate into a corresponding product. "Conversion percentage" refers to the percentage of substrate converted into a product under specified conditions over a period of time. Therefore, the "enzyme activity" or "activity" of an adenosine kinase polypeptide can be expressed as the "conversion percentage" of substrate to product within a specific time period.

[0164] "Strict hybridization conditions" are used herein to refer to the conditions under which nucleic acid hybrids are stable. As is known to those skilled in the art, the stability of a hybrid is reflected in its melting temperature (…). T m The stability of hybrids is generally a function of ionic strength, temperature, G / C ratio, and the presence of a ionizing agent. Polynucleotides... T mThe value can be calculated using known methods for predicting melting temperatures (see, for example, 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 (erroneous, Nucl. Acids Res., (1991, 19:698); Sambrook et al., ibid.); Suggs et al., 1981, 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 to a complementary sequence encoding the engineered adenosine kinase of the present invention under defined conditions (such as moderately stringent or highly stringent conditions).

[0165] "Hybridization strictness" refers to the hybridization conditions in nucleic acid hybridization, such as washing conditions. Typically, hybridization reactions are performed under lower strictness conditions, followed by washing under different but higher strictness conditions (see, for example, 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 "moderate strictness hybridization" refers to conditions that allow target DNA to bind to a complementary nucleic acid that has approximately 60% identity with the target DNA, preferably approximately 75% or 85% identity, and greater than approximately 90% identity with the target polynucleotide. An exemplary moderate strictness condition is equivalent to hybridization at 42°C in 50% formamide, 5× Denhart solution, 5× SSPE, and 0.2% SDS, followed by washing at 42°C in 0.2× SSPE and 0.2% SDS. "High strictness hybridization" generally refers to conditions close to the pyrolysis temperature. T m Conditions of approximately 10°C or lower, such as those used in solution, are employed to determine specific polynucleotide sequences. In some embodiments, high stringency conditions refer to conditions that allow hybridization only of nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C (i.e.,...). ， If the hybrid is unstable in 0.018 M NaCl at 65 °C, it will be unstable under high-toughness conditions (as expected herein). For example, high-toughness conditions can be provided by hybridization at 42 °C in the equivalent of 50% formamide, 5× Denhart solution, 5× SSPE, and 0.2% SDS, followed by washing at 65 °C in 0.1× SSPE and 0.1% SDS. Another high-toughness condition is hybridization in the equivalent of hybridization at 65 °C in 5× SSC containing 0.1% (w / v) SDS, followed by washing at 65 °C in 0.1× SSC containing 0.1% SDS. Other high-toughness hybridization conditions, as well as moderate-toughness conditions, are described in the references cited above.

[0166] "Coding sequence" refers to the amino acid sequence of a nucleic acid (e.g., protein) that encodes a protein. ， Part of the gene.

[0167] "Codon optimization" refers to changing the codons of polynucleotides encoding proteins to codons that are preferentially used in a particular organism, so that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate, because most amino acids are represented by a few codons (called "synonyms"), codon usage in a particular organism is known to be non-random and biased towards specific codon triples. This codon preference may be even greater for a given gene, genes with a common function or ancestral origin, highly expressed proteins versus low-copy-number proteins, and aggregated protein-coding regions of an organism's genome. In some implementations, polynucleotides encoding adenosine kinase can be codon-optimized for optimal production in the selected host organism for expression.

[0168] When used alone or in combination, the terms "preferred," "optimal," and "high codon usage preference" are interchangeable, referring to codons used in protein-coding regions at a higher frequency than other codons encoding the same amino acid. Preferred codons can be determined based on codon usage in a single gene, a group of genes with a common function or origin, high-expression genes, codon frequencies in aggregated protein-coding regions of an entire organism, codon frequencies in aggregated protein-coding regions of related organisms, or combinations thereof. Codons whose frequency increases with gene expression levels are generally considered optimal codons for expression. Several methods are known to be available for determining codon frequencies in a specific organism (e.g., ， Codon usage (relative synonymous codon usage) and codon preference, including multivariate analysis, such as using cluster analysis or correspondence analysis, and the number of effective codons used in the gene (see, for example, GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 1998, 14:372-73; Stenico et al., Nucl. AcidsRes., 1994, 222437-46; and Wright, Gene, 1990, 87:23-29). Codon usage tables for many different organisms are available (see, for example, Wada et al., Nucl. Acids Res., 1992, 20:2111-2118; Nakamura et al., Nucl. Acids Res., 2000, 28:292; Duret et al., ibid.; Henaut and Danchin, published in...). Escherichia coli and SalmonellaNeidhardt et al. (eds.), ASM Press, Washington D.C., pp. 2047-2066 (1996). Data sources used to obtain codon usage can rely on any available nucleotide sequence capable of encoding a protein. These datasets include nucleic acid sequences that actually encode expressed proteins (e.g., complete protein-coding sequences - CDS), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences (see, for example, Mount, “Bioinformatics: Sequence and Genome Analysis,” Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Uberbacher, Meth. Enzymol., 1996, 266:259-281; ​​and Tiwari et al., Comput. Appl. Biosci., 1997, 13:263-270).

[0169] "Control sequence" herein refers to all components that are essential or advantageous for the expression of the polynucleotides and / or polypeptides of this application. Each control sequence may be native or exogenous for the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequence includes a promoter as well as transcription and translation termination signals.

[0170] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) relative to the polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide, and, where appropriate, directs or regulates the encoded polypeptide of interest.

[0171] A "promoter sequence" is a nucleic acid sequence that defines and / or initiates the expression of a polynucleotide of interest (such as a coding sequence). A promoter sequence contains a transcriptional control sequence that mediates the expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to those of the host cell.

[0172] "Heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences (e.g., promoter sequences, signal peptides, terminator sequences, etc.) that originate from different sources and are essentially unrelated.

[0173] "Vector" refers to a polynucleotide construct used to introduce a polynucleotide sequence into a cell. In some embodiments, a vector is an expression vector operatively linked to a suitable control sequence that enables the expression of the polynucleotide of interest in a suitable host, and, where appropriate, the expression of the encoded polypeptide in a suitable host.

[0174] "Expression" includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also covers peptide secretion from cells.

[0175] "Production" refers to the production of proteins and / or other compounds by cells. This term is intended to encompass any steps involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of peptides from cells.

[0176] “Cultivation” refers to the growth of a microbial cell population under any suitable conditions (e.g., using liquid, gel, or solid culture media).

[0177] "Host cell" and "host strain" refer to a suitable host containing an expression vector containing the DNA provided herein (e.g., a polynucleotide encoding a variant of adenosine kinase). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA technology, as is known in the art.

[0178] "pH stability" refers to an adenosine kinase peptide that maintains similar activity (e.g., greater than 60% to 80%) after exposure to high or low pH (e.g., 4.5 to 6 or 8 to 12) for a period of time (e.g., 0.5 hours to 24 hours) compared to untreated enzymes.

[0179] "Thermal stability" refers to an adenosine kinase peptide that maintains similar activity (e.g., greater than 60% to 80%) after being exposed to the same high temperature for a period of time (e.g., 0.5 hours to 24 hours) compared to a wild-type enzyme exposed to high temperatures (e.g., 40°C to 80°C).

[0180] "Solvent stability" refers to an adenosine kinase peptide that maintains similar activity (e.g., greater than 60% to 80%) after exposure to different concentrations (e.g., 5% to 99%) of solvents (ethanol, isopropanol, dimethyl sulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl adenosine, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5 hours to 24 hours) compared to a wild-type enzyme exposed to the same concentration of the same solvent.

[0181] "Thermal stability and solvent stability" refers to adenosine kinase peptides that possess both thermal stability and solvent stability.

[0182] "Breakselectivity" refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, meaning that one stereoisomer is formed more readily than the other, or it can be complete, meaning that only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is called enantiomerism, which is the fraction (usually reported as a percentage) of one enantiomer in the sum of the two enantiomers. This fraction (usually in percentage form) is typically reported in the art as an enantiomer excess ("ee"), which is calculated according to the following formula: [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity is called diastereoselectivity, which is the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers. It is usually reported alternatively as diastereoisomer excess (“de”). Enantiomer excess and diastereoisomer excess are types of stereoisomer excess.

[0183] "Regioselectivity" and "regioselective reaction" refer to a reaction in which one direction of bond formation or breaking is preferred over all other possible directions. If the distinction is complete, the reaction can be completely (100%) regioselective, and if the reaction product at one site is more dominant than the reaction products at other sites, the reaction can be substantially regioselective (at least 75%) or partially regioselective (x%), where the percentage is set according to the reaction of interest.

[0184] "Chemicoselectivity" refers to the phenomenon in chemical or enzymatic reactions where one product is formed preferentially over another.

[0185] "Suitable reaction conditions" refers to the conditions in the enzyme-catalyzed conversion reaction solution (e.g., ...). ，The range includes enzyme loading, substrate loading, temperature, pH, buffer solution, co-solvent, etc., under which conditions the adenosine kinase polypeptide of the present invention can convert the substrate into the desired product compound. Some exemplary “suitable reaction conditions” are provided herein.

[0186] "Loading capacity," such as "compound loading capacity" or "enzyme loading capacity," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0187] In the context of enzymatic conversion processes, "substrate" refers to a compound or molecule acted upon by an engineered enzyme (e.g., an engineered adenosine kinase polypeptide) provided herein.

[0188] In the context of enzymatic conversion reactions, "product" refers to a compound or molecule produced by the action of an enzymatic polypeptide on a substrate.

[0189] The yield of a product (e.g., nucleoside triphosphate or analogue) from a reaction is “increased” when a particular component present during the reaction (e.g., adenosine kinase) produces more products compared to a reaction under the same conditions, using the same substrate and other substituents, but without the component of interest.

[0190] If the amount of a particular enzyme is less than about 2%, about 1%, or about 0.1% (by weight) compared to the amount of other enzymes involved in the catalytic reaction, then that particular enzyme is "substantially absent".

[0191] "Fractional distillation" of liquids (e.g., culture media) refers to the application of separation processes (e.g., salt precipitation, column chromatography, size exclusion, and filtration) or combinations of such processes to provide a solution in which the percentage of the desired protein in the solution is greater than the percentage in the initial liquid product.

[0192] "alkyl" refers to a saturated hydrocarbon group having 1 to 18 carbon atoms, which is straight-chain or branched, more preferably having 1 to 8 carbon atoms, and most preferably having 1 to 6 carbon atoms. Alkyl groups having a specified number of carbon atoms are indicated in parentheses (e.g., (C1-C4)alkyl refers to an alkyl group having 1 to 4 carbon atoms).

[0193] "Alkenyl" refers to a straight-chain or branched group having 2 to 12 carbon atoms (including the terminal value), containing at least one double bond, but optionally more than one double bond.

[0194] "Alynyl" refers to a group having 2 to 12 carbon atoms (including terminal values), being straight-chain or branched, containing at least one triple bond, but optionally more than one triple bond, and additionally optionally containing one or more double bond moieties.

[0195] "Heteroalkyl", "heteroalkenyl", and "heteroynyl" refer to alkyl, alkenyl, and ynyl groups, as defined herein, in which one or more carbon atoms are independently replaced by the same or different heteroatoms or heteroatom groups. Heteroatoms and / or heteroatom groups that may replace carbon atoms include, but are not limited to, -O-, -S-, -SO-, -NR'-, -PH-, -S(O)-, -S(O)2-, -S(O)NR'-, -S(O)2NR'-, and so on, including combinations thereof, wherein each Rα is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0196] "Alkoxy" refers to the group -OR', where R' is an alkyl group as defined above, including other optionally substituted alkyl groups as defined herein.

[0197] "Amino" refers to the -NH group. I The substituted amino group refers to the groups –NHR', NR'R', and NR'R'R', where each R' is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cyclohexaalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, thioalkyl, sulfinyl, sulfonyl, etc. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfonylamino, etc.

[0198] "O" refers to =O.

[0199] "Oxygen group" refers to a divalent group -O-, which can have various substituents to form different oxygen groups, including ethers and esters.

[0200] The "carboxyl group" refers to -COOH.

[0201] "Carbonyl" refers to -C(O)-, which can have a variety of substituents to form different carbonyl groups, including acids, acyl halides, aldehydes, amides, esters, and ketones.

[0202] "Alkoxycarbonyl" refers to -C(O)OR', where R' is an alkyl group that can be optionally substituted as defined herein.

[0203] As used herein, “aminocarbonyl” refers to -C(O)NH2. A substituted aminocarbonyl group refers to –C(O)NR'R', where amino NR'R' is as defined herein.

[0204] "Halogen" and "halogenated group" refer to fluorine, chlorine, bromine and iodine.

[0205] "Hydroxy group" refers to -OH.

[0206] "Cyano" refers to -CN.

[0207] "Aryl" refers to an unsaturated aromatic carbocyclic group having 6 to 12 carbon atoms (including the terminal values) that has a monocyclic (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracene). Exemplary aryl groups include phenyl, pyridyl, naphthyl, and so on.

[0208] "Heterocyclic," "heterocyclic," and the interchangeable "heterocyclic alkyl" refer to a saturated or unsaturated group having a monocyclic or multiple fused rings, comprising 2 to 10 (including terminal) carbon ring atoms and 1 to 4 (including terminal) heterocyclic atoms selected from nitrogen, sulfur, or oxygen. Such heterocyclic groups may have a monocyclic ring (e.g., piperidinyl or tetrahydrofuranyl) or multiple fused rings (e.g., indolinyl, dihydrobenzofuran, or quininecycloyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, inazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthroxaline, isothiazine, phenazine, isoxazine, phenoxazine, phenthiazine, imidazoline, piperidine, piperazine, pyrrole, indoline, etc.

[0209] "Heteroaryl" refers to an aromatic heterocyclic group having one to ten carbon atoms (including terminal values) and one to four heteroatoms (including terminal values) selected from oxygen, nitrogen, and sulfur. Such heteroaryl groups can have a monocyclic ring (e.g., pyridyl or furanyl) or multiple fused rings (e.g., indoleazinyl or benzothiophene).

[0210] "Heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group (i.e., a heteroaryl-alkyl group), preferably having one to six carbon atoms (including the terminal number) on the alkyl moiety and five to twelve ring atoms (including the terminal number) on the heteroaryl moiety. Examples of such heteroarylalkyl groups are pyridylmethyl, etc.

[0211] "Heteroarylene" refers to an alkenyl group substituted with a heteroarylene group (i.e., a heteroarylene-alkenyl group), preferably having 2 to 6 carbon atoms (including the terminal value) and the heteroarylene group having 5 to 12 ring atoms (including the terminal value).

[0212] "Heteroaryleneyl" refers to an alkynyl group substituted with a heteroaryle (i.e., a heteroarylene-alkynyl group), preferably having 2 to 6 carbon atoms (including the terminal value) and the heteroarylene moiety having 5 to 12 ring atoms (including the terminal value).

[0213] The term "membered ring" refers to any ring structure. The number preceding the term "membered" indicates the number of skeleton atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thioran are 6-membered rings, while cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

[0214] As used herein, "phosphate" refers to a functional group consisting of an orthophosphate ion (a phosphorus atom covalently bonded to four oxygen atoms). Orthophosphate ions typically have one or more hydrogen atoms or organic groups. The phosphate group or chain can be modified, as further described herein.

[0215] As used in this article, "phosphorylation" refers to the addition or presence of one or more phosphoryl groups (phosphorus atoms covalently bonded to three oxygen atoms).

[0216] "Thiophosphate" refers to a phosphate group in a phosphodiester bond, NMP, NDP, NTP, or NQP where a non-bridging oxygen is replaced by a sulfur atom. In some embodiments, nucleoside 5'-thiomonophosphate is referred to as NMP-S or NMPαS. In some embodiments, nucleoside-5'-1-thio(diphosphate) and nucleoside-5'-1-thio(triphosphate) are referred to as NDPαS or αS-NDP and NTPαS or αS-NTP, respectively. In some embodiments, nucleoside-5'-2-thio(diphosphate) and nucleoside-5'-2-thio(triphosphate) are referred to as NDPβS and NTPβS, respectively.

[0217] "Dithiophosphate" refers to a situation where two non-bridging oxygens in the phosphate group of a phosphate diester bond, NMP, NDP, NTP, or NQP are replaced by two sulfurs.

[0218] Unless otherwise stated, the hydrogen-occupied positions in the aforementioned groups may be further replaced by substituents, such as, but not limited to, hydroxyl, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluorine, chlorine, bromine, iodine, halogroup, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxyl, alkoxycarbonyl, formamido, substituted formamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamide, substituted sulfonamide, cyano, amino, substituted amino. Alkylamino, dialkylamino, aminoalkyl, acylamino, amido, aminooxime, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalen, arylynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalen, heteroarylynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrroleyl, piperidinyl, morpholino, heterocyclic compounds, (heterocyclic)oxy and (heterocyclic)alkyl; and preferably, the heteroatoms are oxygen, nitrogen and sulfur. It should be understood that when open valence bonds are present on these substituents, these open valence bonds can be further substituted by alkyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, and / or heterocyclic groups. When these open valence bonds are present on carbon, these open valence bonds can be further substituted by halogens and oxygen-bonded, nitrogen-bonded, or sulfur-bonded substituents. Furthermore, when multiple such open valence bonds are present, these groups can form rings by directly forming bonds or by forming bonds with new heteroatoms (preferably oxygen, nitrogen, or sulfur). It should also be understood that the above substitutions can be carried out under the premise that substituting hydrogen with a substituent does not introduce unacceptable instability into the molecules of the present invention, and that such substitution is otherwise chemically reasonable.

[0219] "Optional" and "optionally" mean that the event or situation subsequently described may or may not occur, and the description includes both scenarios in which the event or situation occurs and scenarios in which it does not occur. Those skilled in the art will understand that the description of any molecule containing one or more optional substituents means only of sterically feasible and / or synthetically feasible compounds.

[0220] "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" and "aryl" portions of the molecule may or may not be substituted, and for the series "optionally substituted alkyl, cycloalkyl, aryl, and heteroaryl", the alkyl group, cycloalkyl group, aryl group, and heteroaryl group may or may not be substituted independently of each other.

[0221] Engineered adenosine kinase peptide In one aspect, this disclosure provides an adenosine kinase engineered to have improved properties compared to the parental adenosine kinase. In some embodiments, the engineered adenosine kinase exhibits, in particular, increased activity against nucleoside monophosphate (NMP) substrates, increased thermostability, and / or increased activity against modified NMP substrates. The engineered adenosine kinase can be used to convert nucleoside monophosphate substrates into their corresponding nucleoside diphosphate products, especially in enzyme cascade systems.

[0222] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to amino acid residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 2-1302, 1356-2652, and 2676-3192, or 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 higher sequence identity with the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 2-1302, 1356-2652, and 2676-3192, wherein the amino acid sequence comprises an amino acid sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 83%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0223] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104. The reference sequences corresponding to these sequences have 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 higher sequence identity, wherein the amino acid sequences contain amino acid sequences relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0224] In some embodiments, the engineered adenosine kinase comprises an amino acid sequence that corresponds to a reference sequence of amino acid residues 12-231 of SEQ ID NO: 2, or has 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 higher sequence identity with respect to the reference sequence of amino acid residues 12-231 of SEQ ID NO: 2, or one or more substitutions thereof.

[0225] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 4. An amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or one or more substitutions to the reference sequence corresponding to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2.

[0226] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to amino acid residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, or 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 higher sequence identity with the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2.

[0227] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions at at least the following amino acid positions: 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0228] In some embodiments, the engineered adenosine kinase contains at least the following substitutions or amino acid residues in its amino acid sequence: 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y , 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S , 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71T, 73I / R, 74C / G / Q / S, 75L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81F / I / L / Q / S, 82I / L / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 97I, 98A / G / Q, 99A / C, 100F / S / V, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 110C / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S, 119F / K / L / P / R / S,120G / K / L / R / S / T、122A / H / S、123S、124V、125I、126A / E / S / V、127I / L / P / S、128C / E / I / K / N / R / S、129I / L / P / S、131A / G / V、132G / K / L / T / V、133A / E / F / L / Q / S / V / W、134L、135I / K / M / P / R / S、136A / I / L / S / V / Y、137L、138C / I / M / V、139A / H / L / R、140G、141G / V、142F / L / M / W、143A / C / G / P / R / S / T / V、146D / H / N / R / V、148F / H / M / P / Q / R / S / T、149L、150C / G / L / P / S / V、151A / F / R / Y、152F / H、153C / K / S / V、154Q / R、155A / F / T / W、156A / C / N / T / V、157P / V、161G / L、162G、163G / Q / S、166C / F / L / P / S、168G / L / N / Q / S、169A / D / I / Y、170A / G / H / P / R / S、172A / H / M / S / T、173F / K / R / S / T / V、175F / L / S、178E / G / N / R、179A / C / G / I / L / P / V、180G / H / P、181C / I / V、182A / G / I / L / Q / S、183A / L / Q、184I / K / M / N / R / S / T / V、186L、187G / Y、188G / L、190A / C / E / G / H / N / Q / R、191D / K、192A / H / I / W、194F / L / R / V / Y、195G / I / M / R、196I、197A / L / Q / V、198G、199R、200A、201A / F / K / L / S、202E / G / M / S、203A / E / L / R、204R / S、205L / P、206W、207A / L / T、208E、210V、211A、212C / H / I / L / M / N / P / Q / R / S / W、213L / P / Q / S、214A / E / L / M / P / R / T / W、215A / P / V、216A / D / E / G / H / L / M / N / P / R / S / T / V、217E / G / H / P / T、218A / I / L、219A / F / G / L / P / S / T / W、220E / V、221D、222I / T、223T、224G / S / T、225A / L / Q / R / T / V、226K / L / P / R / S / T、227P、228D / F / G / I / L / P / Q / R / S / T / V、229C / I、230A / M / P / Q or 231A / Q / R / T, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0229] In some embodiments, the engineered adenosine kinase amino acid sequence includes at least the following substitutions: G11D, M13G / S, A14C / E, Y15F, L16M, F18C / E / L, G20A / P / R / T / V, P21L / N / R / S / T / V, P22A / S, K26L, G27C / S, Y29Q / R, A30G / L / S / Y, K31R, I32F / I / L / N, Q34A / E / S / V, E35A / F / L / S, K36A / E / I / L / M / Q / R / S / V / Y, T37F / G / I / L / R / Y, G38C / F / L / R, I39A / M / Q / T, P40C / F / L / S / T. I42T, S43A / C / G / H / N, T44S, D46E / F / L / M / Y, F48L, D50A / C / L / N / Q / R / S / V, I5 1F / G / K / L / M / R / T, V52A / H, K53A / M / S, K54G / H / I / M / Q / R / T, E55G / L / Q / S, N56 E / Q / T, D57H / L / P / S, E58C / G / Y, L59E / G / H / M / P / R / T / V / W / Y, G60A / C / P / S / W, K61A / E / N / P / S / V, K62A / E / G / I / L / S, I63S, K64R, E65A / D / F / K / P / T, I66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W、M67R、E68A / D / G / I / L / Q / R / S / T / V / W / Y、K69F / L / Q / R / Y, G70R, E71T, V73I / R, P74C / G / Q / S, D75L, E76G / L / P, L77A / I / M / Q / S , V78C / G / N / T, N79A / K / L / P / W, E80A / D / G / L / N / P / R / S / V / W, V81F / I / L / Q / S, V 82I / L / P / Q / T, K83G / H / L / S / T, R84A / E / H / M / S, S87A / E / I / K / L / M / R / V / Y, E88 N / R / T, K89A / H / I / L / P / Q / T / V, D90C / E / F / K / S / T / V, C91A / F / G / L / S / V, E92L / S, K93A / E / G / P / S / T / V / Y, G94A / C / F / M / Q / S / T / V / Y, L97I, D98A / G / Q, G99A / C , Y100F / S / V, P101-, R102A / C / N, V104A / F / H / I / L / Q / R / S / W, A105G / K / L / M / R / S, E108M / R / S / V, F109E / Y, L110C / F, D111E / P / R, S112A / C / E / K / M / N / Q / R / T,F113E / W / Y、E115G / K / R、S116A / E / F / L、Q117L / N / S、N118A / G / L / R / S、K119F / K / L / P / R / S、Q120G / K / L / R / S / T、T122A / H / S、A123S、A124V、V125I、L126A / E / S / V、F127I / L / P / S、D128C / E / I / K / N / R / S、V129I / L / P / S、E131A / G / V、D132G / K / L / T / V、V133A / E / F / L / Q / S / W、V134L、V135I / K / M / P / R / S、Q136A / I / L / S / V / Y、R137L、L138C / I / M / V、T139A / H / L / R、S140G、R141G / V、R142F / L / M / W、I143A / C / G / P / R / S / T / V、K146D / H / N / R / V、G148F / H / M / P / Q / R / S / T、R149L、I150C / G / L / P / S / V、Y151A / F / R、N152F / H、M153C / K / S / V、I154Q / R、S155A / F / T / W、L156A / C / N / T / V、P157V、D161G / L、E162G、L163G / Q / S、D166C / F / L / P / S、K168G / L / N / Q / S、V169A / D / I / Y、K170A / G / H / P / R / S、V172A / H / M / S / T、Q173F / K / R / S / T / V、D175F / L / S、K178E / G / N / R、E179A / C / G / I / L / P / V、E180G / H / P、T181C / I / V、V182A / G / I / L / Q / S、R183A / L / Q、H184I / K / M / N / R / S / T / V、Y186L、K187G / Y、V188G / L、L190A / C / E / G / H / N / Q / R、E191D / K、K192A / H / I / W、Q194F / L / R / V / Y、P195G / I / M / R、V196I、I197A / L / Q / V、D198G、Y199R、Y200A、G201A / F / K / L / S、K202E / G / M / S、K203A / E / L / R、G204R / S、I205L / P、L206W、K207A / L / T、R208E、D210V、G211A、T212C / H / I / L / M / N / P / Q / R / S / W、I213L / P / Q / S、G214A / E / L / M / P / R / T / W、I215A / P / V、D216A / D / E / G / H / L / M / N / P / R / S / T / V、N217E / G / H / P / T、V218A / I / L, V219A / F / G / L / P / S / T / W, A220E / V, E221D, V222I / T, L223T, K224G / S / T, I225A / L / Q / R / T / V, I226K / L / P / R / S / T, G227P, W228D / F / G / I / L / P / Q / R / S / T / V, S229C / I, D230A / M / P / Q or K231A / Q / R / T, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0230] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions at at least the following amino acid positions: 13, 18, 29, 30, 31, 32, 34, 35, 48, 50, 53, 55, 59, 60, 61, 62, 66, 68, 77, 78, 80, 83, 87, 100, 104, 105, 109, 118, 119, 127, 128, 133, 136, 139, 142, 143, 150, 151, 155, 157, 168, 170, 175, 184, 186, 190, 201, 207, 212, 213, 214, 216, 217, 218, 224, or 226, or combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0231] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or amino acid residues: 13G / S, 18C / E / L, 29R / Q, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 48F, 50A / C / L / N / Q / R / S / V, 53A / M / S, 55G / L / Q / S, 59E / G / H / L / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 77A / I / M / Q / S, 78C / G / N / T, 80A / D / G / L / N / P / R / S / 119 F / K / L / P / R / S, 127I / L / P / S, 128C / E / I / K / N / R / S, 133A / E / F / L / Q / S / V / W, 136A / I / L / S / V / Y, 139A / H / L / R, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 150C / G / L / P / S / V, 151A / F / R / Y, 155A / F / T / W, 157P / V, 168G / L / N / Q / S, 170A / G / H / P / R / S, 175F / L / S, 184I / K / M / N / R / S / T / V, 186L, 190A / C / E / G / H / N / Q / R, 201A / F / K / L / S, 207A / L / T, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 216A / E / G / H / L / M / N / P / R / S / T / V / W, 217E / G / H / P / T, 218A / I / L, 224G / S / T or 226K / L / P / R / S / T, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0232] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or amino acid residues: 13G, 18L, 29Q, 30G / S, 31R, 32I, 34S, 35A, 48L, 50Q, 53A, 55Q, 59V, 60A, 61S, 62S, 66W, 68L, 77I, 78G, 80A / P, 83L, 87K, 100F, 104A, 105R, 109Y, 118G, 119R, 127I / L, 128E / K, ​​133E, 136A / V, 139L, 142L / R, 143V, 150S, 151F, 155F, 157V, 168G, 170G / S, 175F / L, 184M, 186L, 190Q / R, 201A / S, 207A, 212S, 213Q, 214E, 216E / M / P / R, 217P, 218I, 224G or 226L, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0233] In some embodiments, the engineered adenosine kinase contains at least the following substitutions in its amino acid sequence: M13G, F18L, Y29Q, A30G / S, K31R, R32I, Q34S, E35A, F48L, D50Q, K53A, E55Q, L59V, G60A, K61S, K62S, I66W, E68L, L77I, V78G, E80A / P, K83L, S87K, Y100F, V104A, A105R, F109Y, N118G, K119R, F127I / L, D 128E / K, ​​V133E, Q136A / V, T139L, R142L / R, I143V, I150S, Y151F, S155F, P157V, K168G, K170G / S, D175F / L, H184M, Y186L, L190Q / R, G201A / S, K207A, T212S, I213Q, G214E, D216E / M / P / R, N217P, V218I, K224G or I226L, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0234] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 29, 30, 128, 142, 186, 212, or 213, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 29Q, 30S, 128K, 142L, 186L, 212S, or 213Q, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: Y29Q, A30S, D128K, R142L, Y186L, T212S, or I213Q, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0235] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set at the following amino acid positions: 29 / 30 / 128 / 142 / 186 / 212 / 213, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 29Q / 30S / 128K / 142L / 186L / 212S / 213Q, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following set of substitutions: Y29Q / A30S / D128K / R142L / Y186L / T212S / I213Q, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0236] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 155 or 226, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 155F or 226L, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: S155F or I226L, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0237] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 155 / 226, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 155F / 226L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: S155F / I226L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0238] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 78, 80, or 224, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 78G, 80A, or 224G, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: V78G, E80A, or K224G, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0239] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 78 / 80 / 224, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 78G / 80A / 224G, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: V78G / E80A / K224G, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0240] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions at amino acid positions: 60 or 170, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 60A or 170G, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: G60A or K170G, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0241] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 60 / 170, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 60A / 170G, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: G60A / K170G, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0242] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions at amino acid positions: 55 or 133, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 55Q or 133E, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: E55Q or V133E, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0243] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 55 / 133, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residue: 55Q / 133E, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: E55Q / V133E, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0244] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions at amino acid positions: 61 or 201, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 61S or 201S, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: K61S or G201S, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0245] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 61 / 201, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 61S / 201S, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: K61S / G201S, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0246] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 13, 30, 31, 53, 109, 119, or 168, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 13G, 30G, 31R, 53A, 109Y, 119R, or 168G, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence includes at least the following substitutions: M13G, S30G, K31R, K53A, F109Y, K119R, or K168G, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0247] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set at the following amino acid positions: 13 / 30 / 31 / 53 / 109 / 119 / 168, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 13G / 30G / 31R / 53A / 109Y / 119R / 168G, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following set of substitutions: M13G / S30G / K31R / K53A / F109Y / K119R / K168G, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0248] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 34, 35, or 105, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 34S, 35A, or 105R, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: Q34S, E35A, or A105R, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0249] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 34 / 35 / 105, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 34S / 35A / 105R, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: Q34S / E35A / A105R, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0250] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the substitutions at the following amino acid positions: 59 or 139, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 59V or 139L, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: L59V or T139L, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0251] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 59 / 139, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 59V / 139L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: L59V / T139L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0252] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions at amino acid positions: 48 or 100, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 548L or 100F, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: F48L or Y100F, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0253] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following substitution set: 48 / 100, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following substitution set or amino acid residues: 48L / 100F, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following substitution set: F48L / Y100F, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0254] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 32, 127, or 175, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 32I, 127I, or 175L, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: R32I, F127I, or D175L, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0255] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 32 / 127 / 175, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 32I / 127I / 175L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: R32I / F127I / D175L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0256] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 62, 128, or 190, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 62S, 128E, or 190R, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: K62S, K128E, or L190R, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0257] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 62 / 128 / 190, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 62S / 128E / 190R, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: K62S / K128E / L190R, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0258] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 136, 150, or 217, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 136A, 150S, or 217P, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions: Q136A, I150S, or N217P, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0259] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 136 / 150 / 217, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 136A / 150S / 217P, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: Q136A / I150S / N217P, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0260] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 68, 142, 207, 216, or 218, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 68L, 142R, 207A, 216P, or 218I, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase includes at least the following substitutions: E68L, L142R, K207A, D216P, or V218I, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0261] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following set of substitutions: 68 / 142 / 207 / 216 / 218, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following set of substitutions or amino acid residues: 68L / 142R / 207A / 216P / 218I, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following set of substitutions: E68L / L142R / K207A / D216P / V218I, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0262] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 18, 118, or 170, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 18L, 118G, or 170S, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: F18L, N118G, or G170S, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0263] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 18 / 118 / 170, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 18L / 118G / 170S, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: F18L / N118G / G170S, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0264] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 77, 143, 214, or 216, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 77I, 143V, 214E, or 216R, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: L77I, I143V, G214E, or P216R, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0265] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 77 / 143 / 214 / 216, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 77I / 143V / 214E / 216R, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: L77I / I143V / G214E / P216R, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0266] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 50, 184, or 216, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 50Q, 184M, or 216M, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: D50Q, H184M, or R216M, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0267] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 50 / 184 / 216, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 50Q / 184M / 216M, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: D50Q / H184M / R216M, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0268] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 104, 127, 136, or 175, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 104A, 127L, 136V, or 175F, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: V104A, I127L, A136V, or L175F, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0269] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set at the following amino acid positions: 104 / 127 / 136 / 175, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 104A / 127L / 136V / 175F, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: V104A / I127L / A136V / L175F, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0270] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 151 or 157, or a combination thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 151F or 157V, or a combination thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: Y151F or P157V, or a combination thereof, wherein said amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0271] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 151 / 157, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 151F / 157V, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: Y151F / P157V, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0272] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at least at the following amino acid positions: 83, 190, 201, or 216, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 83L, 190Q, 201A, or 216E, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: K83L, R190Q, S201A, or M216E, or combinations thereof, wherein said amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0273] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: 83 / 190 / 201 / 216, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set or amino acid residues: 83L / 190Q / 201A / 216E, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitution set: K83L / R190Q / S201A / M216E, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. 151 / 157 In some embodiments, the engineered adenosine kinase amino acid sequence contains substitutions or sets of substitutions at at least the following amino acid positions: 29 / 30 / 128 / 142 / 186 / 212 / 213, 18 / 142 / 212 / 213, 29 / 30 / 128 / 142 / 186, 40 / 135 / 214, 18 / 29 / 30 / 127 / 150, 82, 69, 118, 29 / 30, 206, 32, 16, 104, 111 / 135 / 136 / 214, 29 / 128 / 175, 178, 18 / 128 / 142, 32 / 108 / 111 / 156 / 191, 18 / 29 / 128 / 129 / 142 / 186, 32 / 133, 18 / 29 / 30 / 212 / 213 / 222 29 / 65 / 127, 32 / 40 / 43 / 52 / 155 / 156 / 217, 18 / 29 / 128 / 129, 80, 32 / 43 / 138 / 152 / 191 / 214 / 215, 183, 18 / 175 / 186 / 212 / 213, 129, 18 / 29 / 30 / 65 / 129 / 142 / 150 / 186, 127 / 142 / 212 / 213, 30 / 65 / 142 / 222, 40 / 43 / 215, 136, 29 / 150 / 211 / 212 / 213 / 221, 43 / 111 / 136 / 219, 226, 88, 140 / 215 / 216, 32 / 133 / 134 or 18 / 29 / 128 / 186, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0274] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 29Q / 30S / 128K / 142L / 186L / 212S / 213Q, 18L / 142L / 212S / 213Q, 29Q / 30S / 128K / 142L / 186L, 40C / 135P / 214P, 18L / 29Q / 30S / 127L / 150S, 82I, 69Q, 118G, 2 9Q / 30S, 206W, 32N, 16M, 104I, 111E / 135P / 136A / 214P, 29Q / 128K / 175S, 178N, 18L / 128K / 142L, 32N / 108 M / 111E / 156N / 191K、18L / 29Q / 128K / 129I / 142L / 186L、32N / 133L、18L / 29Q / 30S / 212S / 213Q / 222I、29Q / 65K / 127L, 32N / 40C / 43G / 52H / 155F / 156N / 217T, 18L / 29Q / 128K / 129I, 80N, 32N / 43G / 138I / 152H / 191K / 214P / 215V, 183Q, 18L / 175S / 186L / 212S / 213Q, 129I, 18L / 29Q / 30S / 65K / 129I / 142L / 150S / 186L, 127L / 142L / 212S / 213Q, 30S / 65K / 142L / 222I, 40C / 43G / 215V, 136A, 29Q / 150S / 211A / 212S / 213Q / 221D, 43G / 111E / 136A / 219W, 226L, 88N, 140G / 215V / 216A, 32N / 133L / 134L or 18L / 29Q / 128K / 186L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0275] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: Y29Q / A30S / D128K / R142L / Y186L / T212S / I213Q, F18L / R142L / T212S / I213Q, Y29Q / A30S / D128K / R142L / Y186L, P40C / V135P / G214P, F18L / Y29Q / A30S / F127L / I150S, V82I, K69Q, N118G, Y29Q / A30S, L2 06W, R32N, L16M, V104I, D111E / V135P / Q136A / G214P, Y29Q / D128K / D175S, K178N, F18L / D128K / R142L, R32N / E108M / D111E / L156N / E191K, F18L / Y29Q / D128K / V129I / R142L / Y186L, R32N / V133L, F18L / Y29Q / A30S / T212S / I213Q / V222I, Y29Q / E65K / F 127L, R32N / P40C / S43G / V52H / S155F / L156N / N217T, F18L / Y29Q / D128K / V129I, E80N, R32N / S43G / L138I / N152H / E191K / G21 4P / I215V, R183Q, F18L / D175S / Y186L / T212S / I213Q, V129I, F18L / Y29Q / A30S / E65K / V129I / R142L / I150S / Y186L, F127L / R 142L / T212S / I213Q, A30S / E65K / R142L / V222I, P40C / S43G / I215V, Q136A, Y29Q / I150S / G211A / T212S / I213Q / E221D, S43G / D111E / Q136A / V219W, I226L, E88N, S140G / I215V / D216A, R32N / V133L / V134L or F18L / Y29Q / D128K / Y186L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0276] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the amino acid positions shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0277] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes at least one substitution shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0278] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the amino acid positions shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0279] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the substitutions or sets of substitutions of the engineered adenosine kinase variants shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0280] In some implementations, the engineered adenosine kinase comprises substances included in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, and 42. The reference amino acid sequence of the engineered adenosine kinase variant shown in .2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2 and 49.2 has 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 higher sequence identity, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0281] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to residues 12-231 of SEQ ID NO: 4. The reference sequences corresponding to 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104 have amino acid sequences with 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 higher sequence identity.

[0282] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, or 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 higher sequence identity to the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192.

[0283] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to residues 12-231 of SEQ ID NO: 4. An amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0284] In some embodiments, the engineered adenosine kinase comprises a reference sequence corresponding to residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, or 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%, or 99% or higher sequence identity with the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, wherein the amino acid sequence comprises an amino acid sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 83%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to SEQ ID NO: 4. The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0285] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions at at least the following amino acid positions: 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 188, 190 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231, or combinations thereof, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

[0286] In some embodiments, the engineered adenosine kinase contains at least the following substitutions or amino acid residues in its amino acid sequence: 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y , 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S , 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71T, 73I / R, 74C / G / Q / S, 75L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81F / I / L / Q / S, 82I / L / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 97I, 98A / G / Q, 99A / C, 100F / S / V, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 110C / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S, 119F / K / L / P / R / S,120G / K / L / R / S / T、122A / H / S、123S、124V、125I、126A / E / S / V、127I / L / P / S、128C / E / I / K / N / R / S、129I / L / P / S、131A / G / V、132G / K / L / T / V、133F / Q / S / V / W、133A / E / F / L / Q / S / V / W、134L、135I / K / M / P / R / S、136A / I / L / S / V / Y、137L、138C / I / M / V、139A / H / L / R、140G、141G / V、142F / L / M / W、143A / C / G / P / R / S / T / V、146D / H / N / R / V、148F / H / M / P / Q / R / S / T、149L、150C / G / L / P / S / V、151A / F / R / Y、152F / H、153C / K / S / V、154Q / R、155A / F / T / W、156A / C / N / T / V、157P / V、161G / L、162G、163G / Q / S、166C / F / L / P / S、168G / L / N / Q / S、169A / D / I / Y、170A / G / H / P / R / S、172A / H / M / S / T、173F / K / R / S / T / V、175F / L / S、178E / G / N / R、179A / C / G / I / L / P / V、180G / H / P、181C / I / V、182A / G / I / L / Q / S、183A / L / Q、184I / K / M / N / R / S / T / V、186L、187G / Y、188G / L、190A / C / E / G / H / N / Q / R、191D / K、192A / H / I / W、194F / L / R / V / Y、195G / I / M / R、196I、197A / L / Q / V、198G、199R、200A、201A / F / K / L / S、202E / G / M / S、203A / E / L / R、204R / S、205L / P、206W、207A / L / T、208E、210V、211A、212C / H / I / L / M / N / P / Q / R / S / W、213L / P / Q / S、214A / E / L / M / P / R / T / W、215A / P / V、216A / D / E / G / H / L / M / N / P / R / S / T / V、217E / G / H / P / T、218A / I / L、219A / F / G / L / P / S / T / W、220E / V、221D、222I / T、223T、224G / S / T、225A / L / Q / R / T / V、226K / L / P / R / S / T、227P、228D / F / G / I / L / P / Q / R / S / T / V、229C / I、230A / M / P / Q or 231A / Q / R / T, or combinations thereof, wherein the amino acid position corresponds to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4. The reference sequences corresponding to 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

[0287] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions at at least the following amino acid positions: 13, 18, 29, 30, 31, 32, 34, 35, 48, 50, 53, 55, 59, 60, 61, 62, 66, 68, 77, 78, 80, 83, 87, 100, 104, 105, 109, 118, 119, 127, 128, 133, 136, 139, 142, 143, 150, 151, 155, 157, 168, 170, 175, 184, 186, 190, 201, 207, 212, 213, 214, 216, 217, 218, 224, or 226, or combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, or the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

[0288] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or amino acid residues: 13G / S, 18C / E / L, 29R / Q, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 48F, 50A / C / L / N / Q / R / S / V, 53A / M / S, 55G / L / Q / S, 59E / G / H / L / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 77A / I / M / Q / S, 78C / G / N / T, 80A / D / G / L / N / P / R / S / V / W, 83G / H / L / S / T, 87A / E / I / K / L / M / R / V / Y, 100F / S / V, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 109E / Y, 118A / G / L / R / S, 119F / K / L / P / R / S, 127I / L / P / S, 128C / E / I / K / N / R / S, 133A / E / F / L / Q / S / V / W, 136A / I / L / S / V / Y, 139A / H / L / R, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 150C / G / L / P / S / V, 151A / F / R / Y, 155A / F / T / W, 157P / V, 168G / L / N / Q / S, 170A / G / H / P / R / S, 175F / L / S 184I / K / M / N / R / S / T / V, 186L, 190A / C / E / G / H / N / Q / R, 201A / F / K / L / S, 207A / L / T, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 216A / E / G / H / L / M / N / P / R / S / T / V / W, 217E / G / H / P / T, 218A / I / L, 224G / S / T or 226K / L / P / R / S / T, or combinations thereof.The amino acid positions described herein are relative to the reference sequences corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or relative to the reference sequences corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

[0289] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or amino acid residues: 13G, 18L, 29Q, 30G / S, 31R, 32I, 34S, 35A, 48L, 50Q, 53A, 55Q, 59V, 60A, 61S, 62S, 66W, 68L, 77I, 78G, 80A / P, 83L, 87K, 100F, 104A, 105R, 109Y, 118G, 119R, 127I / L, 12 8E / K, ​​133E, 136A / V, 139L, 142L / R, 143V, 150S, 151F, 155F, 157V, 168G, 170G / S, 170S, 175F / L, 184M, 186L, 190Q / R, 201A / S, 207A, 212S, 213Q, 214E, 216E / M / P / R, 217P, 218I, 224G or 226L, or combinations thereof, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

[0290] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 4.

[0291] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 86-372, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0292] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 100 / 101, 94, 179, 198, 57, 78, 68, 93, 77, 62, 14, 51, 118, 91, 163, 65, 173, 224, 66, 109, 60, 40 / 88, 191, 63, 68 / 170, 100, 108, 58, 180, 178, 199, 40, 80 / 203, 40 / 43, 39, 55, 110, 51 / 59, 155 / 226, 80, 219, 166, 202, 59, 38, 90, 80 / 184 / 203, 35, 22 2, 54, 111 / 153 / 155, 112, 175, 74, 105, 170, 117, 128, 61, 119, 155, 79, 220, 226, 108 / 155, 108 / 111, 111, 69 / 88, 43, 43 / 69 / 138, 40 / 43 / 88 / 134 / 178, 214, 80 / 82 / 184 / 219, 88 / 136, 16 / 40 / 43 / 88, 40 / 134, 88, 111 / 155, 223, 115, 190, 201, 126, 56, 216, 76, 69, 120, 116, 203, 194 or 138, wherein the amino acid position is relative to SEQ. The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0293] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 100V / 101-, 94S, 179L, 198G, 57P, 78G, 68R, 93V, 77S, 62L, 14C, 51R, 118R, 91F, 163G, 65P, 173R, 224G, 66E, 109E, 60S, 40C / 88N, 191D, 63S, 68D / 170P, 100S, 51M, 108S, 58G, 57H, 180G, 178R, 199R, 40C, 94 Q, 80N / 203A, 40C / 43G, 39T, 62S, 40L, 55Q, 110C, 51R / 59R, 155F / 226L, 80N, 219T, 166P, 60P, 202S, 59T, 80A, 38L, 94C, 62A, 51K, 90E, 59M, 80N / 184K / 203A, 35S, 222T, 178G, 40T, 54M, 111E / 153K / 155F, 62I, 80G, 112M, 175S, 74S, 14E, 57L, 51G, 94F, 68A, 105R , 170A, 117N, 128S, 40F, 112C, 94A, 61A, 68W, 202G, 180P, 119R, 119R, I66G, 179G, 155F, 59V, 79L, 220E, 51F, 93A, 226L, 108M / 15 5F, 108M / 111E, 111E, 69Q / 88N, 43G, 43G / 69Q / 138I, 40C / 43G / 88N / 134L / 178N, 214P, 80N / 82I / 184K / 219W, 88N / 136A, 16M / 40C / 43G / 88N, 40C / 134L, 88N, 111E / 155F, 223T, 74G, 115R, 39Q, 93E, 119L, 190N, 202M, 55S, 51L, 112Q, 94T, 66A, 201A, 201K, 201L, 66R, 128N, 214L, 126S, 56E, 179I, 216S, 76P, 69Y, 115G, 166C, 120L, 116L, 108V, 203E, 194F, 138M, 178E, or 38C, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0294] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: Y100V / P101-, G94S, E179L, D198G, D57P, V78G, E68R, K93V, L77S, K62L, A14C, I51R, N118R, C91F, L163G, E65P, Q173R, K224G, I66E, F109E, G60S, P40C / E88N, E191D, I63S, E68D / K170P, Y100S, I51M, E108S, E58G, D57H, E180G, K178R, Y199R, P40C, G94Q, E80 N / K203A, P40C / S43G, I39T, K62S, P40L, E55Q, L110C, I51R / L59R, S155F / I2 26L, E80N, V219T, D166P, G60P, K202S, L59T, E80A, G38L, G94C, K62A, I51K, D90E, L59M, E80N / H184K / K203A, E35S, V222T, K178G, P40T, K54M, D111E / M1 53K / S155F, K62I, E80G, S112M, D175S, P74S, A14E, D57L, I51G, G94F, E68A, A105R, K170A, Q117N, K128S, P40F, S112C, G94A, K61A, E68W, K202G, E180P, K119R, K119R, I66G, E179G, S155F, L59V, N79L, A220E, I51F, K93A, I226L, E 108M / S155F, E108M / D111E, D111E, K69Q / E88N, S43G, S43G / K69Q / L138I, P4 0C / S43G / E88N / V134L / K178N, G214P, E80N / V82I / H184K / V219W, E88N / Q136 A. L16M / P40C / S43G / E88N, P40C / V134L, E88N, D111E / S155F, L223T, P74G, E 115R, I39Q, K93E, K119L, L190N, K202M, E55S, I51L, S112Q, G94T, I66A, G20 1A, G201K, G201L, I66R, K128N, G214L, L126S, N56E, E179I, D216S, E76P, K69Y, E115G, D166C, Q120L, S116L, E108V, K203E, Q194F, L138M, K178E or G38C,The amino acid positions mentioned herein are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0295] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or one or more substitutions relative to the reference sequence corresponding to the reference sequence corresponding to SEQ ID NO: 172.

[0296] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 374-444, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or relative to the reference sequence corresponding to SEQ ID NO: 172.

[0297] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 66 / 78, 78 / 80 / 224, 78, 65 / 66, 78 / 224, 78 / 80 / 109, 40 / 60 / 94 / 226, 66 / 68, 78 / 80, 94 / 117 / 118 / 226, 68 / 109, 68, 94 / 179, 51 / 66 / 68 / 224, 94 / 117 51 / 66 / 68 / 78 / 224, 66, 118, 93 / 94 / 180, 109, 68 / 224, 40 / 62 / 93, 93, 51 / 66, 94 / 117 / 118, 198 / 220, 93 / 117, 117 / 118, 93 / 198, 68 / 173, 40 / 117, 224, 40 / 118 / 226, 93 / 94 or 40 / 62 / 118, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or relative to the reference sequence corresponding to SEQ ID NO: 172.

[0298] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 66E / 78G, 78G / 80A / 224G, 78G, 65P / 66E, 78G / 224G, 78G / 80A / 109E, 40F / 60S / 94S / 226I, 66A / 68R, 66E / 68R, 78G / 80A, 94S / 117N / 118R / 226I, 68R / 109E, 68R, 94S / 179L, 51R / 66E / 68R / 224G, 94S / 117N 51R / 66E / 68R / 78G / 224G, 66E, 118R, 93V / 94S / 180P, 109E, 68R / 224G, 40F / 62L / 93V, 93V, 51R / 66E, 94S / 117N / 118R, 198G / 220E, 93V / 117N, 117N / 118R, 93V / 198G, 68R / 173R, 40F / 117N, 224G, 40F / 118R / 226I, 93V / 94S or 40F / 62L / 118R, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 172, or relative to the reference sequence corresponding to SEQ ID NO: 172.

[0299] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: I66E / V78G, V78G / E80A / K224G, V78G, E65P / I66E, V78G / K224G, V78G / E80A / F109E, P40F / G60S / G94S / L226I, I66A / E68R, I66E / E68R, V78G / E80A, G94S / Q117N / N118R / L226I, E68R / F109E, E68R, G94S / E179L, I51R / I66E / E68R / K224G, G94S / Q117N, I51R / I66E / E68R / V78G / K224G, I66E, N118R, K93V / G94S / E180P, F109E, E68R / K224G, P40F / K62L / K93V, K93V, I51R / I66E, G94S / Q117N / N118R, D198G / A220E, K93V / Q117N, Q117N / N118R, K93V / D198G, E68R / Q173R, P40F / Q117N, K224G, P40F / N118R / L226I, K93V / G94S or P40F / K62L / N118R, wherein the amino acid position is relative to SEQ The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 172, or relative to the reference sequence corresponding to SEQ ID NO: 172.

[0300] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376.

[0301] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 446-510, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO: 376.

[0302] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 13, 60, 126, 104, 201, 59, 55, 133, 61, 53, 190, 57, 62, 181, 168, 54, 173, 170, 34, 183, 14, 60 / 62 / 124 / 170, 60 / 170, or 60 / 62, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO: 376.

[0303] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or sets of substitutions or amino acid residues: 13S, 60A, 126E, 104L, 201S, 59E, 55Q, 133E, 61P, 59M, 13G, 53A, 190A, 57S, 62A, 181V, 55G, 168G, 54R, 54Q, 173R, 168Q, 168N, 170P, 34V, 59H, 183L, 173K, 183A, 14E, 60A / 62A / 124V / 170G, 60A / 170G, or 60A / 62A, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO: 376.

[0304] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or sets of substitutions: M13S, G60A, L126E, V104L, G201S, L59E, E55Q, V133E, K61P, L59M, M13G, K53A, L190A, D57S, K62A, T181V, E55G, K168G, K54R, K54Q, Q173R, K168Q, K168N, K170P, Q34V, L59H, R183L, Q173K, R183A, A14E, G60A / K62A / A124V / K170G, G60A / K170G, or G60A / K62A, wherein the amino acid position is relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO: 376.

[0305] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 508.

[0306] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 512-580, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or relative to the reference sequence corresponding to SEQ ID NO: 508.

[0307] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 15, 15 / 68, 15 / 181, 13 / 15, 13 / 15 / 201, 54 / 55, 55 / 133, 55 / 133 / 197, 54 / 55 / 133 / 197, 68, 54 / 133 / 197, 54 / 197, 13 / 15 / 68, 34, 54, 133, 120, 34 / 133, 13 / 15 / 181, 55, 15 / 59, 68 / 181, 201, 181, 66, 15 / 59 / 181, 197, 13 / 15 / 59, 61, 34 / 61, 13, 34 / 69, or 163, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of 508, or relative to the reference sequence corresponding to SEQ ID NO: 508.

[0308] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 15F, 15F / 68A, 15F / 181V, 13S / 15F, 13S / 15F / 201S, 54Q / 55Q, 55Q / 133E, 55Q / 133E / 197A, 54Q / 55Q / 133E / 197A, 68A, 54Q / 133E / 197A, 54Q / 197A, 13S / 15F / 68Y, 34V, 54Q, 133E, 120K, 34V / 133A, 13S / 15F / 181V, 55Q, 15F / 59M, 68A / 181V, 201S, 68Y, 181V, 66S, 15F / 59M / 181V, 197A, 13S / 15F / 59M, 61S, 34V / 61S, 13S, 61A, 34V / 69R or 163S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or relative to the reference sequence corresponding to SEQ ID NO: 508.

[0309] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: Y15F, Y15F / E68A, Y15F / T181V, M13S / Y15F, M13S / Y15F / G201S, K54Q / E55Q, E55Q / V133E, E55Q / V133E / I197A, K54Q / E55Q / V133E / I197A, E68A, K54Q / V133E / I197A, K54Q / I197A, M13S / Y15F / E 68Y, Q34V, K54Q, V133E, Q120K, Q34V / V133A, M13S / Y15F / T181V, E55Q, Y15F / L59M, E68A / T181V, G201S, E68Y, T181V, I66S, Y15F / L59M / T181V, I197A, M13S / Y15F / L59M, K61S, Q34V / K61S, M13S, K61A, Q34V / K69R, or L163S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or relative to the reference sequence corresponding to SEQ ID NO: 508.

[0310] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 524.

[0311] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 582-608, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO: 524.

[0312] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions or sets of substitutions at at least the following amino acid positions: 120, 61 / 201, 34, 36, 105, 112, 31, or 146, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO: 524.

[0313] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or sets of substitutions or amino acid residues: 120R, 61S / 201S, 34S, 36I, 36V, 36A, 36L, 36M, 105S, 34A, 112E, 31R, 146D, or 112R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO: 524.

[0314] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes at least the following substitutions or sets of substitutions: Q120R, K61S / G201S, Q34S, K36I, K36V, K36A, K36L, K36M, A105S, Q34A, S112E, K31R, K146D, or S112R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO: 524.

[0315] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 584.

[0316] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 610-666, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or relative to the reference sequence corresponding to SEQ ID NO: 584.

[0317] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 13 / 30 / 31 / 53 / 109 / 119 / 168, 36 / 126 / 190, 126, 36 / 39 / 126, 126 / 190, 36 / 126, 36 / 190, 39 / 126, 13 / 109 / 119, 109, 36 / 120 / 190, 126 / 148, 190, 108 / 126, 30 / 31, 13 / 118 / 182, 31 / 53, 39, 119, 13 / 119 / 168, 53 / 168, 34, 34 / 65 / 146, 36 / 39, 119 / 168, 34 / 112, or 173, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of 584, or relative to the reference sequence corresponding to SEQ ID NO: 584.

[0318] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 13G / 30G / 31R / 53A / 109Y / 119R / 168G, 36Y / 126E / 190A, 126E, 36Q / 39M / 126E, 126E / 190A, 36Y / 39M / 126E, 36Q / 126E, 36Y / 190A, 39M / 126E, 13G / 109Y / 119R, 109 Y, 36Y / 120G / 190A, 126E / 148T, 190A, 108R / 126E, 30G / 31R, 13G / 118A / 182I, 31R / 53A, 39M, 119R, 13G / 119R / 168G, 53A / 168G, 34S, 34S / 65A / 146D, 36Q / 39M, 119R / 168G, 34S / 112R, 36Y / 39M or 173K, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or relative to the reference sequence corresponding to SEQ ID NO: 584.

[0319] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: M13G / S30G / K31R / K53A / F109Y / K119R / K168G, K36Y / L126E / L190A, L126E, K36Q / I39M / L126E, L126E / L190A, K36Y / I39M / L126E, K36Q / L126E, K36Y / L190A, I39M / L126E, M13G / F109Y / K119R, F109Y, K36Y / Q120G / L190A, L126E / G148T, L190A, E108R / L126E, S30G / K31R, M13G / N118A / V182I, K31R / K53A, I39M, K119R, M13G / K119R / K168G, K53A / K168G, Q34S, Q34S / E65A / K146D, K36Q / I39M, K119R / K168G, Q34S / S112R, K36Y / I39M or Q173K, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or relative to the reference sequence corresponding to SEQ ID NO: 584.

[0320] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610.

[0321] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 668-780, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO: 610.

[0322] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 190, 115 / 126 / 190, 126 / 190, 34 / 35 / 105, 126, 59 / 126, 34 / 105 / 116 / 146, 48, 66, 59, 148, 82, 100, 27, 135, 77, 138, 52, 68, 139, 184, 81, 65, 153, 50, 181, 214, 172, 69, or 154, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO: 610.

[0323] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 190A, 115K / 126E / 190A, 126E / 190A, 34S / 35A / 105R, 126E, 59E / 126E, 34S / 105R / 116E / 146D, 48L, 66F, 59T, 148H, 82T, 59V, 59Y, 59P, 100F, 148F, 27S, 135R, 148T, 77 M, 138C, 148Q, 126A, 52A, 66Q, 59W, 59G, 148M, 59M, 135S, 68L, 139A, 184I, 68R, 184V, 66S, 68Q, 81I, 68V, 65F, 153S, 126V, 68A, 66T, 50C, 138V, 181I, 214T, 139L, 68T, 148S, 172H, 138I, 69L, 81Q, or 154R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO: 610.

[0324] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: L190A, E115K / L126E / L190A, L126E / L190A, Q34S / E35A / A105R, L126E, L59E / L126E, Q34S / A105R / S116E / K146D, F48L, I66F, L59T, G148H, V82T, L59V, L59Y, L59P, Y100F, G148F, G27S, V135R, G148T, L77M, L138 C, G148Q, L126A, V52A, I66Q, L59W, L59G, G148M, L59M, V135S, E68L, T139A, H184I, E68R, H184V, I66S, E68Q, V81I, E68V, E65F, M153S, L126V, E68A, I66T, D50C, L138V, T181I, G214T, T139L, E68T, G148S, V172H, L138I, K69L, V81Q, or I154R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO: 610.

[0325] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 674.

[0326] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 782-868, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO: 674.

[0327] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 81 / 148 / 219, 68 / 148 / 219, 68 / 81 / 219, 148 / 219, 81 / 219, 136 / 138 / 139, 68 / 219, 66 / 68 / 219, 66 / 148 / 219, 66 / 68 / 148, 59, 59 / 135, 219, 59 / 139, 138, 126 / 148 / 219, 59 / 181, 81, 66 / 81 / 219, 126 / 219, 66 / 68 / 81 / 148 / 219, 181, 27 / 148, 135 / 136 / 139, 148, 59 / 135 / 138 / 181, 59 / 135 / 136 / 138 / 139, 59 / 135 / 136 / 138 / 181, 27, 27 / 126 / 148, 135 / 136 / 138, 68 / 81 / 126 / 148 / 219, 136, 68, 126 / 148 or 138 / 139, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO: 674.

[0328] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 81I / 148T / 219L, 68A / 148H / 219L, 68A / 81I / 219L, 148H / 219F, 68A / 148T / 219L, 81I / 219L, 136L / 138V / 139L, 148T / 219F, 1 48H / 219L, E68A / V219L, 66T / 68A / 219L, 66Q / 148H / 219F, 148T / 219L, 66T / 68A / 148H, 59 V, 59V / 135R, 219F, 59V / 139L, 138V, 126A / 148T / 219L, 59V / 181I, 81I, 66Q / 81I / 219L, 2 19L, 126A / 219L, 66Q / 68A / 81I / 148T / 219L, 181I, 27S / 148H, 135R / 136L / 139R, 136L / 13 8V / 139R, 148T, 59V / 135R / 138V / 181I, 59V / 135R / 136L / 138V / 139R, 59V / 135R / 136L / 13 8V / 181I, 66Q / 68G / 81I / 148H / 219L, 27S, 27S / 126A / 148T, 135R / 136L / 138V, 68G / 81I / 126A / 148H / 219L, 136L, 27S / 126A / 148H, 68G, 126A / 148T or 138V / 139R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO: 674.

[0329] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: V81I / G148T / V219L, E68A / G148H / V219L, E68A / V81I / V219L, G148H / V219F, E68A / G148T / V219L, V81I / V219L, Q136L / L138V / T139L, G148T / V219F, G148H / V219L , E68A / V219L, I66T / E68A / V219L, I66Q / G148H / V219F, G148T / V219L, I66T / E68A / G148H, L59V, L59V / V1 35R, V219F, L59V / T139L, L138V, L126A / G148T / V219L, L59V / T181I, V81I, I66Q / V81I / V219L, V219L, L1 26A / V219L, I66Q / E68A / V81I / G148T / V219L, T181I, G27S / G148H, V135R / Q136L / T139R, Q136L / L138V / T139R, G148T, L59V / V135R / L138V / T181I, L59V / V135R / Q136L / L138V / T139R, L59V / V135R / Q136L / L138 V / T181I, I66Q / E68G / V81I / G148H / V219L, G27S, G27S / L126A / G148T, V135R / Q136L / L138V, E68G / V81I / L126A / G148H / V219L, Q136L, G27S / L126A / G148H, E68G, L126A / G148T or L138V / T139R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO: 674.

[0330] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO: 816.

[0331] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 870-1014, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO: 816.

[0332] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 48 / 100 / 148, 48 / 66 / 148 / 219, 48 / 100, 48 / 100 / 135 / 136, 48 / 66 / 77 / 100 / 135 / 136 / 148, 48 / 100 / 136, 48 / 135 / 148, 48 / 148, 48 / 136 / 148, 48 / 135, 48 / 135 / 136, 66 / 100, 48 / 66 / 219, 48 / 135 / 219, 100 / 148, 48 / 136 / 219, 48 / 219, 48 / 135 / 148 / 219, 100 / 135 / 136, 100 / 136, 66 / 77 / 100 / 136 / 219, 100 / 135, 48 / 77 / 100 / 135 / 136, 100, 77 / 100 / 135, 48 / 66 / 100 / 135 / 136 / 148 / 219, 52 / 184 66, 52 / 68, 136 / 148, 126 / 184, 148, 184, 52, 135 / 136 / 148, 126 / 138 / 184, 27 / 184, 27 / 126, 27 / 126 / 148, 27, 27 / 126 / 184, 66 / 100 / 135 / 219, 27 / 68, 52 / 68 / 81 / 126, 48 / 66 / 136 / 148, 66 / 136 / 148, 66 / 135, 66 / 136, 66 / 13 5 / 219, 136, 135 / 148 / 219, 68 / 126, 66 / 148 / 219, 27 / 68 / 126, 27 / 68 / 184, 126, 68 / 81 / 126, 77 / 135, 135 / 136, 77 / 136, 77 / 148, 82, 138 / 184, 77 / 135 / 136, 27 / 138, 81, 81 / 126, 27 / 126 / 138, 68 or 81 / 138, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO: 816.

[0333] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 48L / 100F / 148H, 48L / 66F / 148F / 219L, 48L / 100F, 48L / 100F / 135R / 136L, 48L / 66F / 77M / 100F / 135R / 136L / 148H, 48L / 100F / 136L, 48L / 135R / 148F, 48L / 148F, 48L / 136L / 148F, 48L / 135R, 48L / 135R / 136L, 66F / 100F, 48L / 66Q / 219L, 48L / 135R / 219L, 100F / 148H, 48L / 136L / 219L, 48L / 219L, 48L / 135R / 148F / 219L, 100F / 135R / 136L, 100F / 136L, 66F / 77M / 100F / 136L 13 6L / 148F, 126A / 184I, 148F, 126A / 184V, 184V, 52A, 135R / 136L / 148F, 126A / 138V / 184V, 184I, 27S / 184V, 27S / 126A, 27S / 126A / 148R, 27S, 27S / 126A / 184V, 66Q / 100F / 135R / 219L, 27S / 68A, 52A / 68A / 81L / 126A, 66Q, 48L / 66Q / 136L / 148F, 66F / 136L / 148H, 66Q / 135R, 66Q / 136L, 66 F / 135R / 219L, 136L, 135R / 148F / 219L, 68A / 126A, 66F / 148H / 219L, 27S / 68L / 126A, 27S / 68L / 184V, 126A, 68L / 81L / 126A, 77M / 135R, 135R / 136L, 77M / 136L, 77M / 148F, 82T, 138V / 184V, 77M / 135R / 136L, 27S / 138V, 81L, 81L / 126A, 27S / 126A / 138V, 68A or 81L / 138V, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO: 816.

[0334] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: F48L / Y100F / G148H, F48L / I66F / G148F / V219L, F48L / Y100F, F48L / Y100F / V135R / Q136L, F48L / I66F / L77M / Y100F / V135R / Q136L / G148H, F48L / Y100F / Q136L, F48L / V135R / G148F, F48L / G148F, F48L / Q136L / G148F, F48L / V135R, F48L / V135R / Q136L, I66F / Y10 0F, F48L / I66Q / V219L, F48L / V135R / V219L, Y100F / G148H, F48L / Q136L / V21 9L, F48L / V219L, F48L / V135R / G148F / V219L, Y100F / V135R / Q136L, Y100F / Q 136L, I66F / L77M / Y100F / Q136L / V219L, Y100F / V135R, F48L / L77M / Y100F / V 135R / Q136L, Y100F, L77M / Y100F / V135R, F48L / I66F / Y100F / V135R / Q136L / G 148F / V219L, V52A / H184I, I66F, V52A / E68L, Q136L / G148F, L126A / H184I, G 148F, L126A / H184V, H184V, V52A, V135R / Q136L / G148F, L126A / L138V / H184 V, H184I, G27S / H184V, G27S / L126A, G27S / L126A / G148R, G27S, G27S / L126A / H184V, I66Q / Y100F / V135R / V219L, G27S / E68A, V52A / E68A / V81L / L126A, I6 6Q, F48L / I66Q / Q136L / G148F, I66F / Q136L / G148H, I66Q / V135R, I66Q / Q136 L, I66F / V135R / V219L, Q136L, V135R / G148F / V219L, E68A / L126A, I66F / G14 8H / V219L, G27S / E68L / L126A, G27S / E68L / H184V, L126A, E68L / V81L / L126A , L77M / V135R, V135R / Q136L, L77M / Q136L, L77M / G148F, V82T, L138V / H184V,L77M / V135R / Q136L, G27S / L138V, V81L, V81L / L126A, G27S / L126A / L138V, E68A, or V81L / L138V, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO: 816.

[0335] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874.

[0336] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 1016-1302, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO: 874.

[0337] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 32 / 60 / 135 / 175 / 225, 32 / 81 / 127 / 128 / 135 / 175 / 225, 83 / 175 / 225, 135 / 175 / 225, 54 / 84 / 190, 55 / 190 / 212, 32 / 60 / 81 / 83 / 127 / 128 / 135 / 225, 175 / 225, 32 / 127 / 175, 84 / 190 / 212, 32 / 128 / 175, 81 / 175, 21 / 54, 54 / 55 / 212, 32 / 175 / 225, 128 / 175 / 225, 6 2 / 84 / 190, 81 / 135 / 225, 21 / 54 / 62 / 190 / 212, 60 / 81 / 83 / 175, 55 / 84 / 190, 32 / 81 / 83 / 135 / 225, 175, 62 / 212, 32 / 81 / 83, 32 / 81 / 225, 54 / 55 / 84 / 212, 55 / 19 0, 62 / 179, 54 / 84 / 179 / 212, 54 / 55 / 62 / 84, 81 / 135, 32 / 81 / 135, 179 / 190 / 212, 55 / 62, 100, 21 / 54 / 62 / 190, 190 / 212, 128 / 175, 21 / 62 / 190, 54 / 179 / 190, 5 4 / 190, 21 / 55 / 190, 21 / 54 / 62, 21 / 84, 21 / 55, 21 / 62, 21 / 179, 55 / 62 / 190 / 212, 54 / 62 / 84, 127 / 128 / 175 / 225, 54 / 62, 100 / 104, 54 / 55, 21 / 55 / 212, 62, 55 / 84, 55 / 62 / 212, 21 / 84 / 190, 225, 21 / 54 / 179 / 190, 83 / 135, 32 / 175, 21 / 54 / 78 / 190, 55 / 62 / 179 / 190 / 212, 21 / 54 / 190, 21 / 55 / 84 / 190, 62 / 84, 21 / 55 / 62, 2 1 / 62 / 84, 135, 32 / 60 / 81 / 127, 21 / 54 / 55 / 62 / 78, 54 / 84, 55 / 62 / 179 / 190, 54 / 55 / 62 / 84 / 179 / 190 / 212, 190, 83, 78, 54 / 62 / 190, 21 / 55 / 62 / 179 / 190, 32 / 6 0 / 127, 54 / 62 / 84 / 190 / 212, 21 / 62 / 179, 127 / 128 / 225, 32 / 225, 32 / 60 / 81, 81, 54, 21, 60 / 81 / 128 / 175, 84, 21 / 190 / 212, 60 / 128 / 225, 179, 21 / 55 / 62 / 190,128, 60, 212, 55, 127, 32, 80, 220, 62 / 78 / 179, 55 / 62 / 84, 21 / 55 / 190 / 212, 32 / 127 / 128 / 225, 21 / 54 / 55 / 84 / 179, 21 / 190, 32 / 60 / 81 / 83 / 128 / 135, 127 / 128, or 21 / 55 / 179, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO: 874.

[0338] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 32I / 60S / 135K / 175L / 225A, 32I / 81L / 127I / 128N / 135K / 175L / 225A, 83S / 175F / 225A, 135K / 175L / 225A, 54T / 84H / 190R, 55S / 190H / 212N, 32I / 60S / 81L / 83S / 127I / 128N / 135K / 225A, 175F / 225A, 32I / 127I / 175L, 84A / 190H / 212N, 32I / 128N / 175F, 81L / 1 75L, 81L / 175F, 21R / 54T, 54T / 55S / 212N, 32I / 175L / 225A, 128N / 175F / 225A , 62S / 84A / 190R, 81L / 135M / 225A, 21R / 54T / 62S / 190R / 212N, 60S / 81L / 83G / 175L, 55S / 84A / 190R, 32I / 81L / 83G / 135K / 225A, 175F, 62S / 212N, 32I / 81L / 83S, 32I / 81L / 225A, 54T / 55S / 84A / 212N, 55S / 190R, 62S / 179P, 62E / 84A / 190 H, 54T / 84A / 179P / 212N, 54T / 55S / 62S / 84A, 81L / 135M, 32I / 81L / 135K, 179P / 190H / 212N, 55S / 62E, 100Y, 21R / 54T / 62E / 190H, 190H / 212N, 128N / 175L, 2 1R / 62S / 190H, 54T / 179P / 190H, 54T / 190R, 21R / 55S / 190H, 21R / 54T / 62E, 21 R / 84A, 21R / 55S, 21R / 62S, 21R / 179P, 175L, 55S / 62E / 190H / 212N, 54T / 62E / 8 4A, 127I / 128N / 175L / 225A, 54T / 62E, 100Y / 104F, 55S / 62E / 190R / 212N, 54T / 55S, 21R / 55S / 212N, 62S, 55S / 84A, 55S / 62S / 212N, 21R / 84A / 190H, 225A, 21 R / 54T / 179P / 190R, 83S / 135K, 32I / 175L, 21R / 54T / 78T / 190R, 62E, 55S / 62E / 179P / 190H / 212N, 81L / 135K, 21R / 54T / 190H, 21R / 55S / 84A / 190H, 62S / 84A,21R / 55S / 62E, 21R / 62S / 84A, 135M, 32I / 60S / 81L / 127I, 21R / 54T / 55S / 62E / 78T, 100Y / 104H, 54T / 84A, 55S / 62S / 179P / 190R, 54T / 55S / 62S / 84A / 179P / 190H / 212N, 190R, 190H, 83S, 100Y / 104R, 78T, 54T / 62E / 190R、21R / 55S / 62E / 179P / 190H、78C、32I / 60S / 127I、54T / 62E / 84A / 190H / 212N、21R / 62S / 179P、100Y / 104Q, 127I / 128N / 225A, 32I / 225A, 32I / 60S / 81L, 225V, 81L, 54T, 21R, 60S / 81L / 128N / 175L, 190Q, 84 A. 21R / 190R / 212N, 190C, 135K, 60S / 128N / 225A, 225T, 62G, 225Q, 179P, 225R, 100Y / 104S, 83H, 21R / 55 S / 62E / 190R, 100Y / 104W, 190G, 128N, 84M, 60S, 78N, 84S, 62L, 128E, 212N, 55S, 127I, 21S, 32I, 80G, 84 E, 220V, 62S / 78T / 179P, 190E, 55S / 62E / 84A, 21R / 55S / 190H / 212N, 32I / 127I / 128N / 225A, 21R / 54T / 55S / 84A / 179P, 21R / 190R, 32I / 60S / 81L / 83S / 128N / 135M, 127I / 128N, or 21R / 55S / 179P, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO: 874.

[0339] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: R32I / A60S / V135K / D175L / I225A, R32I / V81L / F127I / K128N / V135K / D175L / I225A, K83S / D175F / I225A, V135K / D175L / I225A, K54T / R84H / L190R, Q55S / L190H / S212N, R32I / A60S / V81L / K83S / F127I / K128N / V135K / I225A, D175F / I225A, R32I / F127I / D175L, R84 A / L190H / S212N, R32I / K128N / D175F, V81L / D175L, V81L / D175F, P21R / K54T , K54T / Q55S / S212N, R32I / D175L / I225A, K128N / D175F / I225A, K62S / R84A / L 190R, V81L / V135M / I225A, P21R / K54T / K62S / L190R / S212N, A60S / V81L / K83 G / D175L, Q55S / R84A / L190R, R32I / V81L / K83G / V135K / I225A, D175F, K62S / S 212N, R32I / V81L / K83S, R32I / V81L / I225A, K54T / Q55S / R84A / S212N, Q55S / L190R, K62S / E179P, K62E / R84A / L190H, K54T / R84A / E179P / S212N, K54T / Q55 S / K62S / R84A, V81L / V135M, R32I / V81L / V135K, E179P / L190H / S212N, Q55S / K62E, F100Y, P21R / K54T / K62E / L190H, L190H / S212N, K128N / D175L, P21R / K6 2S / L190H, K54T / E179P / L190H, K54T / L190R, P21R / Q55S / L190H, P21R / K54T / K62E, P21R / R84A, P21R / Q55S, P21R / K62S, P21R / E179P, D175L, Q55S / K62E / L190H / S212N, K54T / K62E / R84A, F127I / K128N / D175L / I225A, K54T / K62E, F1 00Y / V104F, Q55S / K62E / L190R / S212N, K54T / Q55S, P21R / Q55S / S212N, K62S,Q55S / R84A、Q55S / K62S / S212N、P21R / R84A / L190H、I225A、P21R / K54T / E179P / L190R、K83S / V135K、R32I / D175L、P21R / K54T / G78T / L190R、K62E、Q55S / K62E / E179P / L190H / S212N、V81L / V135K、P21R / K54T / L190H、P21R / Q55S / R84A / L190H、K62S / R84A、P21R / Q55S / K62E、P21R / K62S / R84A、V135M、R32I / A60S / V81L / F127I、P21R / K54T / Q55S / K62E / G78T、F100Y / V104H、K54T / R84A、Q55S / K62S / E179P / L190R、K54T / Q55S / K62S / R84A / E179P / L190H / S212N、L190R、L190H、K83S、F100Y / V104R、G78T、K54T / K62E / L190R、P21R / Q55S / K62E / E179P / L190H、G78C、R32I / A60S / F127I、K54T / K62E / R84A / L190H / S212N、P21R / K62S / E179P、F100Y / V104Q、F127I / K128N / I225A、R32I / I225A、R32I / A60S / V81L、I225V、V81L、K54T、P21R、A60S / V81L / K128N / D175L、L190Q、R84A、P21R / L190R / S212N、L190C、V135K、A60S / K128N / I225A、I225T、K62G、I225Q、E179P、I225R、F100Y / V104S、K83H、P21R / Q55S / K62E / L190R、F100Y / V104W、L190G、K128N、R84M、A60S、G78N、R84S、K62L、K128E、S212N、Q55S、F127I、P21S、R32I、A80G、R84E、A220V、K62S / G78T / E179P、L190E、Q55S / K62E / R84A、P21R / Q55S / L190H / S212N、R32I / F127I / K128N / I225A、P21R / K54T / Q55S / R84A / E179P、P21R / L190R、R32I / A60S / V81L / K83S / K128N / V135M、F127I / K128N or P21R / Q55S / E179P, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO: 874.

[0340] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032.

[0341] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 1356-1552, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0342] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises at least the following amino acid positions: 54 / 80 / 225, 55 / 80 / 180, 54 / 55 / 80 / 208 / 225, 84 / 128 / 155 / 190 / 212, 62 / 155 / 190, 83 / 84 / 155 / 190, 62 / 128 / 135 / 190, 225, 84 / 155 / 190, 128 / 190, 190 / 212, 83 / 84 / 155, 54 / 208 / 225, 84 / 155 / 190, 84 / 212, 84 / 123 / 155 / 212, 62 / 128 / 190, 55 / 80 / 208 / 22 5, 155 / 190, 83 / 84 / 190 / 212, 83 / 128 / 155 / 190 / 212, 55 / 180 / 225, 84 / 190 / 212, 54 / 80 / 180 / 208 / 225, 80 / 208, 135 / 155 / 190, 212, 80 / 180 / 208, 128 / 135 / 190 / 212, 128 / 155 / 212, 80 / 208 / 225, 83 / 212, 62 / 84 / 155 / 190, 54 / 179 / 225, 84, 83 / 84 / 190, 128 / 212, 54 / 55 / 80 / 208, 55, 62 / 128, 54 / 80 / 208 / 225, 62 / 83 / 84 / 190, 179 / 225, 190, 80 / 169 / 208, 62 / 128 / 212, 80, 62, 54 / 55 / 80 / 225, 54 / 80 / 180 / 208, 83 / 84 / 128 / 190 / 212, 62 / 83 / 190, 62 / 190, 80 / 179, 80 / 18 0, 54 / 80, 62 / 83 / 84 / 128 / 190 / 212, 83 / 84 / 135 / 212, 54 / 80 / 179 / 208, 62 / 84 / 128, 11 / 62 / 84 / 128 / 190 / 212, 80 / 179 / 208, 80 / 225, 62 / 84 / 128 / 190 / 212, 6 2 / 84 / 212, 80 / 179 / 180 / 208 / 225, 62 / 135, 55 / 208, 62 / 84, 128 / 135 / 190, 55 / 208 / 225, 84 / 128 / 190, 62 / 84 / 128 / 190, 62 / 135 / 212, 54 / 179 / 180 / 225, 62 / 84 / 135 / 212, 135, 54 / 55 / 225, 55 / 80 / 179 / 180, 84 / 135 / 190, 84 / 128, 54, 84 / 128 / 135, 54 / 179 / 208 / 225, 55 / 179 / 225, 128, 62 / 83 / 84 / 155 / 190, 84 / 13584 / 128 / 155, 55 / 179 / 180, 83 / 84 / 128 / 190, or 54 / 55 / 179 / 225, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0343] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 54T / 80D / 225A, 55S / 80D / 180H, 54T / 55S / 80D / 208E / 225L, 84M / 128E / 155T / 190R / 212N, 62S / 155T / 190H, 83T / 84M / 155T / 190H, 62S / 128E / 135K / 190H, 225A, 84M / 155T / 190R, 128E / 190R, 190R / 212N, 83T / 84M / 155T, 54T / 208E / 225A, 84M / 155T / 190H, 84 H / 212N, 84M / 123S / 155T / 212N, 62S / 128E / 190R, 55S / 80D / 208E / 225A, 155T / 190R、83T / 84M / 190R / 212N、83T / 128E / 155T / 190H / 212N、55S / 180H / 225A、8 4M / 190R / 212N, 54T / 80D / 180H / 208E / 225A, 80D / 208E, 135K / 155T / 190R, 21 2N, 80D / 180H / 208E, 128E / 135K / 190R / 212N, 128E / 155T / 212N, 80D / 208E / 22 5L, 83T / 212N, 62S / 84M / 155T / 190R, 54T / 179V / 225A, 84H, 83T / 84M / 190R, 1 28E / 212N, 54T / 55S / 80D / 208E, 190H / 212N, 55S, 62S / 128E, 54T / 80D / 208E / 225A, 62S / 83T / 84M / 190R, 179A / 225A, 190R, 80D / 169D / 208E, 62S / 128E / 21 2N, 80D, 62S, 54T / 55S / 80D / 225L, 54T / 80D / 180H / 208E, 83T / 84M / 128E / 190H / 212N, 62S / 83T / 190R, 179V / 225A, 62S / 190R, 80D / 179V, 80D / 180H, 54T / 80 D. 62S / 83T / 84H / 128E / 190H / 212N, 83T / 84M / 135K / 212N, 54T / 80D / 179A / 208 E, 62S / 84H / 128E, 11D / 62S / 84M / 128E / 190H / 212N, 80D / 179V / 208E, 80D / 22 5A, 62S / 84M / 128E / 190H / 212N, 62S / 84H / 212N, 80D / 179V / 180H / 208E / 225L,62S / 135K, 55S / 208E, 62S / 84H, 128E / 135K / 190H, 55S / 208E / 225A, 190H, 84H / 128E / 190H, 62S / 84H / 128E / 190R, 62S / 135K / 2 12N, 54T / 179V / 180H / 225A, 128E / 190H, 62S / 84M / 135K / 212N, 135K, 54T / 55S / 225L, 55S / 80D / 179V / 180H, 62S / 84M / 212N, 84 M / 135K / 190R, 84M / 128E, 54T, 84M / 128E / 135K, 54T / 179A / 208E / 225A, 55S / 179V / 225L, 128E, 62S / 83T / 84M / 155T / 190R, 84M / 135K, 84H / 128E / 155T, 55S / 179V / 180H, 83T / 84M / 128E / 190R, 83T / 84M / 190H, 84M or 54T / 55S / 179V / 225A, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0344] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: K54T / A80D / I225A, Q55S / A80D / E180H, K54T / Q55S / A80D / R208E / I225L, R84M / K128E / F155T / L190R / S212N, K62S / F155T / L190H, K83T / R84M / F155T / L190H, K62S / K128E / V135K / L190H, I225A, R84M / F155T / L190R, K128E / L190R, L190R / S212N, K83T / R84 M / F155T, K54T / R208E / I225A, R84M / F155T / L190H, R84H / S212N, R84M / A1 23S / F155T / S212N, K62S / K128E / L190R, Q55S / A80D / R208E / I225A, F155T / L190R, K83T / R84M / L190R / S212N, K83T / K128E / F155T / L190H / S212N, Q55S / E180H / I225A, R84M / L190R / S212N, K54T / A80D / E180H / R208E / I225A, A80 D / R208E, V135K / F155T / L190R, S212N, A80D / E180H / R208E, K128E / V135K / L190R / S212N, K128E / F155T / S212N, A80D / R208E / I225L, K83T / S212N, K6 2S / R84M / F155T / L190R, K54T / E179V / I225A, R84H, K83T / R84M / L190R, K12 8E / S212N, K54T / Q55S / A80D / R208E, L190H / S212N, Q55S, K62S / K128E, K54 T / A80D / R208E / I225A, K62S / K83T / R84M / L190R, E179A / I225A, L190R, A80 D / V169D / R208E, K62S / K128E / S212N, A80D, K62S, K54T / Q55S / A80D / I225L , K54T / A80D / E180H / R208E, K83T / R84M / K128E / L190H / S212N, K62S / K83T / L190R, E179V / I225A, K62S / L190R, A80D / E179V, A80D / E180H, K54T / A80D,K62S / K83T / R84H / K128E / L190H / S212N, K83T / R84M / V135K / S212N, K54T / A80D / E179A / R208E, K62S / R84H / K12 8E、G11D / K62S / R84M / K128E / L190H / S212N、A80D / E179V / R208E、A80D / I225A、K62S / R84M / K128E / L190H / S212 N, K62S / R84H / S212N, A80D / E179V / E180H / R208E / I225L, K62S / V135K, Q55S / R208E, K62S / R84H, K128E / V135K / L190H, Q55S / R208E / I225A, L190H, R84H / K128E / L190H, K62S / R84H / K128E / L190R, K62S / V135K / S212N, K54T / E179V / E180H / I225A, K128E / L190H, K62S / R84M / V135K / S212N, V135K, K54T / Q55S / I225L, Q55S / A80D / E179V / E180H, K62S / R84M / S212N, R84M / V135K / L190R, R84M / K128E, K54T, R84M / K128E / V135K, K54T / E179A / R208E / I225A, Q55S / E179V / I225L, K128E, K62S / K83T / R84M / F155T / L190R, R84M / V135K, R84H / K128E / F155T, Q55S / E179V / E180H, K83T / R84M / K128E / L190R, K83T / R84M / L190H, R84M, or K54T / Q55S / E179V / I225A, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0345] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388.

[0346] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 1554-1786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388 or relative to the reference sequence corresponding to SEQ ID NO: 1388.

[0347] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 143, 228, 136, 82, 156, 205, 226, 150, 219, 217, 218, 68, 142, 212, 213, 79, 230, 207, 155, 227, 129, 210, 133, 151, 43, 204, 182, 82 / 136 / 150 / 217, 136 / 150, 79 / 81 / 143 / 156 / 212 / 228, 82 / 136, 7 9 / 81 / 143 / 212, 79 / 133 / 228, 81 / 143 / 156 / 212, 136 / 219, 82 / 136 / 226, 136 / 150 / 217, 133 / 156, 133 / 143 / 228, 150 / 217, 82 / 136 / 150, 81, 81 / 143, 81 / 133 / 228, 143 / 228, 212 / 228, 79 / 81, 133 / 228, 133 / 212, 136 / 217, 216, 215 or 127, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or relative to the reference sequence corresponding to SEQ ID NO: 1388.

[0348] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 143S, 228G, 136A, 143T, 82Q, 143A, 156C, 205P, 82P, 226P, 228L, 143C, 150G, 150S, 219P, 150P, 217H, 226T, 143G, 218L, 68S, 142R, 212I, 212L, 228P, 213S, 226S, 156T, 79P, 230A, 2 18A, 143R, 228S, 68L, 207T, 228R, 155A, 136S, 150L, ​​150V, 227P, 129L, 230Q, 210V, 205L, 68G, 133W, 155W, 212R, 207 A, 151F, 212P, 218I, 129P, 43A, 230P, 129S, 68A, 142F, 204S, 136Y, 182S, 133S, 228I, 43C, 217G, 129I, 82Q / 136L / 150 G / 217P, 136A / 150S, 79P / 81S / 143A / 156C / 212I / 228G, 82Q / 136A, 79P / 81S / 143A / 212I, 217P, 79P / 133F / 228L, 81S / 143A / 156T / 212I, 136A / 219A, 82Q / 136A / 226P, 136A / 150S / 217P, 136A / 150G, 133F, 133F / 156C, 133F / 143A / 228L, 13 6L, 150S / 217P, 82Q / 136A / 150S, 81S, 81S / 143T, 81S / 133F / 228L, 143A / 228L, 212I / 228G, 79P / 81S, 133F / 228L, 133F / 212M, 136L / 217H, 82P / 136L, 216H, 215P, 143P, 151R, 212M, 213P, 127S, 133Q, 212W, 219G, or 219S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or relative to the reference sequence corresponding to SEQ ID NO: 1388.

[0349] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: I143S, W228G, Q136A, I143T, V82Q, I143A, L156C, I205P, V82P, L226P, W228L, I143C, I150G, I150S, V219P, I150P, N217H, L226T, I143G, V218L, E68S, L142R, S212I, S212L, W228P, Q213S, L226S, L156T, N79P, D230A, V218A, I143R, W228S, E68L, K207T, W228R, F155A, Q136S, I150L, ​​I150V, G227P, V129L, D230Q, D210V, I205L, E68G, E133W, F155W, S212R, K207A, Y151F, S 212P, V218I, V129P, S43A, D230P, V129S, E68A, L142F, G204S, Q136Y, V182S, E133S, W228I, S43C, N217G, V129I, V82Q / Q136L / I150G / N217 P、Q136A / I150S、N79P / V81S / I143A / L156C / S212I / W228G、V82Q / Q136A、N79P / V81S / I143A / S212I、N217P、N79P / E133F / W228L、V81S / I143 A / L156T / S212I, Q136A / V219A, V82Q / Q136A / L226P, Q136A / I150S / N217P, Q136A / I150G, E133F, E133F / L156C, E133F / I143A / W228L, Q136 L, I150S / N217P, V82Q / Q136A / I150S, V81S, V81S / I143T, V81S / E133F / W228L, I143A / W228L, S212I / W228G, N79P / V81S, E133F / W228L, E133F / S212M, Q136L / N217H, V82P / Q136L, D216H, I215P, I143P, Y151R, S212M, Q213P, I127S, E133Q, S212W, V219G or V219S, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or relative to the reference sequence corresponding to SEQ ID NO: 1388.

[0350] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to SEQ ID NO: 1708.

[0351] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 1788-1968, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708 or relative to the reference sequence corresponding to SEQ ID NO: 1708.

[0352] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 127, 143 / 155 / 219 / 228, 219, 127 / 142, 142 / 151 / 207 / 218, 143 / 213, 151 / 218, 127 / 142 / 218, 142 / 151, 143 / 155 / 205 / 213 / 228, 127 / 142 / 207, 127 / 216 / 218, 142 / 151 / 207, 143 / 219, 127 / 207, 127 / 218. 143 / 219 / 228, 127 / 216, 68 / 127 / 207, 143 / 155 / 212 / 228, 151, 143 / 213 / 228, 143 / 155 / 205, 151 / 207 / 216, 143 / 228, 68 / 127 / 207 / 218, 143 / 155, 143, 205 / 213 / 219, 155 / 212 / 219 / 228, 205 / 219 / 228, 143 / 205, 142 / 207, 143 / 205 / 219, 143 / 212 / 213, 127 / 207 / 218, 142, 127 / 142 / 151 / 207 / 218, 143 / 205 / 219 / 228, 205 / 213 / 228, 68 / 151 / 207, 68 / 127 / 142 / 207, 68 / 127, 218, 127 / 142 / 196, 142 / 207 / 218, 68 / 142 / 216, 68 / 127 / 216 / 218, 68 / 127 / 218, 151 / 216 / 218, 143 / 212 / 228, 127 / 142 / 151, 151 / 207, 127 / 142 / 216, 129 / 207 / 218, 68 / 142 / 151 / 207, 68 / 142 / 151, 151 / 207 / 218, 228, 155 / 213, 127 / 142 / 151 / 218, 127 / 151, 155 / 228, 68 / 142 / 207, 80 / 151 / 207, 205 / 228, 68 / 142 / 207 / 216 / 218, 68 / 218, 68 / 207, 216 / 218, 68 / 142, 212, 205 or 142 / 207 / 216 / 218, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or relative to the reference sequence corresponding to SEQ ID NO: 1708.

[0353] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 127P, 143S / 155A / 219P / 228R, 219P, 127P / 142R, 142R / 151A / 207A / 218A, 143G / 213L, 151A / 218A, 127P / 142R / 218I, 142R / 151A, 143S / 155A / 205L / 213L / 228R, 127P / 142R / 207A, 127P / 216P / 218A, 142R / 151A / 207A, 143S / 219P, 127P / 207A, 127P / 218 I. 143G / 219P / 228R, 127P / 216P, 68L / 127P / 207L, 143G / 155A / 212R / 228R, 151A, 143S / 219P / 228R, 143S / 213L / 228R, 143G / 155A / 205L, 151A / 207A / 21 6P, 143G / 228R, 68L / 127P / 207A, 68L / 127P / 207A / 218A, 143G / 155A, 143G, 2 05L / 213L / 219P, 155A / 212R / 219P / 228R, 143S / 228R, 205L / 219P / 228R, 143 G / 205L, 142R / 207A, 143G / 205L / 219P, 143G / 212R / 213L, 127P / 207L / 218I , 142R, 127P / 216P / 218I, 127P / 142R / 151A / 207A / 218I, 143S / 205L / 219P / 2 28R, 205L / 213L / 228R, 143G / 219P, 142R / 207L, 68L / 151A / 207L, 143S / 213L , 142R / 151A / 207L, 127P / 142R / 207L, 68L / 127P / 142R / 207L, 143S / 205L, 68 L / 127P, 218A, 127P / 142R / 196I, 142R / 207L / 218A, 143S / 155A, 143S, 68L / 142R / 216P, 68L / 127P / 216P / 218I, 68L / 127P / 218I, 151A / 216P / 218I, 143S / 212R / 228R, 127P / 142R / 151A, 151A / 207L, 127P / 142R / 216P, 129I / 207A / 2 18A, 142R / 207L / 218I, 68L / 142R / 151A / 207A, 151A / 207A, 68L / 142R / 151A,151A / 207L / 218I, 228R, 155A / 213L, 127P / 142R / 151A / 218I, 127P / 151A, 155A / 228R, 143S / 155A / 205L, 151A / 216P / 218A, 68L / 142R / 207L, 80V / 151A / 207L, 205L / 228R, 68L / 142R / 207A / 216P / 218I, 68L / 218I, 68L / 207A, 216P / 218I, 68L / 142R, 212R, 68L / 207L, 205L or 142R / 207A / 216P / 218I, wherein the amino acid position is relative to SEQ ID The reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or relative to the reference sequence corresponding to SEQ ID NO: 1708.

[0354] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: I127P, I143S / F155A / V219P / W228R, V219P, I127P / L142R, L142R / Y151A / K207A / V218A, I143G / Q213L, Y151A / V218A, I127P / L142R / V218I, L142R / Y151A, I143S / F155A / I205L / Q213L / W228R, I127P / L142R / K207A, I127P / D216P / V218A, L142R / Y151A / K207A, I1 43S / V219P, I127P / K207A, I127P / V218I, I143G / V219P / W228R, I127P / D216 P, E68L / I127P / K207L, I143G / F155A / S212R / W228R, Y151A, I143S / V219P / W2 28R, I143S / Q213L / W228R, I143G / F155A / I205L, Y151A / K207A / D216P, I143 G / W228R, E68L / I127P / K207A, E68L / I127P / K207A / V218A, I143G / F155A, I14 3G, I205L / Q213L / V219P, F155A / S212R / V219P / W228R, I143S / W228R, I205L / V219P / W228R, I143G / I205L, L142R / K207A, I143G / I205L / V219P, I143G / S2 12R / Q213L, I127P / K207L / V218I, L142R, I127P / D216P / V218I, I127P / L142 R / Y151A / K207A / V218I, I143S / I205L / V219P / W228R, I205L / Q213L / W228R, I 143G / V219P, L142R / K207L, E68L / Y151A / K207L, I143S / Q213L, L142R / Y151 A / K207L, I127P / L142R / K207L, E68L / I127P / L142R / K207L, I143S / I205L, E6 8L / I127P, V218A, I127P / L142R / V196I, L142R / K207L / V218A, I143S / F155A, I143S, E68L / L142R / D216P, E68L / I127P / D216P / V218I, E68L / I127P / V218I,Y151A / D216P / V218I, I143S / S212R / W228R, I127P / L142R / Y151A, Y151A / K207L、I127P / L142R / D216P、V129I / K207A / V218A、L142R / K207L / V218 I. E68L / L142R / Y151A / K207A, Y151A / K207A, E68L / L142R / Y151A, Y151A / K207L / V218I, W228R, F155A / Q213L, I127P / L142R / Y151A / V218I, I127P / Y151A, F155A / W228R, I143S / F155A / I205L, Y151A / D216P / V218A, E68L / L142R / K207L, A80V / Y151A / K207L, I205L / W228R, E68L / L142R / K207A / D216P / V218I, E68L / V218I, E68L / K207A, D216P / V218I, E68L / L142R, S212R, E68L / K207L, I205L or L142R / K207A / D216P / V218I, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or relative to the reference sequence corresponding to SEQ ID NO: 1708.

[0355] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952.

[0356] 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%, 96%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 1970-2032, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952 or relative to the reference sequence corresponding to SEQ ID NO: 1952.

[0357] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 18 / 118, 87 / 119 / 192, 119 / 173 / 192, 118, 87 / 119, 18 / 118 / 170, 18, 119, 192, 87, 89, 59, 170, 64, 112, 93, 184, 146, 51, 125, 192, 191, 117, 172, or 163, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or relative to the reference sequence corresponding to SEQ ID NO: 1952.

[0358] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or sets of substitutions or amino acid residues: 18L / 118G, 87M / 119K / 192A, 119K / 173R / 192A, 118G, 87M / 119K, 18L / 118G / 170S, 18L, 119K, 192A, 87E, 87M, 89Q, 192I, 59L, 170S, 64R, 112N, 93E, 184S, 146N, 112K, 51T, 125I, 93S, 192W, 112A, 191D, 51M, 117L, 93Y, 172T, or 163Q, wherein the amino acid position corresponds to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or to the reference sequence corresponding to SEQ ID NO: Regarding the reference sequence corresponding to 1952.

[0359] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: F18L / N118G, S87M / R119K / K192A, R119K / Q173R / K192A, N118G, S87M / R119K, F18L / N118G / G170S, F18L, R119K, K192A, S87E S87M, K89Q, K192I, V59L, G170S, K64R, S112N, K93E, H184S, K146N, S112K, I51T, V125I, K93S, K192W, S112A, E191D, I51M, Q117L, K93Y, V172T or L163Q, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or relative to the reference sequence corresponding to SEQ ID NO: 1952.

[0360] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980.

[0361] 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%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2034-2134, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

[0362] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 87 / 119 / 192, 87 / 192, 87 / 119, 119 / 192, 192, 119, 146 / 192, 87, 59 / 87, 59 / 87 / 112 / 146, 146, 87 / 112 / 119, 74, 75, 66, 181, 81, 73, 77, 188, 216, 82, 136, 214, 21, 217, 154, 133, 71, 212, or 228, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

[0363] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 87E / 119K / 192A, 87E / 192A, 87E / 119K, 119K / 192A, 87M / 119K, 192A, 119K, 146N / 192A, 87E, 87M / 192A, 87M, 59L / 87E, 59L / 87E / 112A / 146N, 146N, 87E / 112A / 119K 74G, 75L, 66A, 181V, 66W, 66G, 66Q, 81F, 66H, 73R, 66N, 77S, 77A, 66D, 66V, 188L, 77Q, 73I, 216E, 82L, 136L, 214L, 188G, 214T, 21S, 66T, 217E, 154R, 216T, 133V, 71T, 214R, 136I, 212H, 214P, or 228Q, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

[0364] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: S87E / R119K / K192A, S87E / K192A, S87E / R119K, R119K / K192A, S87M / R119K, K192A, R119K, K146N / K192A, S87E, S87M / K192A, S87M, V59L / S87E, V59L / S87E / S112A / K146N, K146N, S87E / S112A / R119K, P74G, D75L, I66A, T181V, I66W, I66G, I66Q, V81F, I66H, V73R, I66N, L77S, L77A, I66D, I66V, V188L, L77Q, V73I, P216E, V82L, A136L, G214L, V188G, G214T, P21S, I66T, P217E, I154R, P216T, E133V, E71T, G214R, A136I, S212H, G214P, or W228Q, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

[0365] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072.

[0366] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2136-2264, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

[0367] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions or sets of substitutions at at least the following amino acid positions: 87, 194, 197, 93, 202, 38, 91, 183, 39, 146, 203, 37, 92, 36, 89, 131, 132, 90, 148, 35 / 197, 231, or 56, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

[0368] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 87R, 87K, 87L, 194R, 197L, 87Y, 93G, 202E, 38C, 91V, 194L, 183L, 93T, 39T, 91L, 146H, 203E, 203L, 146N, 37G, 194Y, 37R, 92L, 36R, 36S, 89T, 131A, 132T, 90F, 197Q The amino acid positions are 90V, 148S, 91S, 35E / 197V, 132V, 197A, 197V, 93P, 92S, 36M, 39A, 194V, 87V, 132G, 38F, 90T, 231R, 148F, 91G, 91A, 36I, 87E, 37L, 231T, 231A, 89P, 202S, 36E, 231Q, 87A, 146V, 146R, 56T, 93A, or 203R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

[0369] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: S87R, S87K, S87L, Q194R, I197L, S87Y, K93G, K202E, G38C, C91V, Q194L, R183L, K93T, I39T, C91L, K146H, K203E, K203L, K146N, T37G, Q194Y, T37R, E92L, K36R, K36S, K89T, E131A, D132T, D90F, I197Q, D90V, G148S, C91S, A35E / I197V, D132V, I197A, I197V, K93P, E92S, K36M, I39A, Q194V, S87V, D132G, G38F, D90T, K231R, G148F, C91G, C91A, K36I, S87E, T37L, K231T, K231A, K89P, K202S, K36E, K231Q, S87A, K146V, K146R, N56T, K93A, or K203R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

[0370] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138.

[0371] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2266-2366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

[0372] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 161, 162, 153, 216, 212, 156, 214, 229, 77 / 143 / 214 / 216, 143 / 156 / 216, 77 / 143, 77 / 143 / 216, 77 / 214 / 216, 77 / 216, 143 / 156, 156 / 216, 143, 77, 77 / 214, 77 / 143 / 161 / 162 / 212 / 214, 77 / 162 / 214 / 216, 77 / 143 / 156 / 214 / 216, 77 / 143 / 156 / 162 / 212 / 216, 77 / 143 / 156 / 161 / 214 / 216, 77 / 156 / 162 / 216, 156 / 161 / 214 / 216, 77 / 143 / 162 / 214 / 216, 143 / 216, 214 / 216, 143 / 156 / 214 / 216, 143 / 214 / 216, 77 / 143 / 212 / 216, 143 / 156 / 161 / 162 / 216, 156 / 162 / 214, 77 / 143 / 156 / 161 / 162, 143 / 161 / 214 / 216, 77 / 161 / 216 or 143 / 212 / 214 / 216, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

[0373] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 161G, 162G, 153C, 216L, 212Q, 216H, 156V, 161L, 214W, 229C, 216M, 216V, 214A, 214M, 77I / 143V / 214E / 216R, 143V / 156V / 216R, 77I / 143V, 77I / 143V / 216R, 77I / 21 4E / 216R, 77I / 216L, 143V / 156V, 156V / 216R, 143V, 77I / 214E / 216L, 77I, 77I / 214E, 77I / 143V / 161L / 162G / 212Q / 214E, 77I / 162G / 214E / 216L, 77I / 143V / 156V / 214E / 216L, 77I / 143V / 156V / 162G / 212Q / 216R, 77I / 143 V / 156V / 161L / 214E / 216R, 77I / 216R, 77I / 156V / 162G / 216R, 156V / 161L / 214E / 216L, 77I / 143V / 162G / 214W / 216L、143V / 216L、214E / 216L、143V / 156V / 214E / 216R、143V / 214E / 216L、143V / 214E / 216R、214E、77I / 143V / 212Q / 216R, 143V / 156V / 161L / 162G / 216R, 156V / 162G / 214E, 77I / 143V / 156V / 161L / 162G, 143V / 161L / 214E / 216R, 77I / 161L / 216R, 143V / 212Q / 214E / 216R, 77I / 143V / 214W / 216R, 143V / 216R or 216R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

[0374] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: D161G, E162G, M153C, P216L, S212Q, P216H, L156V, D161L, G214W, S229C, P216M, P216V, G214A, G214M, L77I / I143V / G214E / P216R, I143V / L156V / P216R, L77I / I143V, L77I / I143V / P216R, L77I / G214E / P216R, L 77I / P216L, I143V / L156V, L156V / P216R, I143V, L77I / G214E / P216L, L77I, L77I / G214E, L77I / I143V / D161L / E162G / S212Q / G2 14E, L77I / E162G / G214E / P216L, L77I / I143V / L156V / G214E / P216L, L77I / I143V / L156V / E162G / S212Q / P216R, L77I / I143V / L15 6V / D161L / G214E / P216R, L77I / P216R, L77I / L156V / E162G / P216R, L156V / D161L / G214E / P216L, L77I / I143V / E162G / G214W / P2 16L, I143V / P216L, G214E / P216L, I143V / L156V / G214E / P216R, I143V / G214E / P216L, I143V / G214E / P216R, G214E, L77I / I143V / S212Q / P216R, I143V / L156V / D161L / E162G / P216R, L156V / E162G / G214E, L77I / I143V / L156V / D161L / E162G, I143V / D161L / G214E / P216R, L77I / D161L / P216R, I143V / S212Q / G214E / P216R, L77I / I143V / G214W / P216R, I143V / P216R or P216R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

[0375] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294.

[0376] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2368-2594, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

[0377] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions at at least the following amino acid positions: 80, 105, 228, 50, 175, 170, 82, 173, 65, 53, 184, 122, 195, 68, 89, 34, 118, 119, 87, 179, 117, 113, 54, 190, 216, 88, 36, 166, 38, 169, 66, 142, 136, 131, 187, 40, 74, 127, 55, 148, 215, 64, 155, 116, 90, 132, 60, 94, 112, 120, 37, 108, 35, 51, 61, 212, 172, 59, or 56, or combinations thereof, wherein the amino acid positions are relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

[0378] In some embodiments, the engineered adenosine kinase contains at least the following substitutions or amino acid residues in its amino acid sequence: 80P, 105K, 228S, 50Q, 175F, 170R, 82L, 173S, 65T, 53M, 184V, 122S, 195M, 228L, 68S, 89A, 34E, 118S, 53S, 119F, 87A, 179C, 117S. 113E, 50C, 80G, 122H, 89V, 80R, 54I, 190Q, 216M, 88R, 80D, 36Q, 105S, 195G, 68V, 105L, 1 75D, 184T, 166L, 89H, 38R, 54G, 38F, 89I, 169I, 66C, 142W, 117L, 136Y, 131V, 187Y, 40L, 1 73T, 74C, 228V, 127L, 80L, 55G, 184M, 136V, 228T, 148R, 88T, 228Q, 66S, 215V, 195R, 190 G, 64R, 184R, 122A, 50S, 142M, 155W, 80S, 216G, 116A, 90C, 132K, 105G, 60W, 94Y, 112T, 87 S, 187G, 120T, 87I, 68R, 37Y, 173R, 105M, 108V, 35L, 68I, 166F, 35F, 120S, 184S, 169A, 51L, 61V, 116F, 65D, 54H, 212C, 172S, 55L, 59L, 60C, 228F, or 56Q, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

[0379] In some embodiments, the engineered adenosine kinase contains at least the following substitutions in its amino acid sequence: A80P, R105K, W228S, D50Q, L175F, S170R, V82L, Q173S, E65T, A53M, H184V, T122S, P195M, W228L, L68S, K89A, S34E, G118S, A53S, R119F, K87A, E179C, Q117S, F113E, D50C. , A80G, T122H, K89V, A80R, K54I, R190Q, R216M, E88R, A80D, K36Q, R105S, P195G, L68V, R105L, L175D, H18 4T, D166L, K89H, G38R, K54G, G38F, K89I, V169I, W66C, R142W, Q117L, A136Y, E131V, K187Y, P40L, Q173T, P 74C, W228V, I127L, A80L, Q55G, H184M, A136V, W228T, G148R, E88T, W228Q, W66S, I215V, P195R, R190G, K6 4R, H184R, T122A, D50S, R142M, F155W, A80S, R216G, S116A, D90C, D132K, R105G, A60W, G94Y, S112T, K87S, K187G, Q120T, K87I, L68R, T37Y, Q173R, R105M, E108V, A35L, L68I, D166F, A35F, Q120S, H184S, V169A, I51L, S61V, S116F, E65D, K54H, S212C, V172S, Q55L, V59L, A60C, W228F, or N56Q, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

[0380] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368.

[0381] 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%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2596-2652 or 2676-2714, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368 or relative to the reference sequence corresponding to SEQ ID NO: 2368.

[0382] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 20, 21, 22, 43, 44, 46, 48, 138, 181, 182, 136 / 228, 82 / 142 / 173 / 184 / 216, 184 / 216, 50 / 184 / 216, 127 / 173 / 184, 105 / 136 / 170 / 175 / 228, 105 / 175, 50 / 184, 105 / 170 / 175 / 228, 50 / 127 / 173 / 184, 5 0 / 142 / 184 / 216, 50 / 82 / 127 / 216, 50, 50 / 82 / 173 / 216, 50 / 127 / 142 / 184 / 216, 50 / 82 / 127, 105 / 136, 105 / 228, 105 / 136 / 175 / 228, 173 / 216, 50 / 173, 228, 127 / 173, 105 / 136 / 170 / 195, 50 / 82 / 216, 175 / 228 or 50 / 82 / 127 / 142 / 184 / 216, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

[0383] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 20A, 21L, 21N, 21V, 22A, 43A, 43G, 43H, 44S, 46E, 46F, 46L, 46M, 46Y, 48F, 138M, 138V, 181C, 181V, 182A, 136Y / 228V, 82 L / 142M / 173T / 184T / 216M, 184T / 216M, 50Q / 184M / 216M, 127L / 173R / 184M, 105K / 136V / 170R / 175F / 228T, 105K / 175F, 127L / 173T / 184M, 50Q / 184T, 105K / 170R / 175F / 228V, 50 Q / 127L / 173R / 184M, 50Q / 142M / 184M / 216M, 50Q / 82L / 127L / 216M, 50Q, 50Q / 82L / 173T / 216M、50Q / 127L / 142W / 184M / 216M、50Q / 82L / 127L、136V / 228V、105K / 136V、105K / 228 V, 105K / 136V / 175F / 228V, 173T / 216M, 50Q / 173T, 228V, 127L / 173R, 105K / 136V / 170R / 195G, 50Q / 82L / 216M, 175F / 228V or 50Q / 82L / 127L / 142M / 184M / 216M, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

[0384] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: G20A, P21L, P21N, P21V, P22A, S43A, S43G, S43H, T44S, D46E, D46F, D46L, D46M, D46Y, L48F, L138M, L138V, T181C, T181V, V182A A136Y / W228V, V82L / R142M / Q173T / H184T / R216M, H184T / R216M, D50Q / H184M / R 216M, I127L / Q173R / H184M, R105K / A136V / S170R / L175F / W228T, R105K / L175F, I 127L / Q173T / H184M, D50Q / H184T, R105K / S170R / L175F / W228V, D50Q / I127L / Q1 73R / H184M, D50Q / R142M / H184M / R216M, D50Q / V82L / I127L / R216M, D50Q, D50Q / V 82L / Q173T / R216M, D50Q / I127L / R142W / H184M / R216M, D50Q / V82L / I127L, A136 V / W228V, R105K / A136V, R105K / W228V, R105K / A136V / L175F / W228V, Q173T / R216 M, D50Q / Q173T, W228V, I127L / Q173R, R105K / A136V / S170R / P195G, D50Q / V82L / R216M, L175F / W228V or D50Q / V82L / I127L / R142M / H184M / R216M, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

[0385] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to SEQ ID NO: 2602.

[0386] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 2716-2988, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0387] In some embodiments, the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at at least the following amino acid positions: 18, 18 / 56, 20, 21, 26, 27, 29, 30, 32, 42, 43, 50, 66, 67, 69, 70, 80, 98, 99, 102, 135, 137, 139, 141, 142, 143, 148, 149, 152, 153, 156, 173, 184, or 216, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0388] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 18C / 56T, 18E, 20P, 20R, 20T, 20V, 21L, 21R, 21T, 26L, 27C, 29R, 30L, 30S, 30Y, 32L, 42T, 43N, 50R, 66P, 67R, 69F, 70R, 80G, 80S, 98A, 98 G, 98Q, 99A, 99C, 102A, 102C, 102N, 135I, 137L, 139H, 141G, 141V, 142L, 143T, 148P, 148S, 149L, 152F, 153V, 156A, 173F, 184N, 216D, 216E, 216G, 216L, 216N, 216P, or 216W, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0389] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: L18C / N56T, L18E, G20P, G20R, G20T, G20V, P21L, P21R, P21T, K26L, G27C, Q29R, G30L, G30S, G30Y, I32L, I42T, S43N, Q50R, W66P, M67R, K69F, G70R, P80G, P80S, D98A, D98G, D 98Q, G99A, G99C, R102A, R102C, R102N, V135I, R137L, L139H, R141G, R141V, R142L, V143T, G148P, G148S, R149L, N152F, M153V, L156A, Q173F, M184N, M216D, M216E, M216G, M216L, M216N, M216P, or M216W, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0390] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions or substitution groups at at least the following amino acid positions: 65, 65 / 104, 65 / 104 / 105 / 116 / 127, 65 / 104 / 105 / 127 / 136, 65 / 104 / 136, 65 / 104 / 136 / 170, 65 / 105 / 116 / 136 / 170, 65 / 105 / 127 / 136, 65 / 105 / 127 / 136 / 175, 65 / 105 / 127 / 170, 65 / 105 / 127 / 175, 65 / 105 / 136, 65 / 105 / 136 / 170, 65 / 105 / 170, 65 / 116, 65 / 127 / 136, 65 / 1 27 / 136 / 170, 65 / 127 / 170, 94 / 173, 94 / 187, 104 / 105, 104 / 105 / 127, 104 / 105 / 127 / 136 / 175 / 195, 104 / 105 / 136 / 170, 104 / 127, 104 / 127 / 136 / 170, 104 / 127 / 136 / 175, 105, 105 / 116 / 127 / 136 / 170 / 175, 105 / 127 / 136, 105 / 127 / 136 / 195, 105 / 136, 105 / 136 / 195, 116 / 127 / 136 / 175, 127 / 195, 132, 136 or 170, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0391] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: 65T, 65T / 104A, 65T / 104A / 105K / 116A / 127L, 65T / 104A / 105K / 127L / 136V, 65T / 104A / 136V, 65T / 104A / 136V / 170R, 65T / 105K / 116A / 136V / 170R, 65T / 105K / 127L / 136V, 65T / 105K / 127L / 136V / 175F, 65T / 105K / 127L / 170R, 65T / 105K / 127L / 175F, 65 T / 105K / 136V, 65T / 105K / 136V / 170R, 65T / 105K / 170R, 65T / 116A, 65T / 127L / 136V / 170R, 65T / 127 L / 136Y, 65T / 127L / 170R, 94Y / 173R, 94Y / 187G, 104A / 105K, 104A / 105K / 127L, 104A / 105K / 127L / 136V / 175F / 195G, 104A / 105K / 136V / 170R, 104A / 127L, 104A / 127L / 136V / 170R, 104A / 127L / 136V / 175F, 105K, 105K / 116A / 127L / 136Y / 170R / 175F, 105K / 127L / 136V, 105K / 127L / 136V / 195G, 105K / 136V, 105K / 136V / 195G, 116A / 127L / 136Y / 175F, 127L / 195G, 132K, 136V, or 170R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0392] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: E65T, E65T / V104A, E65T / V104A / R105K / S116A / I127L, E65T / V104A / R105K / I127L / A136V, E65T / V104A / A136V, E65T / V104A / A136V / S170R, E65T / R105K / S116A / A136V / S170R, E65T / R105 K / I127L / A136V, E65T / R105K / I127L / A136V / L175F, E65T / R105K / I127L / S170R, E65T / R105K / I127L / L175F, E65T / R 105K / A136V, E65T / R105K / A136V / S170R, E65T / R105K / S170R, E65T / S116A, E65T / I127L / A136V / S170R, E65T / I127L / A136Y、E65T / I127L / S170R、G94Y / Q173R、G94Y / K187G、V104A / R105K、V104A / R105K / I127L、V104A / R105K / I127L / A 136V / L175F / P195G, V104A / R105K / A136V / S170R, V104A / I127L, V104A / I127L / A136V / S170R, V104A / I127L / A136V / L175F, R105K, R105K / S116A / I127L / A136Y / S170R / L175F, R105K / I127L / A136V, R105K / I127L / A136V / P195G, R105K / A136V, R105K / A136V / P195G, S116A / I127L / A136Y / L175F, I127L / P195G, D132K, A136V, or S170R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0393] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions at at least the following amino acid positions: 36, 37, 40, 50, 59, 89, 117, 120, 128, 168, or 203, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0394] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 36V, 37I, 37L, 40S, 50A, 59T, 59Y, 89P, 117L, 120L, 128I, 168L, or 203L, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0395] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: K36V, T37I, T37L, P40S, Q50A, V59T, V59Y, K89P, Q117L, Q120L, E128I, G168L, or K203L, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0396] In some embodiments, the amino acid sequence of the engineered adenosine kinase includes substitutions at at least the following amino acid positions: 32, 36, 37, 50, 58, 61, 89, 90, 94, 97, 104, 110, 111, 118, 119, 128, 131, 132, 166, 169, 170, 172, 192, 195, or 200, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0397] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 32F, 36S, 37F, 50N, 50V, 58Y, 61E, 61N, 89L, 90S, 94M, 94V, 97I, 104R, 110F, 111P, 111R, 118L, 119P, 128C, 128R, 131G, 132L, 166L, 166S, 169Y, 170G, 172A, 192H, 195I, or 200A, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0398] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: I32F, K36S, T37F, Q50N, Q50V, E58Y, S61E, S61N, K89L, D90S, G94M, G94V, L97I, V104R, L110F, D111P, D111R, G118L, R119P, E128C, E128R, E131G, D132L, D166L, D166S, V169Y, S170G, V172A, K192H, P195I, or Y200A, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

[0399] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832.

[0400] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2990-3032, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or relative to the reference sequence corresponding to SEQ ID NO: 2832.

[0401] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions or sets of substitutions at at least the following amino acid positions: 36, 36 / 50, 36 / 50 / 89 / 93 / 110, 36 / 50 / 89 / 93 / 139, 36 / 50 / 89 / 139 / 170, 36 / 50 / 89 / 172, 36 / 89, 36 / 170, 50, 50 / 89 / 93, 50 / 89 / 93 / 139, 50 / 93, 50 / 170, 89 / 170 / 172, 90 / 104, 90 / 151 / 157, 104 / 151 / 154, 104 / 151 / 154 / 157, 104 / 154 / 157 / 216, 151 / 157, 151 / 216, or 216, wherein said amino acid positions are relative to SEQ. The reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or relative to the reference sequence corresponding to SEQ ID NO: 2832.

[0402] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions: 36S, 36S / 50N, 36S / 50N / 89L / 93V / 110F, 36S / 50N / 89L / 93V / 139R, 36S / 50N / 89L / 139R / 170G, 36S / 50N / 89L / 172A, 36S / 89L, 36S / 170G, 50N, 50N / 89L / 93V, 5 0N / 89L / 93V / 139R, 50N / 93V, 50N / 170G, 89L / 170G / 172A, 90S / 104R, 90S / 151F / 157V, 104R / 151F / 154Q, 104R / 151F / 154Q / 157V, 104R / 154Q / 157V / 216E, 151F / 157V, 151F / 216E or 216D, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or relative to the reference sequence corresponding to SEQ ID NO: 2832.

[0403] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: K36S, K36S / Q50N, K36S / Q50N / K89L / K93V / L110F, K36S / Q50N / K89L / K93V / L139R, K36S / Q50N / K89L / L139R / S170G, K36S / Q50N / K89L / V172A, K36S / K89L, K36S / S170G, Q50N, Q50N / K89L / K93V, Q5 0N / K89L / K93V / L139R, Q50N / K93V, Q50N / S170G, K89L / S170G / V172A, D90S / A104R, D90S / Y151F / P157V, A104R / Y151F / I154Q, A104R / Y151F / I154Q / P157V, A104R / I154Q / P157V / M216E, Y151F / P157V, Y151F / M216E or M216D, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO:2832, or relative to the reference sequence corresponding to SEQ ID NO: 2832.

[0404] 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or one or more substitutions with respect to the reference sequence corresponding to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994.

[0405] 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%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NO. in SEQ ID NO: 3034-3150, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

[0406] In some embodiments, the engineered adenosine kinase amino acid sequence comprises substitutions at at least the following amino acid positions: 50, 58, 59, 68, 74, 76, 79, 80, 83, 90, 112, 113, 119, 157, 170, 172, 182, 184, 217, 224, 226, 228, 229, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

[0407] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions or amino acid residues: 50L, 58C, 59W, 68A, 68E, 68V, 74Q, 76G, 76L, 79A, 79K, 79L, 79P, 79W, 80W, 90K, 112C, 113W, 119S, 170H, 170P, 170R, 172M, 182G, 182L, 182Q, 184R, 217T, 224S, 224T, 226K, 226R, 228D, 230M, or 231R, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

[0408] In some embodiments, the engineered adenosine kinase amino acid sequence comprises at least the following substitutions: Q50L, E58C, V59W, L68A, L68E, L68V, P74Q, E76G, E76L, N79A, N79K, N79L, N79P, N79W, P80W, D90K, S112C, F113W, R119S, S170H, S170P, S170R, V172M, V182G, V182L, V182Q, M184R, P217T, G224S, G224T, L226K, L226R, W228D, D230M, or K231R, or combinations thereof, wherein the amino acid position corresponds to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or to the reference sequence corresponding to SEQ ID NO: 2994. Regarding the reference sequence corresponding to NO: 2994.

[0409] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains substitutions or sets of substitutions at at least the following amino acid positions: 79 / 104 / 157 / 228, 79 / 157 / 228 / 229, 79 / 157 / 229, 79 / 228, 83 / 104, 83 / 104 / 151 / 168 / 173 / 190, 83 / 113, 83 / 173 / 190, 83 / 173 / 190 / 201, 83 / 173 / 201, 83 / 190, 83 / 190 / 201 / 216, 83 / 216, 104 / 157, 157 / 173 / 190 / 216, 157 / 183, 157 / 190, 157 / 190 / 216, 157 / 228, 173 / 216 or 201 / 216, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

[0410] In some embodiments, the engineered adenosine kinase amino acid sequence contains at least the following substitutions or sets of substitutions or amino acid residues: 79W / 104R / 157P / 228V, 79W / 157P / 228V / 229I, 79W / 157P / 229I, 79W / 228V, 83L, 83L / 104R, 83L / 104R / 151Y / 168S / 173T / 190Q, 83L / 113Y, 83L / 173T / 201A, 83L / 173V / 1 90Q, 83L / 173V / 190Q / 201F, 83L / 190Q, 83L / 190Q / 201A / 216E, 83L / 216E, 104R / 157P, 157P, 157P / 173T / 190Q / 216E, 157P / 183L, 157P / 190Q, 157P / 190Q / 216E, 157P / 228V, 173T / 216E, 201F / 216E or 229I, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

[0411] In some embodiments, the amino acid sequence of the engineered adenosine kinase contains at least the following substitutions or sets of substitutions: N79W / A104R / V157P / W228V, N79W / V157P / W228V / S229I, N79W / V157P / S229I, N79W / W228V, K8...

Claims

1. An engineered adenosine kinase comprising a reference sequence corresponding to amino acid residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 2-1302, 1356-2652, and 2676-3192, or 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 higher sequence identity with the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 2-1302, 1356-2652, and 2676-3192, wherein the amino acid sequence comprises an amino acid sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 83%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to SEQ ID NOs. The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

2. The engineered adenosine kinase of claim 1, comprising a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or a reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104. The reference sequences corresponding to these sequences have 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 higher sequence identity, wherein the amino acid sequences contain amino acid sequences relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

3. The engineered adenosine kinase of claim 1, 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 higher sequence identity with respect to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or one or more substitutions relative to the reference sequence corresponding to the reference sequence corresponding to SEQ ID NO:

2.

4. The engineered adenosine kinase of claim 1, comprising the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO:

4. The reference sequences corresponding to 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104 have an amino acid sequence identity of 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 higher, wherein the amino acid sequence comprises the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or one or more substitutions of the reference sequence corresponding to SEQ ID NO:

2.

5. The engineered adenosine kinase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to the reference sequence corresponding to the reference sequence corresponding to the amino acid residues 12-231 of SEQ ID NO: 2, or one or more substitutions thereof.

6. The engineered adenosine kinase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 1 27, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 18 8, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

7. The engineered adenosine kinase of any one of claims 1 to 6, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S, 56E / Q / T, 57H / L / P / S, 58C / G / Y, 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 67R, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 69F / L / Q / R / Y, 70R, 71 T, 73I / R, 74C / G / Q / S, 75L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81F / I / L / Q / S, 82I / L / P / Q / T, 83G / H / L / S / T, 84 A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 97I, 98A / G / Q, 99A / C, 100F / S / V, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 110C / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S, 118A / G / L / R / S,119F / K / L / P / R / S、120G / K / L / R / S / T、122A / H / S、123S、124V、125I、126A / E / S / V、127I / L / P / S、128C / E / I / K / N / R / S、129I / L / P / S、131A / G / V、132G / K / L / T / V、133A / E / F / L / Q / S / V / W、134L、135I / K / M / P / R / S、136A / I / L / S / V / Y、137L、138C / I / M / V、139A / H / L / R、140G、141G / V、142F / L / M / W、143A / C / G / P / R / S / T / V、146D / H / N / R / V、148F / H / M / P / Q / R / S / T、149L、150C / G / L / P / S / V、151A / F / R / Y、152F / H、153C / K / S / V、154Q / R、155A / F / T / W、156A / C / N / T / V、157P / V、161G / L、162G、163G / Q / S、166C / F / L / P / S、168G / L / N / Q / S、169A / D / I / Y、170A / G / H / P / R / S、172A / H / M / S / T、173F / K / R / S / T / V、175F / L / S、178E / G / N / R、179A / C / G / I / L / P / V、180G / H / P、181C / I / V、182A / G / I / L / Q / S、183A / L / Q、184I / K / M / N / R / S / T / V、186L、187G / Y、188G / L、190A / C / E / G / H / N / Q / R、191D / K、192A / H / I / W、194F / L / R / V / Y、195G / I / M / R、196I、197A / L / Q / V、198G、199R、200A、201A / F / K / L / S、202E / G / M / S、203A / E / L / R、204R / S、205L / P、206W、207A / L / T、208E、210V、211A、212C / H / I / L / M / N / P / Q / R / S / W、213L / P / Q / S、214A / E / L / M / P / R / T / W、215A / P / V、216A / D / E / G / H / L / M / N / P / R / S / T / V、217E / G / H / P / T、218A / I / L、219A / F / G / L / P / S / T / W、220E / V、221D、222I / T、223T、224G / S / T、225A / L / Q / R / T / V、226K / L / P / R / S / T、227P、228D / F / G / I / L / P / Q / R / S / T / V、229C / I、230A / M / P / Q or 231A / Q / R / T, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

8. The engineered adenosine kinase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 13, 18, 29, 30, 31, 32, 34, 35, 48, 50, 53, 55, 59, 60, 61, 62, 66, 68, 77, 78, 80, 83, 87, 100, 104, 105, 109, 118, 119, 127, 128, 133, 136, 139, 142, 143, 150, 151, 155, 157, 168, 170, 175, 184, 186, 190, 201, 207, 212, 213, 214, 216, 217, 218, 224 or 226, or combinations thereof, wherein the amino acid positions are relative to SEQ ID. The reference sequence corresponding to amino acid residues 12-231 of NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

9. The engineered adenosine kinase of any one of claims 1 to 5 and 8, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 13G / S, 18C / E / L, 29R / Q, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 48F, 50A / C / L / N / Q / R / S / V, 53A / M / S, 55G / L / Q / S, 59E / G / H / L / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 77A / I / M / Q / S, 78C / G / N / T, 80 118 A / G / L / R / S, 119F / K / L / P / R / S, 127I / L / P / S, 128C / E / I / K / N / R / S, 133A / E / F / L / Q / S / V / W, 136A / I / L / S / V / Y, 139A / H / L / R, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 150C / G / L / P / S / V, 151A / F / R / Y, 155A / F / T / W, 157P / V, 168G / L / N / Q / S, 170A / G / H / P / R / S, 175F / L / S, 184I / K / M / N / R / S / T / V, 186L, 190A / C / E / G / H / N / Q / R, 201A / F / K / L / S, 207A / L / T, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 216A / E / G / H / L / M / N / P / R / S / T / V / W, 217E / G / H / P / T, 218A / I / L, 224G / S / T or 226K / L / P / R / S / T, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

10. The engineered adenosine kinase of any one of claims 1 to 5 and 8, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 13G, 18L, 29Q, 30G / S, 31R, 32I, 34S, 35A, 48L, 50Q, 53A, 55Q, 59V, 60A, 61S, 62S, 66W, 68L, 77I, 78G, 80A / P, 83L, 87K, 100F, 104A, 105R, 109Y, 118G, 11 9R, 127I / L, 128E / K, ​​133E, 136A / V, 139L, 142L / R, 143V, 150S, 151F, 155F, 157V, 168G, 170G / S, 170S, 175F / L, 184M, 186L, 190Q / R, 201A / S, 207A, 212S, 213Q, 214E, 216E / M / P / R, 217P, 218I, 224G, or 226L, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

11. The engineered adenosine kinase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 29 / 30 / 128 / 142 / 186 / 212 / 213, 18 / 142 / 212 / 213, 29 / 30 / 128 / 142 / 186, 40 / 135 / 214, 18 / 29 / 30 / 127 / 150, 82, 69, 118, 29 / 30, 206, 32, 16, 104, 111 / 135 / 136 / 214, 29 / 128 / 175, 178, 18 / 128 / 142, 32 / 108 / 111 / 156 / 191, 18 / 29 / 128 / 129 / 142 / 186, 32 / 133, 18 / 29 / 30 / 212 / 213 / 222, 29 / 65 / 127, 32 / 40 / 43 / 52 / 155 / 156 / 217, 18 / 29 / 128 / 129, 80, 32 / 43 / 138 / 152 / 191 / 214 / 215, 183, 18 / 175 / 186 / 212 / 213, 129, 18 / 29 / 30 / 65 / 129 / 142 / 150 / 18 6, 127 / 142 / 212 / 213, 30 / 65 / 142 / 222, 40 / 43 / 215, 136, 29 / 150 / 211 / 212 / 213 / 221, 43 / 111 / 136 / 219, 226, 88, 140 / 215 / 216, 32 / 133 / 134 or 18 / 29 / 128 / 186, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

12. The engineered adenosine kinase of any one of claims 1 to 5 and 11, wherein the amino acid sequence of the engineered adenosine kinase comprises at least one of the following substitutions or sets of substitutions or amino acid residues: 29Q / 30S / 128K / 142L / 186L / 212S / 213Q, 18L / 142L / 212S / 213Q, 29Q / 30S / 128K / 142L / 186L, 40C / 135P / 214P, 18L / 29Q / 30S / 127L / 150S, 82I, 69Q, 118G, 29Q / 30S, 206W, 32N, 16M, 104I, 111E / 135P / 136A / 214P, 29Q / 128K / 175S, 178N, 18L / 128K / 142L, 32N / 108M / 111E / 156N / 191K, 18L / 29Q / 128K / 129I / 142L / 186L, 32N / 133L, 18L / 29Q / 30S / 212S / 2 13Q / 222I, 29Q / 65K / 127L, 32N / 40C / 43G / 52H / 155F / 156N / 217T, 18L / 29Q / 128K / 129I, 80N, 32N / 43G / 138I / 152H / 191K / 214P / 215V, 183Q, 18L / 175S / 186L / 212S / 213Q, 129I, 18L / 29Q / 30S / 65K / 129I / 142L / 150S / 186 L, 127L / 142L / 212S / 213Q, 30S / 65K / 142L / 222I, 40C / 43G / 215V, 136A, 29Q / 150S / 211A / 212S / 213Q / 221D, 43G / 111E / 136A / 219W, 226L, 88N, 140G / 215V / 216A, 32N / 133L / 134L or 18L / 29Q / 128K / 186L, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

13. The engineered adenosine kinase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least one substitution shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to SEQ ID NO: The reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

14. The engineered adenosine kinase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the amino acids listed in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 2 The substitutions or sets of substitutions of the engineered adenosine kinase variants shown in 7.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

15. The engineered adenosine kinase according to any one of claims 1 to 5, wherein the adenosine kinase comprises, and comprises, those listed in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, and 40.

2. The reference amino acid sequence of the engineered adenosine kinase variant shown in 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2 has 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 higher sequence identity, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

16. The engineered adenosine kinase of claim 1, comprising the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or the reference sequence corresponding to residues 12-231 of SEQ ID NO:

4. The reference sequences corresponding to the numbers 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104 have amino acid sequences with 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 higher sequence identity.

17. The engineered adenosine kinase of claim 1, comprising a reference sequence corresponding to residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652 and 2676-3192, or 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 higher sequence identity to the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652 and 2676-3192.

18. The engineered adenosine kinase of claim 1, comprising the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or the reference sequence corresponding to residues 12-231 of SEQ ID NO:

4. The reference sequences corresponding to 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104 have an amino acid sequence with 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 higher sequence identity, wherein the amino acid sequence comprises an amino acid sequence with respect to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

19. The engineered adenosine kinase of claim 1, comprising the reference sequence corresponding to residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192, or the reference sequence corresponding to even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652, and 2676-3192 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 higher sequence identity with respect to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

20. The engineered adenosine kinase of claim 18 or 19, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 11, 13, 14, 15, 16, 18, 20, 21, 22, 26, 27, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 6 3, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 88, 89, 90, 91, 92, 93, 94, 97, 98, 99, 100, 101, 102, 104, 105, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 12 7, 128, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 162, 163, 166, 168, 169, 170, 172, 173, 175, 178, 179, 180, 181, 182, 183, 184, 186, 187, 18 8, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231, or combinations thereof, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or with respect to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

21. The engineered adenosine kinase of any one of claims 18 to 20, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 11D, 13G / S, 14C / E, 15F, 16M, 18C / E / L, 20A / P / R / T / V, 21L / N / R / S / T / V, 22A / S, 26L, 27C / S, 29Q / R, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 36A / E / I / L / M / Q / R / S / V / Y, 37F / G / I / L / R / Y, 38C / F / L / R, 39A / M / Q / T, 40C / F / L / S / T, 42T, 43A / C / G / H / N, 44S, 46E / F / L / M / Y, 48F / L, 50A / C / L / N / Q / R / S / V, 51F / G / K / L / M / R / T, 52A / H, 53A / M / S, 54G / H / I / M / Q / R / T, 55G / L / Q / S , 56E / Q / T, 57H / L / P / S, 58C / G / Y, 59E / G / H / M / P / R / T / V / W / Y, 60A / C / P / S / W, 6 1A / E / N / P / S / V, 62A / E / G / I / L / S, 63S, 64R, 65A / D / F / K / P / T, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W、67R、68A / D / E / G / I / L / Q / R / S / T / V / W / Y、69F / L / Q / R / Y, 70R, 71T, 73I / R, 74C / G / Q / S, 75L, 76G / L / P, 77A / I / M / Q / S, 78C / G / N / T, 79 A / K / L / P / W, 80A / D / G / L / N / P / R / S / V / W, 81F / I / L / Q / S, 82I / L / P / Q / T, 83G / H / L / S / T, 84A / E / H / M / S, 87A / E / I / K / L / M / R / V / Y, 88N / R / T, 89A / H / I / L / P / Q / T / V, 90C / E / F / K / S / T / V, 91A / F / G / L / S / V, 92L / S, 93A / E / G / P / S / T / V / Y, 94A / C / F / M / Q / S / T / V / Y, 97I, 98A / G / Q, 99A / C, 100F / S / V / Y, 101-, 102A / C / N, 104A / F / H / I / L / Q / R / S / W, 105G / K / L / M / R / S, 108M / R / S / V, 109E / Y, 110C / F, 111E / P / R, 112A / C / E / K / M / N / Q / R / T, 113E / W / Y, 115G / K / R, 116A / E / F / L, 117L / N / S,118A / G / L / R / S、119F / K / L / P / R / S、120G / K / L / R / S / T、122A / H / S、123S、124V、125I、126A / E / S / V、127I / L / P / S、128C / E / I / K / N / R / S、129I / L / P / S、131A / G / V、132G / K / L / T / V、133F / Q / S / V / W、133A / E / F / L / Q / S / V / W、134L、135I / K / M / P / R / S、136A / I / L / S / V / Y、137L、138C / I / M / V、139A / H / L / R、140G、141G / V、142F / L / M / W、143A / C / G / P / R / S / T / V、146D / H / N / R / V、148F / H / M / P / Q / R / S / T、149L、150C / G / L / P / S / V、151A / F / R / Y、152F / H、153C / K / S / V、154Q / R、155A / F / T / W、156A / C / N / T / V、157P / V、161G / L、162G、163G / Q / S、166C / F / L / P / S、168G / L / N / Q / S、169A / D / I / Y、170A / G / H / P / R / S、172A / H / M / S / T、173F / K / R / S / T / V、175F / L / S、178E / G / N / R、179A / C / G / I / L / P / V、180G / H / P、181C / I / V、182A / G / I / L / Q / S、183A / L / Q、184I / K / M / N / R / S / T / V、186L、187G / Y、188G / L、190A / C / E / G / H / N / Q / R、191D / K、192A / H / I / W、194F / L / R / V / Y、195G / I / M / R、196I、197A / L / Q / V、198G、199R、200A、201A / F / K / L / S、202E / G / M / S、203A / E / L / R、204R / S、205L / P、206W、207A / L / T、208E、210V、211A、212C / H / I / L / M / N / P / Q / R / S / W、213L / P / Q / S、214A / E / L / M / P / R / T / W、215A / P / V、216A / D / E / G / H / L / M / N / P / R / S / T / V、217E / G / H / P / T、218A / I / L、219A / F / G / L / P / S / T / W、220E / V、221D、222I / T、223T、224G / S / T、225A / L / Q / R / T / V、226K / L / P / R / S / T、227P、228D / F / G / I / L / P / Q / R / S / T / V, 229C / I, 230A / M / P / Q, or 231A / Q / R / T, or combinations thereof, wherein the amino acid position corresponds to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104 of the reference sequence, or corresponds to SEQ ID NO:

4. The reference sequence corresponding to 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

22. The engineered adenosine kinase of any one of claims 18 to 20, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 13, 18, 29, 30, 31, 32, 34, 35, 48, 50, 53, 55, 59, 60, 61, 62, 66, 68, 77, 78, 80, 83, 87, 100, 104, 10 5, 109, 118, 119, 127, 128, 133, 136, 139, 142, 143, 150, 151, 155, 157, 168, 170, 175, 184, 186, 190, 201, 207, 212, 213, 214, 216, 217, 218, 224 or 226, or combinations thereof, wherein the amino acid position is relative to SEQ ID The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or with respect to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

23. The engineered adenosine kinase of any one of claims 18 to 20 and 22, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 13G / S, 18C / E / L, 29R / Q, 30G / L / S / Y, 31R, 32F / I / L / N, 34A / E / S / V, 35A / E / F / L / S, 48F, 50A / C / L / N / Q / R / S / V, 53A / M / S, 55G / L / Q / S, 59E / G / H / L / M / P / R / T / V / W / Y, 60A / C / P / S / W, 61A / E / N / P / S / V, 62A / E / G / I / L / S, 66A / C / D / E / F / G / H / N / P / Q / R / S / T / V / W, 68A / D / E / G / I / L / Q / R / S / T / V / W / Y, 77A / I / M / Q / S, 78C / G 1 09E / Y, 118A / G / L / R / S, 119F / K / L / P / R / S, 127I / L / P / S, 128C / E / I / K / N / R / S, 133A / E / F / L / Q / S / V / W, 136A / I / L / S / V / Y, 139A / H / L / R, 142F / L / M / W, 143A / C / G / P / R / S / T / V, 150C / G / L / P / S / V, 151A / F / R / Y, 155A / F / T / W, 157P / V, 168G / L / N / Q / S, 170A / G / H / P / R / S, 1 75F / L / S, 184I / K / M / N / R / S / T / V, 186L, 190A / C / E / G / H / N / Q / R, 201A / F / K / L / S, 207A / L / T, 212C / H / I / L / M / N / P / Q / R / S / W, 213L / P / Q / S, 214A / E / L / M / P / R / T / W, 216A / E / G / H / L / M / N / P / R / S / T / V / W, 217E / G / H / P / T, 218A / I / L, 224G / S / T, or 226K / L / P / R / S / T, or combinations thereof.The amino acid positions described herein refer to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

24. The engineered adenosine kinase of any one of claims 18 to 20 and 22, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 13G, 18L, 29Q, 30G / S, 31R, 32I, 34S, 35A, 48L, 50Q, 53A, 55Q, 59V, 60A, 61S, 62S, 66W, 68L, 77I, 78G, 80A / P, 83L, 87K, 100F, 104A, 105R, 109Y, 118G, 119R, 127I / L, 128E / K, ​​133E, 136A / V, 139L, 142L / R, 143V, 150S, 151F, 155F, 157V, 168G, 170G / S, 170S, 175F / L, 184M, 186L, 190Q / R, 201A / S, 207A, 212S, 213Q, 214E, 216E / M / P / R, 217P, 218I, 224G, or 226L, or combinations thereof, wherein the amino acid position is relative to SEQ. The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or with respect to the reference sequence corresponding to SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

25. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or one or more substitutions with respect to the reference sequence corresponding to SEQ ID NO:

4.

26. The engineered adenosine kinase of claim 19, 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%, 98%, 99%, or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 86-372, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO:

4.

27. The engineered adenosine kinase of claim 25 or 26, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 100 / 101, 94, 179, 198, 57, 78, 68, 93, 77, 62, 14, 51, 118, 91, 163, 65, 173, 224, 66, 109, 60, 40 / 88, 191, 63, 68 / 170, 100, 108, 58, 180, 178, 199, 40, 80 / 203, 40 / 43, 39, 55, 110, 51 / 59, 155 / 226, 80, 219, 166, 202, 59, 38, 90, 80 / 184 / 203, 35, 222, 54, 111 / 153 / 155, 112, 175, 74, 105, 170, 117, 128, 61, 119, 155, 79, 220, 226, 108 / 155, 108 / 111, 111, 69 / 88, 43, 43 / 69 / 138, 40 / 43 / 88 / 134 / 178, 214, 80 / 82 / 184 / 219, 88 / 136, 16 / 40 / 43 / 88, 40 / 134, 88, 111 / 155, 223, 115, 190, 201, 126, 56, 216, 76, 69, 120, 116, 203, 194 or 138, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO:

4.

28. The engineered adenosine kinase of any one of claims 25 to 27, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following set of substitutions or amino acid residues: 100V / 101-, 94S, 179L, 198G, 57P, 78G, 68R, 93V, 77S, 62L, 14C, 51R, 118R, 91F, 163G, 65P, 173R, 224G, 66E, 109E, 60S, 40C / 88N, 191D, 63S, 68D / 170P, 100S, 51M, 108S, 58G, 57H, 180G , 178R, 199R, 40C, 94Q, 80N / 203A, 40C / 43G, 39T, 62S, 40L, 55Q, 110C, 51R / 59R, 155F / 226L, 80N, 219T, 166P, 60P, 202S, 59T, 80A, 3 8L, 94C, 62A, 51K, 90E, 59M, 80N / 184K / 203A, 35S, 222T, 178G, 40T, 54M, 111E / 153K / 155F, 62I, 80G, 112M, 175S, 74S, 14E, 57L, 51G, 94F, 68A, 105R, 170A, 117N, 128S, 40F, 112C, 94A, 61A, 68W, 202G, 180P, 119R, 119R, I66G, 179G, 155F, 59V, 79L, 220E, 51F, 93A, 22 6L, 108M / 155F, 108M / 111E, 111E, 69Q / 88N, 43G, 43G / 69Q / 138I, 40C / 43G / 88N / 134L / 178N, 214P, 80N / 82I / 184K / 219W, 88N / 136A, 1 6M / 40C / 43G / 88N, 40C / 134L, 88N, 111E / 155F, 223T, 74G, 115R, 39Q, 93E, 119L, 190N, 202M, 55S, 51L, 112Q, 94T, 66A, 201A, 201K, 201L, 66R, 128N, 214L, 126S, 56E, 179I, 216S, 76P, 69Y, 115G, 166C, 120L, 116L, 108V, 203E, 194F, 138M, 178E, or 38C, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 4, or relative to the reference sequence corresponding to SEQ ID NO:

4.

29. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or with respect to the reference sequence corresponding to SEQ ID NO:

172.

30. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 374-444, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or relative to the reference sequence corresponding to SEQ ID NO:

172.

31. The engineered adenosine kinase of claim 29 or 30, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 66 / 78, 78 / 80 / 224, 78, 65 / 66, 78 / 224, 78 / 80 / 109, 40 / 60 / 94 / 226, 66 / 68, 78 / 80, 94 / 117 / 118 / 226, 68 / 109, 68, 94 / 179, 51 / 66 / 68 / 224, 94 / 117, 51 / 66 / 68 / 78 / 224, 66, 118, 93 / 94 / 180, 109, 68 / 224, 40 / 62 / 93, 93, 51 / 66, 94 / 117 / 118, 198 / 220, 93 / 117, 117 / 118, 93 / 198, 68 / 173, 40 / 117, 224, 40 / 118 / 226, 93 / 94, or 40 / 62 / 118, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 172, or relative to the reference sequence corresponding to SEQ ID NO:

172.

32. The engineered adenosine kinase of any one of claims 29 to 31, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 66E / 78G, 78G / 80A / 224G, 78G, 65P / 66E, 78G / 224G, 78G / 80A / 109E, 40F / 60S / 94S / 226I, 66A / 68R, 66E / 68R, 78G / 80A, 94S / 117N / 118R / 226I, 68R / 109E, 68R, 94S / 179L, 51R / 66E / 68R / 2 24G, 94S / 117N, 51R / 66E / 68R / 78G / 224G, 66E, 118R, 93V / 94S / 180P, 109E, 68R / 224G, 40F / 62L / 93V, 93V, 51R / 66E, 94S / 117N / 118R, 198G / 220E, 93V / 117N, 117N / 118R, 93V / 198G, 68R / 173R, 40F / 117N, 224G, 40F / 118R / 226I, 93V / 94S or 40F / 62L / 118R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO:172, or relative to the reference sequence corresponding to SEQ ID NO:

172.

33. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO:

376.

34. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 446-510, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO:

376.

35. The engineered adenosine kinase of claim 33 or 34, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 13, 60, 126, 104, 201, 59, 55, 133, 61, 53, 190, 57, 62, 181, 168, 54, 173, 170, 34, 183, 14, 60 / 62 / 124 / 170, 60 / 170, or 60 / 62, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or relative to the reference sequence corresponding to SEQ ID NO:

376.

36. The engineered adenosine kinase of any one of claims 33 to 35, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 13S, 60A, 126E, 104L, 201S, 59E, 55Q, 133E, 61P, 59M, 13G, 53A, 190A, 57S, 62A, 181V, 55G, 168G, 54R, 54Q, 173R, 168Q, 168N, 170P, 34V, 59H, 183L, 173K, 183A, 14E, 60A / 62A / 124V / 170G, 60A / 170G, or 60A / 62A, wherein the amino acid position corresponds to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 376, or to SEQ ID NO: Regarding the reference sequence corresponding to 376.

37. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or with respect to the reference sequence corresponding to SEQ ID NO:

508.

38. The engineered adenosine kinase of claim 19, 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 higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 512-580, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or relative to the reference sequence corresponding to SEQ ID NO:

508.

39. The engineered adenosine kinase of claim 37 or 38, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 15, 15 / 68, 15 / 181, 13 / 15, 13 / 15 / 201, 54 / 55, 55 / 133, 55 / 133 / 197, 54 / 55 / 133 / 197, 68, 54 / 133 / 197, 54 / 197, 13 / 15 / 68, 34, 54, 133, 120, 34 / 133, 13 / 15 / 181, 55, 15 / 59, 68 / 181, 201, 181, 66, 15 / 59 / 181, 197, 13 / 15 / 59, 61, 34 / 61, 13, 34 / 69, or 163, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or relative to the reference sequence corresponding to SEQ ID NO:

508.

40. The engineered adenosine kinase of any one of claims 37 to 39, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 15F, 15F / 68A, 15F / 181V, 13S / 15F, 13S / 15F / 201S, 54Q / 55Q, 55Q / 133E, 55Q / 133E / 197A, 54Q / 55Q / 133E / 197A, 68A, 54Q / 133E / 197A, 54Q / 197A, 13S / 15F / 68Y, 34V, 54Q, 133E, 120K, 34V / 133A, 13S / 15F / 181V, 55Q, 15F / 59M, 68A / 181V, 201S, 68Y, 181V, 66S, 15F / 59M / 181V, 197A, 13S / 15F / 59M, 61S, 34V / 61S, 13S, 61A, 34V / 69R, or 163S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 508, or relative to the reference sequence corresponding to SEQ ID NO:

508.

41. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or with respect to the reference sequence corresponding to SEQ ID NO:

524.

42. The engineered adenosine kinase of claim 19, 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 higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 582-608, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO:

524.

43. The engineered adenosine kinase of claim 41 or 42, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 120, 61 / 201, 34, 36, 105, 112, 31, or 146, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO:

524.

44. The engineered adenosine kinase of any one of claims 41 to 43, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 120R, 61S / 201S, 34S, 36I, 36V, 36A, 36L, 36M, 105S, 34A, 112E, 31R, 146D, or 112R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 524, or relative to the reference sequence corresponding to SEQ ID NO:

524.

45. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or with respect to the reference sequence corresponding to SEQ ID NO:

584.

46. ​​The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 610-666, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or relative to the reference sequence corresponding to SEQ ID NO:

584.

47. The engineered adenosine kinase of claim 45 or 46, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 13 / 30 / 31 / 53 / 109 / 119 / 168, 36 / 126 / 190, 126, 36 / 39 / 126, 126 / 190, 36 / 126, 36 / 190, 39 / 126, 1 3 / 109 / 119, 109, 36 / 120 / 190, 126 / 148, 190, 108 / 126, 30 / 31, 13 / 118 / 182, 31 / 53, 39, 119, 13 / 119 / 168, 53 / 168, 34, 34 / 65 / 146, 36 / 39, 119 / 168, 34 / 112 or 173, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or relative to the reference sequence corresponding to SEQ ID NO:

584.

48. The engineered adenosine kinase of any one of claims 45 to 47, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 13G / 30G / 31R / 53A / 109Y / 119R / 168G, 36Y / 126E / 190A, 126E, 36Q / 39M / 126E, 126E / 190A, 36Y / 39M / 126E, 36Q / 126E, 36Y / 190A, 39M / 126E, 13G / 1 09Y / 119R, 109Y, 36Y / 120G / 190A, 126E / 148T, 190A, 108R / 126E, 30G / 31R, 13G / 118A / 182I, 31R / 53A, 39M, 119R, 13G / 119R / 168G, 53A / 168G, 34S, 34S / 65A / 146D, 36Q / 39M, 119R / 168G, 34S / 112R, 36Y / 39M or 173K, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 584, or relative to the reference sequence corresponding to SEQ ID NO:

584.

49. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO:

610.

50. The engineered adenosine kinase of claim 19, 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 higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 668-780, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO:

610.

51. The engineered adenosine kinase of claim 49 or 50, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 190, 115 / 126 / 190, 126 / 190, 34 / 35 / 105, 126, 59 / 126, 34 / 105 / 116 / 146, 48, 66, 59, 148, 82, 100, 27, 135, 77, 138, 52, 68, 139, 184, 81, 65, 153, 50, 181, 214, 172, 69, or 154, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO:

610.

52. The engineered adenosine kinase of any one of claims 49 to 51, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 190A, 115K / 126E / 190A, 126E / 190A, 34S / 35A / 105R, 126E, 59E / 126E, 34S / 105R / 116E / 146D, 48L, 66F, 59T, 148H, 82T, 59V, 59Y, 59P, 100F, 148F, 27S The amino acid positions are 135R, 148T, 77M, 138C, 148Q, 126A, 52A, 66Q, 59W, 59G, 148M, 59M, 135S, 68L, 139A, 184I, 68R, 184V, 66S, 68Q, 81I, 68V, 65F, 153S, 126V, 68A, 66T, 50C, 138V, 181I, 214T, 139L, 68T, 148S, 172H, 138I, 69L, 81Q, or 154R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 610, or relative to the reference sequence corresponding to SEQ ID NO:

610.

53. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO:

674.

54. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 782-868, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO:

674.

55. The engineered adenosine kinase of claim 52 or 54, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 81 / 148 / 219, 68 / 148 / 219, 68 / 81 / 219, 148 / 219, 81 / 219, 136 / 138 / 139, 68 / 219, 66 / 68 / 219, 66 / 148 / 219, 66 / 68 / 148, 59, 59 / 135, 219, 59 / 139, 138, 126 / 148 / 219, 59 / 181, 81, 66 / 81 / 219, 126 / 219, 66 / 68 / 81 / 148 / 219, 181, 27 / 148, 135 / 136 / 139, 148, 59 / 135 / 138 / 181, 59 / 135 / 136 / 138 / 139, 59 / 135 / 136 / 138 / 181, 27, 27 / 126 / 148, 135 / 136 / 138, 68 / 81 / 126 / 148 / 219, 136, 68, 126 / 148 or 138 / 139, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO:

674.

56. The engineered adenosine kinase of any one of claims 52 to 55, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 81I / 148T / 219L, 68A / 148H / 219L, 68A / 81I / 219L, 148H / 219F, 68A / 148T / 219L, 81I / 219L, 136L / 13 8V / 139L, 148T / 219F, 148H / 219L, E68A / V219L, 66T / 68A / 219L, 66Q / 148H / 219F, 148T / 219L , 66T / 68A / 148H, 59V, 59V / 135R, 219F, 59V / 139L, 138V, 126A / 148T / 219L, 59V / 181I, 81I, 6 6Q / 81I / 219L, 219L, 126A / 219L, 66Q / 68A / 81I / 148T / 219L, 181I, 27S / 148H, 135R / 136L / 13 9R、136L / 138V / 139R、148T、59V / 135R / 138V / 181I、59V / 135R / 136L / 138V / 139R、59V / 135R / 136L / 138V / 181I, 66Q / 68G / 81I / 148H / 219L, 27S, 27S / 126A / 148T, 135R / 136L / 138V, 68G / 81I / 126A / 148H / 219L, 136L, 27S / 126A / 148H, 68G, 126A / 148T, or 138V / 139R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 674, or relative to the reference sequence corresponding to SEQ ID NO:

674.

57. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO:

816.

58. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 870-1014, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO:

816.

59. The engineered adenosine kinase of claim 57 or 58, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 48 / 100 / 148, 48 / 66 / 148 / 219, 48 / 100, 48 / 100 / 135 / 136, 48 / 66 / 77 / 100 / 135 / 136 / 148, 48 / 100 / 136, 48 / 135 / 148, 48 / 148, 48 / 136 / 148, 48 / 1 35, 48 / 135 / 136, 66 / 100, 48 / 66 / 219, 48 / 135 / 219, 100 / 148, 48 / 136 / 219, 48 / 219, 48 / 135 / 148 / 219, 100 / 135 / 136, 100 / 136, 66 / 77 / 100 / 136 / 219, 100 / 135, 48 / 77 / 100 / 135 / 136, 100, 77 / 100 / 135, 48 / 66 / 100 / 135 / 136 / 148 / 219, 52 / 184, 66, 52 / 68, 136 / 148, 126 / 184, 148, 184, 52, 135 / 136 / 148, 126 / 138 / 184, 27 / 184, 27 / 126, 27 / 126 / 148, 27, 27 / 126 / 184, 66 / 100 / 135 / 219, 27 / 68, 52 / 68 / 81 / 126, 48 / 66 / 136 / 148, 66 / 136 / 148, 66 / 135, 66 / 136 66 / 135 / 219, 136, 135 / 148 / 219, 68 / 126, 66 / 148 / 219, 27 / 68 / 126, 27 / 68 / 184, 126, 68 / 81 / 126, 77 / 135, 135 / 136, 77 / 136, 77 / 148, 82, 138 / 184, 77 / 135 / 136, 27 / 138, 81, 81 / 126, 27 / 126 / 138, 68 or 81 / 138, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO:

816.

60. The engineered adenosine kinase of any one of claims 57 to 59, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 48L / 100F / 148H, 48L / 66F / 148F / 219L, 48L / 100F, 48L / 100F / 135R / 136L, 48L / 66F / 77M / 100F / 135R / 136L / 148H, 48L / 100F / 136L, 48L / 135R / 148F, 48L / 148F, 48L / 136L / 148F, 48L / 135R, 48L / 135R / 136 L, 66F / 100F, 48L / 66Q / 219L, 48L / 135R / 219L, 100F / 148H, 48L / 136L / 2 19L, 48L / 219L, 48L / 135R / 148F / 219L, 100F / 135R / 136L, 100F / 136L, 66 F / 77M / 100F / 136L / 219L, 100F / 135R, 48L / 77M / 100F / 135R / 136L, 100F , 77M / 100F / 135R, 48L / 66F / 100F / 135R / 136L / 148F / 219L, 52A / 184I, 66 F、52A / 68L、136L / 148F、126A / 184I、148F、126A / 184V、184V、52A、135R / 136L / 148F、126A / 138V / 184V、184I、27S / 184V、27S / 126A、27S / 126A / 148R, 27S, 27S / 126A / 184V, 66Q / 100F / 135R / 219L, 27S / 68A, 52A / 68A / 81L / 126A, 66Q, 48L / 66Q / 136L / 148F, 66F / 136L / 148H, 66Q / 135R, 66Q / 1 36L, 66F / 135R / 219L, 136L, 135R / 148F / 219L, 68A / 126A, 66F / 148H / 219L, 27S / 68L / 126A, 27S / 68L / 184V, 126A, 68L / 81L / 126A, 77M / 135R, 135R / 136L, 77M / 136L, 77M / 148F, 82T, 138V / 184V, 77M / 135R / 136L, 27S / 138V, 81L, 81L / 126A, 27S / 126A / 138V, 68A or 81L / 138V, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 816, or relative to the reference sequence corresponding to SEQ ID NO:

816.

61. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or with respect to the reference sequence corresponding to SEQ ID NO:

874.

62. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 1016-1302, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO:

874.

63. The engineered adenosine kinase of claim 61 or 62, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 32 / 60 / 135 / 175 / 225, 32 / 81 / 127 / 128 / 135 / 175 / 225, 83 / 175 / 225, 135 / 175 / 225, 54 / 84 / 190, 55 / 190 / 212, 32 / 60 / 81 / 83 / 127 / 128 / 135 / 225, 175 / 225, 32 / 127 / 175, 84 / 190 / 212, 32 / 128 / 175, 81 / 175, 21 / 54, 54 / 55 / 212, 3 2 / 175 / 225, 128 / 175 / 225, 62 / 84 / 190, 81 / 135 / 225, 21 / 54 / 62 / 190 / 212, 60 / 81 / 83 / 175, 55 / 84 / 190, 32 / 81 / 83 / 135 / 225, 175, 62 / 212, 32 / 81 / 83, 32 / 8 1 / 225, 54 / 55 / 84 / 212, 55 / 190, 62 / 179, 54 / 84 / 179 / 212, 54 / 55 / 62 / 84, 81 / 135, 32 / 81 / 135, 179 / 190 / 212, 55 / 62, 100, 21 / 54 / 62 / 190, 190 / 212, 128 / 17 5, 21 / 62 / 190, 54 / 179 / 190, 54 / 190, 21 / 55 / 190, 21 / 54 / 62, 21 / 84, 21 / 55, 21 / 62, 21 / 179, 55 / 62 / 190 / 212, 54 / 62 / 84, 127 / 128 / 175 / 225, 54 / 62, 100 / 1 04, 54 / 55, 21 / 55 / 212, 62, 55 / 84, 55 / 62 / 212, 21 / 84 / 190, 225, 21 / 54 / 179 / 190, 83 / 135, 32 / 175, 21 / 54 / 78 / 190, 55 / 62 / 179 / 190 / 212, 21 / 54 / 190, 21 / 55 / 84 / 190, 62 / 84, 21 / 55 / 62, 21 / 62 / 84, 135, 32 / 60 / 81 / 127, 21 / 54 / 55 / 62 / 78, 54 / 84, 55 / 62 / 179 / 190, 54 / 55 / 62 / 84 / 179 / 190 / 212, 190, 83, 78, 54 / 6 2 / 190, 21 / 55 / 62 / 179 / 190, 32 / 60 / 127, 54 / 62 / 84 / 190 / 212, 21 / 62 / 179, 127 / 128 / 225, 32 / 225, 32 / 60 / 81, 81, 54, 21, 60 / 81 / 128 / 175, 84, 21 / 190 / 212,60 / 128 / 225, 179, 21 / 55 / 62 / 190, 128, 60, 212, 55, 127, 32, 80, 220, 62 / 78 / 179, 55 / 62 / 84, 21 / 55 / 190 / 212, 32 / 127 / 128 / 225, 21 / 54 / 55 / 84 / 179, 21 / 190, 32 / 60 / 81 / 83 / 128 / 135, 127 / 128, or 21 / 55 / 179, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO:

874.

64. The engineered adenosine kinase of any one of claims 61 to 63, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 32I / 60S / 135K / 175L / 225A, 32I / 81L / 127I / 128N / 135K / 175L / 225A, 83S / 175F / 225A, 135K / 175L / 225A, 54T / 84H / 190R, 55S / 190H / 212N, 32I / 60S / 81L / 83S / 127I / 128N / 135K / 225A, 175F / 225A, 32I / 127I / 175L, 84A / 190H / 212N, 32I / 128N / 175F, 81L / 175L, 81L / 175F, 21R / 54T, 54T / 55S / 212N, 32I / 175L / 225A, 128N / 175F / 225A, 62S / 84A / 190R, 81L / 135M / 225A, 21R / 54T / 6 2S / 190R / 212N, 60S / 81L / 83G / 175L, 55S / 84A / 190R, 32I / 81L / 83G / 135K / 22 5A, 175F, 62S / 212N, 32I / 81L / 83S, 32I / 81L / 225A, 54T / 55S / 84A / 212N, 55S / 190R, 62S / 179P, 62E / 84A / 190H, 54T / 84A / 179P / 212N, 54T / 55S / 62S / 84A, 8 1L / 135M, 32I / 81L / 135K, 179P / 190H / 212N, 55S / 62E, 100Y, 21R / 54T / 62E / 1 90H, 190H / 212N, 128N / 175L, 21R / 62S / 190H, 54T / 179P / 190H, 54T / 190R, 21 R / 55S / 190H, 21R / 54T / 62E, 21R / 84A, 21R / 55S, 21R / 62S, 21R / 179P, 175L, 55 S / 62E / 190H / 212N, 54T / 62E / 84A, 127I / 128N / 175L / 225A, 54T / 62E, 100Y / 1 04F、55S / 62E / 190R / 212N、54T / 55S、21R / 55S / 212N、62S、55S / 84A、55S / 62S / 212N, 21R / 84A / 190H, 225A, 21R / 54T / 179P / 190R, 83S / 135K, 32I / 175L, 21R / 54T / 78T / 190R, 62E, 55S / 62E / 179P / 190H / 212N, 81L / 135K, 21R / 54T / 190H,21R / 55S / 84A / 190H, 62S / 84A, 21R / 55S / 62E, 21R / 62S / 84A, 135M, 32I / 60S / 81L / 127I, 21R / 54T / 55S / 62E / 78T, 100Y / 104H, 54T / 84A, 55S / 62S / 179P / 190R, 54T / 55S / 62S / 84A / 179P / 190H / 212N, 190R, 190H, 83S, 10 0Y / 104R, 78T, 54T / 62E / 190R, 21R / 55S / 62E / 179P / 190H, 78C, 32I / 60S / 127I, 54T / 62E / 84A / 190H / 212N, 2 1R / 62S / 179P, 100Y / 104Q, 127I / 128N / 225A, 32I / 225A, 32I / 60S / 81L, 225V, 81L, 54T, 21R, 60S / 81L / 128N / 175L, 190Q, 84A, 21R / 190R / 212N, 190C, 135K, 60S / 128N / 225A, 225T, 62G, 225Q, 179P, 225R, 100Y / 104S, 83H, 21R / 55S / 62E / 190R, 100Y / 104W, 190G, 128N, 84M, 60S, 78N, 84S, 62L, 128E, 212N, 55S, 127I, 21S, 32I The amino acid positions are 80G, 84E, 220V, 62S / 78T / 179P, 190E, 55S / 62E / 84A, 21R / 55S / 190H / 212N, 32I / 127I / 128N / 225A, 21R / 54T / 55S / 84A / 179P, 21R / 190R, 32I / 60S / 81L / 83S / 128N / 135M, 127I / 128N, or 21R / 55S / 179P, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 874, or relative to the reference sequence corresponding to SEQ ID NO:

874.

65. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

66. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 1356-1552, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

67. The engineered adenosine kinase of claim 65 or 66, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 54 / 80 / 225, 55 / 80 / 180, 54 / 55 / 80 / 208 / 225, 84 / 128 / 155 / 190 / 212, 62 / 155 / 190, 83 / 84 / 155 / 190, 62 / 128 / 135 / 190, 225, 84 / 155 / 190, 128 / 190, 190 / 212, 83 / 84 / 155, 54 / 208 / 225, 84 / 155 / 190, 84 / 212, 84 / 123 / 155 / 212. 62 / 128 / 190, 55 / 80 / 208 / 225, 155 / 190, 83 / 84 / 190 / 212, 83 / 128 / 155 / 190 / 212, 55 / 180 / 225, 84 / 190 / 212, 54 / 80 / 180 / 208 / 225, 80 / 208, 135 / 155 / 190 212, 80 / 180 / 208, 128 / 135 / 190 / 212, 128 / 155 / 212, 80 / 208 / 225, 83 / 212, 62 / 84 / 155 / 190, 54 / 179 / 225, 84, 83 / 84 / 190, 128 / 212, 54 / 55 / 80 / 208, 55, 6 2 / 128, 54 / 80 / 208 / 225, 62 / 83 / 84 / 190, 179 / 225, 190, 80 / 169 / 208, 62 / 128 / 212, 80, 62, 54 / 55 / 80 / 225, 54 / 80 / 180 / 208, 83 / 84 / 128 / 190 / 212, 62 / 83 / 190, 62 / 190, 80 / 179, 80 / 180, 54 / 80, 62 / 83 / 84 / 128 / 190 / 212, 83 / 84 / 135 / 212, 54 / 80 / 179 / 208, 62 / 84 / 128, 11 / 62 / 84 / 128 / 190 / 212, 80 / 179 / 208, 80 / 225, 62 / 84 / 128 / 190 / 212, 62 / 84 / 212, 80 / 179 / 180 / 208 / 225, 62 / 135, 55 / 208, 62 / 84, 128 / 135 / 190, 55 / 208 / 225, 84 / 128 / 190, 62 / 84 / 128 / 190, 62 / 13 5 / 212, 54 / 179 / 180 / 225, 62 / 84 / 135 / 212, 135, 54 / 55 / 225, 55 / 80 / 179 / 180, 84 / 135 / 190, 84 / 128, 54, 84 / 128 / 135, 54 / 179 / 208 / 225, 55 / 179 / 225, 128,62 / 83 / 84 / 155 / 190, 84 / 135, 84 / 128 / 155, 55 / 179 / 180, 83 / 84 / 128 / 190, or 54 / 55 / 179 / 225, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

68. The engineered adenosine kinase of any one of claims 65 to 67, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 54T / 80D / 225A, 55S / 80D / 180H, 54T / 55S / 80D / 208E / 225L, 84M / 128E / 155T / 190R / 212N, 62S / 155T / 190H, 83T / 84M / 155T / 190H, 62S / 128E / 135K / 190H, 225A, 84M / 155T / 190R, 128E / 190R, 190R / 212N, 83T / 84M / 155T, 54 T / 208E / 225A, 84M / 155T / 190H, 84H / 212N, 84M / 123S / 155T / 212N, 62S / 128 E / 190R, 55S / 80D / 208E / 225A, 155T / 190R, 83T / 84M / 190R / 212N, 83T / 128E / 155T / 190H / 212N, 55S / 180H / 225A, 84M / 190R / 212N, 54T / 80D / 180H / 208E / 2 25A, 80D / 208E, 135K / 155T / 190R, 212N, 80D / 180H / 208E, 128E / 135K / 190R / 212N, 128E / 155T / 212N, 80D / 208E / 225L, 83T / 212N, 62S / 84M / 155T / 190R, 54T / 179V / 225A, 84H, 83T / 84M / 190R, 128E / 212N, 54T / 55S / 80D / 208E, 190H / 212N, 55S, 62S / 128E, 54T / 80D / 208E / 225A, 62S / 83T / 84M / 190R, 179A / 22 5A, 190R, 80D / 169D / 208E, 62S / 128E / 212N, 80D, 62S, 54T / 55S / 80D / 225L, 5 4T / 80D / 180H / 208E, 83T / 84M / 128E / 190H / 212N, 62S / 83T / 190R, 179V / 225 A. 62S / 190R, 80D / 179V, 80D / 180H, 54T / 80D, 62S / 83T / 84H / 128E / 190H / 212 N, 83T / 84M / 135K / 212N, 54T / 80D / 179A / 208E, 62S / 84H / 128E, 11D / 62S / 84M / 128E / 190H / 212N, 80D / 179V / 208E, 80D / 225A, 62S / 84M / 128E / 190H / 212N,62S / 84H / 212N, 80D / 179V / 180H / 208E / 225L, 62S / 135K, 55S / 208E, 62S / 84H, 128E / 135K / 190H, 55S / 208E / 225A, 190H, 84H / 128E / 190H, 62S / 84H / 128E / 190R, 62S / 135K / 212N, 54T / 179V / 180H / 225A, 128E / 190H, 62S / 84M / 135K / 212N, 135K, 54T / 55S / 225L, 55S / 80D / 179V / 1 80H, 62S / 84M / 212N, 84M / 135K / 190R, 84M / 128E, 54T, 84M / 128E / 135K, 54T / 179A / 208E / 225A, 55S / 179V / 225L, 128E, 62S / 83T / 84M / 155T / 190R, 84M / 135K, 84H / 128E / 155T, 55S / 179V / 180H, 83T / 84M / 128E / 190R, 83T / 84M / 190H, 84M or 54T / 55S / 179V / 225A, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

69. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or with respect to the reference sequence corresponding to SEQ ID NO: 1388.

70. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 1554-1786, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or relative to the reference sequence corresponding to SEQ ID NO: 1388.

71. The engineered adenosine kinase of claim 69 or 70, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 143, 228, 136, 82, 156, 205, 226, 150, 219, 217, 218, 68, 142, 212, 213, 79, 230, 207, 155, 227, 129, 210, 133, 151, 43, 204, 182, 82 / 136 / 150 / 217, 136 / 150, 79 / 81 / 143 / 156 / 212 / 2 28, 82 / 136, 79 / 81 / 143 / 212, 79 / 133 / 228, 81 / 143 / 156 / 212, 136 / 219, 82 / 136 / 226, 136 / 150 / 217, 133 / 156, 133 / 143 / 228, 150 / 217, 82 / 136 / 150, 81, 81 / 143, 81 / 133 / 228, 143 / 228, 212 / 228, 79 / 81, 133 / 228, 133 / 212, 136 / 217, 216, 215 or 127, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or relative to the reference sequence corresponding to SEQ ID NO: 1388.

72. The engineered adenosine kinase of any one of claims 69 to 71, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 143S, 228G, 136A, 143T, 82Q, 143A, 156C, 205P, 82P, 226P, 228L, 143C, 150G, 150S, 219P, 150P, 217H, 226T, 143G, 218L, 68S, 142R, 212I, 212L, 228P, 213S, 2 26S, 156T, 79P, 230A, 218A, 143R, 228S, 68L, 207T, 228R, 155A, 136S, 150L, ​​150V, 227P, 129L, 230Q, 210V, 205L, 68G, 13 3W, 155W, 212R, 207A, 151F, 212P, 218I, 129P, 43A, 230P, 129S, 68A, 142F, 204S, 136Y, 182S, 133S, 228I, 43C, 217G, 129I , 82Q / 136L / 150G / 217P, 136A / 150S, 79P / 81S / 143A / 156C / 212I / 228G, 82Q / 136A, 79P / 81S / 143A / 212I, 217P, 79P / 133F / 228L、81S / 143A / 156T / 212I、136A / 219A、82Q / 136A / 226P、136A / 150S / 217P、136A / 150G、133F、133F / 156C、133F / 143A / 228L, 136L, 150S / 217P, 82Q / 136A / 150S, 81S, 81S / 143T, 81S / 133F / 228L, 143A / 228L, 212I / 228G, 79P / 81S, 133F / 228L, 133F / 212M, 136L / 217H, 82P / 136L, 216H, 215P, 143P, 151R, 212M, 213P, 127S, 133Q, 212W, 219G, or 219S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1388, or relative to the reference sequence corresponding to SEQ ID NO: 1388.

73. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or relative to the reference sequence corresponding to SEQ ID NO: 1708.

74. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 1788-1968, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or relative to the reference sequence corresponding to SEQ ID NO: 1708.

75. The engineered adenosine kinase of claim 73 or 74, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 127, 143 / 155 / 219 / 228, 219, 127 / 142, 142 / 151 / 207 / 218, 143 / 213, 151 / 218, 127 / 142 / 218, 142 / 151, 143 / 155 / 205 / 213 / 228, 127 / 142 / 207, 127 / 216 / 218, 142 / 151 / 207, 143 / 219, 127 / 207, 127 / 218, 143 / 219 / 228, 127 / 216, 68 / 127 / 207, 143 / 155 / 212 / 228, 151, 143 / 213 / 228, 143 / 155 / 205, 151 / 207 / 216, 143 / 228, 68 / 127 / 207 / 218, 143 / 155, 143, 205 / 213 / 219, 155 / 212 / 219 / 228, 205 / 219 / 228, 143 / 205, 142 / 207, 143 / 205 / 219, 143 / 212 / 21 3, 127 / 207 / 218, 142, 127 / 142 / 151 / 207 / 218, 143 / 205 / 219 / 228, 205 / 213 / 228, 68 / 151 / 207, 68 / 127 / 142 / 207, 68 / 127, 218, 127 / 142 / 196, 142 / 207 / 218, 68 / 142 / 216, 68 / 127 / 216 / 218, 68 / 127 / 218, 151 / 216 / 218, 143 / 212 / 228, 127 / 142 / 151, 151 / 207, 127 / 142 / 216, 129 / 207 / 218, 68 / 142 / 151 / 207, 68 / 142 / 151, 151 / 207 / 218, 228, 155 / 213, 127 / 142 / 151 / 218, 127 / 151, 155 / 228, 68 / 142 / 207, 80 / 151 / 207, 205 / 228, 68 / 142 / 207 / 216 / 218, 68 / 218, 68 / 207, 216 / 218, 68 / 142, 212, 205 or 142 / 207 / 216 / 218, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or relative to the reference sequence corresponding to SEQ ID NO: 1708.

76. The engineered adenosine kinase of any one of claims 73 to 75, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 127P, 143S / 155A / 219P / 228R, 219P, 127P / 142R, 142R / 151A / 207A / 218A, 143G / 213L, 151A / 218A, 127P / 142R / 218I, 142R / 151A, 143S / 155A / 205L / 213L / 228R, 127P / 142R / 207A, 127P / 216P / 218A, 142R / 151A / 207A, 143 S / 219P, 127P / 207A, 127P / 218I, 143G / 219P / 228R, 127P / 216P, 68L / 127P / 207L, 143G / 155A / 212R / 228R, 151A, 143S / 219P / 228R, 143S / 213L / 228R, 14 3G / 155A / 205L, 151A / 207A / 216P, 143G / 228R, 68L / 127P / 207A, 68L / 127P / 2 07A / 218A, 143G / 155A, 143G, 205L / 213L / 219P, 155A / 212R / 219P / 228R, 143 S / 228R, 205L / 219P / 228R, 143G / 205L, 142R / 207A, 143G / 205L / 219P, 143G / 212R / 213L, 127P / 207L / 218I, 142R, 127P / 216P / 218I, 127P / 142R / 151A / 2 07A / 218I, 143S / 205L / 219P / 228R, 205L / 213L / 228R, 143G / 219P, 142R / 207 L, 68L / 151A / 207L, 143S / 213L, 142R / 151A / 207L, 127P / 142R / 207L, 68L / 12 7P / 142R / 207L, 143S / 205L, 68L / 127P, 218A, 127P / 142R / 196I, 142R / 207L / 218A, 143S / 155A, 143S, 68L / 142R / 216P, 68L / 127P / 216P / 218I, 68L / 127P / 218I, 151A / 216P / 218I, 143S / 212R / 228R, 127P / 142R / 151A, 151A / 207L, 12 7P / 142R / 216P, 129I / 207A / 218A, 142R / 207L / 218I, 68L / 142R / 151A / 207A,151A / 207A, 68L / 142R / 151A, 151A / 207L / 218I, 228R, 155A / 213L, 127P / 142R / 151A / 218I, 127P / 151A, 155A / 228R, 143S / 155A / 205L, 151A / 216P / 218A, 68L / 142R / 207L, 80V / 151A / 207L, 205L / 228R, 68L / 142R / 207A / 216P / 218I, 68L / 218I, 68L / 207A, 216P / 218I, 68L / 142R, 212R, 68L / 207L, 205L, or 142R / 207A / 216P / 218I, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1708, or relative to the reference sequence corresponding to SEQ ID NO: 1708.

77. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or relative to the reference sequence corresponding to SEQ ID NO: 1952.

78. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 1970-2032, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or relative to the reference sequence corresponding to SEQ ID NO: 1952.

79. The engineered adenosine kinase of claim 77 or 78, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 18 / 118, 87 / 119 / 192, 119 / 173 / 192, 118, 87 / 119, 18 / 118 / 170, 18, 119, 192, 87, 89, 59, 170, 64, 112, 93, 184, 146, 51, 125, 192, 191, 117, 172, or 163, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or relative to the reference sequence corresponding to SEQ ID NO: 1952.

80. The engineered adenosine kinase of any one of claims 77 to 79, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 18L / 118G, 87M / 119K / 192A, 119K / 173R / 192A, 118G, 87M / 119K, 18L / 118G / 170S, 18L, 119K, 192A, 87E, 87M, 89Q, 192I, 59L, 170S, 64R, 112N, 93E, 184S, 146N, 112K, 51T, 125I, 93S, 192W, 112A, 191D, 51M, 117L, 93Y, 172T, or 163Q, wherein the amino acid position is relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of SEQ ID NO: 1952, or relative to the reference sequence corresponding to SEQ ID NO: 1952.

81. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or with respect to the reference sequence corresponding to SEQ ID NO: 1980.

82. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2034-2134, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

83. The engineered adenosine kinase of claim 81 or 82, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 87 / 119 / 192, 87 / 192, 87 / 119, 119 / 192, 192, 119, 146 / 192, 87, 59 / 87, 59 / 87 / 112 / 146, 146, 87 / 112 / 119, 74, 75, 66, 181, 81, 73, 77, 188, 216, 82, 136, 214, 21, 217, 154, 133, 71, 212, or 228, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

84. The engineered adenosine kinase of any one of claims 81 to 83, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 87E / 119K / 192A, 87E / 192A, 87E / 119K, 119K / 192A, 87M / 119K, 192A, 119K, 146N / 192A, 87E, 87M / 192A, 87M, 59L / 87E, 59L / 87E / 112A / 146N, 146N, 87E / 112A / 119K, 74G, 75L, 66A, 181V, 66W, 66G, 66Q, 81F, 66H, 73R, 66N, 77S, 77A, 66D, 66V, 188L, 77Q, 73I, 216E, 82L, 136L, 214L, 188G, 214T, 21S, 66T, 217E, 154R, 216T, 133V, 71T, 214R, 136I, 212H, 214P, or 228Q, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 1980, or relative to the reference sequence corresponding to SEQ ID NO: 1980.

85. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

86. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2136-2264, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

87. The engineered adenosine kinase of claim 85 or 86, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 87, 194, 197, 93, 202, 38, 91, 183, 39, 146, 203, 37, 92, 36, 89, 131, 132, 90, 148, 35 / 197, 231, or 56, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

88. The engineered adenosine kinase of any one of claims 85 to 87, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 87R, 87K, 87L, 194R, 197L, 87Y, 93G, 202E, 38C, 91V, 194L, 183L, 93T, 39T, 91L, 146H, 203E, 203L, 146N, 37G, 194Y, 37R, 92L, 36R, 36S, 89T, 131A, 132T, 90F, 197Q, 90V, 148S, 91S, 35E / 197V, 132V, 197A, 197V, 93P, 92S, 36M, 39A, 194V, 87V, 132G, 38F, 90T, 231R, 148F, 91G, 91A, 36I, 87E, 37L, 231T, 231A, 89P, 202S, 36E, 231Q, 87A, 146V, 146R, 56T, 93A, or 203R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2072, or relative to the reference sequence corresponding to SEQ ID NO: 2072.

89. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or with respect to the reference sequence corresponding to SEQ ID NO: 2138.

90. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2266-2366, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

91. The engineered adenosine kinase of claim 89 or 90, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 161, 162, 153, 216, 212, 156, 214, 229, 77 / 143 / 214 / 216, 143 / 156 / 216, 77 / 143, 77 / 143 / 216, 77 / 214 / 216, 77 / 216, 143 / 156, 156 / 216, 143, 77, 77 / 214, 77 / 143 / 161 / 162 / 212 / 214, 77 / 162 / 214 / 216, 77 / 143 / 156 / 214 / 216, 77 / 143 / 156 / 162 / 212 / 216, 77 / 143 / 156 / 161 / 214 / 216, 77 / 156 / 162 / 216, 156 / 161 / 214 / 216, 77 / 143 / 162 / 214 / 216, 143 / 216, 214 / 216, 143 / 156 / 214 / 216, 143 / 214 / 216, 77 / 143 / 212 / 216, 143 / 156 / 161 / 162 / 216, 156 / 162 / 214, 77 / 143 / 156 / 161 / 162, 143 / 161 / 214 / 216, 77 / 161 / 216 or 143 / 212 / 214 / 216, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

92. The engineered adenosine kinase of any one of claims 89 to 91, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 161G, 162G, 153C, 216L, 212Q, 216H, 156V, 161L, 214W, 229C, 216M, 216V, 214A, 214M, 77I / 143V / 214E / 216R, 143V / 156V / 216R, 77I / 143V, 7 7I / 143V / 216R, 77I / 214E / 216R, 77I / 216L, 143V / 156V, 156V / 216R, 143V, 77I / 214E / 216L, 77I, 77I / 214E, 77I / 143V / 161L / 162G / 212Q / 214E, 77I / 162G / 214E / 216L, 77I / 143V / 156V / 214E / 216L, 77I / 143V / 156V / 162G / 212Q / 216R、77I / 143V / 156V / 161L / 214E / 216R、77I / 216R、77I / 156V / 162G / 216R、156V / 161L / 214E / 216L、77I / 143V / 162G / 214W / 216L, 143V / 216L, 214E / 216L, 143V / 156V / 214E / 216R, 143V / 214E / 216L, 143V / 214E / 216R, 214E, 77 I / 143V / 212Q / 216R, 143V / 156V / 161L / 162G / 216R, 156V / 162G / 214E, 77I / 143V / 156V / 161L / 162G, 143V / 161L / 214E / 216R, 77I / 161L / 216R, 143V / 212Q / 214E / 216R, 77I / 143V / 214W / 216R, 143V / 216R or 216R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO:2138, or relative to the reference sequence corresponding to SEQ ID NO: 2138.

93. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

94. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2368-2594, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

95. The engineered adenosine kinase of claim 93 or 94, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 80, 105, 228, 50, 175, 170, 82, 173, 65, 53, 184, 122, 195, 68, 89, 34, 118, 119, 87, 179, 117, 113, 54, 190. 216, 88, 36, 166, 38, 169, 66, 142, 136, 131, 187, 40, 74, 127, 55, 148, 215, 64, 155, 116, 90, 132, 60, 94, 112, 120, 37, 108, 35, 51, 61, 212, 172, 59, or 56, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

96. The engineered adenosine kinase of any one of claims 93 to 95, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 80P, 105K, 228S, 50Q, 175F, 170R, 82L, 173S, 65T, 53M, 184V, 122S, 195M, 228L, 68S, 89A, 34E, 118S, 53S, 119F, 87A, 179C, 117S, 113E, 50C, 80G, 122H, 89V, 80R, 54I, 190Q, 216M, 88R, 80D, 36Q, 105S, 195G, 68V, 105L, 175D, 184T, 166L, 89H, 38R, 54G, 38F, 89I, 169I, 66C, 142W, 117L, 136Y, 131 V, 187Y, 40L, 173T, 74C, 228V, 127L, 80L, 55G, 184M, 136V, 228T, 148R, 88T, 228Q, 66S, 215V , 195R, 190G, 64R, 184R, 122A, 50S, 142M, 155W, 80S, 216G, 116A, 90C, 132K, 105G, 60W, 94Y, 1 12T, 87S, 187G, 120T, 87I, 68R, 37Y, 173R, 105M, 108V, 35L, 68I, 166F, 35F, 120S, 184S, 169A, 51L, 61V, 116F, 65D, 54H, 212C, 172S, 55L, 59L, 60C, 228F, or 56Q, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to amino acid residues 12-231 of SEQ ID NO: 2294, or relative to the reference sequence corresponding to SEQ ID NO: 2294.

97. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

98. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 97%, 98%, 99% or higher sequence identity to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2596-2652 and 2676-2714, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

99. The engineered adenosine kinase of claim 97 or 98, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 136 / 228, 82 / 142 / 173 / 184 / 216, 184 / 216, 50 / 184 / 216, 127 / 173 / 184, 105 / 136 / 170 / 175 / 228, 105 / 175, 50 / 184, 105 / 170 / 175 / 228, 50 / 127 / 173 / 184, 50 / 142 / 184 / 216, 50 / 82 / 127 / 216, 50, 50 / 82 / 173 / 216, 50 / 127 / 142 / 184 / 216, 50 / 82 / 127, 105 / 136, 105 / 228, 105 / 136 / 175 / 228, 173 / 216, 50 / 173, 228, 127 / 173, 105 / 136 / 170 / 195, 50 / 82 / 216, 175 / 228, or 50 / 82 / 127 / 142 / 184 / 216, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

100. The engineered adenosine kinase of any one of claims 97 to 99, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 136Y / 228V, 82L / 142M / 173T / 184T / 216M, 184T / 216M, 50Q / 184M / 216M, 127L / 173R / 184M, 105K / 136V / 170R / 175F / 228T, 105K / 175F, 127L / 173T / 184M, 50Q / 184T, 105K / 170R / 175F / 228V, 50Q / 127L / 173R / 184M, 50Q / 142M / 184M / 216M, 50Q / 82L / 127L / 216M, 50Q, 50Q / 82L / 173T / 216M, 50Q / 127L / 142W / 184M / 216M, 50Q / 82L / 127L, 136V / 228V, 105K / 136V, 105K / 228V, 105K / 136V / 175F / 228V, 173T / 216M, 50Q / 173T, 228V, 127L / 173R, 105K / 136V / 170R / 195G, 50Q / 82L / 216M, 175F / 228V or 50Q / 82L / 127L / 142M / 184M / 216M, wherein the amino acid position is relative to SEQ The reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

101. The engineered adenosine kinase of claim 97 or 98, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 20, 21, 22, 43, 44, 46, 48, 138, 181, or 182, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

102. The engineered adenosine kinase of any one of claims 97, 98, and 101, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 20A, 21L, 21N, 21V, 22A, 43A, 43G, 43H, 44S, 46E, 46F, 46L, 46M, 46Y, 48F, 138M, 138V, 181C, 181V, or 182A, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2368, or relative to the reference sequence corresponding to SEQ ID NO: 2368.

103. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or with respect to the reference sequence corresponding to SEQ ID NO: 2602.

104. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with respect to residues 12-231 of SEQ ID NO: 2602, or with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602.

105. The engineered adenosine kinase of claim 103 or 104, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 18, 18 / 56, 20, 21, 26, 27, 29, 30, 32, 42, 43, 50, 66, 67, 69, 70, 80, 98, 99, 102, 135, 137, 139, 141, 142, 143, 148, 149, 152, 153, 156, 173, 184, or 216, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

106. The engineered adenosine kinase of any one of claims 103, 104, and 105, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 18C / 56T, 18E, 20P, 20R, 20T, 20V, 21L, 21R, 21T, 26L, 27C, 29R, 30L, 30S, 30Y, 32L, 42T, 43N, 50R, 66P, 67R, 69F, 70 R, 80G, 80S, 98A, 98G, 98Q, 99A, 99C, 102A, 102C, 102N, 135I, 137L, 139H, 141G, 141V, 142L, 143T, 148P, 148S, 149L, 152F, 153V, 156A, 173F, 184N, 216D, 216E, 216G, 216L, 216N, 216P, and 216W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

107. The engineered adenosine kinase of claim 103 or 104, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 65, 65 / 104, 65 / 104 / 105 / 116 / 127, 65 / 104 / 105 / 127 / 136, 65 / 104 / 136, 65 / 104 / 13 6 / 170, 65 / 105 / 116 / 136 / 170, 65 / 105 / 127 / 136, 65 / 105 / 127 / 136 / 175, 65 / 105 / 127 / 170, 65 / 105 / 127 / 175, 65 / 105 / 136, 65 / 105 / 136 / 170, 65 / 105 / 170, 65 / 116, 65 / 1 27 / 136, 65 / 127 / 136 / 170, 65 / 127 / 170, 94 / 173, 94 / 187, 104 / 105, 104 / 105 / 127, 104 / 105 / 127 / 136 / 175 / 195, 104 / 105 / 136 / 170, 104 / 127, 104 / 127 / 136 / 170, 104 / 12 7 / 136 / 175, 105, 105 / 116 / 127 / 136 / 170 / 175, 105 / 127 / 136, 105 / 127 / 136 / 195, 105 / 136, 105 / 136 / 195, 116 / 127 / 136 / 175, 127 / 195, 132, 136 or 170, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

108. The engineered adenosine kinase of any one of claims 103, 104, and 108, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 65T, 65T / 104A, 65T / 104A / 105K / 116A / 127L, 65T / 104A / 105K / 127L / 136V, 65T / 104A / 136V, 65T / 104A / 136V / 170R , 65T / 105K / 116A / 136V / 170R, 65T / 105K / 127L / 136V, 65T / 105K / 127L / 136V / 175F, 65T / 105K / 127L / 17 0R、65T / 105K / 127L / 175F、65T / 105K / 136V、65T / 105K / 136V / 170R、65T / 105K / 170R、65T / 116A、65T / 127 L / 136V / 170R, 65T / 127L / 136Y, 65T / 127L / 170R, 94Y / 173R, 94Y / 187G, 104A / 105K, 104A / 105K / 127L, 1 04A / 105K / 127L / 136V / 175F / 195G, 104A / 105K / 136V / 170R, 104A / 127L, 104A / 127L / 136V / 170R, 104A / 1 27L / 136V / 175F, 105K, 105K / 116A / 127L / 136Y / 170R / 175F, 105K / 127L / 136V, 105K / 127L / 136V / 195G, 105K / 136V, 105K / 136V / 195G, 116A / 127L / 136Y / 175F, 127L / 195G, 132K, 136V, and 170R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

109. The engineered adenosine kinase of claim 103 or 104, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 36, 37, 40, 50, 59, 89, 117, 120, 128, 168, or 203, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

110. The engineered adenosine kinase of any one of claims 103, 104, and 109, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 36V, 37I, 37L, 40S, 50A, 59T, 59Y, 89P, 117L, 120L, 128I, 168L, or 203L, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

111. The engineered adenosine kinase of claim 103 or 104, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 32, 36, 37, 50, 58, 61, 89, 90, 94, 97, 104, 110, 111, 118, 119, 128, 131, 132, 166, 169, 170, 172, 192, 195, or 200, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

112. The engineered adenosine kinase of any one of claims 103, 104, and 111, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 32F, 36S, 37F, 50N, 50V, 58Y, 61E, 61N, 89L, 90S, 94M, 94V, 97I, 104R, 110F, 111P, 111R, 118L, 119P, 128C, 128R, 131G, 132L, 166L, 166S, 169Y, 170G, 172A, 192H, 195I, or 200A, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2602, or relative to the reference sequence corresponding to SEQ ID NO: 2602.

113. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or with respect to the reference sequence corresponding to SEQ ID NO: 2832.

114. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with respect to residues 12-231 of SEQ ID NO: 2832, or with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832.

115. The engineered adenosine kinase of claim 113 or 114, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 36, 36 / 50, 36 / 50 / 89 / 93 / 110, 36 / 50 / 89 / 93 / 139, 36 / 50 / 89 / 139 / 170, 36 / 50 / 89 / 172, 36 / 89, 36 / 170, 50, 50 / 89 / 93, 50 / 89 / 93 / 139, 50 / 93, 50 / 170, 89 / 170 / 172, 90 / 104, 90 / 151 / 157, 104 / 151 / 154, 104 / 151 / 154 / 157, 104 / 154 / 157 / 216, 151 / 157, 151 / 216 or 216, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2832, or relative to the reference sequence corresponding to SEQ ID NO: 2832.

116. The engineered adenosine kinase of any one of claims 113 to 115, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 36S, 36S / 50N, 36S / 50N / 89L / 93V / 110F, 36S / 50N / 89L / 93V / 139R, 36S / 50N / 89L / 139R / 170G, 36S / 50N / 89L / 172A, 36S / 89L, 36S / 170G, 50N, 50N / 89L / 93V, 50N / 89L / 93V / 139R, 50N / 93V, 50N / 170G, 89L / 170G / 172A, 90S / 104R, 90S / 151F / 157V, 104R / 151F / 154Q, 104R / 151F / 154Q / 157V, 104R / 154Q / 157V / 216E, 151F / 157V, 151F / 216E or 216D, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO:2832, or relative to the reference sequence corresponding to SEQ ID NO: 2832.

117. The engineered adenosine kinase of claim 18, 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 higher sequence identity to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

118. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with respect to residues 12-231 of SEQ ID NO: 2994, or with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994.

119. The engineered adenosine kinase of claim 117 or 118, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions at least at the following amino acid positions: 50, 58, 59, 68, 74, 76, 79, 80, 90, 112, 113, 119, 170, 172, 182, 184, 217, 224, 226, 228, 230, or 231, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

120. The engineered adenosine kinase of any one of claims 117 to 119, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or amino acid residues: 50L, 58C, 59W, 68A, 68E, 68V, 74Q, 76G, 76L, 79A, 79K, 79L, 79P, 79W, 80W, 90K, 112C, 113W, 119S, 170H, 170P, 170R, 172M, 182G, 182L, 182Q, 184R, 217T, 224S, 224T, 226K, 226R, 228D, 230M, or 231R, or combinations thereof, wherein the amino acid position corresponds to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994. Regarding the reference sequence corresponding to 2994.

121. The engineered adenosine kinase of claim 117 or 118, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 79 / 104 / 157 / 228, 79 / 157 / 228 / 229, 79 / 157 / 229, 79 / 228, 83, 83 / 104, 83 / 104 / 151 / 168 / 173 / 190, 83 / 113, 83 / 1 73 / 190, 83 / 173 / 190 / 201, 83 / 173 / 201, 83 / 190, 83 / 190 / 201 / 216, 83 / 216, 104 / 157, 157, 157 / 173 / 190 / 216, 157 / 183, 157 / 190, 157 / 190 / 216, 157 / 228, 173 / 216, 201 / 216 or 229, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

122. The engineered adenosine kinase of any one of claims 117, 118, and 121, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 79W / 104R / 157P / 228V, 79W / 157P / 228V / 229I, 79W / 157P / 229I, 79W / 228V, 83L, 83L / 104R, 83L / 104R / 151Y / 168S / 173T / 190Q, 83L / 113Y, 83L / 173T / 201A, 83L / 173V / 190Q, 83L / 173V / 190Q / 201F, 83L / 190Q, 83L / 190Q / 201A / 216E, 83L / 216E, 104R / 157P, 157P, 157P / 173T / 190Q / 216E, 157P / 183L, 157P / 190Q, 157P / 190Q / 216E, 157P / 228V, 173T / 216E, 201F / 216E or 229I, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 2994, or relative to the reference sequence corresponding to SEQ ID NO: 2994.

123. The engineered adenosine kinase of claim 18, 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 higher sequence identity with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 3104, or with respect to the reference sequence corresponding to SEQ ID NO: 3104.

124. The engineered adenosine kinase of claim 19, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with respect to residues 12-231 of SEQ ID NO: 3104, or with respect to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 3104.

125. The engineered adenosine kinase of claim 123 or 124, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 104, 104 / 157, 104 / 157 / 228 / 229, 104 / 157 / 229, 104 / 170 / 190 / 228, 104 / 201 / 228, 157 / 228, 157 / 228 / 229, 170 / 190 / 228, 170 / 190 / 228 / 229, 190, 190 / 229, or 201 / 228, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 3104, or relative to the reference sequence corresponding to SEQ ID NO: 3104.

126. The engineered adenosine kinase of any one of claims 123, 124, and 125, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 104R, 104R / 157P, 104R / 157P / 228V / 229I, 104R / 157P / 229I, 104R / 170P / 190R / 228V, 104R / 201S / 228V, 157P / 228V, 157P / 228V / 229I, 170P / 190R / 228V, 170P / 190R / 228V / 229I, 190R, 190R / 229I, or 201S / 228V, wherein the amino acid position is relative to SEQ ID NO: The reference sequence corresponding to residues 12-231 of 3104, or relative to the reference sequence corresponding to SEQ ID NO: 3104.

127. The engineered adenosine kinase of claim 123 or 124, wherein the amino acid sequence of the engineered adenosine kinase comprises substitutions or sets of substitutions at least at the following amino acid positions: 104, 104 / 157 / 190 / 228, 104 / 170 / 190, 104 / 170 / 228, 104 / 190, 104 / 190 / 201, 104 / 190 / 228, 104 / 228 / 229, or 228, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 3104, or relative to the reference sequence corresponding to SEQ ID NO: 3104.

128. The engineered adenosine kinase of any one of claims 123, 124, and 127, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the following substitutions or sets of substitutions or amino acid residues: 104R, 104R / 157P / 190R / 228V, 104R / 170P / 190R, 104R / 170P / 228V, 104R / 190R, 104R / 190R / 201S, 104R / 190R / 228V, 104R / 228V / 229I, or 228V, wherein the amino acid position is relative to the reference sequence corresponding to residues 12-231 of SEQ ID NO: 3104, or relative to the reference sequence corresponding to SEQ ID NO: 3104.

129. The engineered adenosine kinase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least one substitution shown in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid position is relative to SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, corresponding to the reference sequence.

130. The engineered adenosine kinase of any one of claims 1 to 5, wherein the amino acid sequence of the engineered adenosine kinase comprises at least the amino acids listed in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 2 The substitutions or sets of substitutions of the engineered adenosine kinase variants shown in 7.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, wherein the amino acid positions are relative to the reference sequences corresponding to SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

131. The engineered adenosine kinase according to any one of claims 1 to 5, wherein the adenosine kinase comprises, and comprises, those listed in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.1, 13.2, 14.1, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 23.2, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, and 40.

2. The reference amino acid sequence of the replacement or set of replacements of the engineered adenosine kinase variants shown in SEQ ID NO: 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2 has 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 higher sequence identity, wherein the amino acid position relative to SEQ ID NO: 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 83%, 84%, 85%, 96%, 97%, 98%, 99% or higher sequence identity, wherein the amino acid position relative to SEQ ID NO: 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2 has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 96%, 97%, 98%, 99% or higher sequence identity, wherein the amino acid position relative to SEQ ID NO: 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, and 49.2, 41.2, 42 For the reference sequence corresponding to ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104.

132. The engineered adenosine kinase of claim 1, wherein the amino acid sequence of the engineered adenosine kinase comprises residues 12-231 of even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652 and 2676-3192, or comprises even-numbered SEQ ID NOs in SEQ ID NOs: 4-1302, 1356-2652 and 2676-3192, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions.

133. The engineered adenosine kinase of claim 1, wherein the amino acid sequence of the engineered adenosine kinase comprises residues 12-231 of SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or wherein the amino acid sequence comprises SEQ ID NO: 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994 or 3104, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions.

134. The engineered adenosine kinase according to any one of claims 1 to 133, having adenosine kinase activity and at least one improved property compared to a reference adenosine kinase.

135. The engineered adenosine monophosphate kinase of claim 134, wherein the improved properties compared to a reference adenosine monophosphate kinase are selected from: i) increased activity to unmodified nucleoside monophosphates (NMPs), ii) increased stability, iii) increased thermal stability, iv) increased activity to 2'-fluoromodified nucleoside monophosphates, and v) increased activity to 2'-O-methylmodified nucleoside monophosphates, or any combination of i), ii), iii), iv) and v).

136. The engineered adenosine kinase of claim 134 or 135, wherein the reference adenosine kinase has the amino acid sequence corresponding to residues 12-231 of SEQ ID NO: 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104, or SEQ ID NO: The amino acid sequences corresponding to 2, 4, 172, 376, 508, 524, 584, 610, 674, 816, 874, 1032, 1388, 1708, 1952, 1980, 2072, 2138, 2294, 2368, 2602, 2832, 2994, or 3104.

137. The engineered adenosine kinase according to any one of claims 1 to 136, wherein the engineered adenosine kinase is purified.

138. A recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered adenosine kinase as described in any one of claims 1 to 136.

139. The recombinant polynucleotide of claim 138, comprising a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of SEQ ID NO: 1, 3, 171, 375, 507, 523, 583, 609, 673, 815, 873, 1031, 1387, 1707, 1951, 1979, 2071, 2137, 2293, 2367, 2601, 2831, 2993, or 3103, or a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of SEQ ID NO: The reference polynucleotide sequence corresponding to 1, 3, 171, 375, 507, 523, 583, 609, 673, 815, 873, 1031, 1387, 1707, 1951, 1979, 2071, 2137, 2293, 2367, 2601, 2831, 2993, or 3103 has a sequence identity of 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 higher, wherein the recombinant polynucleotide encodes adenosine kinase.

140. The recombinant polynucleotide of claim 138, comprising a reference polynucleotide sequence corresponding to nucleotide residues 34-693 of odd-numbered SEQ ID NOs in SEQ ID NOs: 3-1301, 1355-2651, and 2675-3191, or 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 higher sequence identity to the reference polynucleotide sequence corresponding to odd-numbered SEQ ID NOs in SEQ ID NOs: 3-1301, 1355-2651, and 2675-3191, wherein the recombinant polynucleotide encodes an engineered adenosine kinase.

141. The recombinant polynucleotide of any one of claims 138 to 140, wherein the polynucleotide sequence has a preferred codon or is codon-optimized.

142. The recombinant polynucleotide of claim 138, wherein the polynucleotide sequence comprises nucleotide residues 34-693 of SEQ ID NO: 1, 3, 171, 375, 507, 523, 583, 609, 673, 815, or 873, 1031, 1387, 1707, 1951, 1979, 2071, 2137, 2293, 2367, 2601, 2831, 2993, or 3103, or comprises SEQ ID NO: 1, 3, 171, 375, 507, 523, 583, 609, 673, 815, or 873, 1031, 1387, 1707, 1951, 1979, 2071, 2137, 2293, 2367, 2601, 2831, 2993, or 3103.

143. The recombinant polynucleotide of claim 138, wherein the polynucleotide sequence comprises nucleotide residues 34-693 of the odd-numbered SEQ ID NOs in SEQ ID NOs: 3-1301, 1355-2651 and 2675-3191, or comprises the odd-numbered SEQ ID NOs in SEQ ID NOs: 3-1301, 1355-2651 and 2675-3191.

144. An expression vector comprising any one of claims 138 to 143.

145. The expression vector of claim 144, wherein the recombinant polynucleotide is operatively linked to a control sequence.

146. The expression vector of claim 145, wherein the control sequence comprises at least a promoter.

147. A host cell comprising an expression vector as described in any one of claims 144 to 146.

148. The host cell of claim 147, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

149. The host cell of claim 148, comprising bacterial cells, fungal cells, insect cells or mammalian cells.

150. A method for producing an engineered adenosine kinase polypeptide, comprising culturing a host cell as described in any one of claims 147 to 149 under suitable culture conditions to produce the engineered adenosine kinase polypeptide.

151. The method of claim 150, further comprising recovering or isolating the adenosine kinase polypeptide from the culture and / or host cells.

152. The method of claim 150 or 151, further comprising purifying the adenosine kinase polypeptide.

153. A composition comprising engineered adenosine kinase as described in any one of claims 1 to 137.

154. The composition of claim 153, further comprising a nucleoside monophosphate substrate.

155. The composition of claim 154, wherein the nucleoside is a modified nucleoside monophosphate substrate.

156. A method for converting a nucleoside or a modified nucleoside monophosphate into the corresponding nucleoside diphosphate (NMP), comprising contacting the nucleoside monophosphate with an engineered adenosine kinase as described in any one of claims 1 to 137 in the presence of a phosphate donor NTP and under suitable reaction conditions to convert the nucleoside monophosphate into the corresponding nucleoside diphosphate (NMP).

157. The method of claim 156, wherein the nucleoside monophosphate is an unmodified nucleoside monophosphate.

158. The method of any one of claims 156 or 157, wherein the nucleoside monophosphate comprises a nucleobase selected from: adenine, cytosine, guanine, thymine, uracil, xanthine, hypoxanthine, 2,6-diaminopurine, purine, 6,8-diaminopurine, 5-methylcytosine (m 5 C), 2-thiouridine, pseudouridine, dihydrouridine, inosine, and 7-methylguanosine (m 7 G).

159. The method of any one of claims 156 to 158, wherein the nucleoside monophosphate has an H or OH at the 2'-position of the sugar moiety.

160. The method of claim 159, wherein the nucleoside monophosphate is AMP, GMP, UMP, CMP or TMP, and wherein the nucleoside monophosphate has an OH at the 2'-position of the sugar moiety, thereby generating the corresponding products rADP, rGDP, rUDP, rCDP or rTDP.

161. The method of claim 159, wherein the nucleoside monophosphate is AMP, GMP, UMP, CMP or TMP, and wherein the nucleoside monophosphate has an H at the 2'-position of the sugar moiety, thereby generating the corresponding products dADP, dGDP, dUDP, dCDP or dTDP.

162. The method of claim 156, wherein the nucleoside monophosphate is a modified nucleoside, wherein the modified nucleoside monophosphate has a modified sugar moiety, a modified nucleobase, and / or a modified phosphate.

163. The method of claim 162, wherein the modified nucleoside monophosphate comprises a modified sugar moiety.

164. The method of claim 163, wherein the modified sugar portion is the modified 2'-position of the sugar portion.

165. The method of claim 164, wherein the modified 2'-position 2 is 2'-O-R', wherein R' is an alkyl, aminoalkyl, cyanoalkyl, -C(O)-alkyl, or 2'-halogen.

166. The method of claim 165, wherein the modified 2'-position is a 2'-O-alkyl group, which is 2'-O-CH3, 2'-O-CH2CH3, 2'-O-(2-methoxyethyl), 2'-O-allyl, 2'-O-propynyl, 2'-O-aminoethyl, 2'-O-cyanoethyl, or 2'-O-acetal.

167. The method of claim 165, wherein the 2'-position is a 2'-halogenated group.

168. The method of claim 167, wherein the 2'-position is 2'-F, 2'-CL, or 2'-Br.

169. The method of any one of claims 163 to 168, wherein the modified sugar moiety is modified at the 3'-position.

170. The method of claim 169, wherein the 3'-position of the sugar moiety comprises a capping group, wherein the capping group is a formate, benzoylformate, acetate, propionate, isobutyrate, aminooxy(-ONH2), O-methyl, O-methoxymethyl, O-methylthiomethyl, O-benzyloxymethyl, O-allyl, 3'-O-(2-nitrobenzyl), O-azidomethyl(O-CH2N3), O-tert-butyldithiomethyl, phosphate, diphosphate, or triphosphate.

171. The method of any one of claims 162 to 170, wherein the modified nucleoside monophosphate comprises a modified nucleobase.

172. The method of claim 171, wherein the modified nucleobase is selected from 5-bromouracil, 5-iodouracil, 6-mCEPh-purine, 6-phenylpyrrolocytosine, N2-alkyl-8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, and benzimidazole.

173. The method of any one of claims 162 to 172, wherein the modified nucleoside monophosphate comprises modified phosphate.

174. The method of claim 173, wherein the modified nucleoside monophosphate comprises nucleoside 5'-thiophosphate, thereby producing the product nucleoside 5'-1-thiobisphosphate (NDPαS).

175. The method of claim 174, wherein the product NDPαS is (Rp)-NDPαS, (Sp)-NDPαS, or a mixture of diastereomers of (Rp)-NDPαS and (Sp)-NDPαS.

176. The method of claim 175, further comprising the step of resolving the (Rp)-NDPαS and (Sp)-NDPαS diastereomers.

177. The method of any one of claims 156 to 176, wherein the phosphate donor NTP is rATP or dATP.

178. The method of any one of claims 156 to 176, wherein the phosphate donor NTP has the same nucleotide structure as the nucleoside monophosphate.

179. The method of any one of claims 156 to 178, further comprising an NTP regeneration system.

180. The method of claim 179, wherein the NTP regeneration system comprises creatine kinase, pyruvate kinase, polyphosphokinase and / or acetate kinase.

181. The method of claim 180, wherein the NTP regeneration system is an acetate kinase and the substrate acetyl phosphate.

182. The method of claim 181, further comprising pyruvate oxidase and substrates pyruvate and inorganic phosphate.

183. The method of claim 182, further comprising catalase.

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