Engineered adenylyl-sulfate kinase variants

Abstract

The present invention provides engineered adenylyl-sulfate kinase polypeptides capable of adding a phosphate group to the 3' position of the sugar of a natural or modified nucleoside diphosphate, as well as compositions and methods of utilizing these engineered polypeptides.

Description

ENGINEERED ADENYL YL-SULF ATE KINASE VARIANTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 706,195, filed October 11 , 2024; U.S. Provisional Application No. 63 / 789,779, filed April 16, 2025; and U.S. Provisional Application No. 63 / 826,279, filed June 18, 2025; all of which are incorporated by reference herein.TECHNICAL FIELD

[0002] The present invention provides engineered adenylyl-sulfate kinase polypeptides capable of adding a phosphate group to the 3’ position of the sugar of a natural or modified nucleoside diphosphate, as well as compositions and methods of utilizing these engineered polypeptidesREFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification as an XML file, with a file name of CXI 0-281 WO4_ST26. xml, a creation date of October 9, 2025, and a size of 5,710,959 bytes. The Sequence Listing filed is part of the specification and is incorporated in its entirety by reference herein.BACKGROUND

[0004] Synthetic biology' is becoming established in a diverse range of high value, high growth markets. From food and agriculture to therapeutics, diagnostics, and vaccines; tools such as gene editing, DNA sequencing and gene synthesis are being used to build value-added products with advanced functionality (e g., cell bioreactors, etc.) and desired end products (e.g., drugs, chemicals, etc.). The barrier to widespread implementation of these technologies is the ability to efficiently synthesize RNA, DNA, and other polynucleotides.

[0005] In particular, silencing RNA (siRNA) therapeutics are a promising class of drugs that have the potential to treat numerous difficult to treat conditions in a highly' targeted manner by' binding to known mRNA targets (Hu et al., Sig Transduct Target Ther, 2020, 5: 101 ; Zhang et al., Bioch. Pharmac., 2021, 189: 1 14432) As these therapies become more common and are targeted at larger patient populations, the ability to produce large amounts of the oligonucleotide active pharmaceutical ingredient (API) becomes critical.

[0006] Phosphoramidite chemistry has been developed extensively over the years to synthesize small amounts of DNA and RNA, but suffers from several cost, processing and sustainability issues that are potentially limiting as API demand grows to triple-quadruple digit kilograms per year (Andrews et al., J. Org. Chem., 2021, 86:49-61). Additionally, RNA synthesis using phosphoramidite synthesis chemistry is limited to producing short oligonucleotides of approximately 200 base pairs (Beaucage & Caruthers, Tetrahedron Lett., 1981, 22(20): 1859.)

[0007] New oligonucleotide synthesis techniques are being developed to replace phosphoramidite chemistry to meet the growing demand for large quantities of DNA and RNA necessary' for modem medical andindustrial applications. The most promising of these is template independent oligonucleotide synthesis using various polymerases, including terminal nucleotidyl transferases (TdTs) and polyX polymerases These methods often rely on modified nucleoside triphosphates (NTPs) that incorporate blocking groups or other structural or chemical elements that allow the controlled addition of a defined sequence of NTPs. These modified NTPs include NTPs with blocking groups on the 3’ or 2’ positions of the sugar, as well as NTPs with modified bases or thiol derivates for the formation of more stable oligonucleotide phosphorothioate backbone bonds.

[0008] As these methods mature, a limiting factor is the commercial availability and cost of natural and modified NTPs for synthesis reactions. In particular, NTPs with a phosphate at the 3 ’ position of the sugar (nucleoside tetraphosphates, nucleoside quadriphosphates, pppNps, 3 ’P-NTPs, or NQPs), with or without additional modifications to the nucleobase, sugar, and / or phosphate chain, are useful for emerging template independent synthesis applications However, these NQPs are not widely commercially available and are cost prohibitive at an industrial scale. Therefore, new methods to synthesize natural and modified NTPs and NQPs are necessary to enable production of oligonucleotides on the scale required for modem synthetic biology' applicationsSUMMARY

[0009] The present disclosure provides adenylyl-sulfate kinase polypeptides for the synthesis of nucleoside tetraphosphates, also referred to as nucleoside quadriphosphates (pppNps or NQPs), as well as compositions and methods of utilizing these polypeptides. In the embodiments herein, the adenylyl-sulfate kinase polypeptides can be naturally occurring adenylyl-sulfate kinases or engineered adenylyl-sulfate kinases.

[0010] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having one or more amino acid residue differences at amino acid residues at about 6 angstroms (A) or less from the active site of an adenylyl-sulfate kinase. In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having one or more amino acid residue differences at amino acid residues at about 4 5 A or less from the active site of the adenylyl-sulfate kinase. In some embodiments, the active site is determined from a protein crystal structure and / or by protein homology modeling of a reference adenylyl-sulfate kinase

[0011] In some embodiments, the engineered adenylyl-sulfate kmase comprising an amino acid sequence having one or more amino acid residue differences at amino acid residues that are about 6 A or less from the active site comprises at least an amino acid residue difference at amino acid position 7, 10, 11, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 63, 64, 65, 68, 76, 77, 78, 81 , 82, 85, 86, 89, 106, 107, 108, 109, 1 10, 132, 139, 141, 142, 143, 144, 145, 147, 148, 156, 157, 158, 159, 160, 163, 164, 176, 177, 179, 180, 181, 182, 183, 185, or 186, or any combinations thereof, and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14).

[0012] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having one or more amino acid residue differences in one or more motifs of an adenosine 5 ’-phosphosulfate (APS) kinase domain, wherein the motif is selected from:[VI]-[WF]-[LF]-T-G-L-[SP]-[GAC]-X-G-K (SEQ ID NO: .3325):L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326);[LA]-[GS]-F-[ST];K-G-L-Y-[KA]-[KY]-A (SEQ ID NO: 3327); andF-T-G-[IV]

[0013] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif [LA]-[GS]-F-[ST] , located on the amino side of alpha helix a3 of the APS kinase domain.

[0014] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif K-G-L-Y-[KA]-[KY]-A (SEQ ID NO: 3327), located at alpha helix cc6 of the APS kinase domain

[0015] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif [VI]-[WF]-[LF]-T-G-L-[SP]-[GAC]-X-G- K (SEQ ID NO: 3325), located from beta strand [31 to alpha helix al of the APS kinase domain.

[0016] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326), located from beta strand P2 to alpha helix a2.

[0017] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif F-T-G-[IV] , located between alpha helix a6 and beta strand P5.

[0018] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having one or more amino acid residue differences at amino acid positions that are about 6 A or less from the active site of the adenylyl-sulfate kinase combined with the one or more amino acid residue differences in the motifs described herein to generate engineered adenylyl-sulfate kinase with improved properties. In some embodiments, the engineered adenylyl-sulfate kinase exhibits at least an improved property described herein.

[0019] In some embodiments, the engineered the engineered adenylyl-sulfate kinase comprising an amino acid sequence having one or more amino acid residue differences at amino acid positions that are about 6 A or less from the active site of the adenylyl-sulfate kinase and / or one or more amino acid residue differences in the motifs described herein can have additional amino acid residue differences at other amino acid positions.

[0020] In some embodiments, the engineered adenylyl-sulfate kinases of the present disclosure are variants of the wild-type CysC gene from Thermotoga sp. RQ7. As provided in the detailed description, these engineered adenylyl-sulfate kinases are capable of adding a phosphate group to the 3' position of the sugar of a natural or modified nucleoside diphosphate.

[0021] In some embodiments, the present disclosure provides an engineered adenylyl-sulfate kinase polypeptide, or a functional fragment thereof, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence selected from SEQ ID NOs: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, and 3228, comprising at least one substitution or one substitution set at one or more positions, wherein the positions are numbered with reference to SEQ ID NO:14 and wherein the engineered adenylyl-sulfate kinase polypeptide has increased activity on natural substrates, increased activity on non-natural substrates, increased activity on 2 ’-fluoro modified nucleotides, increased activity on 2’-O-methyl modified nucleotides, increased thermostability, increased substrate promiscuity, increased soluble protein expression, decreased product inhibition and / or decreased byproduct formation, as compared to a wild-type or reference adenylyl-sulfate kinase known to those of skill in the art. These engineered adenylyl-sulfate kinase polypeptides with one or more amino acid substitutions or substitution sets are described, below, in the detailed description of the invention.

[0022] In some embodiments, the engineered adenylyl-sulfate kinase, or a functional fragment thereof, comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 14, 18-258, 272-1358, and 1364-3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 18-258, 272-1358, and 1364-3324, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to a reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0023] In some embodiments, the engineered adenylyl-sulfate kinase, or a functional fragment thereof, comprises an amino acid sequence having at least 50%, 55%>, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%>, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, wherein the ammo acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0024] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 35, 38, 39, 41 , 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 1 13, 1 14, 115, 116, 1 17, 118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 140, 141, 144,146, 147, 148, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO:14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0025] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 11, 16, 18, 19, 27, 42, 50, 51, 53, 67, 73, 76, 77, 78, 79, 85, 89, 94, 95, 97, 105, 109, 116, 118, 124, 139, 141, 147, 150, 156, 157, 158, 164, 171, 176, 178, 180, 181, 182, 183, 188, 193, 195, 200, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0026] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 1 1 , 76, 83, 85, 89, 109, 1 10, 141 , 158, 176, or 180, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0027] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 11, 72, 76, 78, 80, 81, 82, 83, 85, 89, 107, 109, 1 10, 1 15, 141, 146, 154, 155, 176, 182, or 185, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0028] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 33, 39, 45, 67, 72, 76, 81, 82, 85, 86, 89, 106, 115, 141, 149, 153, 155, 156, 157, 164, 180, 182, or 187, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0029] In some embodiments, the engineered adenylyl-sulfate kinase, or a functional fragment thereof, comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%>, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18- 258, 272-1358, and 1364-3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to a reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228.

[0030] In some embodiments, the engineered adenylyl-sulfate kinase, or a functional fragment thereof, comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, wherein the ammo acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228.

[0031] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18,19, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 35, 38, 39, 41 , 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 61, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 12, 1 13, 1 14, 115, 1 16, 117,118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 140, 141, 144,146, 147, 148, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, 01- 3228.

[0032] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 11, 16, 18, 19, 27, 42, 50, 51, 53, 67, 73, 76, 77, 78, 79, 85, 89, 94, 95, 97, 105, 109, 116, 118, 124, 139, 141, 147, 150, 156, 157, 158, 164, 171 , 176, 178, 180, 181, 182, 183, 188, 193, 195, 200, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228.

[0033] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 3 / 1 1, 3 / 11 / 76, 3 / 11 / 76 / 78 / 109, 3 / 11 / 76 / 109, 3 / 11 / 76 / 109 / 158 / 180, 3 / 11 / 76 / 158, 3 / 11 / 78, 3 / 11 / 78 / 109, 3 / 11 / 78 / 109 / 138, 3 / 11 / 78 / 109 / 138 / 176, 3 / 11 / 78 / 109 / 158, 3 / 11 / 78 / 109 / 180, 3 / 11 / 78 / 138, 3 / 11 / 78 / 176, 3 / 11 / 109, 3 / 11 / 109 / 138, 3 / 11 / 109 / 138 / 176, 3 / 11 / 109 / 138 / 180, 3 / 11 / 138, 3 / 76 / 78 / 109, 3 / 76 / 138 / 158, 11 / 76, 11 / 76 / 78, 11 / 76 / 78 / 109, 11 / 76 / 78 / 109 / 138, 11 / 76 / 78 / 109 / 138 / 158, 11 / 76 / 78 / 109 / 176, 11 / 76 / 78 / 158, 1 1 / 76 / 109, 11 / 76 / 109 / 138, 11 / 76 / 109 / 180, 11 / 76 / 138, 11 / 76 / 138 / 158, 11 / 76 / 158 / 180, 11 / 76 / 180, 11 / 78 / 109, 11 / 78 / 109 / 138 / 180, 11 / 78 / 109 / 158, 11 / 78 / 109 / 158 / 180, 11 / 78 / 158, 11 / 109, 11 / 109 / 138, 11 / 109 / 158, 11 / 109 / 158 / 180, 11 / 109 / 176, 11 / 158, 76 / 78 / 109, 76 / 78 / 109 / 138, 76 / 78 / 158, 76 / 109, 78 / 109, or 109, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 28, or relative to the reference sequence corresponding to SEQ ID NO: 28

[0034] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 3, 3 / 39 / 176, 39 / 85 / 158 / 176, 72 / 176, 1 10, 154 / 158 / 176, 158, 158 / 176, or 1767, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 154, or relative to the reference sequence corresponding to SEQ ID NO: 154

[0035] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 7, 8, 13, 30, 35, 41, 47, 64, 69, 71, 72, 73, 74, 75, 77, 79, 80, 81, 89, 95, 105, 107, 108, 112, 113, 1 14, 117, 128, 129, 130, 133, 135, 138, 141, 147, 148, 156, 159, 186, or 190, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

[0036] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 22, 39, 45, 54, 73, 83, 91, 96, 112, 134, 158, 167, 188, 193, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

[0037] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 91 / 158, 91 / 158 / 171, 91 / 158 / 188, 91 / 158 / 193, 91 / 193, 141 / 158, 158, 158 / 177, 158 / 177 / 193, 158 / 188, 158 / 188 / 193, 158 / 193, or 188 / 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or relative to the reference sequence corresponding to SEQ ID NO: 414.

[0038] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2, 4, 7, 12, 19, 22, 27, 51, 55, 83, 94, 96, 105, 124, 130, 141, 149, 150, 177, 188, 190, 191, 193, 200, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 426, or relative to the reference sequence corresponding to SEQ ID NO: 426.

[0039] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 2 / 7 / 83, 4, 4 / 83 / 193, 4 / 191 / 193, 7, 7 / 51 / 83 / 193, 7 / 51 / 141 / 193, 7 / 83, 7 / 83 / 193, 7 / 141, 7 / 188 / 193, 12 / 83, 12 / 83 / 193, 19 / 83 / 188 / 193, 27, 27 / 55, 27 / 150 / 177 / 200 / 203, 27 / 150 / 203, 27 / 200, 27 / 200 / 203, 51, 51 / 83, 51 / 83 / 141 / 193, 51 / 188 / 193, 55, 55 / 105, 55 / 105 / 150, 55 / 149, 55 / 203, 83, 83 / 141, 83 / 141 / 188 / 193, 83 / 188 / 193, 83 / 193, 105, 105 / 124 / 150, 105 / 150, 105 / 150 / 200 / 203, 105 / 200, 105 / 200 / 203, 124 / 149 / 203, 141, 141 / 188 / 193, 149, 149 / 150, 149 / 200, 149 / 203, 150, 150 / 177, 150 / 190, 150 / 200 / 203, 150 / 203, 177, 177 / 200, 188 / 193, 200 / 203, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

[0040] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 67, 76, 78, 79, 80, 137, 144, 157, 160, 163, 164, 170, 171, 180, 181, 183, or 184, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

[0041] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 79, 79 / 137 / 160 / 164, 79 / 137 / 160 / 164 / 193, 79 / 144 / 160, 79 / 144 / 160 / 164, 79 / 144 / 160 / 193, 79 / 160, 79 / 160 / 164, 79 / 164, 137 / 144 / 160 / 164, 137 / 144 / 164, 137 / 160, 137 / 160 / 164 / 181, 137 / 164, 144 / 160, 144 / 160 / 164, or 160 / 164, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to ammo acid residues 1 -200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0042] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 79 / 80, 79 / 137, 157, 157 / 170, 157 / 170 / 181, 157 / 180, 157 / 181, 170 / 181, or 181, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0043] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 5, 16, 18, 54, 55, 73, 76, 89, 106, 1 13, 118, 123, 139, 160, 164, 178, 179, 180, 181, or 182, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522

[0044] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 7, 11, 18, 25, 29, 45, 50, 51, 57, 93, 97, 105, 114, 121, 126, 127, or 130, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0045] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 18, 18 / 76 / 181, 18 / 123, 18 / 180 / 181 , 18 / 181, 54 / 123 / 179 / 180 / 181, 73 / 178, 76 / 179 / 180 / 181 , 76 / 181 , 89 / 118 / 178 / 181, 118, 123, 181 , 181 / 182, or 187, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding toamino acid residues 1-200 of SEQ ID NO: 732, or relative to the reference sequence corresponding to SEQ ID NO: 732.

[0046] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 7, 7 / 25 / 51, 7 / 51, 7 / 51 / 97, 7 / 51 / 97 / 105, 7 / 51 / 105, 7 / 97, 7 / 105, 25 / 51 / 97 / 105, 51, 51 / 97, 97, 97 / 105, or 105, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

[0047] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 22, 50, 51, 67, 105, 132, 170, 193, 195, 201, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848

[0048] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 50, 50 / 57 / 193 / 195, 57 / 193 / 195, 105 / 164 / 193, 164, 193, or 195, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

[0049] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 7, 76, 78, 79, 89, 138, 139, 157, 160, 178, or 202, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

[0050] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 7, 7 / 76 / 79, 7 / 76 / 79 / 138, 7 / 76 / 79 / 157, 7 / 76 / 79 / 202, 7 / 76 / 138, 7 / 76 / 138 / 157 / 178 / 202, 7 / 79, 7 / 79 / 138 / 202, 7 / 79 / 157 / 178, 7 / 79 / 157 / 202, 7 / 79 / 178 / 202, 7 / 79 / 202, 7 / 138, 7 / 138 / 202, 7 / 157 / 178, 76, 76 / 79 / 138 / 178 / 202, 76 / 202, 79 / 138 / 157, 79 / 157 / 178, 79 / 178 / 202, 138, 138 / 157, 138 / 157 / 202, 138 / 178 / 202, 138 / 202, 157, 157 / 178, 157 / 202, or 202, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

[0051] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 42, 43, 44, 67, 76, 78, 83, 118, 146, 147, 149, 150, 152, 153, 178, 193, 201, 202, or 203, 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

[0052] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 31, 38, 46, 97, 104, 110, 116, 117, 120, 182, or 186, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0053] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 42 / 43 / 76 / 78 / 178, 42 / 43 / 76 / 193, 42 / 76 / 78, 42 / 76 / 78 / 178, 42 / 76 / 78 / 193, 42 / 78, 43 / 76, 43 / 76 / 78, 43 / 76 / 78 / 178, 43 / 78, 43 / 178 / 193, 54 / 76 / 78, 76, 76 / 78, 76 / 78 / 178, 78 / 193, 149, 149 / 153, 149 / 153 / 201, 149 / 201, 153, 153 / 201, 178, 193, 201, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0054] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 5, 6, 8, 9, 19, 27, 50, 51, 53, 56, 86, 156, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236

[0055] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 67, 67 / 83, 67 / 83 / 1 16 / 122 / 193, 67 / 116, 67 / 116 / 149, 67 / 122 / 149, 67 / 149, 67 / 153, 67 / 178, 83 / 1 16 / 193, 83 / 149, 83 / 149 / 153 / 193, 83 / 149 / 178, 116, 1 16 / 153, 149, 149 / 153 / 193, 153, or 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0056] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least ammo acid residue difference at ammo acid position 7, 18, 23, 25, 99, 102, 103, 104, 116, 124, 125, 126, 133, 146, 150, 153, 173, 177, 180, 181, 184, 193, 202, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1322, or relative to the reference sequence corresponding to SEQ ID NO: 1322.

[0057] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 3 / 5, 3 / 5 / 8, 3 / 5 / 59, 3 / 8 / 54 / 193, 3 / 8 / 59, 3 / 54, 5, 5 / 8, 5 / 8 / 90, 5 / 193, 8, 8 / 53 / 54, 8 / 53 / 59 / 90 / 153 / 156, 8 / 54, 8 / 54 / 90, 8 / 59 / 153, 8 / 90, 8 / 90 / 193, 8 / 153, 8 / 156, 8 / 193, 53 / 54 / 59, 53 / 54 / 59 / 90 / 153 / 156, 53 / 54 / 90 / 153 / 156, 53 / 54 / 153 / 156, 53 / 59, 53 / 59 / 90, 54, 54 / 59 / 90 / 193, 54 / 90, 59, 59 / 90 / 153 / 156, 59 / 90 / 193, 59 / 193, 90, 90 / 193, 153, or 193, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0058] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 17, 19, 26, 28, 29, 53, 56, 57, 67, 92, 93, 95, 98, 128, 130, 134, 139, 140, 147, 156, 157, 160, 162, 164, 174, 176, 199, or 201 , or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0059] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 16, 50, 51, 59, 73, 77, 78, 79, 117, 118, 120, 121, 185, 186, 188, 189, 190, or 195, or any combinations thereof, wherein the amino acid positions are relative tothe reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0060] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 53, 53 / 59 / 89 / 90 / 153, 53 / 90, 53 / 90 / 180, 53 / 95, 53 / 180, 95, 120, or 180, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0061] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 8, 10, 103, 108, 116, 123, 124, 125, 132, 138, 141, 147, 149, 150, 170, 175, 178, 180, 183, 193, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716

[0062] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 16 / 19 / 53, 16 / 19 / 95, 16 / 199, 53, 53 / 77, 53 / 77 / 95, 53 / 95, 53 / 95 / 134, 53 / 176 / 199, 77 / 95, 95, 156, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

[0063] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 120, 140, 197, or 199, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0064] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 6, 7, 16, 28, 66, 105, 120, 122, 137, 139, 156, 165, 167, 195, 197, 198, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0065] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 50, 50 / 53, 50 / 53 / 73 / 199, 50 / 53 / 77, 50 / 53 / 195, 50 / 73 / 77 / 189 / 199, 50 / 95 / 120 / 195 / 199, 53, S m / Tl , 53 / 73 / 77 / 189, 53 / 73 / 95 / 185, 53 / 73 / 95 / 195, 53 / 73 / 189, 53 / 73 / 195, 53 / 73 / 199, 53 / 95, 53 / 195, 73 / 77 / 189, 73 / 185 / 189, 77 / 95 / 120 / 189, 95, 95 / 185, 95 / 189 / 199, or 95 / 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0066] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 2 / 123 / 193 / 204, 2 / 170, 116, 116 / 170 / 178, 116 / 178, 116 / 193, 116 / 204, 123 / 170 / 204, 123 / 204, 170, 170 / 193, 193, 193 / 204, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acidresidues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0067] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 83, 101, 116, 171, 188, or 195, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0068] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 31, 106, 123, 124, 125, 126, 147, 150, or 157, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946

[0069] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 6, 6 / 7, 6 / 7 / 50, 6 / 7 / 95 / 150, 6 / 7 / 120 / 150, 6 / 50 / 150, 6 / 73 / 95 / 150, 6 / 120 / 150, 6 / 150, 7 / 50 / 95 / 116, 7 / 1 16 / 120 / 150, 50, 50 / 73 / 95, 50 / 95 / 120, 50 / 95 / 132, 50 / 116, 73, 73 / 95, 73 / 1 16 / 120, 73 / 150, 95, 95 / 116, 95 / 120, 95 / 132, 95 / 150, 116, 132, or 132 / 150, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0070] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least ammo acid residue difference at ammo acid position 18, 19, 21, 51, 54, 57, 76, 78, 79, 83, 86, 91, 95, 101, 178, 180, or 181, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0071] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 31, 31 / 73 / 95 / 150, 31 / 73 / 147, 31 / 83, 31 / 95 / 147 / 195, 31 / 95 / 150, 31 / 147 / 195, 73 / 83 / 95 / 147, 73 / 83 / 147 / 182, 73 / 124 / 147, 76 / 95 / 147 / 188, 83, 83 / 95, 83 / 95 / 147, 83 / 124, 83 / 124 / 150, 83 / 147, 83 / 150 / 182, 83 / 150 / 188, 83 / 182 / 188, 95, 95 / 150 / 182, 116 / 120 / 123, 116 / 120 / 123 / 147, 124, 126 / 147, 147, 147 / 182, 182, or 188, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0072] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 16, 17, 18, 19, 50, 69, 76, 78, 79, 95, 117, 120, 121, 132, 156, 160, 176, 178, 179, 180, 181, 182, or 183, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058

[0073] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 51 / 73 / 76 / 83 / 95 / 181 / 182 / 195, 51 / 120 / 178, 73, 73 / 76 / 79 / 83 / 95 / 171 / 188, 73 / 76 / 95, 73 / 76 / 95 / 101 / 188, 73 / 95 / 171 / 195, 73 / 95 / 188, 76 / 79 / 95 / 171 / 188,76 / 83 / 95, 83 / 95, 83 / 95 / 120 / 182 / 188 / 195, 95, 95 / 120 / 178, 95 / 120 / 188 / 195, or 178, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158.

[0074] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 43, 83, 90, 94, 96, 97, 101, 105, 107, 109, 160, 162, 163, or 165, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158.

[0075] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 16 / 18 / 19 / 156 / 181 / 183 / 188, 18, 18 / 73 / 76 / 79 / 83 / 156 / 178 / 181, 18 / 156, 18 / 156 / 181 / 188, 83 / 156, 101 / 156, 120, 120 / 156, or 156 / 181 / 182, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2354, or relative to the reference sequence corresponding to SEQ ID NO: 2354.

[0076] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 56, 59, 116, 124, or 147, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2354, or relative to the reference sequence corresponding to SEQ ID NO: 2354.

[0077] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 90 / 101 / 105, 90 / 105 / 178 / 182, 96 / 101 / 105, 101, 101 / 105 / 178 / 181 / 195, 101 / 105 / 181 / 182, 101 / 105 / 181 / 195, 101 / 178 / 181 / 182, 105, 105 / 120, 105 / 120 / 178 / 181, 105 / 120 / 181, 105 / 120 / 195, 105 / 181 / 182, 105 / 195, 178 / 181, or 181, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2402, or relative to the reference sequence corresponding to SEQ ID NO: 2402.

[0078] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 5, 6, 7, 8, 50, 61, 70, 76, 78, 117, 126, 171, 201, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2402, or relative to the reference sequence corresponding to SEQ ID NO: 2402.

[0079] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 5 / 6 / 76 / 147 / 178, 5 / 50, 6 / 76 / 178 / 182 / 201, 6 / 124 / 147 / 178 / 182, 6 / 182, or 59 / 139 / 178, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2472, or relative to the reference sequence corresponding to SEQ ID NO: 2472.

[0080] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 4, 27, 28, 29, 54, 58, 59, 67, 73, 90, 91, 93, 95,138, 170, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2472, or relative to the reference sequence corresponding to SEQ ID NO: 2472.

[0081] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 2 / 5 / 50, 2 / 5 / 50 / 139, 5 / 50, 50 / 76 / 196 / 201, 50 / 126 / 139, 50 / 126 / 139 / 171 / 178, 50 / 126 / 171 / 196, 50 / 139 / 196, 50 / 171 / 201, 76, 76 / 171, 126 / 139 / 196, 139 / 171, 139 / 196 / 201, 171, or 201, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2558, or relative to the reference sequence corresponding to SEQ ID NO: 2558.

[0082] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 6, 27, 31, 42, 47, 51 , 53, 58, 59, 67, 70, 73, 76, 78, 80, 83, 91, 92, 93, 94, 96, 139, 147, 166, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2558, or relative to the reference sequence corresponding to SEQ ID NO: 2558.

[0083] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 2 / 5 / 27, 2 / 5 / 138, 4, 4 / 5, 4 / 5 / 27, 4 / 5 / 27 / 138 / 170, 5 / 27, 27, 27 / 138 / 170, 138, or 170, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2624, or relative to the reference sequence corresponding to SEQ ID NO: 2624.

[0084] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kmase comprises at least ammo acid residue difference at ammo acid position 18, 25, 45, 103, 104, 112, 124, 157, 158, 160, 191, 192, 193, 196, 197, 198, 199, 200, 201, 202, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2624, or relative to the reference sequence corresponding to SEQ ID NO: 2624.

[0085] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 27 / 42 / 94, 27 / 80, 27 / 93, 27 / 204, 31 / 94, 42 / 47, or 67 / 94, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2730, or relative to the reference sequence corresponding to SEQ ID NO: 2730.

[0086] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 15, 17, 27, 45, 46, 52, 55, 56, 59, 61, 65, 67, 70, 77, 86, 89, 97, 100, 118, 121, 139, 183, 191, 193, 194, 199, 201, or 202, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2730, or relative to the reference sequence corresponding to SEQ ID NO: 2730.

[0087] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 54 / 78, 54 / 90, 54 / 93, 90 / 91 / 204, 90 / 204, 93, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2824, or relative to the reference sequence corresponding to SEQ ID NO: 2824.

[0088] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 51, 76, 79, 83, 94, 103, 104, 105, 1 13, 117, 120, 124, 141, 170, 181, 184, 188, 189, 192, 195, 197, 198, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2824, or relative to the reference sequence corresponding to SEQ ID NO: 2824.

[0089] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 45 / 121, 45 / 121 / 197, 45 / 191, 90, 90 / 100 / 121 / 197 / 204, 90 / 100 / 121 / 204, 90 / 121 / 191, 90 / 191, 100 / 121, 104 / 191 / 197, 121 / 191 / 204, 121 / 197 / 204, 191 / 197, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2924, or relative to the reference sequence corresponding to SEQ ID NO: 2924.

[0090] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 4, 5, 6, 7, 13, 21, 22, 25, 27, 31, 43, 90, 121, 157, 158, 182, 183, 186, 191 , or 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2924, or relative to the reference sequence corresponding to SEQ ID NO: 2924.

[0091] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position (s) 79, 181, or 181 / 197, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 3034, or relative to the reference sequence corresponding to SEQ ID NO: 3034.

[0092] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 18, 51, 73, 79, 83, 94, 1 12, 130, 141, 164, 171, 176, 181, 199, 202, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3034, or relative to the reference sequence corresponding to SEQ ID NO: 3034.

[0093] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 4, 4 / 90, 4 / 157, 25, 79 / 182, 90, or 182, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO : 311 , or relative to the reference sequence corresponding to SEQ ID NO: 3116.

[0094] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 17, 23, 27, 31, 59, 97, 121, 147, 157, 172, 191, or 201, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3116, or relative to the reference sequence corresponding to SEQ ID NO: 3116.

[0095] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 4 / 141, 4 / 141 / 147, 4 / 141 / 176, 4 / 147 / 176, 141 / 147, 141 / 176, or 147, or any combinations thereof, wherein the amino acid positions are relative to thereference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3168, or relative to the reference sequence corresponding to SEQ ID NO: 3168.

[0096] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 4, 54, 69, 73, 76, 86, 102, 116, 117, 123, 126, 128, 170, 193, 195, 196, 197, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3168, or relative to the reference sequence corresponding to SEQ ID NO: 3168.

[0097] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 59 / 97 / 157, 59 / 139 / 191, 59 / 157 / 191, 59 / 176, 97, 97 / 139 / 157, 97 / 176, 97 / 191, 139, 139 / 191, 157 / 176, 164, 182, or 191, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3228, or relative to the reference sequence corresponding to SEQ ID NO: 3228

[0098] In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any even-numbered sequence selected from SEQ ID NO: 12-258, 272-1358, and 1364-3324.

[0099] In some embodiments, the engineered adenylyl-sulfate kmase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to ammo acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 14, 18-258, 272-1358, and 1364- 3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 18- 258, 272-1358, and 1364-3324.

[0100] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0101] In some embodiments, the engineered adenylyl-sulfate kmase comprises an amino acid sequence comprising amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364- 3324.

[0102] In some embodiments, the engineered adenylyl-sulfate kmase comprises an amino acid sequence comprising ammo acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or comprising SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848,880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0103] In some embodiments, the engineered adenylyl-sulfate kinase has activity in the conversion of a natural or modified NDP to a nucleoside diphosphate with an additional phosphate at the 3’ position of the sugar moiety'. In some embodiments, the engineered adenylyl-sulfate kinase has at least one improved property, as compared to a wild-type or reference adenylyl-sulfate kinase having an amino acid sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or an amino acid sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228

[0104] In some embodiments, the improved property of the engineered adenylyl-sulfate kinase comprises increased activity on natural substrates, increased activity on non-natural substrates, increased activity on 2’- fluoro modified nucleotides, increased activity on 2’-O-methyl modified nucleotides, increased thermostability, increased substrate promiscuity, increased soluble protein expression, decreased product inhibition and / or decreased byproduct formation, as compared to a wild-type or reference adenylyl-sulfate kinase of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0105] In another aspect, the present disclosure further provides a recombinant polynucleotide comprising a polynucleotide sequence encoding at least one engineered adenylyl-sulfate kinase disclosed herein

[0106] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 1-600 of SEQ ID NO: 13, 27, 153, 239, 413, 425, 473, 521, 731, 847, 879, 961, 1031, 1235, 1321, 1363, 1643, 1833, 1945, 1953, 2057, 2157, 2353, 2401, 2471, 2557, 2623, 2729, 2823, 2923, 3033, 3115, 3167, or 3227, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 13, 27, 153, 239, 413, 425, 473, 521, 731, 847, 879, 961, 1031, 1235, 1321, 1363, 1643, 1833, 1945, 1953, 2057, 2157, 2353, 2401, 2471, 2557, 2623, 2729, 2823, 2923, 3033, 3115, 3167, or 3227, wherein the polynucleotide sequence encodes an adenylyl-sulfate kinase.

[0107] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity' to a reference polynucleotide sequence corresponding to nucleotide residues 1-600 of an odd-numbered SEQ ID NO of SEQ ID NOs: 17- 257, 271-1357, and 1363-3323, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-257, 271-1357, and 1363-3323, wherein the polynucleotide sequence encodes an adenylyl-sulfate kinase.

[0108] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon- optimized for expression in a host cell.

[0109] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding an adenylyl-sulfate kinase disclosed herein.

[0110] In a further aspect, the present disclosure also provides a host cell comprising an expression vector encoding an adenylyl-sulfate kinase. In some embodiments, the host cell comprises a bacterial cell, fungal cell, insect cell, or mammalian cell.[oni] In another aspect, the host cells are used in a method for producing an engineered adenylyl-sulfate kinase polypeptide, the method comprising culturing a host cell comprising an expression vector encoding an engineered adenylyl-sulfate kinase, under suitable culture conditions such that the engineered adenylyl-sulfate kinase is produced.

[0112] In some embodiments, the method further comprises recovering at least one adenylyl-sulfate kinase from the culture and / or host cells. In some embodiments, the method further comprises purifying the expressed engineered adenylyl-sulfate kinase

[0113] In some embodiments, the adenylyl-sulfate kinase is used to add a phosphate group to the 3 ’ position of the sugar of a natural or modified nucleoside diphosphate, the method comprising contacting an adenylyl- sulfate kinase with an NDP substrate under suitable reaction conditions such that an NDP with a phosphate group at the 3’ position of the sugar is produced. In some embodiments of the method, the adenylyl-sulfate kinase comprises an engineered adenylyl-sulfate kinase described herein.

[0114] In some embodiments of the method, the NDP substrate comprises a modification at the 2 ’-position of the sugar moiety, a modified nucleobase, and / or a modified phosphate. In some embodiments, the 2’- modification comprises a 2’-fluoro or a 2’-O-methyl. In some embodiments, the modified phosphate on the NDP substrate comprises an alpha-thiophosphate (e.g., alpha-thiophosphate NDP). In some embodiments, the alpha-thiophosphate NDP is an (Sp) or (Rp) diastereomer, or a mixture of (Sp) and (Rp) diastereomers. In some embodiments, different ratios of (Sp) and (Rp) diastereomers of the alpha-thiophosphate NDP substrate may be used.

[0115] In some embodiments of the method, the suitable reaction conditions comprise an NTP co-substrate as a phosphate donor for the adenylyl-sulfate kinase. In some embodiments, the NTP co-substrate comprises the same structure for the NDP portion as the NDP substrate.DESCRIPTION OF THE INVENTION

[0116] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein, and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry', analytical chemistry and nucleic acid chemistry' described below are those well-known and commonly employed in the art Such techniques are well-known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

[0117] Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the invention as a whole.

[0118] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein

[0119] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise Thus, for example, reference to “a polypeptide” includes more than one polypeptide. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting

[0120] It is to be understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.” It is to be further understood that where descriptions of various embodiments use the term “optional” or “optionally” the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. It is to be understood that both the foregoing general description, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure. The section headings used herein are for organizational purposes only and not to be constmed as limiting the subject matter described.Abbreviations

[0121] The abbreviations used for the genetically encoded amino acids are conventional and are as follows:

[0122] When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (Ca). For example, whereas “Ala” designates alanine without specifying the configuration about the a-carbon, “D-Ala” and “L-Ala” designate D-alamne and L-alamne, respectively.

[0123] When the one-letter abbreviations are used, upper case letters designate ammo acids in the L- configuration about the a-carbon and lower case letters designate ammo acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanme. When polypeptide sequences are presented as a string of one-letter or three -letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0124] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A): guanosine (G): cytidine (C); thymidine (T); and uridine (U). These abbreviations are also used interchangeably for nucleosides and nucleotides (nucleosides with one or more phosphate groups). Unless specifically delineated, the abbreviated nucleosides or nucleotides may be either ribonucleosides (or ribonucleotides) or 2'-deoxyribonucleosides (or 2'-deoxyribonucleotides). The nucleosides or nucleotides may also be modified at the 3 ’ position The nucleosides or nucleotides may be specified as being either ribonucleosides (or ribonucleotides) or 2’-deoxyribonucleosides (or 2’-deoxyribonucleotides) on an individual basis or on an aggregate basis When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates arc not indicatedDefinitions

[0125] In reference to the present invention, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terns are intended to have the following meanings.

[0126] “EC” number refers to the I m / vine Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0127] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains

[0128] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0129] “Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc ). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids.

[0130] “Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one -letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0131] “NTP:NDP 3 ’-phosphotransferase,” “NTP:NDP phosphotransferase,” or “NTP:NDP 3 ’-transferase” refers to an enzyme that catalyzes the phosphorylation of the 3 ’-OH group of the sugar moiety of a nucleoside diphosphate to produce a nucleoside diphosphate with a 3 ’-phosphate moiety. In some embodiments, the NTP:NDP 3 ’-phosphotransferase has the activity of a 3’-O-kinase: phosphorylation of the 3 ’-OH group of the sugar moiety of a nucleoside or nucleotide to produce a nucleoside or nucleotide with a 3’-phosphate moiety

[0132] “AdenylyLsulfate kinase,” “adenosine 5'-phosphosulfate kinase,” or “APS kinase” is an enzyme that catalyzes the reversible conversion of an ATP and a 5’-adenylyl sulfate to an ADP and 3'-phosphoadenylyl sulfate. In some embodiments, the adenylyl-sulfate kinase has the activity of a 3’-O-kinase: phosphorylation of the 3 ’-OH group of the sugar moiety of a nucleoside or nucleotide to produce a nucleoside or nucleotide with a 3 ’-phosphate moiety. In some embodiments, the adenylyl-sulfate kinase is capable of the reversible conversion of a NTP and a nucleoside diphosphate to a NDP and a nucleoside diphosphate with a 3 ’- phosphate moiety on the sugar moiety. In some embodiments, the adenylyl-sulfate kinase is an enzyme classified in EC 2.7. 1.25. In some embodiments, the adenylyl-sulfate kinase has the activity of a 3 ’-O-kinase: phosphorylation of the 3 ’-OH group of the sugar moiety of a nucleoside or nucleotide to produce a nucleoside or nucleotide with a 3 ’-phosphate moiety.

[0133] “Domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of related proteins, generally evolutionarily related proteins. Amino acids at other positions can vary between homologues, orthologues, and paralogues, but amino acids that are conserved at specific positions in the related proteins indicate amino acids that likely influence structure, stability and / or function of a protein.

[0134] “Motif’ or “consensus sequence” as used herein refers to a short, conserved region in the sequence of related proteins. In some instances, motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain. A motif or consensus sequence can be represented by conventional nomenclature, e g., one letter amino acid residues (e.g., A, K, M, etc.), brackets [ ] for variations at a defined amino acid position, X for occurrence of any amino acid, curly braces { } for excluded / disallowed amino acids, and a repeating pattern specified with number or range in parenthesis (n) In some embodiments, each amino acid position in the motif is separated by a hyphen In some embodiments, each amino acid position in the motif is presented without a hyphen. As with descriptions ofproteins and peptides herein, the motif or consensus amino acid sequence is presented from the amino (N-) to carboxy (C-) terminus.

[0135] “Polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably herein and refer to two or more nucleosides or nucleotides that are covalently linked together The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2' deoxyribonucleotides (i.e., DNA), wholly comprised of other synthetic nucleotides or comprised of mixtures of synthetic, ribo- and / or 2' deoxyribonucleotides. The polynucleotides may also include modified nucleotides with substitutions, including 2’ substitutions (e.g., 2’-flouro, 2’-O-methyl, 2’-O-methoxyethyl, locked or constrained ethyl modifications, and others known to those skilled in the art). Nucleosides will be linked together via standard phosphodiester linkages or via one or more non-standard linkages, including but not limited to phosphorothioate linkages The polynucleotide may be single-stranded or double-stranded or may include both single-stranded regions and double-stranded regions Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino-acid sequences. Nucleobases that are modified or synthetic may comprise any known or hypothetical or future discovered modification or structure that would be recognized by one of skill in the art as a modified or synthetic nucleobase. Similarly, the terms “polynucleotide,” “oligonucleotide,” and “nucleic acid’ ’ are intended to comprise any modified or synthetic structure that is now known or discovered in the future that would be recognized by one of skill in the art as being or having the function of a “polynucleotide," “oligonucleotide,” or “nucleic acid. ” An example of a modified or synthetic structure having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid” is PNA or peptide nucleic acid.

[0136] “NTP,” “NDP,” “NMP,” “NQP,” “nucleoside,” “nucleotide” or similar terms are intended to generically refer to ribo, deoxyribo, and / or 2’ modified (modifications other than H or OH) versions of these molecules, unless specifically defined in a specific instance. These molecules may also have additional modifications at other positions, and these terms arc intended to encompass these variations, absent any specific definition to the contrary.

[0137] “Template-independent synthesis” refers to synthesis of an oligonucleotide or a polynucleotide without the use of template strand as a guide for synthesis of a complementary' oligo or polynucleotide strand. Thus, template -independent synthesis refers to an iterative process, whereby, successive NTPs are added to a growing oligo or nucleotide chain or acceptor substrate. Template -independent synthesis may be in a sequence defined manner or may be random, as is the case with the wild-type TdT in creating antigen receptor diversity. Processes for template-independent synthesis are further described herein

[0138] “Coding sequence” refers to that portion of a nucleic acid (e g., a gene) that encodes an amino acid sequence of a protein

[0139] “Naturally -occurring” or “wild-type” refers to the form found in nature For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0140] “Recombinant,” “engineered,” and “non-naturally occurring” when used with reference to a cell, nucleic acid, or polypeptide, refer to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature. In some embodiments, the cell, nucleic acid or polypeptide is identical a naturally occurring cell, nucleic acid or polypeptide, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Nonlimiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0141] “Percentage of sequence identity'” and “percentage homology'” are used interchangeably herein to refer to comparisons among polynucleotides or polypeptides and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i e., gaps) as compared to the reference sequence for optimal alignment of the two sequences The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or ammo acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad Sci USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g , GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2 0 algorithms, which are described by Altschul et al. (See, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucl. Acids Res., 1977, 3389-3402, respectively) Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity' and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WT), using default parameters provided

[0142] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. For instance, a “reference sequence based on SEQ ID NO: 14 having at the residue corresponding to XI 3 a tyrosine” or XI 3 Y refers to a reference sequence in which the corresponding residue at XI 3 in SEQ ID NO: 14, which is a glycine, has been changed to tyrosine.

[0143] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows

[0144] “Substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity, at least between 89 to 95 percent sequence identity, or more usually, at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. In some specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptidesequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity', preferably at least 89 percent sequence identity', at least 95 percent sequence identity' or more (e.g., 99 percent sequence identity). In some embodiments, residue positions that are not identical in sequences being compared differ by conservative amino acid substitutions.

[0145] “Corresponding to,” “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered adenylyl- sulfate kinase polypeptide, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned

[0146] “Amino acid difference” or “residue difference” refers to a change in the ammo acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X25 as compared to SEQ ID NO: 14” refers to a change of the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO: 14. Thus, if the reference polypeptide of SEQ ID NO: 14 has a glycine at position 25, then a “residue difference at position X25 as compared to SEQ ID NO: 14" an amino acid substitution of any residue other than glycine at the position of the polypeptide corresponding to position 25 of SEQ ID NO: 14. In most instances herein, the specific amino acid difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.c., the different residue than in the reference polypeptide). In some embodiments, more than one ammo acid can appear in a specified residue position (i.e., the alternative amino acids can be listed in the form XnY / Z, where Y and Z represent alternate amino acid residues). In some instances (e.g., in Tables 8.2, 9.2, 10.2, 11.2,12.2. 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 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, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2,51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2,70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, and 78.2) the present invention also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. Furthermore, in some instances, a polypeptide of the present invention can include one or more amino acid differences relative to a reference sequence, which is indicated by a list of the specified positions where changes are made relative to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative and non-conservative amino acid substitutions.

[0147] “Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another ammo acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively Exemplary conservative substitutions are provided in Table 1 below

[0148] “Non-conseivative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0149] “Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more ammo acids, 2 or more amino acids, 5 or more ammo acids, 10 or more ammo acids, 15 or more ammo acids, or 20 or more ammo acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered adenylyl- sulfate kinase enzyme. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0150] “Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. In some embodiments, the improved engineered adenylyl-sulfate kinase enzymes comprise insertions of one or more amino acids to the naturally occurring polypeptide as well as insertions of one or more amino acids to other improved adenylyl-sulfate kinase polypeptides. Insertions can be in theinternal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the ammo acids in the naturally occurring polypeptide.

[0151] “Fragment” as used herein refers to a polypeptide that has an amino-terminal and / or carboxy -terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length adenylyl-sulfate kinase polypeptide, for example the polypeptide of SEQ ID NO: 14 or an adenylyl-sulfate kinase provided in the even-numbered sequences of SEQ ID NOs: 18-258.

[0152] “Functional fragment” and “biologically active fragment” are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e g , a full-length engineered adenylyl-sulfate kinase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0153] “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally -occurring environment or expression system (e.g., host cell or in vitro synthesis). The engineered adenylyl-sulfate kinase enzymes may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the engineered adenylyl-sulfate kinase enzyme can be an isolated polypeptide.

[0154] “Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition) and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure adenylyl-sulfate kinase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity ( .e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated engineered adenylyl-sulfate kinase polypeptide is a substantially pure polypeptide composition

[0155] “Improved enzyme property” refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered adenylyl-sulfate kinase polypeptides that exhibit an improvement in any enzyme property as compared to a reference adenylyl-sulfate kinase polypeptide and / or a wild-type adenylyl-sulfate kinase polypeptide, and / or another engineered adenylyl-sulfate kmase polypeptide. For the engineered adenylyl-sulfate kinase polypeptides described herein, the comparison is generally made to the wild-type enzyme from which the adenylyl-sulfate kmase is derived, although in some embodiments, thereference enzyme can be another improved engineered adenylyl-sulfate kinase Thus, the level of “improvement” can be determined and compared between various adenylyl-sulfate kinase polypeptides, including wild-type, as well as engineered adenylyl-sulfate kinases. Improved properties include, but are not limited, to such properties as enzymatic activity (which can be expressed in terms of percent conversion of the substrate), thermostability, solvent stability, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g., substrate or product inhibition), activity at elevated temperatures, increased soluble expression, decreased by-product formation, increased specific activity substrates, and / or increased activity (including enantioselectivity).

[0156] “Increased enzymatic activity” refers to an improved property of the adenylyl-sulfate kinase polypeptides, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of adenylyl-sulfate kinase) as compared to the reference adenylyl-sulfate kinase enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vmaxor kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.2 times the enzymatic activity of the corresponding wild-type enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more enzymatic activity than the naturally occurring or another engineered adenylyl-sulfate kinase from which the adenylyl-sulfate kinase polypeptides were derived. Adenylyl-sulfate kinase activity can be measured by any one of standard assays, such as by monitoring changes in properties of substrates, cofactors, or products. In some embodiments, a paired or coupled reaction may be used to determine activity. In some embodiments, the amount of products generated can be measured by Liquid Chromatography -Mass Spectrometry (LC-MS), HPLC, capillary electrophoresis (CE), or other methods, as known in the art. Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates arc compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates

[0157] “Conversion” refers to the enzymatic conversion of the substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of an adenylyl-sulfate kinase polypeptide can be expressed as “percent conversion” of the substrate to the product

[0158] “Thermostable” refers to an adenylyl-sulfate kinase polypeptide that maintains similar activity (more than 60% to 80% for example) after exposure to elevated temperatures (e.g., 40-80 °C) for a period of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same elevated temperature.

[0159] “Solvent stable” refers to an adenylyl-sulfate kinase polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to varying concentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butylacetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same concentration of the same solvent.

[0160] “Thermo- and solvent stable” refers to an adenylyl-sulfate kinase polypeptide that is both thermostable and solvent stable.

[0161] The tenn “stringent hybridization conditions” is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. In general, the stability' of a hybrid is a function of ion strength, temperature, G / C content, and the presence of chaotropic agents. The 7„, values for polynucleotides can be calculated using known methods for predicting melting temperatures (See e.g., Baldino et al., Meth. Enzymol., 1989, 168:761-777; Bolton et al , Proc. Natl. Acad. Sci. USA, 1962, 48:1390; Bresslauer et al., Proc. Natl. Acad. Sci. USA, 1986, 83:8893-8897; Freier et al , Proc. Natl. Acad. Sci. USA, 1986, 83:9373-9377; Kierzek et al , Biochem , 1986, 25:7840-7846; Rychlik et al , Nucl Acids Res , 1990, 18:6409-6412 (erratum, Nucl Acids Res , 1991 , 19:698); Suggs et al . In Developmental Biology Using Purified Genes, Brown et al , eds , pp. 683-693, Academic Press, Cambridge, MA (1981); and Wetmur, Cnt. Rev Biochem. Mol. Biol., 1991, 26:227-259). In some embodiments, the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered adenylyl-sulfate kinase enzyme of the present invention.

[0162] “Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term “moderately stringent hybridization” refers to conditions that permit target -DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target -polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5*SSPE, 0.2% SDS at 42 °C, followed by washing in 0.2*SSPE, 0.2% SDS, at 42 °C. “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tmas determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (z.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5 ' Denhart's solution, 5*SSPE, 0.2% SDS at 42 °C, followed by washing in 0.1 *SSPE, and 0.1% SDS at 65 °C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0 1 % (w: v) SDS at 65 °C and washing in 0 lx SSC containing 0.1% SDS at 65 °C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0163] “Heterologous” polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0164] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the adenylyl-sulfate kinase enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0165] “Preferred, optimal, high codon usage bias codons” refers interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinfonn , 1998, 14:372-73; Stenico et al., Nucl. Acids Res., 1994, 222437-46; Wright, Gene, 1990, 87:23-29). Codon usage tables are available for many different organisms (See e.g., Wada et al , Nucl Acids Res., 1992, 20:2111-2118; Nakamura et al., Nucl. Acids Res., 2000, 28:292; Henaut and Danchin, In Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p. 2047-2066 (1996)). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding scqucnccs-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (See e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory' Press, Cold Spring Harbor, N.Y. (2001); Uberbacher, Meth. Enzymol., 1996, 266:259-281 ; and Tiwari et al., Comput. Appl. Biosci., 1997, 13:263-270).

[0166] “Control sequence” is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide Such control sequences include, but are not limited to, a leader, poly adenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0167] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e. , in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

[0168] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0169] “Suitable reaction conditions” refer to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, co-solvents, etc.) under which an adenylyl-sulfate kinase polypeptide of the present invention is capable of converting one or more substrate compounds to a product compound Exemplary “suitable reaction conditions” are provided in the present invention and illustrated by the Examples

[0170] “Composition” refers to a mixture or combination of one or more substances, wherein each substance or component of the composition retains its individual properties. As used herein, a biocatalytic composition refers to a combination of one or more substances useful for biocatalysis.

[0171] “Loading”, such as in “compound loading” or “enzyme loading” or “cofactor loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0172] “Substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst. For example, an adenylyl-sulfate kinase biocatalyst used in the synthesis processes disclosed herein acts on a natural or modified nucleoside diphosphate.

[0173] “Product” in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst. For example, an exemplary product for an adenylyl-sulfate kinase biocatalyst used in a process disclosed herein is a 3 ’-phosphorylated natural or modified nucleoside diphosphate

[0174] “Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms inclusively, either straight chained or branched, more preferably from 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbon atoms inclusively. An alkyl with a specified number of carbon atoms is denoted in parenthesis (e.g., (Ci-C6)alkyl refers to an alkyl of 1 to 6 carbon atoms).

[0175] “Alkenyl” refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but optionally containing more than one double bond.

[0176] “Alkynyl” refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties.

[0177] “Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer respectively, to alkyl, alkenyl and alkynyl as defined herein in w hich one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups Heteroatoms and / or heteroatomic groups which can replace the carbon atoms include, but are not limited to-O-, -S-, -S-O-, -NRY-, -PH-, -S(O)-, -S(O)2-, -S(O) NRY-, -S(O)2NR7, and the like, including combinations thereof, where each RTis independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0178] “Amino" refers to the group -NH2. Substituted amino refers to the group -NHR’1, NR’iR’i, and NR'iR'iR11, where each R11is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy -sulfamino, and the like.

[0179] “ Aminoalky 1" refers to an alkyl group in which one or more of the hydrogen atoms are replaced with one or more amino groups, including substituted amino groups.

[0180] “ Aminocarbonyl’' refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NR’1R’1, where the amino group NR,|R11is as defined herein.

[0181] “Oxy" refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0182] “Alkoxy” or “alkyloxy” are used interchangeably herein to refer to the group -OR--. wherein R7is an alkyl group, including optionally substituted alkyl groups.

[0183] “Carboxy" refers to -COOH.

[0184] “Carbonyl” refers to -C(O)-, which may have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0185] “Carboxy alkyl" refers to an alkyl in which one or more of the hydrogen atoms are replaced with one or more carboxy groups.

[0186] “Aminocarbonylalkyl" refers to an alkyl substituted with an aminocarbonyl group, as defined herein.

[0187] “Halogen" or “halo” refers to fluoro, chloro, bromo and iodo.

[0188] “Haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl” is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the expression “(Ci - C2) haloalkyl” includes 1 -fluoromethyl, difluoromethyl, trifluoromethyl, 1 -fluoroethyl, 1,1 -difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl, perfluoroethyl, etc.

[0189] “Hydroxy” refers to -OH.

[0190] “Hydroxyalkyl” refers to an alkyl group in which in which one or more of the hydrogen atoms are replaced with one or more hydroxy groups.

[0191] “Thiol” or “sulfanyl" refers to -SH. Substituted thiol or sulfanyl refers to -S-R'1, where R’1is an alkyl, aryl or other suitable substituent.

[0192] “Sulfonyl” refers to -SO2-. Substituted sulfonyl refers to -SO2-R’1, where R11is an alkyl, aryl or other suitable substituent.

[0193] “Alkylsulfonyl" refers to -SO2-RR where R^ is an alkyl, which can be optionally substituted. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0194] “Phosphate" as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms). The orthophosphate ion is commonly found withone or more hydrogen atoms or organic groups. A phosphate group or chain may be modified, as further described herein

[0195] “Phosphorylated’’ as used herein refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).

[0196] “Thiophosphate’’ refers to an instance where a non-bridging oxygen in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP is replaced with a sulfur.

[0197] “Dithiophosphate” refers to an instance where two non-bridging oxy' gens in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP are replaced with two sulfurs.

[0198] “Optionally substituted” as used herein with respect to the foregoing chemical groups means that positions of the chemical group occupied by hydrogen can be substituted with another atom (unless otherwise specified) exemplified by, but not limited to carbon, oxygen, nitrogen, or sulfur, or a chemical group, exemplified by, but not limited to, hydroxy', oxo, nitro, methoxy, ethoxy', alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxy carbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamide, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyd, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, hetero aryl alkyl, heteroaiylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, prpendmyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; where preferred heteroatoms are oxygen, nitrogen, and sulfur. Additionally, where open valences exist on these substitute chemical groups they can be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and / or heterocycle groups, that where these open valences exist on carbon they can be further substituted by halogen and by oxygen-, nitrogen-, or sulfur-bonded substituents, and where multiple such open valences exist, these groups can be joined to form a ring, either by direct fonnation of a bond or by fonnation of bonds to a new heteroatom, preferably ox ' gen, nitrogen, or sulfur. It is further contemplated that the above substitutions can be made provided that replacing the hydrogen with the substituent docs not introduce unacceptable instability to the molecules of the present invention and is otherwise chemically reasonable. One of ordinary skill in the art would understand that with respect to any chemical group described as optionally substituted, only sterically practical and / or synthetically feasible chemical groups are meant to be included. “Optionally substituted” as used herein refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl,” the “alkyl” portion and the “aryl” portion of the molecule may or may not be substituted, and for the series “optionally substituted alkyl, cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups, independently of the others, may or may not be substituted.

[0199] “Reaction” as used herein refers to a process in which one or more substances or compounds or substrates is converted into one or more different substances, compounds, or processes.Adenylyl-Sulfate Kinase Mediated NQP Synthesis3’-O-Kinases with NTP:NDP 3’ Phosphotransferase Activity

[0200] Several enzymes are known to act as 3 ’-O-kinases on nucleoside or nucleotide substrates, that is, phosphorylation of the 3 ’-OH group of the sugar moiety of a nucleoside or nucleotide to produce a nucleoside or nucleotide with a 3 ’-phosphate moiety. Among these, adenylyl-sulfate kinase catalyzes the conversion of adenosine 5'-phosphosulfate to 3 '-phosphoadenylyl sulfate using ATP as a co-factor (Satishchandran et al., J. Biol. Chem., 1989, 264(25), 15012-15021), raising the prospect that natural or engineered adenylyl-sulfate kinases may have NTP:NDP 3’ phosphotransferase activity.

[0201] Indeed, a recent report describes the adenylyl-sulfate kinase encoded by the CysC gene of Thermotoga sp. RQ7, together with other enzymes having 3’-O-kinase activity useful for the conversion of natural and modified nucleosides and nucleotides to NQPs (PCT / US2023 / 076694). In that report, the wildtype adenylyl-sulfate kinase was found to have soluble expression and activity in the conversion of nucleosides to NQPs in a one-pot system with acetate kinase, adenylate kinase, and adenosine kinase.

[0202] The present disclosure provides engineered adenylyl-sulfate kinases to synthesize natural and modified NQPs (pppNp) In some embodiments, the present disclosure provides adenylyl-sulfate kinase enzymes for the conversion of a nucleoside diphosphate to a nucleoside diphosphate with a phosphate group at the 3’ position of the sugar, as depicted in Scheme 1.Scheme 1

[0203] The adenylyl-sulfate kinase reaction may be optionally coupled to a NTP regeneration system to recycle the NTP co-substrate, depicted in Scheme 1 as an acetate kinase / acetyl phosphate NTP regeneration system.

[0204] It is to be understood that when the adenylyl-sulfate kinase reaction depicted in Scheme 1 comprises a NDP substrate and a NTP co-substrate having the same nucleotide structure (e.g. ADP and ATP), the net reaction catalyzed by the adenylyl-sulfate kinase comprises NTP to nucleoside diphosphate with a phosphategroup at the 3’ position of the sugar (e.g. ATP to 3’P-ADP). However, initiation of the reaction requires a NDP in addition to the NTP co-substrate. Therefore, in some embodiments, the present disclosure provides for conversion of a NTP to a 3’P-NDP.

[0205] The reaction depicted in Scheme 1 may be further coupled to a nucleoside diphosphate kinase, such as an acetate kinase, to generate a nucleoside triphosphate with a phosphate at the 3 ’ position of the sugar moiety, also referred to as NQP (an NTP with a phosphate group at the 3’ position of the sugar), as exemplified, below, in Scheme 2.Scheme 2

[0206] In Scheme 2, the NDP is converted by a NTP:NDP 3’ phosphotransferase (depicted as CysC or adenylyl-sulfate kinase) to a NDP with a phosphate group at the 3’ position of the sugar moiety (3'P-NDP). The 3’P-NDP is then converted by a nucleoside diphosphate kinase (such as an acetate kinase) to a NTP with a phosphate group at the 3 ’ position of the sugar moiety (NQP)

[0207] Although Scheme 1 and Scheme 2 depict canonical nucleobases (adenine, cytosine, uracil, and guanine), the NDP substrate may comprise any nucleobase and may comprise a modified nucleobase or a nucleobase optionally substituted at various positions.

[0208] Similarly, the R at the 2’ position of the sugar moiety may comprise a H or OH or may comprise a modification, as described herein and as known in the art.NTP Recycling

[0209] The adenylyl-sulfate kinase uses a NTP as a co-substrate and phosphate donor, simultaneously producing a NDP. As depicted in Scheme 1 and Scheme 2, a nucleoside diphosphate kinase, such as an acetate kmase (ACK), may be used to recycle the NTP donor from NDP using acetyl-phosphate as a phosphate donor substrate that is converted to acetate.

[0210] Various NTP regenerating systems that may be used with the present invention are known in the art including pyruvate kinase / phosphoenol pyruvate, acetate kinase / acetyl phosphate, and creatine kinase / creatine phosphate (see, e.g., Lian et al., Appl Biochem Biotechnol., 2014, 174:2351-2367).

[0211] The nucleoside diphosphate kinase used for NTP recycling may be the same as or different than the nucleoside diphosphate kinase used for conversion of 3’P-NDP to NQP. The nucleoside diphosphate kinaseused for conversion of 3’P-NDP to NQP may have increased activity on a 3’P-NDP substrate as compared to a reference nucleoside diphosphate kinase or a nucleoside diphosphate kinase used for NTP recycling.

[0212] Use of a phosphate donor NTP that has the same nucleotide structure as the substrate NTP / NDP for the adenylyl-sulfate kinase provides for generation of the 3 ’-phosphorylated product without contamination from using a phosphate donor NTP with a nucleotide structure different from the substrate NTP.NDP Substrate

[0213] In some embodiments, the NDP substrate and the NTP co-substrate for the adenylyl-sulfate kinase reaction are generated enzymatically, such as from a nucleoside, nucleoside monophosphate, and / or nucleoside diphosphate.

[0214] In some embodiments, the NDP substrate is an unmodified NDP. In some embodiments, the unmodified NDP substrate is any naturally occurring NDP

[0215] In some embodiments, the NDP substrate has at the 2’ position of the sugar moiety a H (e.g., deoxy) or OH (e.g., ribo).

[0216] In some embodiments, the nucleobase of the NDP substrate is, among others, adenine, cytosine, guanine, thymine, uracil, xanthine, hypoxanthine, 2,6-diaminopurine, purine, 6,8-diaminopurine, 5- methylcytosine (m’C), 2-thiouridine, pseudouridine, dihydrouridine, inosine, or 7-methyl guanosine (m'G)

[0217] In some embodiments of the method, the NDP substrate is ADP, GDP, UDP, CDP, or TDP, and wherein the NDP has at the 2 ’-position of the sugar moiety an OH, thereby by resulting in corresponding product rAQP, rGQP, rUQP, rCQP, or rTQP, respectively.

[0218] In some embodiments, of the method, the NDP substrate is ADP, GDP, UDP, CDP, or TDP, and wherein the NDP has at the 2 ’-position of the sugar moiety a H, thereby by resulting in corresponding product dAQP, dGQP, dUQP, dCQP, or dTQP, respectively

[0219] In some embodiments, the NDP substrate is a modified NDP, thereby resulting a modified NQP product In some embodiments, the modified NDP comprises a modified sugar moiety, modified nucleobase, or modified phosphate, or combinations thereof.

[0220] In some embodiments, the NDP substrate comprises a modified sugar moiety. In some embodiments, the modified sugar moiety' is modified at the 2’-position of the sugar moiety. In some embodiments, the modified 2’-position of the sugar moiety of the NDP substrate is halo, 2’-O-R’, or 2’-O-COR’, where R’ is an alkyd, alkyloxy alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a Ci-C4alkyl. In some embodiments, the modified 2’-position is a 2’-O-R’, wherein in R’ is alkyloxy alkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl. In some embodiments, tire 2 ’-position of the sugar moiety' of the NDP substrate is -O-R’, wherein R’ is -CI h or -CH2CH3 or - CH2CH2OCH3. In some embodiments, the modified 2’-position is 2 ’-O-(2 -methoxy ethyl), 2’-O-allyl, 2’-O- propargyl, 2’-O-ethylamine, 2’-O-cy anoethyl, or 2’-O-acetalester.

[0221] In some embodiments, the modified 2’-position of the sugar moiety of the NDP substrate is halo In some embodiments, the modified 2’-position of the sugar moiety is F (i.e., 2’-F) or Br (i.e., 2’-Br).

[0222] In some embodiments, the modified sugar moiety of the NDP substrate is a “locked” nucleotide (e.g., locked NDP). In some embodiments, the locked NDP is a locked ADP, locked GDP, locked CDP, lockedTDP, or locked UDP In some embodiments, the ribose moiety of the locked nucleotide is in the C3’-endo (beta-D-LNA) or C2’-endo (alpha-L-LNA) conformation.

[0223] In some embodiments, the modified NDP substrate comprises a modified nucleobase. In some embodiments, the modified nucleobase on the NDP substrate is 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh- purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.

[0224] In some embodiments, the nucleobase of the NDP substrate is, among others, 5-methylcytosine, pyridm-4-one, pyridin-2-one, phenyl, pseudouracil, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5- alkylcytidines, 5-alkyluridines, 5-halouridines, 6-azapyrimidines, 6-alkylpyrimidines, 5-propynyl-uracil, 2- thio-5-propynyl-uracil, quesosine, 2 -thiouridine, 4-thiouridine, 4-acetyltidme, 5- (carboxyhydroxymethy l)uridine, 5-carboxymethylaminomethyl-2 -thiouridine, 5- carboxymethylaminomethyluridine, -D-galactosylqueosine, 1-methyladenosme, 1 - methylinosine, 2,2- dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6-methyladenosine, 7- methylguanosine, 5-methoxyaminomethy 1-2- thiouridine, 5-methylaminomethy luridine, 5-methylcarbon ylmethyluridine, 5- methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, -D- mannosylqueosine, uridine-5-oxy cetic acid, 2-thiocytidine, N1 -methyl-adenine, N6-methyl- adenine, 8'- azido-adenine, N,N-dimethy 1- adenosine, aminoally 1 -adenosine, 5'-methyl -uridine, pseudouridine, Nl- methy 1-pseudoundme, 5'-hydroxy-methy 1-undme, 2'-thio-undme, 4'-thio- uridine, hypoxanthine, xanthine, 5'-methyl-cytidine, 5'-hydroxy-methyl-cytidine, 6'-thio- guanine, or N7-methyl-guanine.

[0225] In some embodiments, the modified nucleobase of the NDP substrate comprises a removable tag, a cleavable linker, or a radio, a photo, and / or a chemical sensor.

[0226] In some embodiments, the modified NDP substrate comprises a modified phosphate. In some embodiments, the modified NDP substrate comprises a 5’-O-l-thio(diphosphate) (NDPaS). In some embodiments, the modified NDP substrate comprises a 5’-O-2-thio(diphosphate) (NDP[3S). In some embodiments, the modified NDPaS comprises an ADPaS, GDPaS, CDPaS, TDPaS, or UDPaS. In some embodiments, the modified NDPaS comprises a rADPaS, rGDPaS, rCDPaS, rTDPaS, or rUDPaS. In some embodiments, the modified NDPaS comprises a dADPaS, dGDPaS, dCDPaS, dTDPaS, or dUDPaS As described herein, the modified NDP substrate having a thiophosphate group, can also have a modified sugar moiety (e.g., 2’-position) and / or modified nucleobase.

[0227] In some embodiments, the modified NDP substrate comprises an NDPaS, wherein the NDPaS is (Rp) or (Sp) diastereomer, or a mixture of (Rp) and (Sp) diastereomers In some embodiments, the NDPaS is the (Rp) diastereomer. In some embodiments, the NDPaS is the (Sp) diastereomer. In some embodiments, the NDPaS is a mixture of (Rp) and (Sp) diastereomers. In some embodiments, the (Rp) or (Sp) diastereomer is provided for each of the rNTPaS and dNTPaS described herein.

[0228] In some embodiments, the method further comprises contacting a nucleoside monophosphate (NMP) with a nucleoside monophosphate kinase in presence of a phosphate donor NTP under suitable reaction conditions such that a product nucleoside diphosphate (NDP) is produced for reaction with the nucleosidediphosphate kinase and / or NTP:NDP 3’ phosphotransferase. In these embodiments, any of the modifieations described in the above paragraphs may be present in the nucleoside monophosphate substrate.

[0229] In some embodiments, the method further comprises contacting a nucleoside with a nucleoside kinase in presence of a phosphate donor NTP under reaction conditions such that a product nucleoside monophosphate (NMP) is produced for reaction with the nucleoside monophosphate kinase and / or NTP:NDP 3’ phosphotransferase. In these embodiments, any of the modifications described in the above paragraphs may be present in the nucleoside substrate.

[0230] In certain embodiments where the NTP:NDP 3’ phosphotransferase / adenylyl-sulfate kinase is coupled with an AcK recycling enzyme, the AcK enz me may be further coupled with a pyruvate oxidase enzyme (POx) to generate acetyl phosphate from pyruvate. In certain embodiments, the POx enzyme transiently generates acetyl phosphate from pyruvate, atmospheric oxygen, and potassium phosphate buffer. These embodiments have the advantage of generating an unstable, moisture sensitive, and expensive substrate (acetyl phosphate) from stable, readily available, and inexpensive reagents (pyruvate, atmospheric oxygen, and potassium phosphate buffer). In some embodiments, the pyruvate oxidase is further coupled with a catalase enzyme to degrade hydrogen peroxide to water and oxygen.NTP:NDP 3' phosphotransferases for NQP Synthesis

[0231] For purposes described herein, any suitable NTP:NDP 3’ phosphotransferase may be used in the present invention. In some embodiments, a suitable naturally occurring NTP:NDP 3’ phosphotransferase may be used In some embodiments, an engineered NTP:NDP 3’ phosphotransferase can be used having the relevant activity in converting NDP to a NDP with a phosphate group at the 3’ position of the sugar and / or a NQP or pppNp.

[0232] In some embodiments, the present disclosure provides novel adenylyl-sulfate kinases that have improved activity in the conversion of NDP to a NDP with a phosphate group at the 3 ' position of the sugar and / or a NQP or pppNp (an NTP with a phosphate group at the 3’ position of the sugar). In some embodiments, the suitable NTP:NDP 3’ phosphotransferase is an adenylyl-sulfate kinase, including naturally occurring adenylyl-sulfate kinase and engineered variants thereof.

[0233] In particular, the engineered adenylyl-sulfate kinase polypeptides of the present disclosure have been engineered for efficient synthesis of NQPs, in the processes depicted in Scheme 1 and Scheme 2, above, and Scheme 3, below, among others. A variety of suitable reaction conditions are known to those skilled in the art, as detailed below and in the Examples.

[0234] In some embodiments, the present disclosure provides enzymes useful in a one -pot method for conversion of nucleosides to NQPs, as depicted in Scheme 3 In some embodiments, the one-pot method for conversion of nucleosides to NQPs occurs in one step.

[0235] In some embodiments, the one -pot method for conversion of nucleosides to NQPs occurs in more than one step. In some embodiments, the one-pot method for conversion of nucleosides to NQPs occurs in two steps. In some embodiments, the two step method for conversion of nucleosides to NQPs occurs in two pots.

[0236] In some embodiments, the first step comprises conversion of a nucleoside to an NTP, and the second step comprises conversion of the NTP to an NQP (as depicted in Scheme 2, above).

[0237] The first step of the two-step method for conversion of nucleosides to NQPs comprises use of a 5’-O- kinase, a nucleotide monophosphate kinase (NMPK), and an acetate kinase (AcK) to sequentially add three phosphates (or modified phosphates or phosphate substitutes) to a nucleoside to generate an NTP.

[0238] The second step of the two-step method for conversion of nucleosides to NQPs comprises use of an adenylyl-sulfate kinase to convert NDP and the natural or modified NTP generated in step one to the NQP product The second step of the two-step method conversion of nucleosides to NQPs, is described above and depicted in Scheme 2.

[0239] As depicted in Scheme 3, the kinases in the one pot or two pot reaction may use a NTP regeneration system as described above, shown here as ACK / acetyl phosphate and further coupled to pyruvate oxidaseEngineered Adenylyl-Sulfate Kinase Polypeptides

[0240] In one aspect, the present disclosure provides engineered adenylyl-sulfate kinase polypeptides useful in the synthesis of NQPs, as well as compositions and methods of utilizing these engineered polypeptides.

[0241] In some embodiments, the present invention provides adenylyl-sulfate kinase polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods for using the polypeptides. Where the description relates to polypeptides, it is to be understood that it can describe the polynucleotides encoding the polypeptides.

[0242] Suitable reaction conditions under which the above -described improved properties of the engineered polypeptides carry out the desired reaction can be determined with respect to concentrations or amounts of polypeptide, substrate, co-substrate, buffer, solvent, pH, conditions including temperature and reaction time, and / or conditions with the polypeptide immobilized on a solid support.

[0243] In some embodiments, an engineered adenylyl-sulfate kinase, or a functional fragment thereof, comprises an amino acid sequence having at least one or more amino acid residue differences at amino acid residues that are about 6 angstroms (A) or less from the active site of an adenylyl-sulfate kinase, wherein the active site is determined from a protein crystal structure and / or by protein homology modeling of a reference adenylyl-sulfate kinase The engineered adenylyl-sulfate kinase is at least capable of converting NDP to a NDP with a phosphate group at the 3’ position of the sugar moiety. As further discussed below, in some embodiments, the engineered adenylyl-sulfate kinase displays an improved property compared to a reference a adenylyl-sulfate kinase

[0244] In some embodiments, the reference adenylyl-sulfate kinase is the adenylyl-sulfate kinase or adenosine 5-phosphosulfate kmase of Arabidopsis thaliana (see, e.g., Poyraz et al., PLOS ONE, 2015, DOI: 10 1371 / joumal. pone.0121494).]0245] In some embodiments, the engineered adenylyl-sulfate kmase comprising an ammo acid sequence having one or more amino acid residue differences at amino acid residues that are about 6 A or less from the active site comprises at least an amino acid residue difference at amino acid position 7, 10, 11, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 63, 64, 65, 68, 76, 77, 78, 81, 82, 85, 86, 89, 106, 107, 108, 109, 110, 132, 139, 141, 142, 143, 144, 145, 147, 148, 156, 157, 158, 159, 160, 163, 164, 176, 177, 179, 180, 181, 182, 183, 185, or 186, or any combinations thereof, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14), and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase.

[0246] In some embodiments, the engineered adenylyl-sulfate kmase comprises an amino acid sequence having at least one or more amino acid residue differences at amino acid residues that arc about 4.5 A or less from the active site of the reference adenylyl-sulfate kmase. In some embodiments, the engineered adenylyl- sulfate kinase comprising an amino acid sequence having one or more amino acid residue differences at amino acid residues that are about 4.5 A or less from the active site comprises at least an amino acid residue difference at amino acid position 7, 10, 11, 36, 37, 38, 39, 40, 41, 42, 43, 44, 64, 65, 68, 77, 82, 85, 89, 107, 108, 109, 110, 142, 143, 144, 145, 147, 156, 157, 158, 159, 160, 164, 177, 181 , 182, 183, or 186, or any combinations thereof, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14), and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase.

[0247] In some embodiments, the engineered adenylyl-sulfate kinase having at least one or more amino acid residue differences at amino acid residues that are about 6 A or less from the active site of the reference adenylyl-sulfate kinase comprises at least an amino acid residue difference at amino acid position 7, 11, 42, 76, 77, 78, 85, 89, 109, 139, 141, 147, 156, 157, 158, 164, 176, 181, 182, or 183, or any combinations thereof, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14), and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase.

[0248] In some embodiments, the engineered adenylyl-sulfate kinase having at least one or more amino acid residue differences at amino acid residues that are about 6 A or less from the active site of the reference adenylyl-sulfate kinase comprises at least an amino acid residue difference selected from amino acid residue 7A / F / G / H / I / L / M / P / Q / S / T / VAV, 11F / Q, 42T, 76A / E / F / H / K / L / N / Q / R / S / T / VAV / Y, 77H / L / W, 78A / C / D / E / G / H / M / Q, 85A / S / V, 89A / L / M / T, 109G, 139C / V, 141A / K / P / R / S / T / W, 147A / C / F / G / I / M / Q / R / S / T / V, 156A / L / M / V, 157 A / D / G / H / I / M / N / Q / R / S / T / V / W, 158D / M / N / P / W, 164G / I / L / N / R / S / V, 176G / I / L / M / R, 181 A / C / D / F / G / L / M / Q / S / T / V / W, 182A / E / G / K / L / M / P / R / T / V / Y, 183A / L / P / R / V, and combinations thereof, and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase. In some embodiments, the one or more amino acid residue differences are selected from 7F / G / M / W, 1 IQ, 42T, 76E / W, 77W, 78A / E, 85S, 89M, 109G, 139V, 141K, 147A / M / V, 156L, 157 A / T, 158P, 164S, 176G, 181L / M / Q, 182E, 183 V, and any combinations thereof, and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase.

[0249] In some embodiments, the homologous, orthologous, or paralogous adenylyl-sulfate kinase is the adenylyl-sulfate kinase of Thermotoga sp. KOL6, Thermotoga sp. SGI, Thermotoga sp. (RKX51929.1), Thermotoga sp. (HDG62185. 1), Thermosipho femreducens (WP_207567147. 1), Petrotoga sp 9PW.55.5.1 (WP_1 13075358.1), Defluviitoga tunisiensis (MFY9404473.1), Petrotoga sibirica (WP_103876974. 1), Mycobacterim tuberculosis (BAQ05257), human bifunctional 3 '-phospho adenosine 5'-phosphosulfate synthetase 1 (PAPS synthetase 1) (PAPSS 1), human bifunctional 3 '-phospho adenosine 5'-phosphosulfate synthetase 2 (PAPSS2), Arabidopsis thaliana, Penicillium chrysogenum, Thiobacillus dentrificans, Aquifex acolicus, or Acropyrum pcmix.

[0250] In some embodiments, an engineered adenylyl-sulfate kinase comprises an amino acid sequence having one or more amino acid residue differences in one or more motifs present in the adenosine 5’- phospho sulfate (APS) kinase domain of an adenylyl-sulfate kinase. Methods for the alignment of sequences for comparison are known in the art, including GAP, BESTFIT, BLAST, FASTA and TFASTA, such as described herein For example, GAP uses the algorithm of Needleman and Wunsch, J Mol Biol., 1970, 48:443-453) to find the global (i e spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps The BLAST algorithm (Altschul et al , J Mol Biol., 1990, 215:403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the defaultpairwise alignment parameters, and a scoring method in percentage For local alignments, the Smith- Waterman algorithm can be used (Smith and Waterman, J. Mol. Biol., 1981, 147(1): 95-7).

[0251] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having one or more amino acid residue differences in one or more motifs:[VI]-[WF]-[LF]-T-G-L-[SP]-[GAC]-X-G-K (SEQ ID NO: 3325);L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326);[LA]-[GS]-F-[ST];K-G-L-Y-[KA]-[KY]-A (SEQ ID NO: 3327); andF-T-G-[IV]; of the APS kinase domain.

[0252] In the embodiments, the motifs and locations thereof are obtained from sequence alignments of adenylyl-sulfate kinases, such as disclosed in Poyraz et al , PLOS ONE, 2015,DOI: 10.1371 / journal. pone 0121494, incorporated by reference herein In some embodiments, the motif [VI]- [WF]-[LF]-T-G-L-[SP]-[GAC]-X-G-K (SEQ ID NO: 3325) is located from beta strand pi to alpha helix al ; the motif L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326) is located from beta strand P2 to alpha helix a2; the motif [LA]-[GS]-F-[ST] is located on the amino side of alpha helix a3; the motif K-G-L-Y-[KA]-[KY]-A (SEQ ID NO: 3327) is located at alpha helix a6; and the motif F-T-G-[IV] is located between alpha helix 6 and beta strand P5 of the APS kinase domain.

[0253] The corresponding sequence of the above motifs in the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14) arc: V-W-L-T-G-L-S-G-A-G-K (SEQ ID NO: 3329); L-D-G-D-N-I-R (SEQ ID NO: 3330); L-G-F-S (SEQ ID NO: 3331); KGLYKKA (SEQ ID NO: 3332); and F-T-G-I (SEQ ID NO: 3333), respectively The amino acid differences described herein can be applied to the motifs to the adenylyl-sulfate kinase of Thermotoga sp. RQ7.

[0254] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif [LA]-[GS]-F-[ST], In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence comprising at least an amino acid residue difference in [GS]-F of the motif [LA]-[GS]-F-[ST] In some embodiments, the [GS] is substituted with E, or F is substituted with W, or combination thereof.

[0255] In some embodiments, the engineered adenylyl-sulfate kmase comprises an amino sequence comprising at least an amino acid residue difference in the motif K-G-L-Y-[KA]-[KY]-A (SEQ ID NO: 3327). In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif K-G-L-Y (SEQ ID NO: 3328). In some embodiments, the L of K-G-L-Y (SEQ ID NO: 3328) is substituted with A.

[0256] In some embodiments, the engineered adenylyl-sulfate kmase comprises an ammo sequence comprising at least an ammo acid residue difference in the motif [VI]-[WF]-[LF]-T-G-L-[SP]-[GAC]-X-G- K (SEQ ID NO: 3325). In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference at [VI], [LF], G, [GAC], or K, or any combinations thereof, in the motif [VI]-[WF]-[LF]-T-G-L-[SP]-[GAC]-X-G-K (SEQ ID NO: 3325). In someembodiments, the [VI] is substituted with L: [LF] is substituted with Y; G is substituted with W; or Kis substituted with P, R, or V, or any combinations thereof

[0257] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326). In some embodiments, the engineered adenylyl-sulfate kinase comprises at least an amino acid residue difference at G, [NW], or [LIVA], or any combination thereof, in the motif L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326). In some embodiments, the G is substituted with A, [NW] is substituted with R, or [LIVA] is substituted with F, M, or Q: or any combinations thereof.

[0258] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino sequence comprising at least an amino acid residue difference in the motif F-T-G-[IV]. The engineered adenylyl- sulfate kinase comprises an amino acid sequence comprising at least an amino acid residue difference at F or T, or any combinations thereof, of the motif F-T-G-[IV] In some embodiments, the F is substituted with S; or T is substituted with A, E, G, H, P, S, or V; or any combinations thereof

[0259] Tn some embodiments, the one or more amino acid residue differences at ammo acid positions that are about 6 A or less from the active site of the adenylyl-sulfate kinase can be combined with the one or more amino acid residue differences in the motifs described herein to generate engineered adenylyl-sulfate kinase with improved properties In some embodiments, the engineered adenylyl-sulfate kinase exhibits at least an improved property described herein.

[0260] In some embodiments, the engineered the engineered adenylyl-sulfate kinase comprising an amino acid sequence having one or more ammo acid residue differences at amino acid positions that are 6 A or less from the active site of the adenylyl-sulfate kmase and / or one or more ammo acid residue differences in the motifs described herein can have additional amino acid residue differences at other amino acid positions of the adenylyl-sulfate kinase polypeptide.

[0261] In some embodiments, the engineered polypeptides of the present disclosure are variants of SEQ ID NO: 14, the wild-type adenylyl-sulfate kinase encoded by the CysC gene of Thermotoga sp. RQ7. These engineered adenylyl-sulfate kinases are capable of improved activity in the production of 3’P-NDPs and corresponding NQPs, using the methods described herein.

[0262] In some embodiments, the present invention provides an engineered adenylyl-sulfate kinase polypeptide comprising an amino acid sequence having at least 80% sequence identity' to an amino acid reference sequence of SEQ ID NO: 14 and further comprising one or more ammo acid residue differences as compared to the reference amino acid sequence, wherein the engineered adenylyl-sulfate kinase polypeptide comprises one or more of the following improved properties: increased activity on natural substrates, increased activity on non-natural substrates, increased activity on 2 ’-fluoro modified nucleotides, increased activity on 2’-O-methyl modified nucleotides, increased thermostability', increased substrate promiscuity, increased soluble protein expression, decreased product inhibition and / or decreased byproduct formation, as compared to a wild-type or reference adenylyl-sulfate kinase

[0263] In some embodiments, the exemplary engineered adenylyl-sulfate kinase comprises an amino acid sequence that has one or more amino acid residue differences as compared to SEQ ID NO: 14 at the residue positions indicated in Tables 8.2, 9.2, 10.2, 11.2, 12.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2,23.2, 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, 39.2, 40.2, 41.2,42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 57.2, 58.2, 59.2, 60.2,61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, and 78.2.

[0264] The structure and function information for the exemplary engineered polypeptides of the present disclosure are based on the conversion of a natural or modified NDP to a natural or modified 3 ’P-NDP and corresponding NQP, the results of which are shown below in Tables 8.2, 9.2, 10.2, 11.2, 12.2, 14.2, 15.2,16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 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, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2,54.2, 55.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2,73.2, 74.2, 75.2, 76.2, 77.2, and 78.2, as further described in the Examples. The odd numbered sequence identifiers (i e., SEQ ID NOs) in these Tables refer to the nucleotide sequence encoding the amino acid sequence provided by the even numbered SEQ ID NOs in these Tables Exemplary sequences are provided in the electronic sequence listing file accompanying this invention, which is hereby incorporated by reference herein. The amino acid residue differences are based on comparison to the reference sequence of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0265] Enzymes with activity in the 3 ’ phosphorylation of nucleosides and nucleotides include homologs of CysC enzymes (adenylyl-sulfate kmase) and CoaE (dephospho-CoA kinase). Adenylyl-sulfate kmase enzymes catalyze the conversion of adenosine 5 '-phospho sulfate to 3 '-phosphoadenylyl sulfate using ATP as a cofactor. Similarly, CoaE enzymes are known to catalyze the conversion of 3'-dephospho-CoA to CoA, using ATP as a co-factor. These enzymes have been reported to have activity in the 3 ’ phosphorylation of the sugar moiety of nucleosides or nucleotides (PCT / US2023 / 076694). As used herein, the term 3’-O-kinase refers to any of these enzymes and any enzyme capable of phosphor lation of the 3’ position of a natural or modified NTP, NDP, NMP, or nucleoside. Enzymes capable of 3’ phosphorylation of the sugar moiety of a natural or modified NDP are referred to herein as NTP:NDP 3’ phosphotransferases. Accordingly, an enzyme may act both as a 3’O-kinasc and as a NTP:NDP 3’ phosphotransferase or may have cither, both, or only one of these activities depending on the specific substrates and reactions conditions that are present. Thus, an engineered adenylyl-sulfate kinase of the present invention may have activity7as a 3’O-kinase and an NTP:NDP 3’ phosphotransferase in the conversion of a NDP or modified NDP to a 3 ’P-NDP or modified 3 'P-NDP.

[0266] In the present disclosure, the wild-type adenylyl-sulfate kinase encoded by the CysC gene from Thermotoga sp. RQ7 (SEQ ID NO: 14) was selected for evolution. The adenylyl-sulfate kinase polypeptides of the present disclosure are engineered variants of SEQ ID NO: 14.

[0267] The polypeptides of the present disclosure have residue differences that result in improved properties necessary to develop an efficient NTP:NDP 3’ phosphotransferase enzyme, capable of biocatalytic synthesis of NQPs. Various residue differences, at both conserved and non-conserved positions, have been discovered to be related to improvements in various enzymes properties, including increased activity on natural substrates, increased activity on non -natural substrates, increased activity on 2 ’-fluoro modified nucleotides, increased activity on 2’-O-methyl modified nucleotides, increased thermostability, increased substrate promiscuity, increased soluble protein expression, decreased product inhibition and / or decreased byproduct formation, ascompared to a wild-type or reference enzyme. Increased activity of the adenylyl-sulfate kinase polypeptide may be evidenced by increased % conversion of substrate to product. The activity of each engineered adenylyl-sulfate kinase relative to the reference polypeptide of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, was determined as conversion of the substrates described in the Examples herein. In some embodiments, a shake flask purified enzyme (SFP) is used to assess the properties of the engineered adenylyl-sulfate kinase, the results of which are provided in the Examples

[0268] In some embodiments, the specific enzyme properties are associated with the residues differences as compared to SEQ ID NO: SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228 at the residue positions indicated herein In some embodiments, residue differences affecting polypeptide expression can be used to increase expression of the engineered adenylyl-sulfate kinase

[0269] In light of the guidance provided herein, it is further contemplated that any of the exemplaiy engineered polypeptides comprising the even-numbered sequences of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, find use as the starting amino acid sequence for synthesizing other adenylyl-sulfate kinase polypeptides, for example by subsequent rounds of evolution that incorporate new combinations of various amino acid residue differences from other polypeptides in Tables 8.2, 9.2, 10.2, 11.2, 12.2, 14.2, 15.2, 16.2,17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 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, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2,55.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2,74.2, 75.2, 76.2, 77.2, and 78.2, and other residue positions described herein Further improvements may be generated by including amino acid residue differences at residue positions that had been maintained as unchanged throughout earlier rounds of evolution

[0270] In some embodiments, the engineered adenylyl-sulfate kinase comprises a polypeptide sequence having at least 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence selected from SEQ ID NO: SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or a functional fragment thereof, and one or more amino acid residue differences relative to the reference sequence.

[0271] In some embodiments, a reference sequence, as well as any specified amino acid sequence herein, can be described without the amino acid residues of a His-tag when present. For example, a polypeptide sequence of an engineered adenylyl-sulfate kinase comprises residues 1 -200 of an engineered adenylyl-sulfate kinase referenced by its SEQ ID NO , where the sequence of the SEQ ID NO includes a C-terminal His-tag It is also to be understood that the range of residues can be adapted to account for any ammo acid deletions within the sequence of the adenylyl-sulfate kinase polypeptide sequence

[0272] As such, in some embodiments, the present disclosure provides an engineered adenylyl-sulfate kinase comprising an amino acid or polypeptide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to a reference sequence corresponding to amino acid residues 1 -200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 18-258, 272-1358, and 1364-3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 18-258, 272-1358, and 1364-3324, wherein the amino acid sequence comprises one or more amino acid residue differences relative to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to a reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0273] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, wherein the ammo acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228

[0274] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: 14, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0275] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, wherein the ammo acid sequence comprises one ormore amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0276] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0277] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least a substitution at amino acid position 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 15, 16, 17, 18, 19, 21 , 22, 23, 25, 26, 27, 28, 29, 30, 31 , 33, 35, 38, 39, 41 , 42, 43, 44, 45, 46, 47, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 61 , 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 1 12, 113, 114, 115, 1 16, 117, 118, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 140, 141, 144, 146, 147, 148,149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0278] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2A / G / I / K / M / P / R / S, 3F / G / R / S / T / Y, 4G / M / N / P / R, 5G / K / M / NZP / Q / R / T / Y, 6D / E / G / P / R / S / T, 7A / F / G / H / I / L / M / P / Q / S / T / V / W, 8L / T / V, 9Q, 10M / P / Y, 11F / Q, 12S, 13S / Y, 151, 16F / L / P / R / S, 17C / Q / R, 18A / C / G / I / L / M / N / P / Q / S / T / V / W, 19A / C / EZL / M / Q / R / S, 21C / G, 22G / R / S / V, 23I / M / R, 25A / Q / R / S, 26S, 27G / H / L / Q / R / T, 28A, 29G / L, 30L, 31I / L, 33Y, 35W, 38A, 39S / V, 41P / R / V, 42T, 43A / Q / S, 44L, 45S / V, 46N / Q, 47D / G, 50A / F / I / K / L / Q / R / S / Y, 51E / K / L / Q / S / T / V / Y, 521, 53G / L / M / V / Y, 54F / G / H / L / N / Q / R / S / T / V, 55A / C / G / L / P / S, 56H / N / Q / R / S, 57L / R / S, 58F / S / V / W, 59G / P / S / T, 61I / T, 64A, 65L, 66R, 67F / L / M / Q / V, 69A / Q / T, 70D / L / T / Y, 71K, 72C / E / G, 73A / D / E / K / M / V, 74S, 75P, 76A / E / F / H / K / L / N / Q / R / S / T / V / W / Y, 77H / L / W, 78A / C / D / E / G / H / M / Q, 79A / E / K / L / P / Q / R / T / V, 80A / G / P / V, 81C / GZL / S, 82T / V, 83A / E / G / Q / R / S / T / V, 85A / S / V, 86L / P / V, 89A / L / M / T, 90L / M / T / V, 91R / S / T / W, 92F / T, 93G / S / T / V, 94A / C / H / M / T / V, 95A / G / H / M / S / T / V, 96I / L / T / V, 97L / Q / S / V / W, 98Q, 99S, 100A, 101F / V, 102V, 103L / S / V, 104L / V, 105A / K / L / S / V, 106G / S, 107H / M / S, 108C / V, 109G, 1 10G / Q / V, 112G / H / L / S, 113A / H / P / R, 114L / M / S / V, 115L / Q / T7W, 116A / E / H / L / M / Q / R / S, 117D / E / G / K / L / M / Q / S / T / V, 118L / M / V, 120E / G / L / Q / R / S / Y, 121 C / F / G / M / Q / R / S / T / V / W, 122C / M / V, 123G / P / R / S, 124A / G / I / K / L / P / R / S / T / V, 125D / E / H, 126A / CZD / L / M / S, 127W, 128 A / K / R / T / V, 129L / V, 130A / IZL / S / T, 132C / I / L / M, 133E / G / L / Y, 134A / L / T, 135W, 137E / G, 138E / L / T, 139C / V, 140L / Q, 141A / K / P / R / S / T / W, 144A / S / T, 146DZM / N / T, 147A / C / F / G / I / M / Q / R / S / T / V, 148R, 149A / G / H / L / P / Q / S / T, 150A / E / F / G / L / M / P / Q / S / V, 152T, 153D / Q / R, 154Q, 155K / S, 156A / L / M / V, 157A / D / G / H / 1 / M / N / Q / R / S / T / V / W, 158D / M / N / P / W, 159S, 160A / E / G / H / P / S / V,162V, 163N / T, 164G / I / L / N / R / S / V, 165DZE / S, 166F, 167Q, 170G / L / M / N / P / Q / V, 171A / E / N / Q / T / V, 172A, 173S / T, 174Y, 175L, 176G / I / L / M / R, 177L / P / S / V, 178M / S, 179A / G, 180G / L / M / R / T / V / W, 181A / C / D / F / GZLZM / Q / S / T / V / W, 182A / E / GZKZL / M / P / R / T / V / Y, 183A / L / P / R / V, 184A / G / P / R / V, 185D / G, 186A / E / M / N / V, 187M / T, 188E / F / L / Q / R, 189G / R, 190C / F / T / V, 191F / M / Q / R / V / W, 192E / G / M / S / V, 193A / C / E / F / G / H / I / L / M / N / Q / R / S / T / V / W, 194M, 195E / H / I / L / M / S / T / W, 196D / G / M / T / W, 197D / G / L / Q / R / S / V / W, 198E / W / Y, 199A / F / G / L / M / S / T / V / Y, 200L / T, 201E / F / H / I / L / M / N / P / Q / R / S / T, 202A / E / I / K / L / M / P / R / S / T / V, 203A / F / G / L / M / Q / T / V, or 204A / D / E / F / H / K / L / P / R / S / T / V / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0279] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue N2A / G / I / K / M / P / R / S, P3F / G / R / S / T / Y, K4G / M / N / P / R, A5G / K / M / N / P / Q / R / T / Y, K6D / E / G / P / R / S / T, N7A / F / G / H / I / L / M / P / Q / S / T / VAV, I8L / T / V, V9Q, W10M / P / Y, Hl 1F / Q, E12S, G13S / Y, V15I, K16F / L / P / R / S, K17C / Q / R, E 18A / C / G / I / L / M / NZP / Q / S / T / V / W, D19A / C / E / L / M / Q / R / S, E21C / G, K22G / R / S / V, L23IZM / R, G25A / Q / R / S, Q26S, K27G / H / L / Q / R / T, G28A, V29G / L, V30L, V31I / L, L33Y, G35W, G38A, A39S / V, K41P / R / V, S42T, T43A / Q / S, I44L, A45S / V, H46N / Q, E47D / G, W50A / F / 1 / K / L / Q / R / S / Y, R51E / K / L / Q / S / T / V / W / Y, L521, L53G / M / V / Y, E54F / G / H / L / N / Q / R / S / T / V, M55A / C / G / L / P / S, G56H / N / Q / R / S, K57L / R / S, L58F / S / V / W, A59G / P / S / F, V61I / T, G64A, D65L, N66R, I67F / L / M / Q / V, H69A / Q / T, G70D / L / T / Y, L71K, N72C / E / G, S73A / D / E / K / M / V, D74S, L75P, G76A / E / F / H / K / L / N / Q / R / S / T / V / W / Y, F77H / L / W, S78A / C / D / E / G / H / M / Q, P79A / E / K / L / Q / R / T / V, E80A / G / P / V, D81C / G / L / S, R82T / V, K83A / E / G / Q / R / S / T / V, N85A / S / V, I86L / P / V, I89AZLZM / T, S90L / M / T / V, E91R / S / T / W, V92F / T, A93G / S / T / V, K94A / C / H / M / T / V, L95A / G / H / M / S / T / V, F96I / L / T / V, A97L / Q / S / V / W, D98Q, A99S, G100A, L101F / V, I102V, T103L / S / V, I104L / V, T105A / K / L / S / V, Al 06G / S, F 107H / M / S, 1108C / V, S 109G , P 11 OG / Q / V, R 112G / H / L / S, E 113 A / H / P / R, D 114L / M / S / V, R1 15L / Q / T / W, K116A / H / E / L / M / QZR / S, R117DZE / GZK / L / M / Q / S / T / V, Al 18L / M / V, N120E / G / L / Q / R / S / Y, L121C / F / G / M / Q / R / S / T / V / W, L122C / M / V, K123G / P / R / S, E 124A / G / I / K / L / P / R / S / T / V, G125D / E / H, E 126A / C / D / L / M / S, F127W, I128A / K / R / T / V, E129L / V, VI 30A / I / L / S / T, V132C / I / L / M, K133E / G / L / Y, C134A / L / T, P135W, D137E / G, V138E / L / T, L139C / V, I140L / Q, E141A / K / P / R / S / T / W, P144A / S / T, G146D / M / N / T, L147A / C / F / G / I / M / Q / R / S / T / V, Y148R, K149A / G / H / L / P / Q / S / T, K150A / E / F / G / L / M / P / Q / S / V, L152T, A153D / Q / R, G154Q, E155K / S, I156A / L / M / V, K157A / D / G / H / I / M / N / R / Q / S / T / V / W, E158D / M / N / P / W, F159S, T 160A / E / G / H / P / S / V, I162V, S163N / T, A164G / I / L / N / R / S / V, P165D / E / S, Y166F, E167Q, E170G / L / M / N / P / Q / V, D171A / E / N / Q / T / V, P172A, E173S / T, L 174Y, V175L, V176G / I / L / M / R, E177L / P / S / V, T178M / S, D179A / G, K180G / L / M / R / T / V / W, El 81 A / C / D / F / G / L / M / Q / S / T / V / W, S182A / E / G / K / L / M / P / R / T / V / Y, I183A / L / P / R / V, E184A / G / P / R / V, E185D / G, S 186 A / E / M / N / V, V187M / T, N188E / F / L / Q / R, K189G / R, I190C / F / T / V, L191F / Q / M / R / V / W, N192E / G / M / S / V, Y193A / C / E / F / G / H / I / L / M / N / Q / R / S / T / V / W, L194M, R195E / H / I / L / M / S / T / W, E 196D / G / M / T / W, K197D / G / L / Q / R / S / V / W, G198E / W / Y, I199A / F / G / L / M / S / T / V / Y, I200L / T, G201E / FZH / I / L / M / N / P / Q / R / S / T, G202A / E / I / K / L / M / P / R / S / T / V, S203A / F / G / L / M / Q / T / V, or G204A / D / E / F / H / K / L / P / R / S / T / V / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding toamino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0280] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 11, 16, 18, 19, 27, 42, 50, 51, 53, 67, 73, 76, 77, 78, 79, 85, 89, 94, 95, 97, 105, 109, 116, 118, 124, 139, 141, 147, 150, 156, 157, 158, 164, 171, 176, 178, 180, 181, 182, 183, 188, 193, 195, 200, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0281] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2A / G / I / K / MZP / R / S, 3F / G / R / S / T / Y, 4G / M / N / P / R, 5G / K / M / N / P / Q / R / T / Y, 6D / E / G / P / R / S / T, 7A / F / G / H / I / L / M / P / Q / S / T / V / W, 11F / Q, 16F / L / P / R / S, 18A / C / G / I / L / M / N / P / Q / S / T / V / W, 19A / C / E / L / M / Q / R / S, 27G / H / L / Q / R / T, 42T, 50A / F / I / K / L / Q / R / S / Y, 51E / K / E / Q / S / T / V / Y, 53G / E / M / V / Y, 67F / E / M / Q / V, 73A / D / E / K / M / V, 76A / E / F / H / K / I. / N / Q / R / S / T / V / W / Y, 77H / L / W, 78A / C / D / E / G / H / M / Q, 79A / E / K / L / P / Q / R / T / V, 85A / S / V, 89A / L / M / T, 94A / C / H / M / T / V, 95A / G / H / M / S / T / V, 97L / Q / S / V / W, 105A / K / L / S / V, 109G, 116A / E / H / L / M / QZR / S, 118L / M / V, 124A / G / I / K / L / P / R / S / T / V, 139C / V, 141A / K / PZR / S / T / W, 147A / C / F / G / I / M / Q / R / S / T / V, 150A / E / F / G / L / M / P / Q / S / V, 156A / L / M / V, 157A / D / G / H / I / M / N / Q / R / S / T / V / W, 158D / M / N / P / W, 164G / 1 / L / N / R / S / V, 171 A / E / N / Q / T / V, 176G / 1 / L / M / R, 178M / S, 180G / L / M / R / T / V / W, 181A / C / D / F / G / L / M / Q / S / T / V / W, 182A / E / G / K / L / M / P / R / T / V / Y, 183A / L / P / R / V, 188E / F / L / Q / R, 193A / C / EZF / G / H / I / LZM / N / Q / R / S / T / V / W, 195E / H / I / L / M / S / T / W, 200L / T, 202A / E / I / K / L / M / P / R / S / T / V, 203A / F / G / L / M / Q / T / V, or 204A / D / E / F / H / K / L / P / R / S / T / V / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0282] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2R, 3F / S / T / Y, 4N, 5R, 6R / S / T, 7F / G / M7W, 1 IQ, 16S, 18A, 19M, 27H / Q, 42T, 50F, 5 IK, 53 Y, 67L, 73A / E, 76E / W, 77W, 78A / E, 79R, 85S, 89M, 94M, 95A / H / M, 97Q, 105A / L / V, 109G, 116E, 118L, 124K / V, 139V, 141K / S, 147A / M / V, 150L, 156L, 157A / T, 158P, 164S, 171N, 176G / V, 178S, 181L / M / Q, 182E, 183V, 188E, 193A / E / T, 195E, 200L, 202P, 203V, or 204E / F / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0283] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue N2A / G / I / K / M / P / R / S, 3F / G / R / S / T / Y, K4G / M / N / P / R, A5G / K / M / N / P / Q / R / T / Y, K6D / E / G / P / R / S / T, N7A / F / G / H / I / E / M / P / Q / S / T / V / W, Hl 1F / Q, K16F / L / P / R / S, E18A / C / G / I / L / M / N / P / Q / S / T / V / W, D19A / C / E / L / M / Q / R / S, K27G / H / L / Q / R / T, S42T, W50A / F / I / K / L / Q / R / S / Y, R51E / K / L / Q / S / T / V / Y, L53G / L / M / V / Y, I67F / L / M / Q / V, S73A / D / E / K / M / V, G76A / E / F / H / K / L / N / Q / R / S / T / V / W / Y, F77H / L / W, S78A / CZDZE / GZH / M / Q, P79A / E / K / L / P / Q / R / T / V, N85A / S / V, I89A / L / M / T, K94A / C / H / M / T / V, L95A / G / H / M / S / T / V, A97L / Q / S / V / W, T105A / K / L / S / V, S109G, KI 16A / E / H / L / M / Q / R / S, Al 18L / M / V, E124A / G / I / K / L / P / R / S / T / V, L139C / V, E141A / K / P / R / S / T / W,L147A / C / F / G / I / M / Q / R / S / T / V, K150A / E / F / G / L / M / P / Q / S / V, I156A / L / M / V, K157A / D / G / H / I / M / N / Q / R / S / T / VAV, E158D / M / N / P / W, A164G / I / L / NZR / S / V, D171A / E / N / Q / T / V, V176G / I / LZM / R, T178M / S, K 180G / L / M / R / T / V / W, E181A / C / D / F / G / L / M / Q / S / T / V V, S182A / E / G / K / L / M / P / R / T / V / Y, I183A / L / P / R / V, N188E / F / L / Q / R, Y193A / C / E / F / G / H / I / L / M / N / Q / R / S / T / VAV, R195E / H / I / L / M / S / T / W, I200L / T, G202A / E / I / K / L / M / P / R / S / T / V, S203A / F / G / L / M / Q / T / V, or G204A / D / E / F / H / K / L / P / R / S / T / V V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0284] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 11, 76, 83, 85, 89, 109, 110, 141, 158, 176, or 180, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0285] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 3F / S / T / Y, 11 Q, 76W, 83S, 85S, 89M, 109G, 1 10Q, 141 W, 158M, 176R, or 180W, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0286] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kmase comprises at least amino acid residue difference, or amino acid residue P3F / S / T / Y, Hl IQ, G76W, K83S, N85S, I89M, S109G, Pl 10Q, E141 W, E158M, V176R, or K180W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0287] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 11, 72, 76, 78, 80, 81, 82, 83, 85, 89, 107, 109, 110, 115, 141, 146, 154, 155, 176, 182, and 185, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0288] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 3F, 1 IQ, 72C, 76F, 76 , 78A, 80P, 80V, 81 C, 82T, 83S, 85S, 89M, 107H, 109G, 110Q, 110V, 115T, 141R, 146T, 154Q, 155S, 176R, 182T, 182V, or 185G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0289] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue P3F, Hl IQ, N72C, G76F, G76W, S78A, E80P, E80V, D81C, R82T, K83S, N85S, I89M, F107H, S109G, Pl 10Q, P110V, R115T, E141R, G146T, G154Q, E155S, V176R, S182T, S182V, and E185G, or any combinations thereof, wherein the amino acid positionsare relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0290] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 33, 39, 45, 67, 72, 76, 81, 82, 85, 86, 89, 106, 115, 141, 149, 153, 155, 156, 157, 164, 180, 182, or 187, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0291] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 3S, 33Y, 39V, 45V, 67L, 72G, 76F, 76R, 76W , 81G, 82V, 85A, 85S, 85V, 86P, 89M, 106G, 115L, 115Q, 115W, 141R, 149G, 153R, 155K, 156M, 157R, 164G, 180W, 182L, 182T, or 187T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0292] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue S48E, P3S, L33Y, A39V, A45V, I67L, N72G, G76F, G76R, G76W, D81G, R82V, N85A, N85S, N85V, I86P, I89M, A106G, R115L, R115Q, R115W, E141R, K 149G, A153R, E155K, I 156M, K157R, A164G, K180W, S182L, S182T, or V187T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0293] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least a substitution at an amino acid position provided in Tables 8.2, 9.2, 10.2, 11.2, 12.2, 14.2, 15.2, 16.2,17.2. 18.2, 19.2, 20.2, 21.2, 22.2, 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, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2,55.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2,74.2, 75.2, 76.2, 77.2, and 78.2, wherein the amino acid positions are relative to tire reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0294] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least one substitution provided in Tables 8.2, 9.2, 10.2, 11.2, 12.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2,21.2, 22.2, 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, 39.2,40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 57.2, 58.2,59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, and78.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0295] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least a substitution or substitution set at amino acid position! s) provided in Tables 8.2, 9.2, 10.2, 11.2, 12.2,14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 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, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2,52.2, 53.2, 54.2, 55.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2,71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, and 78.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0296] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least a substitution or substitution set provided in Tables 8.2, 9.2, 10.2, 11.2, 12.2, 14.2, 15.2, 16.2, 17.2,18.2, 19.2, 20.2, 21.2, 22.2, 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, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2,56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2,75.2, 76.2, 77.2, and 78.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14

[0297] Tn some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 9.2, 10.2, 11.2, 12.2, 14.2, 15.2,16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 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, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2,54.2, 55.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2,73.2, 74.2, 75.2, 76.2, 77.2, and 78.2, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

[0298] In the above embodiments, the engineered adenylyl-sulfate kinase polypeptide may additionally comprise improved conversion of a NDP to a 3'-P-NDP, as compared to the reference sequence of SEQ ID NO: 14.

[0299] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 14, 18-258, 272-1358, and 1364- 3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 18- 258, 272-1358, and 1364-3324.

[0300] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 8, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to the reference sequence corresponding to SEQ ID NO: 8, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0301] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228

[0302] In some embodiments, the engineered adenylyl-sulfate kmase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228

[0303] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18,19, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 35, 38, 39, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 61, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117,118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 140, 141, 144,146, 147, 148, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193,194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236,1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0304] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2A / G / IZKZMZP / R / S, 3F / GZR / S / T / Y, 4G / K / M / NZPZR, 5G / K / M / N / P / Q / R / T / Y, 6D / E / G / P / R / S / T, 7A / F / G / I I / I / L / M / P / Q / S / T / V / W, 8L / T / V, 9Q, 10M / P / Y, 11F / Q, 12S, 13S / Y, 151, 16F / LZPZR / S, 17C / Q / R, 18A / C / G / I / L / M / N / P / Q / S / T / V / W, 19A / CZE / LZM / Q / R / S, 21C / G, 22G / R / S / V, 23I / MZR, 25A / Q / R / S, 26S, 27G / H / L / Q / R / T, 28A, 29G / L, 30L, 31I / L, 33Y, 35W, 38A, 39S / V, 41P / R / V, 42S / T, 43A / Q / S, 44L, 45S / V, 46N / Q, 47D / G, 50A / F / I / K / L / Q / R / S / W / Y, 51E / K / L / QZR / S / T / V / W / Y, 521, 53G / L / M / V / Y, 54F / G / H / L / N / Q / R / S / T / V, 55A / C / G / L / P / S, 56H / N / Q / R / S, 57L / R / S, 58F / S / V / W, 59G / P / S / T, 61I / T, 64A, 65L, 66R, 67F / L / M / Q / V, 69A / Q / T, 70D / L / T / Y, 7 IK, 72C / E / G, 73A / D / E / K / M / S / V, 74S, 75P, 76A / E / F / G / H / K / L / N / Q / R / S / T / V / W / Y, 77F / H / I7W, 78A / C / D / E / G / H / M / Q / S, 79A / E / K / L / P / Q / R / T / V, 80A / G / P / V, 81C / G / L / S, 82T / V, 83A / E / G / Q / R / S / T / V, 85A / S / V, 86L / P / V, 89A / L / M / T, 90L / M / T / V, 91R / S / T / W, 92F / T, 93G / S / T / V, 94A / C / H / M / T / V, 95A / G / H / L / M / S / T / V, 96I / L / T / V, 97A / L / Q / S / V / W, 98Q, 99S, 100A, 101F / V, 102V, 103L / S / V, 104L / V, 105A / K / L / S / V, 106G / S, 107H / M / S, 108C / V, 109G / S, 110G / Q / V, 112GZH / L / S, 113A / H / P / R, 114L / M / S / V, 115L / Q / T / W, 1 16A / H / E / L / M / Q / R / S, 117D / E / G / K / L / M / Q / S / T / V, 118A / L / M / V, 120E / G / L / Q / R / S / Y, 121C / F7G / M / Q / R / S / T / V / W, 122C / M / V, 123G / P / R / S, 124A / G / 1 / K / L / P / R / S / T / V, 125D / EZH, 126A / C / D / L / M / S, 127W, 128A / K / R / T / V, 129L / V, 130A / I / L / S / T, 132C / I / L / M, 133E / G / L / Y, 134A / L / T, 135W, 137E / G, 138E / L / T, 139C / V, 140L / Q, 141A / K / P / R / S / T / W, 144A / S / T, 146DZM / N / T, 147A / C / F / G / I / L / M / Q / R / S / T / V, 148R, 149A / G / H / L / P / Q / S / T, 150A / E / F / G / LZMZP / Q / S / V, 152T, 153D / Q / R, 154Q, 155K / S, 156A / L / M / V, 157A / D / G / H / I / M / N / R / Q / S / T / V / W, 158DZM / NZP / W, 159S, 160A / E / G / H / P / S / V, 162V, 163N / T, 164A / G / I / L / N / R / S / V, 165D / E / S, 166F, 167Q, 170G / L / M / N / P / Q / V, 171A / E / N / Q / T / V, 172A, 173S / T, 174Y, 175L, 176G / I / L / M / R / V, 177L / P / S / V, 178M / S / T, 179A / G, 180G / L / M / R / T / V / W, 181A / C / D / E / F / G / L / M / Q / S / T / V / W, 182A / E / G / K / L / M / P / R / S / T / V / Y, 183 A / L / P / R / V, 184A / G / P / R / V, 185D / G, 186A / E / M / N7V, 187M / T, 188E / F / L / Q / R, 189G / R, 190C / F / T / V, 191F / Q / M / R / V / W, 192E / G / M / S / V, 193A / C / E / F / G / H / I / L / M / N / Q / R / S / T / V / W / Y, 194M, 195E / H / I / L / M / R / S / T / W, 196D / G / M / T / W, 197D / G / L / Q / R / S / V / W, 198E / W / Y, 199A / F / G / L / M / S / T / V / Y, 200I / L / T, 201E / F / H / I / L / M / N / P / Q / R / S / T, 202A / E / G / I / K / L / M / P / R / S / T / V, 203A / F / G / L / M / Q / T / V, or 204A / D / E / F / G / H / K / L / P / R / S / T / V / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0305] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 11, 16, 18, 19, 27, 42, 50, 51, 53, 67, 73, 76, 77, 78, 79, 85, 89, 94, 95, 97, 105, 109, 1 16, 118, 124, 139, 141, 147, 150, 156, 157, 158, 164, 171 , 176, 178, 180, 181, 182, 183, 188, 193, 195, 200, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 ofSEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0306] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2A / G / I / K / MZP / R / S, 3F / G / R / S / T / Y, 4G / K / M / NZP / R, 5G / K / M / N / P / Q / R / T / Y, 6D / E / G / P / R / S / T, 7A / F / G / H / I / L / M / P / Q / S / T / V / W, 11F / Q, 16F / L / P / R / S, 18A / C / G / I / L / M / N / P / Q / S / T / V / W, 19A / C / E / L / M / Q / R / S, 27G / H / L / Q / R / T, 42S / T, 50A / F / I / K / L / Q / R / S / W / Y, 51E / K / L / Q / R / S / T / V / W / Y, 53G / L / M / V / Y, 67F / L / M / Q / V, 73A / D / E / K / M / S / V, 76A / E / F / G / H / K / L / N / Q / R / S / T / VAV / Y, 77F / H / L / W, 78A / C / D / E / G / H / M / Q / S, 79A / E / K / L / P / Q / R / T / V, 85A / S / V, 89A / L / M / T, 94A / C / H / M / T / V, 95A / G / H / L / M / S / T / V, 97A / E / Q / S / V / W, 105 A / K / L / S / V, 109G / S, 116A / H / E / L / M / Q / R / S, 118A / L / M / V, 124A / G / I / K / L / P / R / S / T / V, 139C / V, 141A / K / P / R / S / T / W, 147A / C / F / G / I / L / M / Q / R / S / T / V, 150A / E / F / G / L / M / P / Q / S / V, 156AZL / M / V, 157A / D / G / H / I / M / N / R / Q / S / T / V / W, 158D / M / N / P / W, 164A / G / I / L / N / R / S / V, 171A / E / N / Q / T / V, 176G / I / L / M / R / V, 178M / S / T, 180G / L / MZR / T / V / W, 181A / C / D / E / F / G / L / M / Q / S / T / V / W, 182AZE / G / K / L / M / P / R / S / T / V / Y, 183A / L / P / R / V, 188E / F / L / Q / R, 193 A / C / E / F / G / H / l / L / M / N / Q / R / S / 'f / V / W / Y, 195E / H / 1 / L / M / R / S / T / W, 200I / L / T , 202A / E / G / I / K / L / M / P / R / S / T / V, 203A / F / G / L / M / Q / T / V, or 204A / D / E / F / G / H / K / L / P / R / S / T / V / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0307] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2R, 3F / S / T / Y, 4K / N, 5R, 6R / S / T, 7F / G / M / W, 11 Q, 16S, 18A, 19M, 27H / Q, 42S / T, 50F, 51K, 53Y, 67L, 73A / E, 76E / W, 77W, 78A / E, 79R, 85S, 89M, 94M, 95A / H / M, 97Q, 105A / L / V, 109G, 116E, 118L, 124K / V, 139V, 141K / S, 147A / M / V, 150L, 156L, 157 A / T, 158P, 164S, 171N, 176G / V, 178S / T, 181E / L / M / Q, 182E, 183V, 188E, 193A / E / T, 195E, 200L, 202P, 203 V, or 204E / F / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

[0308] In some embodiments, the engineered adenylyl-sulfate kmase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to ammo acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 128-238, or to a reference sequencecorresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 128-238, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, or relative to the reference sequence corresponding to SEQ ID NO: 28.

[0309] In some embodiments, the engineered adenylyl-sulfate kinase comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity' to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, or to the reference sequence corresponding to SEQ ID NO: 28, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, or relative to the reference sequence corresponding to SEQ ID NO: 28

[0310] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 3 / 1 1, 3 / 11 / 76, 3 / 11 / 76 / 78 / 109, 3 / 1 1 / 76 / 109, 3 / 1 1 / 76 / 109 / 158 / 180, 3 / 1 1 / 76 / 158, 3 / 1 1 / 78, 3 / 1 1 / 78 / 109, 3 / 1 1 / 78 / 109 / 138, 3 / 11 / 78 / 109 / 138 / 176, 3 / 1 1 / 78 / 109 / 158, 3 / 1 1 / 78 / 109 / 180, 3 / 1 1 / 78 / 138, 3 / 1 1 / 78 / 176, 3 / 1 1 / 109, 3 / 11 / 109 / 138, 3 / 11 / 109 / 138 / 176, 3 / 11 / 109 / 138 / 180, 3 / 11 / 138, 3 / 76 / 78 / 109, 3 / 76 / 138 / 158, 11 / 76, 1 1 / 76 / 78, 11 / 76 / 78 / 109, 1 1 / 76 / 78 / 109 / 138, 1 1 / 76 / 78 / 109 / 138 / 158, 11 / 76 / 78 / 109 / 176, 11 / 76 / 78 / 158, 11 / 76 / 109, 1 1 / 76 / 109 / 138, 11 / 76 / 109 / 180, 11 / 76 / 138, 1 1 / 76 / 138 / 158, 11 / 76 / 158 / 180, 11 / 76 / 180, 11 / 78 / 109, 11 / 78 / 109 / 138 / 180, 11 / 78 / 109 / 158, 11 / 78 / 109 / 158 / 180, 11 / 78 / 158, 1 1 / 109, 1 1 / 109 / 138, 1 1 / 109 / 158, 1 1 / 109 / 158 / 180, 11 / 109 / 176, 11 / 158, 76 / 78 / 109, 76 / 78 / 109 / 138, 76 / 78 / 158, 76 / 109, 78 / 109, or 109, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, or relative to the reference sequence corresponding to SEQ ID NO: 28.

[0311] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 3F / 1 IQ, 3F / 11Q / 76W / 78A / 109G, 3F / 11Q / 78A, 3F / 11Q / 78A / 109G / 138L, 3F / 11Q / 78A / 109G / 138L / 176G, 3F / 11Q / 109G / 138L, 3F / 76W / 138L / 158M, 3T / 11Q, 3T / 11Q / 76W, 3T / 11Q / 76W / 78A / 109G, 3T / 11Q / 76W / 109G,3T / 11Q / 76W / 109G / 158M / 180W, 3T / 11Q / 76W / 158M, 3T / 11Q / 78A / 109G, 3T / 11Q / 78A / 109G / 158M, 3T / 11Q / 78A / 138L, 3T / 11Q / 78A / 176G, 3T / 1 1Q / 109G, 3T / 11Q / 109G / 138L, 3T / 11Q / 109G / 138L / 176G, 3T / 11Q / 109G / 138L / 180W, 3T / 11Q / 138L, 3T / 76W / 78A / 109G, 3Y / 11Q / 78A / 109G / 138L,3Y / 11Q / 78A / 109G / 180W, 11Q / 76W, 11Q / 76W / 78A, 11Q / 76W / 78A / 109G, 11Q / 76W / 78A / 109G / 138L, 11Q / 76W / 78A / 109G / 138L / 158D, 11Q / 76W / 78A / 109G / 176G, 11Q / 76W / 78A / 158M, 1 1Q / 76W / 109G, 11Q / 76W / 109G / 138L, 11Q / 76W / 109G / 180W, 11Q / 76W / 138L, 11Q / 76W / 138L / 158M, 11Q / 76W / 158M / 180W, 11Q / 76W / 180W, 11 Q / 78A / 109G, 1 1Q / 78A / 109G / 138L / 180W,11Q / 78A / 109G / 158M, 11Q / 78A / 109G / 158M / 180W, 11 Q / 78A / 158M, 11Q / 109G, 11Q / 109G / 138L,11Q / 109G / 158M, 11 Q / 109G / 158M / 180W, 11Q / 109G / 176G, 1 1Q / 158M, 76W / 78A / 109G, 76W / 78A / 109G / 138L, 76W / 78A / 158M, 76W / 109G, 78A / 109G, or 109G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, or relative to the reference sequence corresponding to SEQ ID NO: 28.

[0312] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) P3F / H1 IQ,Hl 1 Q / G76W / S78A / S109G / V138L, Hl 1 Q / G76W / S78A / S 109G / V138L / E 158D,Hl 1 Q / G76W / S78A / S109G / V176G, H 11 Q / G76W / S78A / E158M, Hl 1 Q / G76W / S109G,Hl 1 Q / G76W / S 109G / V138L, H 11 Q / G76W / S109G / K 180 W, H 11 Q / G76W / V138L,H 11 Q / G76 W / Vl 38L / E 158M, Hl 1 Q / G76W / E 158M / K 180W, H 1 1 Q / G76 W / K 180W, H 11 Q / S78A / S 109G, H 11 Q / S78A / S 109G / V 138L / K 180W, Hl 1 Q / S78A / S 109G / E 158M, H 11 Q / S78A / S 109G / E 158M / K 180 W, H 11 Q / S78A / E 158M, H 11 Q / S 109G, H 11 Q / S 109G / V138L, H 11 Q / S 109G / E 158M,H 11 Q / S 109G / E 158M / K 180 W, H 1 1 Q / S 109G / V 176G, H 11 Q / E 158M, G76 W / S78A / S 109G, G76W / S78A / S109G / V138L, G76W / S78A / E158M, G76W / S109G, S78A / S109G, or S109G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, or relative to the reference sequence corresponding to SEQ ID NO: 28.

[0313] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 240-258, or to a reference sequence corresponding to an cvcn-numbcrcd SEQ ID NO. of SEQ ID NOs: 240-258, wherein the amino acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 154, or relative to the reference sequence corresponding to SEQ ID NO: 154.

[0314] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 154, or to the reference sequence corresponding to SEQ ID NO: 154, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 154, or relative to the reference sequence corresponding to SEQ ID NO: 154.

[0315] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 3, 3 / 39 / 176, 39 / 85 / 158 / 176, 72 / 176, 110, 154 / 158 / 176, 158, 158 / 176, or 1767, or any combinations thereof, wherein the amino acid positions arerelative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 154, or relative to the reference sequence corresponding to SEQ ID NO: 154

[0316] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference (s), or amino acid residue(s) 3F, 3T, 3T / 39V / 176V, 39V / 85A / 158M / 176V, 72G / 176V, 110Q, 154Q / 158M / 176V, 158M, 158M / 176V, or 176V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 154, or relative to the reference sequence corresponding to SEQ ID NO: 154.

[0317] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) P3F, P3T, P3T / A39V / G176V, A39V / S85A / E158M / G176V, N72G / G176V, P110Q, G154Q / E158M / G176V, E158M, E158M / G176V, or G176V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 154, or relative to the reference sequence corresponding to SEQ ID NO: 154.

[0318] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 272-414, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 272-414, wherein the ammo acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

[0319] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or to the reference sequence corresponding to SEQ ID NO: 240, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

[0320] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 7, 8, 13, 30, 35, 41, 47, 64, 69, 71, 72, 73, 74, 75, 77, 79, 80, 81, 89, 95, 105, 107, 108, 112, 113, 114, 117, 128, 129, 130, 133, 135, 138, 141, 147, 148, 156, 159, 186, or 190, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240

[0321] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2M, 71, 8T, 13Y, 30L, 35W, 41P, 41R, 41 V, 47D, 64A, 69A, 71K, 72E, 73E, 74S, 75P, 77L, 79K, 80G, 81L, 81S, 89M, 95T, 105K, 105L, 107S, 108C, 112G, 1 13P, 1 13R, 1 14M, 114V, 117D, 117G, 128K, 128R, 128T, 129L, 129V, 130L, 133E, 133G, 133L, 135W,138L, 141P, 147G, 147Q, 148R, 156V, 159S, 186V, or 190F, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

[0322] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue N2M, N7I, I8T, G13 Y, V30L, G35W, K41P, K41R, K41 V, E47D, G64A, H69A, L71K, N72E, S73E, D74S, L75P, F77L, P79K, E80G, D81L, D81S, I89M, L95T, T105K, T105L, F107S, I108C, R112G, El 13P, El 13R, DI 14M, DI 14V, R117D, R117G, I128K, I128R, I128T, E129L, E129V, V130L, K133E, K133G, K133L, P135W, V138L, E141P, L147G, L147Q, Y148R, I156V, F159S, S186V, or I190F, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240

[0323] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 22, 39, 45, 54, 73, 83, 91 , 96, 1 12, 134, 158, 167, 188, 193, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

[0324] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 22S, 22V, 39S, 45S, 54F, 73K, 83G, 91R, 96V, 1 12S, 134L, 158P, 158W, 167Q, 188R, 193L, or 197R, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

[0325] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue K22S, K22V, A39S, A45S, E54F, S73K, K83G, E91R, F96V, R112S, C134L, E158P, E158W, E167Q, N188R, Y193L, or K197R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

[0326] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity’ to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 416-440, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 416-440, wherein the ammo acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or relative to the reference sequence corresponding to SEQ ID NO: 414

[0327] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or to the reference sequence corresponding to SEQ ID NO: 414, wherein the amino acid sequence comprises one or more amino acid residue differences relative to thereference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or relative to the reference sequence corresponding to SEQ ID NO: 414.

[0328] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 91 / 158, 91 / 158 / 171, 91 / 158 / 188, 91 / 158 / 193, 91 / 193, 141 / 158, 158, 158 / 177, 158 / 177 / 193, 158 / 188, 158 / 188 / 193, 158 / 193, or 188 / 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or relative to the reference sequence corresponding to SEQ ID NO: 414.

[0329] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 91R / 158P, 91R / 158P / 171E, 91R / 158P / 188R, 91R / 158P / 193L, 91R / 193L, 141K / 158P, 158P, 158P / 177P, 158P / 177P / 193L, 158P / 188R, 158P / 188R / 193L, 158P / 193L, or 188R / 193L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or relative to the reference sequence corresponding to SEQ ID NO: 414.

[0330] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) E91R / E158P, E91R / E158P / D17 IE, E91R / E158P / N188R, E91R / E158P / Y193L, E91R / Y193L, E141K / E158P, E158P, E158P / E177P, E158P / E177P / Y193L, E158P / N188R, E158P / N188R / Y193L, E158P / Y193L, or N188R / Y193L, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or relative to the reference sequence corresponding to SEQ ID NO: 414.

[0331] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 442-514, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 442-514, wherein the amino acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 426, or relative to the reference sequence corresponding to SEQ ID NO: 426.

[0332] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 426, or to the reference sequence corresponding to SEQ ID NO: 426, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 426, or relative to the reference sequence corresponding to SEQ ID NO: 426.

[0333] Tn some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 4, 7, 12, 19, 22, 27, 51, 55, 83, 94, 96, 105, 124, 130, 141, 149, 150, 177, 188, 190, 191 , 193, 200, or 203, or any combinations thereof, wherein the aminoacid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 426, or relative to the reference sequence corresponding to SEQ ID NO: 426.

[0334] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 21, 4G, 4P, 7W, 12S, 19A, 19C, 19S, 22 V, 27L, 51E, 51K, 51V, 55A, 55G, 55L, 55P, 83S, 94C, 96T, 105S, 124S, 130L, 141 S, 141T, 149L, 150L, 177S, 188L, 190T, 19 IQ, 193A, 193E, 193N, 200L, 203T, or 203V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 426, or relative to the reference sequence corresponding to SEQ ID NO: 426.

[0335] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue N2I, K4G, K4P, N7W, E12S, D19A, D19C, D19S, K22V, K27L, R51E, R51K, R51 V, M55A, M55G, M55L, M55P, K83S, K94C, F96T, T105S, E124S, V130L, K141 S, K141T, K149L, K150L, E 177S, N188L, I190T, L 191Q, Y193A, Y193E, Y193N, I200L, S203T, or S203 V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 426, or relative to the reference sequence corresponding to SEQ ID NO: 426.

[0336] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 516-696, or to a reference sequence corresponding to an even -numbered SEQ ID NO. of SEQ ID NOs: 516-696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

[0337] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or to the reference sequence corresponding to SEQ ID NO: 474, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

[0338] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 2 / 7 / 83, 4, 4 / 83 / 193, 4 / 191 / 193, 7, 7 / 51 / 83 / 193, 7 / 51 / 141 / 193, 7 / 83, 7 / 83 / 193, 7 / 141, 7 / 188 / 193, 12 / 83, 12 / 83 / 193, 19 / 83 / 188 / 193, 27, 27 / 55, 27 / 150 / 177 / 200 / 203, 27 / 150 / 203, 27 / 200, 27 / 200 / 203, 51, 51 / 83, 51 / 83 / 141 / 193, 51 / 188 / 193, 55, 55 / 105, 55 / 105 / 150, 55 / 149, 55 / 203, 83, 83 / 141, 83 / 141 / 188 / 193, 83 / 188 / 193, 83 / 193, 105, 105 / 124 / 150, 105 / 150, 105 / 150 / 200 / 203, 105 / 200, 105 / 200 / 203, 124 / 149 / 203, 141, 141 / 188 / 193, 149, 149 / 150, 149 / 200, 149 / 203, 150, 150 / 177, 150 / 190, 150 / 200 / 203, 150 / 203, 177, 177 / 200, 188 / 193, 200 / 203, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

[0339] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 2I / 7W / 83S, 4G / 83S / 193N, 4G / 191Q / 193Y, 4P, 7W, 7W / 51E / 83S / 193Y, 7W / 51E / 141T / 193Y, 7W / 83S, 7W / 83S / 193Y, 7W / 141S, 7W / 188L / 193A, 12S / 83S, 12S / 83S / 193A, 19A / 83S / 188L / 193Y, 27L, 27L / 55L, 27L / 150L / 177S / 200L / 203V, 27L / 150L / 203V, 27L / 200L, 27L / 200L / 203V, 51E, 51E / 83S, 51E / 83S / 141T / 193N, 51K / 188L / 193N, 55A, 55A / 105S, 55A / 105S / 150L, 55L, 55L / 149L, 55L / 203T, 83S, 83S / 141 S, 83S / 141S / 188L / 193A, 83S / 188L / 193N, 83S / 193A, 105S, 105S / 124S / 150L, 105S / 150L, 105S / 150L / 200L / 203V, 105S / 200L, 105S / 200L / 203V, 124S / 149L / 203V, 141S, 141S / 188L / 193A, 149L, 149L / 150L, 149L / 200L, 149L / 203V, 150L, 150L / 177S, 150L / 190T, 150L / 200L / 203T, 150L / 200L / 203V, 150L / 203T, 177S, 177S / 200L, 188L / 193N, 200L / 203V, or 203 V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474

[0340] Tn some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) N2I / N7W / K83S, K4G / K83S / E193N, K4G / L 191Q / E193Y, K4P, N7W, N7W / R51E / K83S / E193Y, N7W / R51E / K141T / E193Y, N7W / K83S, N7W / K83S / E193Y, N7W / K141S, N7W / N188L / E193A, E12S / K83S, E12S / K83S / E193A, D19A / K83S / N188L / E193Y, K27L, K27L / M55L, K27L / K 150L / E177S / I200L / S203V, K27L / K150L / S203V, K27L / 1200L, K27L / 1200L / S203V, R51E, R51E / K83S, R51E / K83S / K141T / E193N, R51K / N188L / E193N, M55A, M55A / T105S, M55A / T105S / K150L, M55L, M55L / K149L, M55L / S203T, K83S, K83S / K141 S, K83S / K141 S / N188L / E193A, K83S / N188L / E193N, K83S / E193A, T105S, T 105S / E124S / K150L, T105S / K150L, T105S / K150L / I200L / S203V, T105S / I200L, T105S / I200L / S203V, E124S / K149L / S203V, K141S, K141S / N188L / E193A, K149L, K149L / K150L, K149L / I200L, K149L / S203V, K150L, K150L / E177S, K150L / I190T, K150L / I200L / S203T, K150L / I200L / S203V, K150L / S203T, E177S, E177S / I200L, N188L / E193N, I200L / S203V, or S203V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474

[0341] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 67, 76, 78, 79, 80, 137, 144, 157, 160, 163, 164, 170, 171, 180, 181, 183, or 184, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

[0342] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 6TV, 76G, 76L, 76S, 76T, 78S, 79R, 80P, 137E, 144A, 144S, 144T, 157A, 157G, 157M, 157R, 157V, 157W, 160P, 160V, 163T, 164E, 170M, 171 A, 180R, 180V, 181L, 183L, 183R, 184A, 184P, or 184V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

[0343] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue(s) I67V, W76G, W76L, W76S, W76T, A78S,P79R, E80P, D137E, P144A, P144S, P144T, K157A, K157G, K157M, K157R, K157V, K157W, T160P, T160V, S163T, A164L, E170M, D171A, K180R, K180V, E181L, I183L, I183R, E184A, E184P, or E184V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

[0344] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 698-846, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 698-846, wherein the amino acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0345] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or to the reference sequence corresponding to SEQ ID NO: 522, wherein the amino acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0346] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 79, 79 / 137 / 160 / 164, 79 / 137 / 160 / 164 / 193, 79 / 144 / 160, 79 / 144 / 160 / 164, 79 / 144 / 160 / 193, 79 / 160, 79 / 160 / 164, 79 / 164, 137 / 144 / 160 / 164, 137 / 144 / 164, 137 / 160, 137 / 160 / 164 / 181, 137 / 164, 144 / 160, 144 / 160 / 164, or 160 / 164, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0347] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 79R, 79R / 137E / 160V / 164L, 79R / 137E / 160V / 164L / 193Y, 79R / 144A / 160V, 79R / 144A / 160V / 164L, 79R / 144A / 160V / 193Y, 79R / 160V, 79R / 160V / 164L, 79R / 164L, 137E / 144A / 160V / 164L, 137E / 144A / 164L, 137E / 160V, 137E / 160V / 164L / 181L, 137E / 164L, 144A / 160V, 144A / 160V / 164L, or 160V / 164L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522

[0348] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) P79R, P79R / D137E / T160V / A164L, P79R / D 137E / T 160 V / A 164L / E 193 Y, P79R / P 144 A / T 160 V, P79R / P 144A / T 160 V / A 164E, P79R / P144A / T160V / E193Y, P79R / T160V, P79R / T160V / A164L, P79R / A164L, D137E / P144A / T160V / A164L, D137E / P144A / A164L, D137E / T160V, D137E / T 160V / A164L / E181L, D137E / A164L, P144A / T160V, P144A / T 160V / A164L, or T160V / A164L, or any combinations thereof,wherein the amino aeid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0349] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 79 / 80, 79 / 137, 157, 157 / 170, 157 / 170 / 181, 157 / 180, 157 / 181, 170 / 181, or 181, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0350] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 79R / 80P, 79R / 137E, 157A / 170M / 181L, 157A / 180V, 157A / 181L, 157M, 157M / 170M, 157M / 180V, 157M / 181L, 157W, 170M / 181L, 181L, or 181V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522

[0351] Tn some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) P79R / E80P, P79R / D137E, K157A / E170M / E181L, K157A / K 180V, K157A / E181L, K157M, K157M / E170M, K 157M / K180V, K157M / E181L, K157W, E170M / E181L, E181L, or E181 V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0352] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least ammo acid residue difference at ammo acid position 5, 16, 18, 54, 55, 73, 76, 89, 106, 113, 1 18, 123, 139, 160, 164, 178, 179, 180, 181, or 182, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522

[0353] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 6~IN, 76G, 76L, 76S, 76T, 78S, 79R, 80P, 137E, 144A, 144S, 144T, 157 A, 157G, 157M, 157R, 157V, 157W, 160P, 160V, 163T, 164L, 170M, 171A, 180R, 180V, 181L, 183L, 183R, 184A, 184P, or 184V, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0354] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue I67V, W76G, W76L, W76S, W76T, A78S, P79R, E80P, D137E, P144A, P144S, P144T, K157A, K157G, K157M, K157R, K157V, K157W, T160P, T160V, S163T, A164L, E170M, D171A, K180R, K180V, E181L, I183L, I183R, E184A, E184P, or E184V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0355] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 7, 11, 18, 25, 29, 45, 50, 51, 57, 93, 97, 105, 114,121, 126, 127, or 130, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0356] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 711, 1 IF, 18T, 25S, 29L, 45S, 50F, 50S, 51K, 51V, 57R, 93G, 97Q, 105L, 114L, 114S, 121T, 126L, 127W, 1301, or 130T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0357] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue N7H, QI IF, E18T, G25S, V29L, A45S, W50F, W50S, R51K, R51 V, K57R, A93G, A97Q, T105L, DI 14L, DI 14S, L121T, E126L, F127W, V130I, or VI 30T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

[0358] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 848-878, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 848-878, wherein the ammo acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 732, or relative to the reference sequence corresponding to SEQ ID NO: 732.

[0359] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 732, or to the reference sequence corresponding to SEQ ID NO: 732, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 732, or relative to the reference sequence corresponding to SEQ ID NO: 732.

[0360] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 18, 18 / 76 / 181, 18 / 123, 18 / 180 / 181, 18 / 181, 54 / 123 / 179 / 180 / 181, 73 / 178, 76 / 179 / 180 / 181 , 76 / 181, 89 / 118 / 178 / 181, 118, 123, 181, 181 / 182, or 187, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 732, or relative to the reference sequence corresponding to SEQ ID NO: 732

[0361] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 18W, 18W / 76Y / 181T, 18W / 123P, 18W / 180V / 181E, 18W / 181 T, 54Q / 123P / 179A / 180V / 181E, 73D / 178S, 76Y / 179A / 180V / 181E, 76Y / 181E, 89M / 118L / 178S / 181E, 118L, 123P, 181E, 181E / 182R, 181 S, or 187M, or any combinations thereof, whereinthe amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 732, or relative to the reference sequence corresponding to SEQ ID NO: 732.

[0362] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) E18W, E18W / W76Y / L 181T, E18W / K123P, E18W / K180V / L181E, E18W / L 181T, E54Q / K123P / D179A / K180V / L181E, E73D / T178S, W76Y / D179A / K180V / L181E, W76Y / L181E, I89M / A118L / T178S / L181E, Al 18L, K123P, L181E, L 181 E / S 182R, L 181 S, or VI 87M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 732, or relative to the reference sequence corresponding to SEQ ID NO: 732.

[0363] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 880-952, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 880-952, wherein the amino acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

[0364] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or to the reference sequence corresponding to SEQ ID NO: 848, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

[0365] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 7, 7 / 25 / 51, 7 / 51, 7 / 51 / 97, 7 / 51 / 97 / 105, 7 / 51 / 105, 7 / 97, 7 / 105, 25 / 51 / 97 / 105, 51, 51 / 97, 97, 97 / 105, or 105, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

[0366] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 7H, 7H / 25S / 51K, 7H / 97Q, 7W, 7W / 51K, 7W / 51K / 97Q, 7W / 51K / 97Q / 105L, 7W / 51K / 105L, 7W / 97Q, 7W / 105L, 25S / 51K / 97Q / 105L, 51K, 51K / 97Q, 97Q, 97Q / 105L, or 105L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848

[0367] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) N7H, N7H / G25S / R51K, N7H / A97Q, N7W, N7W / R51K, N7W / R51K / A97Q, N7W / R51K / A97Q / T105L, N7W / R51K / T105L, N7W / A97Q, N7W / T105L, G25S / R51K / A97Q / T105L, R51K, R51K / A97Q, A97Q, A97Q / T105L, or T105L, or any combinations thereof,wherein the amino aeid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

[0368] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 22, 50, 51, 67, 105, 132, 170, 193, 195, 201, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

[0369] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue(s) 22R, 50A, 50L, 50Q, 51Q, 67L, 105A, 105S, 132M, 170L, 193A, 193F, 193L, 193T, 195L, 195M, 195T, 201Q, 203T, 204P, or 204V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848

[0370] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue K22R, W50A, W50L, W50Q, R51 Q, I67L, T105A, T105S, V132M, E170L, E193A, E193F, E193L, E193T, R195L, R195M, R195T, G201Q, V203T, G204P, or G204V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

[0371] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even -numbered SEQ ID NO. of SEQ ID NOs: 954-1022, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 954-1022, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

[0372] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or to the reference sequence corresponding to SEQ ID NO: 848, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

[0373] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 50, 50 / 57 / 193 / 195, 57 / 193 / 195, 105 / 164 / 193, 164, 193, or 195, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

[0374] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 50A, 50L, 50L / 57R / 193F / 195M, 57R / 193L / 195M, 105A / 164S / 193T, 164S, 193L, 193T, or 195M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

[0375] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) W50A, W50L, W50L / K57R / E193F / R195M, K57R / E193L / R195M, L105A / A164S / E193T, A164S, E193L, E193T, or R195M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

[0376] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 7, 76, 78, 79, 89, 138, 139, 157, 160, 178, or 202, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

[0377] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 7F, 7G, 7L, 7M, 7P, 76A, 76E, 76G, 76K, 76L, 76T, 78D, 79R, 89A, 138E, 139V, 157T, 160A, 160H, 178T, 202A, 202E, 2021, 202P, 202R, or 202T, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

[0378] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue W7F, W7G, W7L, W7M, W7P, W76A, W76E, W76G, W76K, W76L, W76T, A78D, P79R, M89A, V138E, L139V, A157T, T160A, T160H, S178T, G202A, G202E, G202I, G202P, G202R, or G202T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848

[0379] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1024-1178, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1024-1178, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962

[0380] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding toamino acid residues 1-200 of SEQ ID NO: 962, or to the reference sequence corresponding to SEQ ID NO: 962, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

[0381] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 7, 7 / 76 / 79, 7 / 76 / 79 / 138, 7116 / 191X51 , 7 / 76 / 79 / 202, 7 / 76 / 138, 7 / 76 / 138 / 157 / 178 / 202, 7 / 79, 7 / 79 / 138 / 202, 7 / 79 / 157 / 178, 7 / 79 / 157 / 202, 7 / 79 / 178 / 202, 7 / 79 / 202, 7 / 138, 7 / 138 / 202, 7 / 157 / 178, 76, 76 / 79 / 138 / 178 / 202, 76 / 202, 79 / 138 / 157, 79 / 157 / 178, 79 / 178 / 202, 138, 138 / 157, 138 / 157 / 202, 138 / 178 / 202, 138 / 202, 157, 157 / 178, 157 / 202, or 202, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962

[0382] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 7F, 7F / 79R / 157T / 178T, 7M, 7M / 76E / 79A / 202P, 7M / 76E / 79R, 7M / 76E / 79R / 138E, 7M / 76E / 79R / 157T, 7M / 76E / 138E, 7M / 76G / 79R / 202P, 7M / 76G / 138E / 157T / 178T / 202A, 7M / 79A / 178T / 202P, 7M / 79A / 202P, 7M / 79R, 7M / 79R / 138E / 202P, 7M / 79R / 157T / 202P, 7M / 138E, 7M / 138E / 202P, 7M / 157T / 178T, 76E, 76E / 79R / 138E / 178T / 202P, 76E / 202P, 79A / 138E / 157T, 79R / 157T / 178T, 79R / 178T / 202P, 138E, 138E / 157T, 138E / 157T / 202P, 138E / 178T / 202P, 138E / 202P, 1571’, 157T / 178T, 157T / 202P, or 202A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

[0383] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) W7F / P79R / A157T / S178T, W7M, W7M / W76E / P79A / G202P, W7M / W76E / P79R, W7M / W76E / P79R / V138E, W7M / W76E / P79R / A157T, W7M / W76E / V138E, W7M / W76G / P79R / G202P, W7M / W76G / V138E / A 157T / S 178T / G202A, W7M / P79A / S178T / G202P, W7M / P79A / G202P, W7M / P79R, W7M / P79R / V138E / G202P, W7M / P79R / A157T / G202P, W7M / V138E, W7M / V138E / G202P, W7M / A157T / S178T, W76E, W76E / P79R / V138E / S178T / G202P, W76E / G202P, P79A / V138E / A157T, P79R / A157T / S178T, P79R / S178T / G202P, V138E, V138E / A157T, V138E / A157T / G202P, V138E / S178T / G202P, V138E / G202P, A157T, A157T / S178T, A157T / G202P, or G202A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

[0384] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 42, 43, 44, 67, 76, 78, 83, 118, 146, 147, 149, 150, 152, 153, 178, 193, 201 , 202, or 203, 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

[0385] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 42T, 43Q, 43S, 44L, 67L, 76A, 76G, 76L, 76R, 76T, 78E, 83 A, 118A, 118V, 146D, 147M, 147V, 149A, 149H, 149P, 149Q, 149S, 149T, 150E, 150F, 150P,150V, 152T, 153D, 153Q, 178T, 193A, 193M, 193V, 201L, 201M, 201Q, 201S, 202K, 202P, 202R, 203A, 203F, 204H, 204L, or 204S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

[0386] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue S42T, T43Q, T43S, I44L, I67L, W76A, W76G, W76L, W76R, W76T, A78E, K83A, LI 18A, LI 18V, G146D, L147M, L147V, K149A, K149H, K149P, K149Q, K149S, K149T, L150E, L150F, L150P, L150V, L152T, A153D, A153Q, S178T, T193A, T193M, T193V, G201L, G201M, G201Q, G201 S, G202K, G202P, G202R, V203A, V203F, G204H, G204L, or G204S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962

[0387] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1 180-1272, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1 180-1272, wherein the amino acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0388] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or to the reference sequence corresponding to SEQ ID NO: 1032, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0389] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 31, 38, 46, 97, 104, 110, 116, 117, 120, 182, or 186, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0390] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 311, 38A, 46Q, 97V, 104L, HOG, 116E, 116L, 116R, 116S, 1 17G, 117K, 117M, 117V, 120S, 182G, 182P, or 186M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0391] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue V3 II, G38A, H46Q, Q97V, I104L, Pl 10G,KI 16E, KI 16L, KI 16R, KI 16S, R117G, R117K, R117M, R117V, N120S, S182G, S182P, or S186M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0392] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 42 / 43 / 76 / 78 / 178, 42 / 43 / 76 / 193, 42 / 76 / 78, 42 / 76 / 78 / 178, 42 / 76 / 78 / 193, 42 / 78, 43 / 76, 43 / 76 / 78, 43 / 76 / 78 / 178, 43 / 78, 43 / 178 / 193, 54 / 76 / 78, 76, 76 / 78, 76 / 78 / 178, 78 / 193, 149, 149 / 153, 149 / 153 / 201, 149 / 201, 153, 153 / 201, 178, 193, 201, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032

[0393] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 42T / 43S / 76E / 78F7178T, 42T / 43S / 76E / 193A, 42T / 76E / 78E, 42T / 76E / 78E / 193A, 42T / 76R / 78E / 178T, 42T / 78E, 43S / 76E, 43S / 76E / 78E / 178T, 43S / 76R / 78E, 43S / 78E, 43S / 178T / 193A, 54V / 76E / 78E, 76E / 78E, 76E / 78E / 178T, 76R, 78E / 193A, 149A / 153Q, 149Q, 149Q / 153Q, 149Q / 153Q / 201 S, 149Q / 201S, 149S / 201S, 153D, 153Q, 153Q / 201 S, 178T, 193 A, 20 I S, or 204L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0394] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) S42T / T43S / W76E / A78E / S178T, S42T / T43S / W76E / T193A, S42T / W76E / A78E, S42T / W76E / A78E / T193A, S42T / W76R / A78E / S178T, S42T / A78E, T43S / W76E, T43S / W76E / A78E / S178T, T43S / W76R / A78E, T43S / A78E, T43S / S178T / T193A, E54V / W76E / A78E, W76E / A78E, W76E / A78E / S178T, W76R, A78E / T193A, K149A / A153Q, K149Q, K149Q / A153Q, K149Q / A153Q / G201S, K149Q / G201S, K149S / G201S, A153D, A153Q, A153Q / G201S, S178T, T193A, G201 S, or G204L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

[0395] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1274-1358, or to a reference sequence corresponding to an even-numbered SEQ ID NO of SEQ ID NOs: 1274-1358, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0396] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding toamino acid residues 1-200 of SEQ ID NO: 1236, or to the reference sequence corresponding to SEQ ID NO: 1236, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0397] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 5, 6, 8, 9, 19, 27, 50, 51, 53, 56, 86, 156, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0398] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2K, 3G, 5M, 5N, 5P, 5Q, 5T, 5Y, 6G, 8V, 9Q, 19Q, 27R, 50Y, 51Q, 51R, 53M, 53Y, 56N, 86V, 156L, 199M, or 199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0399] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue N2K, P3G, A5M, A5N, A5P, A5Q, A5T, A5Y, K6G, I8V, V9Q, D19Q, K27R, W50Y, K51Q, K51R, L53M, L53 Y, G56N, I86V, I156L, I199M, or I199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0400] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 67, 67 / 83, 67 / 83 / 1 16 / 122 / 193, 67 / 116, 67 / 116 / 149, 67 / 122 / 149, 67 / 149, 67 / 153, 67 / 178, 83 / 116 / 193, 83 / 149, 83 / 149 / 153 / 193, 83 / 149 / 178, 116, 116 / 153, 149, 149 / 153 / 193, 153, or 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0401] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 67L, 67L / 83A, 67L / 83A / 116L / 122V / 193V, 67L / 116E, 67L / 116E / 149Q, 67L / 116L, 67L / 122V / 149Q, 67L / 149Q, 67L / 153D, 67L / 178T, 83A / 116L / 193V, 83A / 149Q, 83A / 149Q / 153D / 193V, 83A / 149Q / 178T, 1 16E / 153D, 116L, 149Q, 149Q / 153D / 193V, 153D, or 193 V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0402] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) I67L, I67L / K83A,I67L / K83A / K116L / L122V / A193V, I67L / K1 16E, I67L / K116E / K149Q, I67L / K116L, I67L / L122V / K149Q, I67L / K149Q, I67L / A153D, I67L / S178T, K83A / K1 16L / A193V, K83A / K149Q,K83 AZK 149Q / A 153D / A 193 V, K83 A / K 149Q / S 178T, K 116E / A 153D, K 116L, K 149Q, K149Q / A153D / A193 V, A153D, or A193V, or any combinations thereof, wherein the ammo acid positions arerelative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

[0403] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity’ to a reference sequence corresponding to amino acid residues 1 -200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1364-1462, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1364-1462, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1322, or relative to the reference sequence corresponding to SEQ ID NO: 1322.

[0404] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1322, or to the reference sequence corresponding to SEQ ID NO: 1322, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1322, or relative to the reference sequence corresponding to SEQ ID NO: 1322.

[0405] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least ammo acid residue difference at ammo acid position 7, 18, 23, 25, 99, 102, 103, 104, 1 16, 124, 125, 126, 133, 146, 150, 153, 173, 177, 180, 181, 184, 193, 202, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1322, or relative to the reference sequence corresponding to SEQ ID NO: 1322.

[0406] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 7A, 7F, 7G, 7L, 7Q, 7S, 7V, 7W, 71, 7P, 18P, 18V, 23R, 25R, 25S, 99S, 102V, 103L, 104V, 116A, 124A, 1241, 124K, 124P, 124T, 124V, 125E, 125H, 126L, 133Y, 146M, 146N, 150M, 153Q, 173S, 173T, 177L, 177P, 177V, 180G, 180L, 181C, 181T, 181V, 184V, 1931, 193R, 202L, 202V, or 203A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1322, or relative to the reference sequence corresponding to SEQ ID NO: 1322.

[0407] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least ammo acid residue difference M7A, M7F, M7G, M7L, M7Q, M7S, M7V, M7W, M7I, M7P, E18P, E18V, L23R, G25R, G25S, A99S, I102V, T103L, I104V, E116A, E124A, E124I, E124K, E124P, E124T, E124V, G125E, G125H, E126L, K133Y, G146M, G146N, L150M, A153Q, E173S, E173T, E177L, E177P, E177V, K180G, K180L, E181C, E181 T, E181 V, E184V, A193I, A193R, P202L, P202V, or V203A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1322, or relative to the reference sequence corresponding to SEQ ID NO: 1322.

[0408] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1464-1654, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1464-1654, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0409] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or to the reference sequence corresponding to SEQ ID NO: 1364, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0410] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 3 / 5, 3 / 5 / 8, 3 / 5 / 59, 3 / 8 / 54 / 193, 3 / 8 / 59, 3 / 54, 5, 5 / 8, 5 / 8 / 90, 5 / 193, 8, 8 / 53 / 54, 8 / 53 / 59 / 90 / 153 / 156, 8 / 54, 8 / 54 / 90, 8 / 59 / 153, 8 / 90, 8 / 90 / 193, 8 / 153, 8 / 156, 8 / 193, 53 / 54 / 59, 53 / 54 / 59 / 90 / 153 / 156, 53 / 54 / 90 / 153 / 156, 53 / 54 / 153 / 156, 53 / 59, 53 / 59 / 90, 54, 54 / 59 / 90 / 193, 54 / 90, 59, 59 / 90 / 153 / 156, 59 / 90 / 193, 59 / 193, 90, 90 / 193, 153, or 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0411] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 3G / 5P, 3G / 5P / 8V, 3G / 5P / 59G, 3G / 8V / 54Q / 193V, 3G / 8V / 59G, 3G / 54Q, 5P, 5P / 8V, 5P / 8V / 90M, 5P / 193V, 8V, 8V / 53Y / 54Q, 8V / 53Y / 59G / 90M / 153D / 156L, 8V / 54Q, 8V / 54Q / 90M, 8V / 59G / 153D, 8V / 90M, 8V / 90M / 193V, 8V / 153D, 8V / 156L, 8V / 193V, 53Y / 54Q / 59G, 53Y / 54Q / 59G / 90M / 153D / 156L, 53Y / 54Q / 90M / 153D / 156L, 53Y / 54Q / 153D / 156L, 53Y / 59G, 53Y / 59G / 90M, 54Q, 54Q / 59G / 90M / 193V, 54Q / 90M, 59G, 59G / 90M / 153D / 156L, 59G / 90M / 193V, 59G / 193V, 90M, 90M / 193V, 153D, or 193V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0412] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) P3G / A5P, P3G / A5P / I8V, P3G / A5P / A59G, P3G / I8V / E54Q / A193V, P3G / I8V / A59G, P3G / E54Q, A5P, A5P / I8V, A5P / I8V / S90M, A5P / A193V, I8V, I8V / L53Y / E54Q, I8V / L53Y / A59G / S90M / A153D / I156L, I8V / E54Q, I8V / E54Q / S90M, I8V / A59G / A153D, I8V / S90M, I8V / S90M / A193V, I8V / A153D, I8V / I156L, I8V / A193V, L53Y / E54Q / A59G, L53Y / E54Q / A59G / S90M / A153D / I156L, L53Y / E54Q / S90M / A153D / I156L, L53Y / E54Q / A153D / I156L, L53Y / A59G, L53Y / A59G / S90M, E54Q, E54Q / A59G / S90M / A193V, E54Q / S90M, A59G, A59G / S90M / A153D / I156L, A59G / S90M / A193 V, A59G / A193V, S90M, S90M / A193V, A153D, or A193V, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequencecorresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to tire reference sequence corresponding to SEQ ID NO: 1364.

[0413] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 17, 19, 26, 28, 29, 53, 56, 57, 67, 92, 93, 95, 98, 128, 130, 134, 139, 140, 147, 156, 157, 160, 162, 164, 174, 176, 199, or 201, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0414] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 17C, 19E, 19M, 19Q, 19R, 26S, 28A, 29L, 53G, 56H, 57L, 57S, 67M, 92T, 93G, 95G, 95H, 95S, 98Q, 128A, 128K, 128T, 130A, 130L, 130S, 130T, 134A, 134T, 139V, 140L, 147A, 147C, 1471, 147M, 147R, 147S, 147T, 147V, 156V, 157D, 157N, 157S, 160A, 162V, 164N, 164R, 164V, 174Y, 176L, 176M, 199A, 199F, 199G, 199L, 199S, 199V, 199Y, or 201Q, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0415] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue K17C, D19E, D19M, D19Q, D19R, Q26S, G28A, V29L, L53G, G56H, K57L, K57S, L67M, V92T, A93G, L95G, L95H, L95S, D98Q, I128A, I128K, 1128T, V130A, V130L, V130S, V130T, C134A, C134T, L139V, 1140L, L 147A, L 147C, L1471, L 147M, L 147R, L 147S, L 147T, L147V, I156V, T157D, T157N, T157S, T160A, I162V, S164N, S164R, S164V, L 174Y, V176L, V176M, I199A, I199F, I199G, I199L, I199S, I199V, I199Y, or G201Q, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

[0416] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1656-1732, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1656-1732, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0417] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or to the reference sequence corresponding to SEQ ID NO: 1644, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0418] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 16, 50, 51, 59, 73, 77, 78, 79, 117, 118, 120, 121, 185, 186, 188, 189, 190, or 195, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0419] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 16L, 16R, 50F, 51R, 59P, 59S, 73A, 77W, 78C, 78G, 78S, 79L, 79T, 117E, 117T, 117V, 118V, 120E, 120G, 120Y, 121R, 185D, 186E, 186N, 188F, 189R, 190C, 190V, 1951, or 195S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0420] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue K16L, K16R, W50F, K51R, A59P, A59S, E73A, F77W, E78C, E78G, E78S, R79E, R79T, R1 17E, R 1 17T, R1 17V, El 18V, N120E, N120G, N120Y, E121R, E185D, S186E, S186N, N188F, K189R, I190C, I190V, R195I, or R195S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0421] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 53, 53 / 59 / 89 / 90 / 153, 53 / 90, 53 / 90 / 180, 53 / 95, 53 / 180, 95, 120, or 180, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0422] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 53 Y, 53Y / 59G / 89A / 90M / 153D, 53Y / 90M, 53Y / 90M / 180L, 53Y / 95A, 53Y / 180L, 95A, 120S, or 180L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0423] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) L53Y, L53Y / A59G / M89A / S90M / A153D, L53Y / S90M, L53Y / S90M / K180L, L53Y / L95A, L53Y / K180L, L95A, N120S, and K180L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

[0424] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 1734-1850, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1734-1850, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequencecorresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to tire reference sequence corresponding to SEQ ID NO: 1716.

[0425] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity’ to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or to the reference sequence corresponding to SEQ ID NO: 1716, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

[0426] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 8, 10, 103, 108, 1 16, 123, 124, 125, 132, 138, 141 , 147, 149, 150, 170, 175, 178, 180, 183, 193, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

[0427] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2A, 8L, 10M, 10P, 10Y, 103 V, 108V, 1 16A, 123R, 123S, 124A, 125D, 1321, 138T, 141A, 147A, 147F, 149G, 150A, 150G, 150Q, 170Q, 170V, 175L, 178T, 180T, 183L, 193C, 193L, 193M, 193R, 193S, 193T, 202A, 202G, 202L, 202M, 203L, 203Q, 203T, 204D, 204E, 204R, 204S, or 204 V, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

[0428] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue N2A, I8L, W10M, W10P, W10Y, T103V, I108V, El 16A, K123R, K123S, E124A, G125D, V132I, V138T, K141A, M147A, M147F, K149G, L150A, L 150G, L150Q, E170Q, E170V, V175L, S178T, K180T, I183L, A193C, A193L, A193M, A193R, A193S, A193T, P202A, P202G, P202L, P202M, V203L, V203Q, V203T, G204D, G204E, G204R, G204S, or G204V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

[0429] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 16 / 19 / 53, 16 / 19 / 95, 16 / 199, 53, 53 / 77, 53 / 77 / 95, 53 / 95, 53 / 95 / 134, 53 / 176 / 199, 77 / 95, 95, 156, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716

[0430] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 16R / 19M / 53Y, 16R / 19M / 95H, 16R / 199F, 53Y, 53Y / 77W, 53Y / 77W / 95H, 53Y / 95H, 53Y / 95S, 53Y / 95S / 134A, 53 Y / 176L / 199F, 77W / 95S, 95H, 156V, or 199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequencecorresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to tire reference sequence corresponding to SEQ ID NO: 1716.

[0431] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) K16R / D19M / L53Y, K16R / D19M / A95H, K16R / I199F, L53Y, L53Y / F77W, L53Y / F77W / A95II, L53Y / A95II, L53Y / A95S, L53Y / A95S / C134A, L53Y / V176L / I199F, F77W / A95S, A95H, I156V, or I199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

[0432] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 1852-1952, or to a reference sequence corresponding to an even-numbered SEQ ID NO of SEQ ID NOs: 1852- 1952, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0433] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or to the reference sequence corresponding to SEQ ID NO: 1834, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0434] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 120, 140, 197, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0435] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 120S, 140Q, 197L, or 199Y, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0436] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue N120S, I140Q, K 197L, or I199Y, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0437] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 50, 50 / 53, 50 / 53 / 73 / 199, 50 / 53 / 77, 50 / 53 / 195, 50 / 73 / 77 / 189 / 199, 50 / 95 / 120 / 195 / 199, 53, S m / Tl, 53 / 73 / 77 / 189, 53 / 73 / 95 / 185, 53 / 73 / 95 / 195, 53 / 73 / 189, 53 / 73 / 195, 53 / 73 / 199, 53 / 95, 53 / 195, 73 / 77 / 189, 73 / 185 / 189, 77 / 95 / 120 / 189, 95, 95 / 185, 95 / 189 / 199, or 95 / 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0438] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 50F, 50F / 53L, 50F / 53L / 73A / 199V, 50F / 53L / 77F, 50F / 53L / 195I, 50F / 73A / 77F / 189R / 199V, 50F / 95S / 120G / 1951 / 199V, 53L, 53L / 73A / 77F, 53L / 73A / 77F / 189R, 53L / 73A / 95A / 185D, 53L / 73A / 95A / 1951, 53L / 73A / 189R, 53L / 73A / 195I, 53L / 73A / 199V, 53L / 95A, 53L / 195I, 73A / 77F / 189R, 73A / 185D / 189R, 77F / 95S / 120G / 189R, 95S, 95S / 185D, 95S / 189R / 199V, or 95S / 199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0439] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) W50F, W50F / Y53L, W50F / Y53L / E73A / I 199V, W50F / Y53L / W77F, W50F / Y53L / R195I, W50F / E73A / W77F / K 189R / I199V, W50F / H95S / N120G / R1951 / 1199V, Y53L, Y53L / E73A / W77F, Y53L / E73A / W77F / K189R, Y53L / E73A / H95A / E185D, Y53L / E73A / H95A / R195I, Y53L / E73A / K189R, Y53L / E73A / R195I, Y53L / E73A / I199V, Y53L / H95A, Y53L / R195I, E73A / W77F / K189R, E73A / E185D / K189R, W77F / H95S / N120G / K189R, H95S, H95S / E185D, H95S / K189R / I199V, or H95S / I199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0440] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 6, 7, 16, 28, 66, 105, 120, 122, 137, 139, 156, 165, 167, 195, 197, 198, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0441] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 6R, 6T, 7F, 7H, 7T, 7 V, 7W, 16P, 28A, 66R, 105L, 120R, 122C, 122M, 137G, 139V, 156A, 165E, 167Q, 195W, 197S, 198W, or 199T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0442] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue K6R, K6T, G7F, G7H, G7T, G7V, G7W, K16P, G28A, N66R, A105L, N120R, L122C, L 122M, D137G, L 139V, I156A, P165E, E167Q, R195W, K197S,G198W, or I199T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

[0443] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 1954-2030, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1954-2030, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946

[0444] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or to the reference sequence corresponding to SEQ ID NO: 1946, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0445] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 2 / 123 / 193 / 204, 2 / 170, 1 16, 1 16 / 170 / 178, 116 / 178, 116 / 193, 1 16 / 204, 123 / 170 / 204, 123 / 204, 170, 170 / 193, 193, 193 / 204, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0446] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 2A / 170V, 2R / 123R / 193T / 204S, 116A, 116A / 170Q / 178T, 116A / 178T, 116A / 193T, 116A / 204D, 116A / 204E, 123R / 170V / 204D, 123R / 204D, 170 V, 170V / 193T, 193T, 193T / 204D, 193T / 204E, 204D, or 204S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0447] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) N2A / E170V, N2R / K123R / A193T / G204S, E 116A, E 116A / E 170Q / S 178T, E 116 A / S 178T, E 116A / A 193 T, E 116A / G204D, E 116A / G204E, K123R / E170V / G204D, K123R / G204D, E170V, E170V / A193T, A193T, A193T / G204D, A193T / G204E, G204D, or G204S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0448] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 83, 101, 116, 171, 188, or 195, or anycombinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0449] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 83R, 101F, 1 16Q, 171N, 188E, or 195E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0450] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue K83R, L 101F, El 16Q, D171N, N188E, or R195E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946

[0451] Tn some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 31, 106, 123, 124, 125, 126, 147, 150, or 157, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0452] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least ammo acid residue difference, or ammo acid residue 31L, 106S, 123G, 124P, 124V, 125D, 126A, 126C, 147A, 147L, 147 V, 150A, 150S, 157N, 157Q, or 157S, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0453] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue V31L, A106S, K123G, E124P, El 24V, G125D, E126A, E126C, M147A, M147L, M147V, L 150A, L150S, T157N, T157Q, or T157S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

[0454] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 2032-2152, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2032-2152, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0455] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or to the reference sequence corresponding to SEQ ID NO: 1954, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or to the reference sequence corresponding to SEQ ID NO: 1954.

[0456] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 6, 6 / 7, 6 / 7 / 50, 6 / 7 / 95 / 150, 6 / 7 / 120 / 150, 6 / 50 / 150, 6 / 73 / 95 / 150, 6 / 120 / 150, 6 / 150, 7 / 50 / 95 / 116, 7 / 116 / 120 / 150, 50, 50 / 73 / 95, 50 / 95 / 120, 50 / 95 / 132, 50 / 116, 73, 73 / 95, 73 / 116 / 120, 73 / 150, 95, 95 / 116, 95 / 120, 95 / 132, 95 / 150, 116, 132, or 132 / 150, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954

[0457] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 6T, 6T / 7F / 50F, 6T / 7F / 95S / 150A, 6T / 7H, 6T / 7H / 120R / 150A, 6T / 50F / 150A, 6T / 73A / 95S / 150A, 6T / 120G / 150A, 6T / 150A, 7F / 50F / 95S / 116A, 7F / 116A / 120G / 150A, 50F, 50F / 73A / 95S, 50F / 95S / 120R, 50F / 95S / 132I, 50F / 116A, 73A, 73A / 95S, 73A / 116A / 120G, 73A / 150A, 95S, 95S / 1 16A, 95S / 120G, 95S / 132I, 95S / 150A, 1 16A, 1321, or 132I / 150A, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence con’esponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0458] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) R6T, R6T / G7F / W50F,R6T / G7F / H95S / L 150A, R6T / G7H, R6T / G7H / N120R / L150A, R6T / W50F / L 150A, R6T / E73A / H95S / L150A, R6T / N120G / L150A, R6I7L150A, G7F / W50F / H95S / E116A, G7F / E116A / N120G / L150A, W50F, W50F / E73A / H95S, W50F / H95S / N120R, W50F / H95S / V132I, W50F / E116A, E73A, E73A / H95S,E73A / E116A / N120G, E73A / L 150A, H95S, H95S / E1 16A, H95S / N120G, H95S / V132I, H95S / L150A, El 16A, V132I, or V132I / L150A, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0459] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 18, 19, 21, 51, 54, 57, 76, 78, 79, 83, 86, 91, 95, 101, 178, 180, or 181, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence con’esponding to SEQ ID NO: 1954

[0460] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 18C, 18G, 18P, 19A, 19E, 21 C, 51R, 54E, 54S, 57R, 76G, 78A, 79K, 79L, 79P, 79Q, 79T, 83Q, 83R, 86V, 91W, 95L, 95M, 101F, 178T, 180M, 181A, 181F, 181 L, 181 Q, 181 S, 181 V, or 181 W, or any combinations thereof, wherein the amino acid positions are relativeto the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0461] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue E18C, E18G, E18P, D19A, D19L, E21C, K51R, E54L, E54S, K57R, E76G, E78A, R79K, R79L, R79P, R79Q, R79T, K83Q, K83R, I86V, E91 W, II95L, H95M, L101F, S178T, K180M, E181A, E181F, E181L, E181Q, E181S, E181 V, or E181W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

[0462] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1 -200 of an even-numbered SEQ ID NO of SEQ ID NOs: 2154-2324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2154-2324, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0463] In some embodiments, the engineered adenylyl-sulfate kmase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or to the reference sequence corresponding to SEQ ID NO: 2058, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0464] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 31, 31 / 73 / 95 / 150, 31 / 73 / 147, 31 / 83, 31 / 95 / 147 / 195, 31 / 95 / 150, 31 / 147 / 195, 73 / 83 / 95 / 147, 73 / 83 / 147 / 182, 73 / 124 / 147, 76 / 95 / 147 / 188, 83, 83 / 95, 83 / 95 / 147, 83 / 124, 83 / 124 / 150, 83 / 147, 83 / 150 / 182, 83 / 150 / 188, 83 / 182 / 188, 95, 95 / 150 / 182, 1 16 / 120 / 123, 116 / 120 / 123 / 147, 124, 126 / 147, 147, 147 / 182, 182, or 188, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0465] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 3 IL, 31L / 73A / 95S / 150A, 31L / 73A / 147V, 31L / 83R, 31L / 95S / 147V / 195E, 31L / 95S / 150A, 31L / 147V / 195E, 73A / 83R / 95S / 147V, 73A / 83R / 147V / 182A, 73A / 124 V / 147 V, 76K / 95S / 147V / 188E, 83R, 83R / 95S, 83R / 95S / 147V, 83R / 124V, 83R / 124V / 150A, 83R / 147V, 83R / 150A / 182A, 83R / 150A / 188E, 83R / 182A / 188E, 95S, 95S / 150A / 182A, 116A / 120G / 123R, 1 16A / 120G / 123R / 147V, 124V, 126A / 147V, 147V, 147V / 182A, 182A, or 188E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0466] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) V3 IL, V31L / E73A / H95S / L150A, V31L / E73A / M147V, V31LZK83R, V31L / H95S / M147V / R195E, V31L / H95S / L150A, V31L / M147V / R195E, E73A / K83R / H95S / M147V, E73A / K83R / M147V / S182A, E73A / E124V / M147V, E76K / H95S / M147V / N188E, K83R, K83R / II95S, K83R / 1195 SZM 147V, K83R / E124V, K83R / E124V / L 150A, K83R / M147V, K83R / L 150A / S 182A, K83R / L 150A / N188E, K83R / S 182A / N188E, H95S, H95S / L 150A / S 182A, El 16A / N120G / K123R, El 16A / N120G / K123R / M147V, E124V, E126A / M147V, M147V, M147V / S182A, SI 82 A, or N188E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0467] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 16, 17, 18, 19, 50, 69, 76, 78, 79, 95, 117, 120, 121 , 132, 156, 160, 176, 178, 179, 180, 181 , 182, or 183, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0468] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 16R, 16S, 17R, 18A, 18C, 181, 18N, 18Q, 18S, 18T, 19M, 19Q, 50S, 69Q, 76G, 76Q, 76V, 76Y, 78S, 79E, 79K, 79L, 79T, 79V, 95A, 95L, 95T, 117L, 1 17M, 117T, 1 17V, 120G, 120L, 120R, 121 V, 132C, 132L, 156L, 160A, 160E, 160G, 160P, 176L, 178M, 178T, 179G, 180L, 181D, 181L, 181W, 182E, 182M, 182P, 182Y, 183L, or 183V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0469] In some embodiments, the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue K16R, K16S, K17R, E18A, E18C, E18I, E18N, E18Q, E18S, E18T, D19M, D19Q, F50S, H69Q, E76G, E76Q, E76V, E76Y, E78S, R79E, R79K, R79L, R79T, R79V, II95A, II95L, II95T, R117L, R117M, R117T, R117V, N120G, N120L, N120R, L121 V, V132C, V132L, I156L, T160A, T160E, T160G, T160P, V176L, S178M, S178T, D179G, K180L, E181D, E181L, E181 W, S182E, S182M, S182P, S182Y, I183L, or I183V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

[0470] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 2326-2396, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2326-2396, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158.

[0471] In some embodiments, the engineered adenylyl-sulfate kinase polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2158, or to the reference sequence corresponding to SEQ ID NO: 2158, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158.

[0472] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 51 / 73 / 76 / 83 / 95 / 181 / 182 / 195, 51 / 120 / 178, 73, 73 / 76 / 79 / 83 / 95 / 171 / 188, 73 / 76 / 95, 73 / 76 / 95 / 101 / 188, 73 / 95 / 171 / 195, 73 / 95 / 188, 76 / 79 / 95 / 171 / 188, 76 / 83 / 95, 83 / 95, 83 / 95 / 120 / 182 / 188 / 195, 95, 95 / 120 / 178, 95 / 120 / 188 / 195, or 178, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158

[0473] In some embodiments, the ammo acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 51R / 73E / 76G / 83R / 95M / 181 Q / 182A / 195E, 51R / 120G / 178T, 73E, 73E / 76G / 79P / 83R / 95S / 171N / 188E, 73E / 76G / 95M / 101F / 188E, 73E / 76K / 95M, 73E / 95M / 171N / 195E, 73E / 95M / 188E, 76G / 79P / 95M / 171N / 188E, 76G / 83R / 95S, 83R / 95M, 83R / 95M / 120G / 182A / 188E / 195E, 95M, 95M / 120G / 178T, 95M / 1...

Claims

1. CLAIMSWhat is claimed is:1 An engineered adenylyl-sulfate kinase comprising an amino acid sequence having one or more amino acid residue differences in one or more motifs of adenosine 5 ’-phosphosulfate (APS) kinase domain:[VI]-[WF]-[LF]-T-G-L-[SP]-[GAC]-X-G-K (SEQ ID NO: 3325):L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326);[LA]-[GS]-F-[ST];K-G-L-Y-[KA]-[KY]-A (SEQ ID NO: 3327); andF-T-G-[IV]2 The engineered adenylyl-sulfate kinase of Claim 1, wherein the amino sequence comprises at least an amino acid residue difference in the motif [LA]-[GS]-F-[ST], located on the amino side of alpha helix a3.3 The engineered adenylyl-sulfate kinase of Claim 2, wherein the amino acid sequence comprises at least an amino acid residue difference in [GS]-F of the motif [LA]-[GS]-F-[ST].4 The engineered adenylyl-sulfate kinase of Claim 3, wherein [GS] is substituted with E, or F is substituted with W, or combination thereof.5 The engineered adenylyl-sulfate kinase of Claim 1, wherein the amino sequence comprises at least an amino acid residue difference in the motif K-G-L-Y-[KA]-[KY]-A (SEQ ID NO: 3327), located at alpha helix a6.6 The engineered adenylyl-sulfate kinase of Claim 5, wherein the amino sequence comprises at least an ammo acid residue difference in the motif K-G-L-Y (SEQ ID NO: 3328).7 The engineered adenylyl-sulfate kinase of Claim 6, wherein L of K-G-L-Y (SEQ ID NO: 3328) is substituted with A.8 The engineered adenylyl-sulfate kinase of Claim 1, wherein the amino sequence comprises at least an ammo acid residue difference in the motif [VI]-[WF]-[LF]-T-G-L-[SP]-[GAC]-X-G-K (SEQ ID NO: 3325), located from beta strand pi to alpha helix al.9 The engineered adenylyl-sulfate kinase of Claim 8, wherein the amino sequence comprises at least an ammo acid residue difference in the motif [VI]-[WF]-[LF]-T-G-L-[SP]-[GAC]-X-G-K (SEQ ID NO: 3325) is at [VI], [LF], G, [GAC], or K; or any combinations thereof.

10. The engineered adenylyl-sulfate kinase of Claim 9, wherein [VI] is substituted with L; [LF] is substituted with Y: G is substituted with W; or K is substituted with P / R / V; or any combinations thereof11. The engineered adenylyl-sulfate kinase of Claim 1, wherein the amino sequenee comprises at least an amino acid residue difference in the motif L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326), located from beta strand P2 to alpha helix a2.

12. The engineered adenylyl-sulfate kmase of Claim 1 1, wherein the ammo acid sequence comprises at least an ammo acid residue difference in the motif L-D-G-D-[NW]-[LIVA]-R (SEQ ID NO: 3326) is at G, [NW], or [LIVA], or any combination thereof.

13. The engineered adenylyl-sulfate kinase of Claim 12, wherein G is substituted with A, [NW] is substituted with R, or [LIVA] is substituted with F, M, or Q, or any combinations thereof.

14. The engineered adenylyl-sulfate kinase of Claim 1, wherein the amino sequence comprises at least an amino acid residue difference in the motif F-T-G-[IV], located between alpha helix a6 and beta strand )35.

15. The engineered adenylyl-sulfate kinase of Claim 14, wherein the amino acid sequence comprises at least an amino acid residue difference at F or T, or any combinations thereof.

16. The engineered adenylyl-sulfate kinase of Claim 15, wherein F is substituted with S; or T is substituted with A, E, G, H, P, S, or V; or any combinations thereof.

17. An engineered adenylyl-sulfate kinase, or a functional fragment thereof, comprising an amino acid sequence having at least one or more amino acid residue differences at amino acid residues that are about 6 angstroms (A) or less from the active site of an adenylyl-sulfate kmase, wherein the active site is determined from a protein crystal structure and / or by protein homology modeling of a reference adenylyl- sulfate kinase.

18. The engineered adenylyl-sulfate kinase of Claim 17, wherein the reference adenylyl-sulfate kinase is the adenosine 5-phosphosulfate kinase of Arabidopsis thaliana19. The engineered adenylyl-sulfate kinase of Claim 18, wherein the amino acid sequence comprises at least an amino acid residue difference at amino acid position 7, 10, 11 , 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 63, 64, 65, 68, 76, 77, 78, 81 , 82, 85, 86, 89, 106, 107, 108, 109, 1 10, 132, 139, 141 , 142, 143, 144, 145, 147, 148, 156, 157, 158, 159, 160, 163, 164, 176, 177, 179, 180, 181, 182, 183, 185, or 186, or any combinations thereof, and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14).20 The engineered adenylyl-sulfate kinase of Claim 18, wherein the amino acid sequence comprises an amino acid sequence having at least one or more amino acid residue differences at amino acid residues that are about 4.5 A or less from the active site of the reference adenylyl-sulfate kinase.

21. The engineered adenylyl-sulfate kinase of Claim 20, wherein the amino aeid sequence comprises at least an amino acid residue difference at amino acid position 7, 10, 11, 36, 37, 38, 39, 40, 41, 42, 43, 44;64, 65, 68, 77, 82, 85, 89, 107, 108, 109, 110, 142, 143, 144, 145, 147, 156, 157, 158, 159, 160, 164, 177, 181, 182, 183, or 186, or any combinations thereof, and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14).

22. The engineered adenylyl-sulfate kinase of any one of Claims 17-21, wherein the amino acid sequence comprises at least an ammo acid residue difference at amino acid position 7, 11, 42, 76, 77, 78, 85, 89, 109, 139, 141, 147, 156, 157, 158, 164, 176, 181, 182, or 183, or any combinations thereof, and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14).

23. The engineered adenylyl-sulfate kinase of any one of Claims 17-22, wherein the equivalent positions are of a homologous, orthologous, or paralogous adenylyl-sulfate kinase of Thennotoga sp. KOL6, Thennotoga sp. SGI, Thennotoga sp. (RKX51929.1), Thermotoga sp. (HDG62185.1), Thermosipho fen'ireducens (WP_207567147.1), Petrotoga sp. 9PW.55.5 1 (WP_113075358.1), Defluviitoga tunisiensis (MFY9404473.1), Pctrotoga sibirica (WP_103876974. 1), Mycobactcrim tuberculosis (BAQ05257), human bifunctional 3 '-phospho adenosine 5'-phosphosulfate synthetase 1 (PAPS synthetase 1) (PAPSS 1), human bifunctional 3 '-phospho adenosine 5'-phosphosulfate synthetase 2 (PAPSS2), Arabidopsis thaliana, Pemcillium chrysogenum, Thiobacillus dentrificans, Aquifex aeolicus, or Aeropyrum pemix.

24. The engineered adenylyl-sulfate kinase of Claim 22, wherein the one or more amino acid residue differences are selected from amino acid residue 7A / F / G / II / IZLZM / P / Q / S / T / V / W, 11F / Q, 42T, 76A / E / F / H / K / L / N / Q / R / S / T / V / W / Y, 77H / L / W, 78A / C / D / E / G / H / M / Q, 85A / S / V, 89A / L / M / T, 109G, 139C / V, 141A / K / P / R / S / T / W, 147A / C / F / G / I / M / Q / R / S / T / V, 156A / L / M / V, 157 A / D / G / H / I / M / N / Q / R / S / T / V / W, 158D / M / NZP / W, 164G / I / L / N / R / S / V, 176G / I / L / M / R, 181 A / C / D / F / G / L / M / Q / S / T / V / W, 182A / E / G / K / L / M / P / R / T / V / Y, 183 A / L / P / R / V, and any combinations thereof, and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp RQ7 (SEQ ID NO: 14)25. The engineered adenylyl-sulfate kinase of Claim 24, wherein the one or more ammo acid residue differences are selected from 7F / G / M / W, 1 IQ, 42T, 76E / W, 77 W, 78A / E, 85S, 89M, 109G, 139V, 141K, 147A / M / V, 156E, 157A / T, 158P, 164S, 176G, 181L / M / Q, 182E, 183 V, and any combinations thereof, and at equivalent positions thereof of a homologous, orthologous, or paralogous adenylyl-sulfate kinase, wherein the amino acid positions are relative to the adenylyl-sulfate kinase of Thermotoga sp. RQ7 (SEQ ID NO: 14)26. An engineered adenylyl-sulfate kinase, or a functional fragment thereof, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequencecorresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 14, 18-258, 272-1358, or 1364-3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 18-258, 272-1358, or 1364-3324, wherein the amino acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

27. The engineered adcnylyl-sulfatc kinase of Claim 26, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034,3116, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 322828. The engineered adenylyl-sulfate kinase of Claim 26 or 27, comprising an ammo acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14, wherein the ammo acid sequence comprises one or more ammo acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14.

29. The engineered adenylyl-sulfate kinase of Claim 26 or 27, comprising an ammo acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to the reference sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402,2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, wherein the amino aeid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

30. The engineered adenylyl-sulfate kinase of Claim 26, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364- 3324, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

31. The engineered adenylyl-sulfate kinase of any one of Claims 26-30, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 35, 38, 39, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 64, 65, 66, 67, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 140, 141 , 144, 146, 147, 148, 149, 150, 152, 153, 154, 155,156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

32. The engineered adenylyl-sulfate kinase of any one of Claims 26-31, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2A / G / I / K / M / P / R / S, 3F / G / R / S / T / Y, 4G / M / N / P / R, 5G / K / M / N / P / Q / R / T / Y, 6D / E / G / P / R / S / T, 7A / F / G / H / I / L / M / P / Q / S / T / V / W, 8E / T / V, 9Q, 10M / P / Y, 11F / Q, 12S, 13S / Y, 151, 16F / L / P / R / S, 17C / Q / R, 18A / C / G / IA / M / N7P / Q / S / T / V / W, 19A / C / E / L / M / Q / R / S, 21C / G, 22G / R / S / V, 23I / M / R, 25A / Q / R / S, 26S, 27G / H / L / Q / R / T, 28A, 29G / L, 30L, 31I / L, 33Y, 35W, 38A, 39S / V, 41P / R / V, 42T, 43A / Q / S, 44L, 45S / V, 46N / Q, 47D / G, 50A / F / I / K / L / Q / R / S / Y, 51E / K / L / Q / S / T / V / Y, 521, 53G / L / M / V / Y, 54F / G / H / L / N / Q / R / S / T / V, 55A / C / G / L / P / S, 56H / N / Q / R / S, 57L / R / S, 58F / S / V / W, 59G / P / S / T, 61I / T, 64A, 65L, 66R, 67F / L / M / Q / V, 69A / Q / T, 70D / L / T / Y, 7 IK, 72C / E / G, 73A / D / E / K / M / V, 74S, 75P, 76A / E / F / H / K / L / N / Q / R / S / T / VAV / Y, 77H / L / W, 78A / C / D / E / G / H / M / Q, 79A / E / K / L / P / Q / R / T / V, 80A / G / P / V, 81C / G / L / S, 82T / V, 83A / E / G / Q / R / S / T / V, 85A / S / V, 86L / P / V, 89A / L / M / T, 90L / M / T / V, 91R / S / T / W, 92F / T, 93G / S / T / V, 94A / C / H / M / T / V, 95A / G / H / M / S / T / V, 96I / L / T / V, 97L / Q / S / V / W, 98Q, 99S, 100A, 101F / V, 102V, 103L / S / V, 104L / V, 105A / K / L / S / V, 106G / S, 107H / M / S, 108C / V, 109G, 110G / Q / V, 112G / H / L / S, 113A / H / P / R, 114L / M / S / V, 115L / Q / T / W, 1 16A / E / H / L / M / Q / R / S, 117D / E / G / K / L / M / Q / S / T / V, 118L / M / V,120E / G / L / Q / R / S / Y, 121C / F / G / M / Q / R / S / T / V / W, 122C / M / V, 123G / P / R / S, 124A / G / I / K / L / P / R / S / T / V, 125D / E / H, 126A / C / D / L / M / S, 127W, 128A / K / R / T / V, 129L / V, 130A / IZL / S / T, 132C / I / L / M, 133E / G / L / Y, 134A / L / T, 135W, 137E / G, 138E / L / T, 139C / V, 140L / Q, 141A / K / P / R / S / T / W, 144A / S / T, 146D / M / N / T, 147A / C / F / G / I / M / Q / R / S / T / V, 148R, 149A / G / H / L / P / Q / S / T, 150A / E / F / G / L / M / P / Q / S / V, 152T, 153D / Q / R, 154Q, 155K / S, 156A / L / M / V, 157 A / D / G / H / I / M / N / Q / R / S / T / V / W, 158D / M / N / P / W, 159S, 160A / E / G / H / P / S / V, 162V, 163N / T, 164G / I / L / N / R / S / V, 165D / E / S, 166F, 167Q, 170G / L / M / N / P / Q / V, 171A / E / N / Q / T / V, 172A, 173S / T, 174Y, 175L, 176G / I / L / M / R, 177L / P / S / V, 178M / S, 179A / G, 180G / L / M / R / T / V / W, 181A / C / D / F / G / L / M / Q / S / T / V / W, 182A / E / G / K / L / M / P / R / T / V / Y, 183A / L / P / R / V, 184A / G / P / R / V, 185D / G, 186 A / E / M / N / V, 187M / T, 188E / F / L / Q / R, 189G / R, 190C / F / T / V, 191F / M / Q / R / V / W, 192E / G / M / S / V, 193A / C / E / F / G / H / I / L / M / N / Q / R / S / T / V / W, 194M, 195E / H / I / L / M / S / T / W, 196D / G / M / T / W, 197D / G / L / Q / R / S / V / W, 198E / W / Y, 199 A / F / G / L / M / S / T / V / Y, 200L / T, 201E / F / H / I / I, / M / N / P / Q / R / S / T, 202A / E / I / K / L / M / P / R / S / T / V, 203A / F / G / L / M / Q / T / V, or 204A / D / E / F / H / K / L / P / R / S / T / V / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.33 The engineered adenylyl-sulfate kinase of any one of Claims 26-31, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 1 1, 16, 18, 19, 27, 42, 50, 51, 53, 67, 73, 76, 77, 78, 79, 85, 89, 94, 95, 97, 105, 109, 116, 118, 124, 139, 141, 147, 150, 156, 157, 158, 164, 171, 176, 178, 180, 181, 182, 183, 188, 193, 195, 200, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

34. The engineered adenylyl-sulfate kinase of any one of Claims 26-31 and 33, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 2A / G / I / K / M / P / R / S, 3F / G / R / S / T / Y, 4G / M / N / P / R, 5G / K / M / N / P / Q / R / T / Y, 6D / E / G / P / R / S / T, 7A / F / G / H / I / L / M / P / Q / S / T / V / W, 11F / Q, 16F / L / P / R / S, 18A / C / G / I / L / M / N / P / Q / S / T / V / W, 19A / C / E / L / M / Q / R / S, 27G / H / L / Q / R / T, 42T, 50A / F / I / K / L / Q / R / S / Y, 51E / K / L / Q / S / T / V / Y, 53G / L / M / V / Y, 67F / L / M / Q / V, 73A / D / E / K / M / V, 76A / E / F / H / K / L / N / Q / R / S / T / V / W / Y, 77H / L / W, 78A / C / D / E / G / HZM / Q, 79A / E / K / L / P / Q / R / T / V, 85A / S / V, 89A / L / M / T, 94A / C / H / M / T / V, 95A / G / H / M / S / T / V, 97L / Q / S / V / W, 105A / K / L / S / V, 109G, 116A / E / H / L / M / Q / R / S, 118L / M / V, 124A / G / IZKZL / PZR / S / T / V, 139C / V, 141A / K / P / R / S / T / W, 147A / C / F / G / I / M / Q / R / S / T / V, 150AZE / F / GZLZMZP / Q / S / V, 156A / L / M / V,157 A / D / G / H / I / M / N / Q / R / S / T / V / W, 158D / M / N / P / W, 164G / I / L / N / R / S / V, 171A / E / N / Q / T / V, 176G / I / L / M / R, 178M / S, 180G / L / M / R / T / V / W, 181A / C / D / F / G / L / M / Q / S / T / V / W, 182A / E / G / K / L / M / P / R / T / V / Y, 183A / L / P / R / V, 188E / F / L / Q / R, 193A / C / E / F / G / H / I / L / M / N / Q / R / S / T / V / W, 195E / H / I / L / M / S / T / W, 200L / T, 202A / E / I / K / L / M / P / R / S / T / V, 203A / F / G / L / M / Q / T / V, or 204A / D / E / F / II / K / L / P / R / S / T / V / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

35. The engineered adenylyl-sulfate kinase of any one of Claims 26-31, wherein the amino aeid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 11, 76, 83, 85, 89, 109, 110, 141, 158, 176, or 180, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

36. The engineered adenylyl-sulfate kinase of any one of Claims 26-31 and 35, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference or amino acid residue 3F / S / T / Y, 1 IQ, 76W, 83S, 85S, 89M, 109G, 110Q, 141 W, 158M, 176R, or 180W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

37. The engineered adenylyl-sulfate kinase of any one of Claims 26-31, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 11, 72, 76, 78, 80, 81, 82, 83, 85, 89, 107, 109, 110, 115, 141, 146, 154, 155, 176, 182, or 185, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

38. The engineered adenylyl-sulfate kinase of any one of Claims 26-31 and 37, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 3F, 1 IQ, 72C, 76F, 76W, 78A, 80P, 80V, 81C, 82T, 83S, 85S, 89M, 107H, 109G, 110Q, 110V, 115T, 141R, 146T, 154Q, 155S, 176R, 182T, 182V, or 185G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

39. The engineered adenylyl-sulfate kinase of any one of Claims 26-31, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 3, 33, 39, 45, 67, 72, 76, 81, 82, 85, 86, 89, 106, 115, 141, 149, 153, 155, 156, 157, 164, 180, 182, or 187, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

40. The engineered adenylyl-sulfate kinase of any one of Claims 26-31 and 39, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 3S, 33Y, 39V, 45V, 67L, 72G, 76F, 76R, 76W, 81G, 82V, 85A, 85S, 85V, 86P, 89M, 106G, 115L, 115Q, 115W, 141R, 149G, 153R, 155K, 156M, 157R, 164G, 180W, 182L, 182T, or 187T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.

41. The engineered adenylyl-sulfate kinase of Claim 26, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 14, 18-258, 272-1358, and 1364- 3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 18- 258, 272-1358, and 1364-3324.

42. The engineered adenylyl-sulfate kinase of Claim 26, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

43. The engineered adenylyl-sulfate kinase of Claim 26, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of an even-numbered SEQ ID NO of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228.44 The engineered adenylyl-sulfate kinase of Claim 26, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or to a reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, orrelative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

45. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43, and 44, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 33, 35, 38, 39, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 85, 86, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 140, 141, 144, 146, 147, 148, 149, 150,152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

46. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 2A / G / I / K / M / P / R / S, 3F / G / R / S / T / Y, 4G / K / M / N / P / R, 5G / K / M / N / P / Q / R / T / Y, 6D / E / G / P / R / S / T, 7A / F / G / H / I / E / M / P / Q / S / T / V / W, 8E / T / V, 9Q, 10M / P / Y, 11F / Q, 12S, 13S / Y, 151, 16FZL / PZR / S, 17C / Q / R, 18A / C / G / I / LZM / N / P / Q / S / T / V / W, 19A / C / E / L / M / Q / R / S, 21C / G, 22G / R / S / V, 23I / M / R, 25A / Q / R / S, 26S, 27G / H / L / Q / R / T, 28A, 29G / L, 30L, 31I / L, 33Y, 35W, 38A, 39S / V, 41P / R / V, 42S / T, 43A / Q / S, 44L, 45S / V, 46N / Q, 47D / G, 50A / F / I / K / L / Q / R / S / W / Y, 51E / K / L / QZR / S / T / V / W / Y, 521, 53G / L / M / V / Y, 54F / G / H / L / N / Q / R / S / T / V, 55A / C / G / L / P / S, 56H / N / Q / R / S, 57L / R / S, 58F / S / V / W, 59G / P / S / T, 61I / T, 64A, 65L, 66R, 67F / L / M / Q / V, 69A / Q / T, 70D / L / T / Y, 71K, 72C / E / G, 73A / D / E / K / M / S / V, 74S, 75P, 76A / E / F / G / H / K / L / N / Q / R / S / T / V / W / Y, 77F / H / L / W, 78A / C / D / E / G / H / M / Q / S, 79A / E / K / L / P / Q / R / T / V, 80A / G / P / V, 81C / GZL / S, 82T / V, 83A / E / G / Q / R / S / T / V, 85A / S / V, 86L / P / V, 89A / L / M / T, 90L / M / T / V, 91R / S / T / W, 92F / T, 93G / S / T / V, 94A / C / H / M / T / V, 95A / G / H / L / M / S / T / V, 96I / L / T / V, 97A / L / Q / S / V / W, 98Q, 99S, 100A, 101F / V, 102V, 103L / S / V, 104L / V, 105A / K / L / S / V, 106G / S, 107H / M / S, 108C / V, 109G / S, 110G / Q / V, 112G / H / L / S, 113A / H / P / R, 114L / M / S / V, 115L / Q / T / W, 116A / H / E / L / M / Q / R / S,117D / E / G / K / L / M / Q / S / T / V, 118A / L / M / V, 120E / G / L / Q / R / S / Y, 121C / F / G / M / Q / R / S / T / V / W, 122C / M / V, 123G / P / R / S, 124A / G / I / K / L / P / R / S / T / V, 125DZE / H, 126A / CZD / LZM / S, 127W, 128A / K / R / T / V, 129L / V, 130A / I / L / S / T, 132C / I / L / M, 133E / G / L / Y, 134A / L / T, 135W, 137E / G, 138E / L / T, 139C7V, 140L / Q, 141A / K / P / R / S / T / W, 144A / S / T, 146D / M / N / T, 147A / C / F / G / I / L / M / Q / R / S / T / V, 148R, 149A / G / H / L / P / Q / S / T, 150A / E / F / G / L / M / P / Q / S / V, 152T, 153D / Q / R, 154Q, 155K / S, 156A / L / M / V, 157A / D / G / H / I / M / N / R / Q / S / T / V / W, 158D / M / N / P / W, 159S, 160 A / E / G / H / P / S / V, 162 V, 163N / T,164A / G / I / L / N / R / S / V, 165DZE / S, 166F, 167Q, 170GZL / M / N / P / Q / V, 171A / E / N / Q / T / V, 172A, 173S / T, 174Y, 175L, 176G / I / L / M / R / V, 177L / P / S / V, 178M / S / T, 179A / G, 180G / L / M / R / T / V / W, 181A / C / D / E / F / G / L / M / Q / S / T / V / W, 182A / E / G / K / L / M / P / R / S / T / V / Y, 183A / L / P / R / V, 184A / G / PZR / V, 185D / G, 186A / E / M / N / V, I 87M / T. 188E / F / L / Q / R, 189G / R, 190C / F / T / V, 191F / QZM / R / V / W, 192E / G / M / S / V, 193A / C / E / F / G / H / I / L / M / N / Q / R / S / T / V / W / Y, 194M, 195E / H / I / L / M / R / S / T / W, 196D / G / M / T / W, 197D / G / L / Q / R / S / V / W, 198E / W / Y, 199A / F / G / L / M / S / T / V / Y, 200I / L / T, 201E / F / H / I / L / M / N / P / Q / R / S / T, 202A / E / G / EK / L / M / P / R / S / T / V, 203A / F / G / L / M / Q / T / V, or 204A / D / E / F / G / H / K / L / P / R / S / T / V / W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

47. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at ammo acid position 2, 3, 4, 5, 6, 7, 11, 16, 18, 19, 27, 42, 50, 51, 53, 67, 73, 76, 77, 78, 79, 85, 89, 94, 95, 97, 105, 109, 116, 118, 124, 139, 141, 147, 150, 156, 157, 158, 164, 171, 176, 178, 180, 181, 182, 183, 188, 193, 195, 200, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

48. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 47, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 2A / G / I / K / M / P / R / S, 3F / G / R / S / T / Y, 4G / K / M / N / P / R, 5G / K / M / N / P / Q / R / T / Y, 6D / E / G / P / R / S / T, 7A / F / GZH / I / L / M / P / Q / S / T / V / W, 11F / Q, 16F / L / P / R / S, 18A / C / G / I / L / M / N / P / Q / S / T / V / W, 19A / C / E / L / M / Q / R / S, 27G / H / L / Q / R / T, 42S / T, 50A / F / I / K / L / Q / R / S / W / Y, 51E / K / L / Q / R / S / T / V / W / Y, 53G / L / M / V / Y, 67F / L / M / Q / V, 73A / D / E / K / M / S / V, 76A / E / F / G / H / K / L / N / Q / R / S / T / V / W / Y, 77F / H / L / W, 78A / C / D / E / G / H / M / Q / S, 79A / E / K / L / P / Q / R / T / V, 85A / S / V, 89A / L / M / T, 94A / C / H / M / T / V, 95A / G / H / L / M / S / T / V, 97A / L / Q / S / V / W, 105A / K / L / S / V, 109G / S, 116A / H / E / L / M / Q / R / S, 118A / L / M / V, 124A / G / I / K / L / P / R / S / T / V, 139C / V, 141A / K / PZR / S / T / W, 147A / C / F / G / I / L / M / Q / R / S / T / V, 150A / E / F / G / L / M / P / Q / S / V, 156A / L / M / V, 157A / D / G / H / I / M / N / R / Q / S / T / V / W, 158D / M / N / P / W, 164A / G / I / L / N / R / S / V, 171A / E / N / Q / T / V, 176G / I / LZMZR / V, 178M / S / T, 180G / L / M / R / T / V / W, 181A / C / D / E / F / G / L / M / Q / S / T / V / W, 182A / E / G / K / L / M / P / R / S / T / V / Y, 183A / L / P / R / V, 188E / F / L / Q / R, 193A / C / E / F / G / H / I / L / M / N / Q / R / S / T / V / W / Y, 195E / H / I / L / M / R / S / T / W, 200IZL / T, 202A / E / G / I / K / L / M / P / R / S / T / V, 203A / F / G / L / M / Q / T / V, or 204A / D / E / F / G / H / K / L / P / R / S / T / V / W, or anycombinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

49. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 47, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 2R, 3F, 4K / N, 5R, 6R / S / T, 7F / G / M / W, 1 IQ, 16S, 18A, 19M, 27H / Q, 42S / T, 50F, 51K, 53Y, 67L, 73A / E, 76E / W, 77W, 78A / E, 79R, 85S, 89M, 94M, 95A / H / M, 97Q, 105A / L / V, 109G, 116E, 118L, 124K / V, 139V, 141K / S, 147A / M / V, 150L, 156L, 157A / T, 158P, 164S, 171N, 176G / V, 178S / T, 181E / L / M / Q, 182E, 183V, 188E, 193A / E / T, 195E, 200L, 202P, 203V, or 204E / F / W, or any combinations thereof, w'herein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 31 16, 3168, or 3228, or relative to the reference sequence corresponding to SEQ ID NO: 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

50. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 3 / 11 , 3 / 11 / 76, 3 / 11 / 76 / 78 / 109, 3 / 11 / 76 / 109, 3 / 11 / 76 / 109 / 158 / 180,3 / 1 1 / 76 / 158, 3 / 1 1 / 78, 3 / 1 1 / 78 / 109, 3 / 1 1 / 78 / 109 / 138, 3 / 1 1 / 78 / 109 / 138 / 176, 3 / 1 1 / 78 / 109 / 158, 3 / 1 1 / 78 / 109 / 180, 3 / 1 1 / 78 / 138, 3 / 1 1 / 78 / 176, 3 / 1 1 / 109, 3 / 11 / 109 / 138, 3 / 11 / 109 / 138 / 176, 3 / 11 / 109 / 138 / 180, 3 / 11 / 138, 3 / 76 / 78 / 109, 3 / 76 / 138 / 158, 11 / 76, 11 / 76 / 78, 11 / 76 / 78 / 109, 1 1 / 76 / 78 / 109 / 138, 11 / 76 / 78 / 109 / 138 / 158, 1 1 / 76 / 78 / 109 / 176, 11 / 76 / 78 / 158, 11 / 76 / 109, 11 / 76 / 109 / 138, 11 / 76 / 109 / 180, 11 / 76 / 138, 11 / 76 / 138 / 158, 1 1 / 76 / 158 / 180, 11 / 76 / 180, 11 / 78 / 109, 11 / 78 / 109 / 138 / 180, 11 / 78 / 109 / 158, 11 / 78 / 109 / 158 / 180, 11 / 78 / 158, 1 1 / 109, 11 / 109 / 138, 11 / 109 / 158, 11 / 109 / 158 / 180, 1 1 / 109 / 176, 11 / 158, 76 / 78 / 109, 76 / 78 / 109 / 138, 76 / 78 / 158, 76 / 109, 78 / 109, or 109, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, or relative to the reference sequence corresponding to SEQ ID NO: 28.

51. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 50, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) or ammo acid residue(s) 3F / 1 IQ, 3F / 11Q / 76W / 78A / 109G, 3F / 11Q / 78A,3F / 11Q / 78A / 109G / 138L, 3F / 11Q / 78A / 109G / 138L / 176G, 3F / 11Q / 109G / 138L, 3F / 76W / 138L / 158M, 3T / 11Q, 3T / 1 1Q / 76W, 3T / 1 1Q / 76W / 78A / 109G, 3T / 11Q / 76W / 109G, 3T / 11Q / 76W / 109G / 158M / 180W, 3T / 1 1Q / 76W / 158M, 3T / 1 1Q / 78A / 109G, 3T / 11Q / 78A / 109G / 158M, 3T / 1 1Q / 78A / 138L, 3T / 1 1Q / 78A / 176G, 3T / 1 1Q / 109G, 3T / 11Q / 109G / 138L, 3T / 11Q / 109G / 138L / 176G, 3T / 11Q / 109G / 138L / 180W, 3T / 11Q / 138L,3T / 76W / 78A / 109G, 3Y / 11Q / 78A / 109G / 138L, 3Y / 11Q / 78A / 109G / 180W, 11Q / 76W, 11Q / 76W / 78A, 11 Q / 76W / 78A / 109G, 11 Q / 76W / 78A / 109G / 138L, 11 Q / 76W / 78A / 109G / 138L / 158D,11Q / 76W / 78A / 109G / 176G, 11Q / 76W / 78A / 158M, 11Q / 76W / 109G, 11Q / 76W / 109G / 138L, 11Q / 76W / 109G / 180W, 11Q / 76W / 138L, 11Q / 76W / 138E / 158M, 11Q / 76W / 158M / 180W, 11Q / 76W / 180W, 11Q / 78A / 109G, 11Q / 78A / 109G / 138L / 180W, 11Q / 78A / 109G / 158M, 11Q / 78A / 109G / 158M / 180W,11 Q / 78A / 158M, 11Q / 109G, 11Q / 109G / 138L, 11Q / 109G / 158M, 11Q / 109G / 158M / 180W, 11 Q / 109G / 176G, 11Q / 158M, 76W / 78A / 109G, 76W / 78A / 109G / 138L, 76W / 78A / 158M, 76W / 109G, 78A / 109G, or 109G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 28, or relative to the reference sequence corresponding to SEQ ID NO: 2852. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 3, 3 / 39 / 176, 39 / 85 / 158 / 176, 72 / 176, 110, 154 / 158 / 176, 158, 158 / 176, or 1767, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 154, or relative to the reference sequence corresponding to SEQ ID NO: 15453. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 52, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) or ammo acid residue(s) 3F, 3T, 3T / 39V / 176V, 39V / 85A / 158M / 176V, 72G / 176V, 110Q, 154Q / 158M / 176V, 158M, 158M / 176V, or 176V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO:154, or relative to the reference sequence corresponding to SEQ ID NO: 15454. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 7, 8, 13, 30, 35, 41, 47, 64, 69, 71, 72, 73, 74, 75, 77, 79, 80, 81, 89, 95, 105, 107, 108, 112, 113, 114, 117, 128, 129, 130, 133, 135, 138, 141, 147, 148, 156, 159, 186, or 190, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

55. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 54, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 2M, 71, 8T, 13Y, 30L, 35W, 41P, 41R, 41V, 47D, 64A, 69A, 71K, 72E, 73E, 74S, 75P, 77L, 79K, 80G, 81L, 81 S, 89M, 95T, 105K, 105L, 107S, 108C, 112G, 113P, 1 13R, 1 14M, 114V, 117D, 117G, 128K, 128R, 128T, 129L, 129V, 130L, 133E, 133G, 133L, 135W, 138L, 141P, 147G, 147Q, 148R, 156V, 159S, 186V, or 190F, or any combinations thereof, wherein the ammo acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

56. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 22, 39, 45, 54, 73, 83, 91, 96, 112, 134, 158, 167, 188, 193, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

57. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 56, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 22S, 22V, 39S, 45S, 54F, 73K, 83G, 91R, 96V, 112S, 134L, 158P, 158W, 167Q, 188R, 193L, or 197R, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 240, or relative to the reference sequence corresponding to SEQ ID NO: 240.

58. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 91 / 158, 91 / 158 / 171, 91 / 158 / 188, 91 / 158 / 193, 91 / 193, 141 / 158, 158, 158 / 177, 158 / 177 / 193, 158 / 188, 158 / 188 / 193, 158 / 193, or 188 / 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or relative to the reference sequence corresponding to SEQ ID NO: 414.

59. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 58, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 91R / 158P, 91R / 158P / 171E, 91R / 158P / 188R, 91R / 158P / 193L, 91R / 193L, 141K / 158P, 158P, 158P / 177P, 158P / 177P / 193E, 158P / 188R, 158P / 188R / 193E, 158P / 193L, or 188R / 193L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 414, or relative to the reference sequence corresponding to SEQ ID NO: 414.

60. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at ammo acid position 2, 4, 7, 12, 19, 22, 27, 51, 55, 83, 94, 96, 105, 124, 130, 141, 149, 150, 177, 188, 190, 191, 193, 200, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 426, or relative to the reference sequence corresponding to SEQ ID NO: 426.

61. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 60, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 21, 4G, 4P, 7W, 12S, 19A, 19C, 19S, 22V, 27L, 51E, 51K, 51V, 55A, 55G, 55L, 55P, 83S, 94C, 96T, 105S, 124S, 130E, 141 S, 141T, 149L, 150E, 177S, 188L, 190T, 191Q, 193A, 193E, 193N, 200L, 203T, or 203 V, or any combinations thereof, wherein the amino acid positions are relative to thereference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 426, or relative to the reference sequence corresponding to SEQ ID NO: 426.

62. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 2 / 7 / 83, 4, 4 / 83 / 193, 4 / 191 / 193, 7, 7 / 51 / 83 / 193, 7 / 51 / 141 / 193, 7 / 83, 7 / 83 / 193, 7 / 141, 7 / 188 / 193, 12 / 83, 12 / 83 / 193, 19 / 83 / 188 / 193, 27, 27 / 55, 27 / 150 / 177 / 200 / 203, 27 / 150 / 203, 27 / 200, 27 / 200 / 203, 51, 51 / 83, 51 / 83 / 141 / 193, 51 / 188 / 193, 55, 55 / 105, 55 / 105 / 150, 55 / 149, 55 / 203, 83, 83 / 141, 83 / 141 / 188 / 193, 83 / 188 / 193, 83 / 193, 105, 105 / 124 / 150, 105 / 150, 105 / 150 / 200 / 203, 105 / 200, 105 / 200 / 203, 124 / 149 / 203, 141, 141 / 188 / 193, 149, 149 / 150, 149 / 200, 149 / 203, 150, 150 / 177, 150 / 190, 150 / 200 / 203, 150 / 203, 177, 177 / 200, 188 / 193, 200 / 203, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

63. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 62, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) or ammo acid residue(s) 2I / 7W / 83S, 4G / 83S / 193N, 4G / 191Q / 193Y, 4P, 7W, 7W / 51E / 83S / 193Y, 7W / 51E / 141T / 193Y, 7W / 83S, 7W / 83S / 193Y, 7W / 141S, 7W / 188L / 193A, 12S / 83S, 12S / 83S / 193A, 19A / 83S / 188E / 193Y, 27L, 27L / 55L, 27L / 150E / 177S / 200L / 203V, 27L / 150L / 203V, 27E / 200L, 27E / 200L / 203V, 51E, 51E / 83S, 51E / 83S / 141T / 193N, 51K / 188L / 193N, 55A, 55A / 105S, 55A / 105S / 150L, 55L, 55E / 149E, 55L / 203T, 83S, 83S / 141 S, 83S / 141S / 188E / 193A, 83S / 188L / 193N, 83S / 193A, 105S, 105S / 124S / 150L, 105S / 150L, 105S / 150L / 200L / 203V, 105S / 200E, 105S / 200L / 203V, 124S / 149E / 203V, 141S, 141S / 188L / 193A, 149L, 149E / 150E, 149E / 200L, 149L / 203V, 150L, 150E / 177S, 150L / 190T, 150E / 200E / 203T, 150L / 200L / 203V, 150E / 203T, 177S, 177S / 200E, 188E / 193N, 200E / 203V, or 203 V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

64. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at ammo acid position 67, 76, 78, 79, 80, 137, 144, 157, 160, 163, 164, 170, 171 , 180, 181 , 183, or 184, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

65. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 64, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 67V, 76G, 76L, 76S, 76T, 78S, 79R, 80P, 137E, 144A, 144S, 144T, 157A, 157G, 157M, 157R, 157V, 157W, 160P, 160V, 163T, 164L, 170M, 171A, 180R, 180V, 181L, 183L, 183R, 184 A, 184P, or 184V, or any combinations thereof, wherein the amino acid positions are relative to thereference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 474, or relative to the reference sequence corresponding to SEQ ID NO: 474.

66. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 79, 79 / 137 / 160 / 164, 79 / 137 / 160 / 164 / 193, 79 / 144 / 160, 79 / 144 / 160 / 164, 79 / 144 / 160 / 193, 79 / 160, 79 / 160 / 164, 79 / 164, 137 / 144 / 160 / 164, 137 / 144 / 164, 137 / 160, 137 / 160 / 164 / 181, 137 / 164, 144 / 160, 144 / 160 / 164, or 160 / 164, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

67. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 66, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 79R, 79R / 137E / 160V / 164L, 79R / 137E / 160V / 164L / 193Y, 79R / 144A / 160V, 79R / 144A / 160V / 164L, 79R / 144A / 160V / 193Y, 79R / 160V, 79R / 160V / 164L, 79R / 164L, 137E / 144A / 160V / 164E, 137E / 144A / 164L, 137E / 160V, 137E / 160V / 164L / 181L, 137E / 164E, 144A / 160V, 144A / 160V / 164L, or 160V / 164L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

68. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 79 / 80, 79 / 137, 157, 157 / 170, 157 / 170 / 181, 157 / 180, 157 / 181, 170 / 181, or 181, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

69. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 68, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 79R / 80P, 79R / 137E, 157A / 170M / 181L, 157A / 180V, 157A / 181L, 157M, 157M / 170M, 157M / 180V, 157M / 181L, 157W, 170M / 181L, 181L, or 181V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

70. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 5, 16, 18, 54, 55, 73, 76, 89, 106, 113, 118, 123, 139, 160, 164, 178, 179, 180, 181, or 182, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

71. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 70, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 67V, 76G, 76L, 76S, 76T, 78S, 79R, 80P, 137E, 144A, 144S, 144T, 157A, 157G, 157M, 157R, 157V, 157W, 160P, 160V, 163T, 164L, 170M, 171A, 180R, 180V, 181L, 183L, 183R, 184A, 184P, or 184V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

72. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 7, 11, 18, 25, 29, 45, 50, 51, 57, 93, 97, 105, 114, 121, 126, 127, or 130, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

73. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 73, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 7H, I IP, 18T, 25S, 29L, 45S, 50F, 50S, 51K, 51V, 57R, 93G, 97Q, 105L, 114L, 114S, 121T, 126L, 127W, 1301, or 130T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 522, or relative to the reference sequence corresponding to SEQ ID NO: 522.

74. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 18, 18 / 76 / 181, 18 / 123, 18 / 180 / 181, 18 / 181, 54 / 123 / 179 / 180 / 181, 73 / 178, 76 / 179 / 180 / 181, 76 / 181, 89 / 118 / 178 / 181, 118, 123, 181, 181 / 182, or 187, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 732, or relative to the reference sequence corresponding to SEQ ID NO: 732.

75. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 74, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 18W, 18W / 76Y / 181T, 18W / 123P, 18W / 180V / 181E, 18W / 181T, 54Q / 123P / 179A / 180V / 181E, 73D / 178S, 76Y / 179A / 180V / 181E, 76Y / 181E, 89M / 118L / 178S / 181E, 118L, 123P, 18 IE, 181E / 182R, 181 S, or 187M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 732, or relative to the reference sequence corresponding to SEQ ID NO: 73276. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 7, 7 / 25 / 51, 7 / 51, 7 / 51 / 97, 7 / 51 / 97 / 105, 7 / 51 / 105, 7 / 97, 7 / 105, 25 / 51 / 97 / 105, 51, 51 / 97, 97, 97 / 105, or 105, or any combinations thereof, wherein the amino acid positionsare relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

77. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 77, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) or ammo acid residue(s) 7H, 7H / 25S / 51K, 7H / 97Q, 7W, 7W / 51K, 7W / 51K / 97Q, 7W / 51K / 97Q / 105L, 7W / 51K / 105L, 7W / 97Q, 7W / 105L, 25S / 51K / 97Q / 105L, 51K, 51K / 97Q, 97Q, 97Q / 105L, or 105L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

78. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 22, 50, 51, 67, 105, 132, 170, 193, 195, 201, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

79. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 78 wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 22R, 50A, 50L, 50Q, 51Q, 67L, 105A, 105S, 132M, 170L, 193A, 193F, 193L, 193T, 195L, 195M, 195T, 201Q, 203T, 204P, or 204V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 848, or relative to the reference sequence corresponding to SEQ ID NO: 848.

80. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 50, 50 / 57 / 193 / 195, 57 / 193 / 195, 105 / 164 / 193, 164, 193, or 195, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

81. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 80, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 50A, 50L, 50L / 57R / 193F / 195M, 57R / 193L / 195M, 105A / 164S / 193T, 164S, 193L, 193T, or 195M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

82. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residuedifference at amino acid position 7, 76, 78, 79, 89, 138, 139, 157, 160, 178, or 202, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

83. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 82, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 7F, 7G, 7L, 7M, 7P, 76A, 76E, 76G, 76K, 76L, 76T, 78D, 79R, 89A, 138E, 139V, 157T, 160A, 160H, 178T, 202A, 202E, 2021, 202P, 202R, or 202T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 880, or relative to the reference sequence corresponding to SEQ ID NO: 880.

84. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 7, 7 / 76 / 79, 7 / 76 / 79 / 138, 7 / 76 / 79 / 157, 7 / 76 / 79 / 202, 7 / 76 / 138, 7 / 76 / 138 / 157 / 178 / 202, 7 / 79, 7 / 79 / 138 / 202, 7 / 79 / 157 / 178, 7 / 79 / 157 / 202, 7 / 79 / 178 / 202, 7 / 79 / 202, 7 / 138, 7 / 138 / 202, 7 / 157 / 178, 76, 76 / 79 / 138 / 178 / 202, 76 / 202, 79 / 138 / 157, 79 / 157 / 178, 79 / 178 / 202, 138, 138 / 157, 138 / 157 / 202, 138 / 178 / 202, 138 / 202, 157, 157 / 178, 157 / 202, or 202, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

85. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 84, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 7F, 7F / 79R / 157T / 178T, 7M, 7M / 76E / 79A / 202P, 7M / 76E / 79R, 7M / 76E / 79R / 138E, 7M / 76E / 79R / 157T, 7M / 76E / 138E, 7M / 76G / 79R / 202P, 7M / 76G / 138E / 157T / 178T / 202A, 7M / 79A / 178T / 202P, 7M / 79A / 202P, 7M / 79R, 7M / 79R / 138E / 202P, 7M / 79R / 157T / 202P, 7M / 138E, 7M / 138E / 202P, 7M / 157T / 178T, 76E, 76E / 79R / 138E / 178T / 202P, 76E / 202P, 79A / 138E / 157T, 79R / 157T / 178T, 79R / 178T / 202P, 138E, 138E / 157T, 138E / 157T / 202P, 138E / 178T / 202P, 138E / 202P, 157T, 157T / 178T, 157T / 202P, or 202A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 96286. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at ammo acid position 42, 43, 44, 67, 76, 78, 83, 118, 146, 147, 149, 150, 152, 153, 178, 193, 201, 202, or 203, 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 96287. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 86, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 42T, 43Q, 43S, 44L, 67L, 76A, 76G, 76E, 76R, 76T, 78E, 83A, 118A,118V, 146D, 147M, 147V, 149A, 149H, 149P, 149Q, 149S, 149T, 150E, 150F, 150P, 150V, 152T, 153D, 153Q, 178T, 193A, 193M, 193V, 201L, 201M, 201Q, 201S, 202K, 202P, 202R, 203A, 203F, 204H, 204L, or 204S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 962, or relative to the reference sequence corresponding to SEQ ID NO: 962.

88. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 31, 38, 46, 97, 104, 110, 116, 1 17, 120, 182, or 186, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

89. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 88, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 311, 38A, 46Q, 97V, 104L, 1 10G, 1 16E, 116L, 116R, 116S, 117G, 117K, 117M, 117V, 120S, 182G, 182P, or 186M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

90. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 42 / 43 / 76 / 78 / 178, 42 / 43 / 76 / 193, 42 / 76 / 78, 42 / 76 / 78 / 178, 42 / 76 / 78 / 193, 42 / 78, 43 / 76, 43 / 76 / 78, 43 / 76 / 78 / 178, 43 / 78, 43 / 178 / 193, 54 / 76 / 78, 76, 76 / 78, 76 / 78 / 178, 78 / 193, 149, 149 / 153, 149 / 153 / 201, 149 / 201, 153, 153 / 201, 178, 193, 201, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 1032.

91. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 90, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue diffcrcncc(s), or ammo acid rcsiduc(s) 42T / 43S / 76E / 78E / 178T, 42T / 43S / 76E / 193A, 42T / 76E / 78E, 42T / 76E / 78E / 193A, 42T / 76R / 78E / 178T, 42T / 78E, 43S / 76E, 43S / 76E / 78E / 178T, 43S / 76R / 78E, 43S / 78E, 43S / 178T / 193A, 54V / 76E / 78E, 76E / 78E, 76E / 78E / 178T, 76R, 78E / 193A, 149A / 153Q, 149Q, 149Q / 153Q, 149Q / 153Q / 201 S, 149Q / 201S, 149S / 201 S, 153D, 153Q, 153Q / 201 S, 178T, 193A, 201S, or 204L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1032, or relative to the reference sequence corresponding to SEQ ID NO: 103292. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 5, 6, 8, 9, 19, 27, 50, 51, 53, 56, 86, 156, or 199, or any combinationsthereof, wherein the amino aeid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

93. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 92, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 2K, 3G, 5M, 5N, 5P, 5Q, 5T, 5Y, 6G, 8V, 9Q, 19Q, 27R, 50 Y, 51Q, 51R, 53M, 53 Y, 56N, 86V, 156L, 199M, or 199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

94. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 67, 67 / 83, 67 / 83 / 116 / 122 / 193, 67 / 116, 67 / 1 16 / 149, 67 / 122 / 149, 67 / 149, 67 / 153, 67 / 178, 83 / 116 / 193, 83 / 149, 83 / 149 / 153 / 193, 83 / 149 / 178, 116, 116 / 153, 149, 149 / 153 / 193, 153, or 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

95. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 94, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 67L, 67E / 83A, 67L / 83A / 116L / 122V / 193V, 67L / 116E,67E / 116E / 149Q, 67L / 116L, 67E / 122V / 149Q, 67E / 149Q, 67L / 153D, 67L / 178T, 83A / 116L / 193V, 83A / 149Q, 83A / 149Q / 153D / 193V, 83A / 149Q / 178T, 116E / 153D, 116L, 149Q, 149Q / 153D / 193V, 153D, or 193 V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1236, or relative to the reference sequence corresponding to SEQ ID NO: 1236.

96. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 7, 18, 23, 25, 99, 102, 103, 104, 116, 124, 125, 126, 133, 146, 150, 153, 173, 177, 180, 181, 184, 193, 202, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1322, or relative to the reference sequence corresponding to SEQ ID NO: 1322.

97. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 96, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 7A, 7F, 7G, 7L, 7Q, 7S, 7V, 7W, 71, 7P, 18P, 18V, 23R, 25R, 25S, 99S, 102V, 103L, 104V, 116A, 124A, 1241, 124K, 124P, 124T, 124V, 125E, 125H, 126L, 133Y, 146M, 146N, 150M, 153Q, 173S, 173T, 177L, 177P, 177V, 180G, 180L, 181C, 181T, 181V, 184V, 1931, 193R, 202L, 202V, or 203A, or any combinations thereof, wherein the amino acid positions are relative to the referencesequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1322, or relative to the reference sequence corresponding to SEQ ID NO: 1322.

98. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 3 / 5, 3 / 5 / 8, 3 / 5 / 59, 3 / 8 / 54 / 193, 3 / 8 / 59, 3 / 54, 5, 5 / 8, 5 / 8 / 90, 5 / 193, 8, 8 / 53 / 54, 8 / 53 / 59 / 90 / 153 / 156, 8 / 54, 8 / 54 / 90, 8 / 59 / 153, 8 / 90, 8 / 90 / 193, 8 / 153, 8 / 156, 8 / 193, 53 / 54 / 59, 53 / 54 / 59 / 90 / 153 / 156, 53 / 54 / 90 / 153 / 156, 53 / 54 / 153 / 156, 53 / 59, 53 / 59 / 90, 54, 54 / 59 / 90 / 193, 54 / 90, 59, 59 / 90 / 153 / 156, 59 / 90 / 193, 59 / 193, 90, 90 / 193, 153, or 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

99. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 98, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 3G / 5P, 3G / 5P / 8V, 3G / 5P / 59G, 3G / 8V / 54Q / 193 V, 3G / 8V / 59G, 3G / 54Q, 5P, 5P / 8V, 5P / 8V / 90M, 5P / 193V, 8V, 8V / 53Y / 54Q, 8V / 53Y / 59G / 90M / 153D / 156L, 8V / 54Q, 8V / 54Q / 90M, 8V / 59G / 153D, 8V / 90M, 8V / 90M / 193V, 8V / 153D, 8V / 156L, 8V / 193V, 53Y / 54Q / 59G, 53Y / 54Q / 59G / 90M / 153D / 156L, 53Y / 54Q / 90M / 153D / 156L, 53Y / 54Q / 153D / 156L, 53Y / 59G, 53Y / 59G / 90M, 54Q, 54Q / 59G / 90M / 193V, 54Q / 90M, 59G, 59G / 90M / 153D / 156L, 59G / 90M / 193V, 59G / 193V, 90M, 90M / 193V, 153D, or 193 V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

100. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 17, 19, 26, 28, 29, 53, 56, 57, 67, 92, 93, 95, 98, 128, 130, 134, 139, 140, 147, 156, 157, 160, 162, 164, 174, 176, 199, or 201, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

101. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 100, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 17C, 19E, 19M, 19Q, 19R, 26S, 28A, 29L, 53G, 56H, 57L, 57S, 67M, 92T, 93G, 95G, 95H, 95S, 98Q, 128A, 128K, 128T, 130A, 130L, BOS, 130T, 134A, 134T, 139V, 140L, 147A, 147C, 1471, 147M, 147R, 147S, 147T, 147V, 156V, 157D, 157N, 157S, 160A, 162V, 164N, 164R, 164V, 174Y, 176L, 176M, 199A, 199F, 199G, 199L, 199S, 199V, 199 Y, or 20 IQ, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1364, or relative to the reference sequence corresponding to SEQ ID NO: 1364.

102. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residuedifference at amino acid position 16, 50, 51, 59, 73, 77, 78, 79, 117, 118, 120, 121, 185, 186, 188, 189, 190, or 195, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

103. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 102, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 16L, 16R, 50F, 51R, 59P, 59S, 73A, 77W, 78C, 78G, 78S, 79L, 79T, 117E, 117T, 117V, 118V, 120E, 120G, 120Y, 121R, 185D, 186E, 186N, 188F, 189R, 190C, 190V, 1951, or 195S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

104. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 53, 53 / 59 / 89 / 90 / 153, 53 / 90, 53 / 90 / 180, 53 / 95, 53 / 180, 95, 120, or 180, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

105. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 104, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 53 Y, 53Y / 59G / 89A / 90M / 153D, 53Y / 90M, 53Y / 90M / 180L, 53Y / 95A, 53Y / 180L, 95A, 120S, or 180L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1644, or relative to the reference sequence corresponding to SEQ ID NO: 1644.

106. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 8, 10, 103, 108, 116, 123, 124, 125, 132, 138, 141, 147, 149, 150, 170, 175, 178, 180, 183, 193, 202, 203, or 204, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

107. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 106, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 2A, 8L, 10M, 10P, 10Y, 103 V, 108V, 116A, 123R, 123S, 124A, 125D, 1321, 138T, 141A, 147A, 147F, 149G, 150A, 150G, 150Q, 170Q, 170V, 175E, 178T, 180T, 183L, 193C, 193L, 193M, 193R, 193S, 193T, 202A, 202G, 202L, 202M, 203E, 203Q, 203T, 204D, 204E, 204R, 204S, or 204V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequencecorresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to tire reference sequence corresponding to SEQ ID NO: 1716.

108. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 16 / 19 / 53, 16 / 19 / 95, 16 / 199, 53, 53 / 77, 53 / 77 / 95, 53 / 95, 53 / 95 / 134, 53 / 176 / 199, 77 / 95, 95, 156, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

109. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 108, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 16R / 19M / 53Y, 16R / 19M / 95H, 16R / 199F, 53Y, 53Y / 77W, 53Y / 77W / 95H, 53Y / 95H, 53Y / 95S, 53Y / 95S / 134A, 53Y / 176L / 199F, 77W / 95S, 95H, 156V, or 199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1716, or relative to the reference sequence corresponding to SEQ ID NO: 1716.

110. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 120, 140, 197, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.11 1. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 1 10, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 120S, 140Q, 197L, or 199Y, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

112. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 50, 50 / 53, 50 / 53 / 73 / 199, 50 / 53 / 77, 50 / 53 / 195, 50 / 73 / 77 / 189 / 199, 50 / 95 / 120 / 195 / 199, 53, 53 / 73 / 77, 53 / 73 / 77 / 189, 53 / 73 / 95 / 185, 53 / 73 / 95 / 195, 53 / 73 / 189, 53 / 73 / 195, 53 / 73 / 199, 53 / 95, 53 / 195, 73 / 77 / 189, 73 / 185 / 189, 77 / 95 / 120 / 189, 95, 95 / 185, 95 / 189 / 199, or 95 / 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

113. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 112, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residuedifference(s), or ammo acid residue(s) 50F, 50F / 53L, 50F / 53L / 73A / 199V, 50F / 53L / 77F, 5OF / 53L / 195I, 50F / 73A / 77F / 189R / 199V, 50F / 95S / 120G / 195I / 199V, 53L, 53L / 73A / 77F, 53L / 73A / 77F / 189R, 53E / 73A / 95A / 185D, 53L / 73A / 95A / 195I, 53E / 73A / 189R, 53L / 73A / 195I, 53L / 73A / 199V, 53E / 95A, 53L / 195I, 73A / 77F / 189R, 73A / 185D / 189R, 77F / 95S / 120G / 189R, 95S, 95S / 185D, 95S / 189R / 199V, or 95S / 199V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

114. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at ammo acid position 6, 7, 16, 28, 66, 105, 120, 122, 137, 139, 156, 165, 167, 195, 197, 198, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

115. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 92, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 6R, 6T, 7F, 7H, 7T, 7V, 7W, 16P, 28A, 66R, 105L, 120R, 122C, 122M, 137G, 139V, 156A, 165E, 167Q, 195W, 197S, 198W, or 199T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1834, or relative to the reference sequence corresponding to SEQ ID NO: 1834.

116. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 2 / 123 / 193 / 204, 2 / 170, 1 16, 116 / 170 / 178, 116 / 178, 116 / 193, 116 / 204, 123 / 170 / 204, 123 / 204, 170, 170 / 193, 193, 193 / 204, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

117. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 116, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 2A / 170V, 2R / 123R / 193T / 204S, 116A, 116A / 170Q / 178T, 116A / 178T, 116A / 193T, 116A / 204D, 116A / 204E, 123R / 170V / 204D, 123R / 204D, 170V, 170V / 193T, 193T, 193T / 204D, 193T / 204E, 204D, or 204S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

118. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 83, 101, 116, 171, 188, or 195, or any combinations thereof, wherein theamino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

119. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 1 18, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue 83R, 10 IF, 1 16Q, 17 IN, 188E, or 195E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

120. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 31, 106, 123, 124, 125, 126, 147, 150, or 157, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

121. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 120, wherein the ammo acid sequence of the engineered adenylyl-sulfate kmase comprises at least ammo acid residue difference, or ammo acid residue 3 IL, 106S, 123G, 124P, 124V, 125D, 126A, 126C, 147A, 147L, 147V, 150A, 150S, 157N, 157Q, or 157S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1946, or relative to the reference sequence corresponding to SEQ ID NO: 1946.

122. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 6, 6 / 7, 6 / 7 / 50, 6 / 7 / 95 / 150, 6 / 7 / 120 / 150, 6 / 50 / 150, 6 / 73 / 95 / 150, 6 / 120 / 150, 6 / 150, 7 / 50 / 95 / 116, 7 / 1 16 / 120 / 150, 50, 50 / 73 / 95, 50 / 95 / 120, 50 / 95 / 132, 50 / 116, 73, 73 / 95, 73 / 116 / 120, 73 / 150, 95, 95 / 1 16, 95 / 120, 95 / 132, 95 / 150, 116, 132, or 132 / 150, or any combinations thereof, combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

123. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 122, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 6T, 6T / 7F / 50F, 6T / 7F / 95S / 150A, 6T / 7H, 6T / 7H / 120R / 150A, 6T / 50F / 150A, 6T / 73A / 95S / 150A, 6T / 120G / 150A, 6T / 150A, 7F / 50F / 95S / 116A, 7F / 116A / 120G / 150A, 50F, 50F / 73A / 95S, 50F / 95S / 120R, 50F / 95S / 132I, 50F / 1 16A, 73A, 73A / 95S, 73A / 116A / 120G, 73A / 150A, 95S, 95S / 116A, 95S / 120G, 95S / 132I, 95S / 150A, 116A, 1321, or 132I / 150A, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

124. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 18, 19, 21, 51, 54, 57, 76, 78, 79, 83, 86, 91, 95, 101, 178, 180, or 181, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

125. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 124, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 18C, 18G, 18P, 19A, 19E, 21C, 51R, 54L, 54S, 57R, 76G, 78A, 79K, 79E, 79P, 79Q, 79T, 83Q, 83R, 86V, 91W, 95L, 95M, 101F, 178T, 180M, 181A, 181F, 181L, 181 Q, 181 S, 181V, or 181 W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 1954, or relative to the reference sequence corresponding to SEQ ID NO: 1954.

126. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue diffcrcncc(s) at amino acid position(s) 31, 31 / 73 / 95 / 150, 31 / 73 / 147, 31 / 83, 31 / 95 / 147 / 195, 31 / 95 / 150, 31 / 147 / 195, 73 / 83 / 95 / 147, 73 / 83 / 147 / 182, 73 / 124 / 147, 76 / 95 / 147 / 188, 83, 83 / 95, 83 / 95 / 147, 83 / 124, 83 / 124 / 150, 83 / 147, 83 / 150 / 182, 83 / 150 / 188, 83 / 182 / 188, 95, 95 / 150 / 182, 116 / 120 / 123, 116 / 120 / 123 / 147, 124, 126 / 147, 147, 147 / 182, 182, or 188, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058127. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 126, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 31L, 31E / 73A / 95S / 150A, 31L / 73A / 147V, 31E / 83R, 31E / 95S / 147V / 195E, 31E / 95S / 150A, 31L / 147V / 195E, 73A / 83R / 95S / 147V, 73A / 83R / 147V / 182A,73A / 124 V / 147 V, 76K / 95S / 147V / 188E, 83R, 83R / 95S, 83R / 95S / 147V, 83R / 124V, 83R / 124V / 150A, 83R / 147V, 83R / 150A / 182A, 83R / 150A / 188E, 83R / 182A / 188E, 95S, 95S / 150A / 182A, 116A / 120G / 123R, 116A / 120G / 123R / 147V, 124V, 126A / 147V, 147V, 147V / 182A, 182A, or 188E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

128. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at ammo acid position 16, 17, 18, 19, 50, 69, 76, 78, 79, 95, 117, 120, 121, 132, 156, 160, 176, 178, 179, 180, 181, 182, or 183, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

129. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 128, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 16R, 16S, 17R, 18A, 18C, 181, 18N, 18Q, 18S, 18T, 19M, 19Q, 50S, 69Q, 76G, 76Q, 76V, 76Y, 78S, 79E, 79K, 79L, 79T, 79V, 95A, 95L, 95T, 117L, 117M, 117T, 117V, 120G, 120L, 120R, 121V, 132C, 132L, 156L, 160A, 160E, 160G, 160P, 176L, 178M, 178T, 179G, 180L, 181D, 181L, 181 W, 182E, 182M, 182P, 182Y, 183L, or 183 V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2058, or relative to the reference sequence corresponding to SEQ ID NO: 2058.

130. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 51 / 73 / 76 / 83 / 95 / 181 / 182 / 195, 51 / 120 / 178, 73, 73 / 76 / 79 / 83 / 95 / 171 / 188, 73 / 76 / 95, 73 / 76 / 95 / 101 / 188, 73 / 95 / 171 / 195, 73 / 95 / 188, 76 / 79 / 95 / 171 / 188, 76 / 83 / 95, 83 / 95, 83 / 95 / 120 / 182 / 188 / 195, 95, 95 / 120 / 178, 95 / 120 / 188 / 195, or 178, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158.

131. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 130, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 51R / 73E / 76G / 83R / 95M / 181Q / 182A / 195E, 51R / 120G / 178T, 73E, 73E / 76G / 79P / 83R / 95S / 171N / 188E, 73E / 76G / 95M / 101F / 188E, 73E / 76K / 95M, 73E / 95M / 171N / 195E, 73E / 95M / 188E, 76G / 79P / 95M / 171N / 188E, 76G / 83R / 95S, 83R / 95M, 83R / 95M / 120G / 182A / 188E / 195E, 95M, 95M / 120G / 178T, 95M / 120G / 188E / 195E, or 178T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158.

132. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 43, 83, 90, 94, 96, 97, 101, 105, 107, 109, 160, 162, 163, or 165, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158.

133. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 132, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 43A, 83G, 83S, 90L, 90M, 90V, 94A, 96L, 97E, 97S, 97W, 101 V, 105V, 107M, 109S, 160S, 162V, 163N, 165D, or 165S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2158, or relative to the reference sequence corresponding to SEQ ID NO: 2158.

134. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 16 / 18 / 19 / 156 / 181 / 183 / 188, 18, 18 / 73 / 76 / 79 / 83 / 156 / 178 / 181, 18 / 156, 18 / 156 / 181 / 188, 83 / 156, 101 / 156, 120, 120 / 156, or 156 / 181 / 182, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2354, or relative to the reference sequence corresponding to SEQ ID NO: 2354.

135. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 134, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 16S / 18A / 19M / 156E / 181Q / 183V / 188E, 18A, 18A / 73E / 76G / 79L / 83R / 156L / 178T / 181Q, 18A / 156L, 18A / 156L / 181Q / 188E, 83R / 156L, 101F / 156L, 120G / 156L, 120R, or 156L / 181Q / 182A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2354, or relative to the reference sequence corresponding to SEQ ID NO: 2354.

136. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 56, 59, 116, 124, or 147, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2354, or relative to the reference sequence corresponding to SEQ ID NO: 2354.

137. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 136, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 56R, 56S, 59S, 116M, 124A, 124G, 124K, 124S, 147 A, or 147M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2354, or relative to the reference sequence corresponding to SEQ ID NO: 2354.

138. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue diffcrcncc(s) at ammo acid position(s) 90 / 101 / 105, 90 / 105 / 178 / 182, 96 / 101 / 105, 101, 101 / 105 / 178 / 181 / 195, 101 / 105 / 181 / 182, 101 / 105 / 181 / 195, 101 / 178 / 181 / 182, 105, 105 / 120, 105 / 120 / 178 / 181, 105 / 120 / 181, 105 / 120 / 195, 105 / 181 / 182, 105 / 195, 178 / 181, or 181, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2402, or relative to the reference sequence corresponding to SEQ ID NO: 2402.

139. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 138, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 90V / 101 V / 105V, 90V / 105V / 178T / 182E, 96L / 101 V / 105V, 101 V,101 V / 105V / 178T / 181D / 195E, 101 V / 105V / 181D / 182R, 101 V / 105V / 181L / 195E, 101 V / 178T / 181D / 182A, 105V, 105V / 120G, 105V / 120G / 178T / 181E, 105V / 120G / 181D, 105V / 120G / 181L, 105V / 120G / 195E,105V / 181D / 182E, 105V / 195E, 178T / 181D, or 181D, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2402, or relative to the reference sequence corresponding to SEQ ID NO: 2402.

140. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 2, 3, 5, 6, 7, 8, 50, 61, 70, 76, 78, 117, 126, 171, 201, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2402, or relative to the reference sequence corresponding to SEQ ID NO: 2402.

141. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 140, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or amino acid residue(s) 2G, 2P, 2R, 2S, 3G, 3R, 5K, 5R, 5T, 6D, 6E, 6G, 6R, 6S, 7G, 7E, 8L, 50A, 501, 50K, 50L, 50W, 61T, 70T, 76H, 76N, 76S, 78H, 78Q, 117K, 126D, 171A, 171N, 171T, 171V,20 IP, 204G, or 204K, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2402, or relative to the reference sequence corresponding to SEQ ID NO: 2402.

142. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 5 / 6 / 76 / 147 / 178, 5 / 50, 6 / 76 / 178 / 182 / 201, 6 / 124 / 147 / 178 / 182, 6 / 182, or 59 / 139 / 178, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2472, or relative to the reference sequence corresponding to SEQ ID NO: 2472.

143. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 142, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 5R / 6S / 76S / 147A / 178T, 5R / 50R, 6S / 76S / 178T / 182E / 201E, 6S / 124K / 147A / 178T / 182E, 6S / 182E, or 59S / 139V / 178T, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2472, or relative to the reference sequence corresponding to SEQ ID NO: 2472.

144. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 4, 27, 28, 29, 54, 58, 59, 67, 73, 90, 91, 93, 95, 138, 170, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2472, or relative to the reference sequence corresponding to SEQ ID NO: 2472.

145. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 144, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 4R, 27H, 27T, 28A, 29G, 54G, 54H, 54N, 54R, 58S, 59S, 67F, 73 V, 90T, 91 S, 93G, 93S, 93T, 95V, 138L, 170L, 170Q, 203L, or 203M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2472, or relative to the reference sequence corresponding to SEQ ID NO: 2472.

146. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 2 / 5 / 50, 2 / 5 / 50 / 139, 5 / 50, 50 / 76 / 196 / 201, 50 / 126 / 139, 50 / 126 / 139 / 171 / 178, 50 / 126 / 171 / 196, 50 / 139 / 196, 50 / 171 / 201, 76, 76 / 171, 126 / 139 / 196, 139 / 171, 139 / 196 / 201, 171, or 201, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2558, or relative to the reference sequence corresponding to SEQ ID NO: 2558.

147. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 146, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue diffcrcncc(s), or ammo acid rcsiduc(s) 2R / 5T / 50I, 2R / 5T / 50R / 139V, 5T / 50E, 501 / 126D / 139V / 171N / 178S, 50I / 126D / 171N / 196D, 501 / 139 V / 196D, 50L / 171N / 201E, 50R / 76N / 196D / 201E, 50R / 126D / 139V, 76S, 76S / 171N, 126D / 139V / 196D, 139V / 171N, 139V / 196D / 201E, 171N, or 20 IE, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2558, or relative to the reference sequence corresponding to SEQ ID NO: 2558.

148. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 6, 27, 31, 42, 47, 51, 53, 58, 59, 67, 70, 73, 76, 78, 80, 83, 91, 92, 93, 94, 96, 139, 147, 166, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2558, or relative to the reference sequence corresponding to SEQ ID NO: 2558.

149. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 148, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue(s) 6R, 27Q, 3 IL, 42S, 47G, 5 IL, 5 IT, 51 Y, 53V, 58F, 58V, 58W, 59S, 67Q, 70L, 70Y, 73V, 76L, 78M, 80A, 83S, 91 S, 91T, 92F, 93S, 93V, 94M, 961, 139V, 147C, 147V, 166F, 204L, 204S, or 204W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2558, or relative to the reference sequence corresponding to SEQ ID NO: 2558150. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 2 / 5 / 27, 2 / 5 / 138, 4, 4 / 5, 4 / 5 / 27, 4 / 5 / 27 / 138 / 170, 5 / 27, 27, 27 / 138 / 170,138, or 170, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2624, or relative to the reference sequence corresponding to SEQ ID NO: 2624.

151. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 150, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 2R / 5R / 27H, 2R / 5R / 138L, 4R, 4R / 5R, 4R / 5R / 27H, 4R / 5R / 27T / 138L / 170Q, 5T / 27H, 27H, 27H / 138L / 170Q, 138L, or 170Q, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 2624, or relative to the reference sequence corresponding to SEQ ID NO: 2624.

152. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at ammo acid position 18, 25, 45, 103, 104, 1 12, 124, 157, 158, 160, 191, 192, 193, 196, 197, 198, 199, 200, 201, 202, or 203, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2624, or relative to the reference sequence corresponding to SEQ ID NO: 2624.

153. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 152, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 18L, 18M, 18S, 25A, 45S, 103 V, 104L, 104V, 112L, 124L, 124R, 124S, 157G, 1571, 158N, 160V, 191F, 191V, 191W, 192E, 192S, 193L, 193R, 193W, 196G, 196T, 196W, 197D, 197Q, 197 V, 198E, 199S, 2001, 200T, 201 T, 202R, 202S, 202T, 203A, or 203L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2624, or relative to the reference sequence corresponding to SEQ ID NO: 2624.

154. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 27 / 42 / 94, 27 / 80, 27 / 93, 27 / 204, 31 / 94, 42 / 47, or 67 / 94, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2730, or relative to the reference sequence corresponding to SEQ ID NO: 2730.

155. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 154, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 27Q / 42S / 94M, 27Q / 80A, 27Q / 93S, 27Q / 204W, 31L / 94M, 42S / 47G, or 67 Q / 94M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2730, or relative to the reference sequence corresponding to SEQ ID NO: 2730.

156. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 15, 17, 27, 45, 46, 52, 55, 56, 59, 61, 65, 67, 70, 77, 86, 89, 97, 100, 118, 121, 139, 183, 191, 193, 194, 199, 201, or 202, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2730, or relative to the reference sequence corresponding to SEQ ID NO: 2730.

157. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 156, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 151, 17Q, 27G, 27R, 45S, 46N, 521, 55C, 55S, 56Q, 56R, 59G, 59S, 59T, 611, 65E, 67M, 70D, 70T, 77H, 86V, 89E, 97 A, 100A, 118M, 121C, 121G, 121M, 139C, 183L, 191F, 193S, 194M, 199S, 199T, 201F, 201H, 2011, 201M, 201S, 202M, 202S, or 202T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2730, or relative to the reference sequence corresponding to SEQ ID NO: 2730.

158. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue diffcrcncc(s) at amino acid position(s) 54 / 78, 54 / 90, 54 / 93, 90 / 91 / 204, 90 / 204, 93, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2824, or relative to the reference sequence corresponding to SEQ ID NO: 2824.

159. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 158, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 54T / 78M, 54T / 90T, 54T / 93S, 90T / 91T / 204W, 90T / 204W, 93S, or 204W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2824, or relative to the reference sequence corresponding to SEQ ID NO: 2824.

160. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 51, 76, 79, 83, 94, 103, 104, 105, 113, 117, 120, 124, 141, 170, 181, 184, 188, 189, 192, 195, 197, 198, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2824, or relative to the reference sequence corresponding to SEQ ID NO: 2824.

161. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 160, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 51L, 51 S, 51T, 76A, 79L, 79P, 79T, 83E, 83Q, 83T, 94H, 103S, 103V, 104L, 105L, 113A, 117S, 117T, 120Q, 124L, 141T, 170P, 181F, 181G, 181M, 181S, 184A, 184G, 184R, 188Q, 189G, 189R, 192G, 192M, 192S, 192V, 195H, 1951, 195L, 195R, 197G, 197W, 198Y, 203G, 204F,204L, or 204R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2824, or relative to the reference sequence corresponding to SEQ ID NO: 2824.

162. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 45 / 121, 45 / 121 / 197, 45 / 191, 90, 90 / 100 / 121 / 197 / 204, 90 / 100 / 121 / 204, 90 / 121 / 191, 90 / 191, 100 / 121, 104 / 191 / 197, 121 / 191 / 204, 121 / 197 / 204, 191 / 197, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2924, or relative to the reference sequence corresponding to SEQ ID NO: 2924.

163. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 163, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 45S / 121M, 45S / 121M / 197Q, 45S / 191 V, 90T, 90T / 100A / 121M / 197Q / 204E, 90T / 100A / 121M / 204E, 90T / 121M / 191 V, 90T / 191V, 100A / 121M, 104L / 191 V / 197Q, 121M / 191 V / 204E, 121M / 197Q / 204E, 191V / 197Q, or 197Q, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2924, or relative to the reference sequence corresponding to SEQ ID NO: 2924.

164. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 4, 5, 6, 7, 13, 21, 22, 25, 27, 31, 43, 90, 121, 157, 158, 182, 183, 186, 191, or 193, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2924, or relative to the reference sequence corresponding to SEQ ID NO: 2924.

165. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 165, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 4N, 5G, 6P, 7P, 13S, 21G, 22G, 25Q, 27T, 31L, 43S, 90V, 121G, 121 Q, 121R, 121S, 121W, 157 A, 158N, 182P, 182S, 183A, 183P, 186A, 186V, 191R, 193G, 193L, 193S, 193V, or 193 Y, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 2924, or relative to the reference sequence corresponding to SEQ ID NO: 2924.

166. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue diffcrcncc(s) at amino acid position(s) 79, 181, or 181 / 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 3034, or relative to the reference sequence corresponding to SEQ ID NO: 3034.

167. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 166, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 79T, 181M, or 181M / 197Q, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 3034, or relative to the reference sequence corresponding to SEQ ID NO: 3034.

168. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at ammo acid position 18, 51, 73, 79, 83, 94, 112, 130, 141, 164, 171, 176, 181, 199, 202, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3034, or relative to the reference sequence corresponding to SEQ ID NO: 3034.

169. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 168, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 18T, 18V, 51R, 73M, 79L, 79V, 83V, 94T, 94V, 112H, 1301, 141A, 141S, 164A, 171 Q, 1761, 181L, 199V, 202G, 204R, or 204S, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3034, or relative to the reference sequence corresponding to SEQ ID NO: 3034.

170. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 4, 4 / 90, 4 / 157, 25, 79 / 182, 90, or 182, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3116, or relative to the reference sequence corresponding to SEQ ID NO: 3116.

171. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 170, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 4N, 4N / 90T, 4N / 157A, 25Q, 79P / 182P, 90T, or 182P, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO : 3116, or relative to the reference sequence corresponding to SEQ ID NO: 3116.

172. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 17, 23, 27, 31, 59, 97, 121, 147, 157, 172, 191, or 201, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 31 16, or relative to the reference sequence corresponding to SEQ ID NO: 3116.

173. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 172, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 17R, 231, 23M, 27R, 31L, 59S, 97 A, 121F, 147L, 147M, 157H, 157S, 172A, 191M, 201L, 201M, 201N, 201P, or 201R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3116, or relative to the reference sequence corresponding to SEQ ID NO: 3116.

174. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at amino acid position(s) 4 / 141, 4 / 141 / 147, 4 / 141 / 176, 4 / 147 / 176, 141 / 147, 141 / 176, or 147, or any combinations thereof, wherein the amino acid positions arc relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3168, or relative to the reference sequence corresponding to SEQ ID NO: 3168.

175. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 174, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or ammo acid residue(s) 4K / 141A, 4K / 141A / 147V, 4K / 141 S, 4K / 141 S / 147V, 4K / 141S / 176I, 4K / 147V / 176I, 141A / 147V, 141 S / 147V, 141 S / 176I, or 147V, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO: 3168, or relative to the reference sequence corresponding to SEQ ID NO: 3168.

176. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference at amino acid position 4, 54, 69, 73, 76, 86, 102, 116, 117, 123, 126, 128, 170, 193, 195, 196, 197, 203, or 204, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1 -200 of SEQ ID NO : 3168, or relative to the reference sequence corresponding to SEQ ID NO: 3168.

177. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 176, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference, or ammo acid residue 4M, 54T, 69T, 73S, 76G, 86L, 102V, 116A, 116H, 116L, 117Q, 123R, 126A, 126M, 126S, 128V, 170G, 170N, 193H, 193L, 193Q, 195L, 195R, 195T, 196M, 197G, 197R, 197S, 197 V, 197W, 203L, 204A, or 204T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3168, or relative to the reference sequence corresponding to SEQ ID NO: 3168.

178. The engineered adenylyl-sulfate kinase of any one of Claims 26 and 43-45, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s) at ammo acid position(s) 59 / 97 / 157, 59 / 139 / 191, 59 / 157 / 191, 59 / 176, 97, 97 / 139 / 157, 97 / 176, 97 / 191, 139, 139 / 191, 157 / 176, 164, 182, or 191, or any combinations thereof, wherein the amino acidpositions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3228, or relative to the reference sequence corresponding to SEQ ID NO: 3228.

179. The engineered adenylyl-sulfate kinase of any one of Claims 26, 43-45, and 179, wherein the amino acid sequence of the engineered adenylyl-sulfate kinase comprises at least amino acid residue difference(s), or amino acid residue(s) 59S / 97A / 157H, 59S / 139C / 191M, 59S / 157H / 191M, 59S / 176L 97 A, 97A / 139C / 157H, 97A / 176I, 97A / 191M, 139C, 139C / 191M, 157H / 176I, 1641, 182K, or 191M, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 1-200 of SEQ ID NO: 3228, or relative to the reference sequence corresponding to SEQ ID NO: 3228.

180. The engineered adenylate-sulfate kinase of Claim 26, wherein the engineered adenylyl- sulfate kinase comprises an amino acid sequence comprising amino acid residues 1-200 of an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364-3324, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1364-3324181. The engineered adenylate-sulfate kmase of Claim 26, wherein the engineered adenylyl- sulfate kinase comprises an amino acid sequence comprising amino acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or comprising SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

182. The engineered adenylyl-sulfate kinase of any one of Claims 1-181, wherein the engineered adenylyl-sulfate kinase has activity in the conversion of a natural or modified NDP to a nucleoside diphosphate with an additional phosphate at the 3 ’ position of the sugar.

183. The engineered adenylyl-sulfate kinase of any one of Claims 1-182, comprising at least one improved property, as compared to a wild-type or reference adenylyl-sulfate kinase having an amino acid sequence corresponding to ammo acid residues 1-200 of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228, or an amino acid sequence corresponding to SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

184. The engineered adenylyl-sulfate kinase of Claim 183, wherein the improved property comprises increased activity on natural substrates, increased activity on non-natural substrates, increased activity on 2 '-fluoro modified nucleotides, increased activity on 2’-O-methyl modified nucleotides, increased thermostability, increased substrate promiscuity, increased soluble protein expression, decreased product inhibition and / or decreased byproduct formation, as compared to a wild-type or reference adenylyl-sulfate kmase of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364,1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

185. The engineered adenylyl-sulfate kinase of any one of Claims 1-183, wherein the adenylyl- sulfate kinase comprises increased soluble protein expression, as compared to a wild-type or reference adenylyl-sulfate kinase of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

186. The engineered adenylyl-sulfate kmase of any one of Claims 1-183, wherein the adenylyl- sulfate kinase comprises increased activity in the conversion of a natural or modified NDP to a nucleoside diphosphate with an additional phosphate at the 3 ’ position of the sugar, as compared to a wild-type or reference adenylyl-sulfate kmase of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

187. The engineered adenylyl-sulfate kmase of any one of Claims 1-183, wherein the adenylyl- sulfate kinase comprises increased activity at an elevated temperature in the conversion of a natural or modified NDP to a nucleoside diphosphate with an additional phosphate at the 3 ’ position of the sugar, as compared to a wild-type or reference adenylyl-sulfate kinase of SEQ ID NO: 14, 28, 154, 240, 414, 426, 474, 522, 732, 848, 880, 962, 1032, 1236, 1322, 1364, 1644, 1834, 1946, 1954, 2058, 2158, 2354, 2402, 2472, 2558, 2624, 2730, 2824, 2924, 3034, 3116, 3168, or 3228.

188. The engineered adenylyl-sulfate kinase of any one of Claims 1-187, wherein the adenylyl- sulfate kinase is purified.

189. The engineered adenylyl-sulfate kinase of any one of Claims 1-187, wherein the engineered adenylyl-sulfate kinase is immobilized on a support medium.

190. A recombinant polynucleotide comprising a polynucleotide sequence encoding at least one engineered adenylyl-sulfate kinase of any one of Claims 1 -187191. The recombinant polynucleotide of Claim 190, comprising a polynucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 1-600 of SEQ ID NO: 13, 27, 153, 239, 413, 425, 473, 521, 731, 847, 879, 961, 1031, 1235, 1321, 1363, 1643, 1833, 1945, 1953, 2057, 2157, 2353, 2401, 2471, 2557, 2623, 2729, 2823, 2923, 3033, 3115, 3167, or 3227, or corresponding to SEQ ID NO: 13, 27, 153, 239, 413, 425, 473, 521, 731, 847, 879, 961, 1031, 1235, 1321 , 1363, 1643, 1833, 1945, 1953, 2057, 2157, 2353, 2401, 2471, 2557, 2623, 2729, 2823, 2923, 3033, 3115, 3167, or 3227, wherein the polynucleotide sequence encodes an adenylyl-sulfate kinase.

192. The recombinant polynucleotide of Claim 190, wherein the polynucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 1-600 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17- 257, 271-1357, and 1363-3323, or corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272-1358, and 1363-3323, wherein the polynucleotide sequence encodes an adenylyl-sulfate kinase.

193. The recombinant polynucleotide of any one of Claims 190-192, wherein the polynucleotide sequence is codon-optimized.

194. The recombinant polynucleotide of Claim 190, wherein polynucleotide sequence comprises nucleotide residues 1-600 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 17-257, 271-1357, and 1363- 3323, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 18-258, 272- 1358, and 1363-3323.

195. An expression vector comprising at least one recombinant polynucleotide of any one of Claims 190-194.

196. The expression vector of Claim 195, comprising at least a control sequence.

197. A host cell comprising at least an expression vector of Claim 195 or 196198. The host cell of Claim 197, comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.

199. A method of producing an engineered adenylyl-sulfate kinase polypeptide in a host cell comprising culturing a host cell of Claim 197 or 198, under suitable culture conditions, such that at least one engineered adenylyl-sulfate kinase is produced.

200. The method of Claim 199, further comprising recovering at least one adenylyl-sulfate kinase from the culture and / or host cells.

201. The method of Claim 199 or 200, further comprising purifying the at least one engineered adenylyl-sulfate kinase202. A composition comprising at least one engineered adenylyl-sulfate kinase of any one of Claims 1-189.

203. The composition of Claim 202, further comprising an NDP substrate.

204. The composition of Claim 202 or 203, further comprising an NTP co-substrate.

205. The composition of any one of Claims 202-204, further comprising an acetate kinase.

206. The composition of Claim 203 or 204, wherein the NDP substrate or NTP co-substrate, or both, comprise at least one modification of the 2’ position of the sugar moiety, the nucleobase, or the phosphate moieties(s).

207. A method of producing a NDP with a phosphate group at the 3 ’ position of the sugar moiety, comprising contacting an adenylyl-sulfate kinase enzyme with an NDP substrate under suitable reaction conditions such that an NDP with a phosphate group at the 3 ’ position of the sugar is produced.

208. The method of Claim 207, wherein the adenylyl-sulfate kinase enzyme is a CysC enzyme or homolog thereof.

209. The method of Claim 208, wherein the adenyl-sulfate kinase comprises an engineered adenyl-sulfate kinase of any one of Claims 1-188.

210. The method of any one of Claims 207-209, wherein the NDP comprises a modification at the 2’ position of the sugar moiety, a modification at the nucleobase, and / or modification of the phosphate chain.

211. The method of any one of Claims 207-210, wherein the suitable reaction conditions comprise a NTP co-substrate.

212. The method of Claim 211, w herein the NTP co-substrate has the same nucleotide structure as the NDP substrate.

213. The method Claim 211 or 212 further comprising regenerating or recycling the NTP co- substrate using an NTP regeneration system.

214. The method of Claim 213, wherein the NTP regeneration system for regenerating or recycling the NTP co-substrate comprises an acetate kinase and acetyl phosphate.

215. The method of Claim 214, further comprising use of a pyruvate oxidase enzyme to generate acetyl phosphate for use by the NTP regeneration system.

216. The method of any one of Claims 207-215, wherein the adenylyl-sulfate kinase enzyme is immobilized on a support medium.

217. A method of producing an NDP with a phosphate group at the 3’ position of the sugar, comprising contacting the adenylyl-sulfate kinase enzyme of any one of Claims 1-188 with an NDP under reaction conditions that cause a phosphate group to be added to an NDP at the 3 ’ position of a sugar of the NDP.

218. The method of Claim 217, wherein the NDP has a 2’ modification.

219. The method of Claim 207, 217, or 218, wherein the reaction conditions comprise NTP and are substantially free of 2’-modified NTP.

220. The method of any one of Claims 207 and 217-219, wherein the eontacting eomprises combining the adenylyl-sulfate kinase enzyme with the NDP

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