Engineered DNA ligase variants

Abstract

The present disclosure relates to engineered DNA ligase polypeptides and compositions thereof, and polynucleotides encoding the engineered DNA ligase polypeptides. The disclosure also provides methods of using the engineered DNA ligase polypeptides, or compositions thereof, in diagnostics and as molecular biology tools.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 481,158, filed January 23, 2023, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing, table or computer program

[0004] The sequence listing, filed simultaneously with the specification, with the file name CX9-235WO2_ST26.xml, a creation date of January 22, 2024, and a file size of 2,686,547 bytes, is part of this specification and is incorporated herein by reference. Technical Field

[0005] The present disclosure provides engineered DNA ligase polypeptides and compositions thereof, polynucleotides encoding the engineered DNA ligase polypeptides, and methods of using the engineered DNA ligases, such as for the synthesis of polynucleotides, molecular biology tools, and diagnostic applications. Background Art

[0006] DNA ligase is an enzyme family that catalyzes the covalent joining of DNA molecules by forming a phosphodiester bond between the 3' hydroxyl end of a DNA substrate and the 5' phosphorylated end of another DNA substrate. DNA ligase participates in maintaining genomic integrity by repairing single-strand breaks in duplex DNA during replication, repair, and recombination. Some ligases, such as T4 bacteriophage and eukaryotic DNA ligase, use ATP, while other ligases, such as E. coli ligase, use NAD as a cofactor. DNA ligase can join dsDNA fragments with blunt ends or ends with complementary single-stranded overhangs with complete base pairing. It has been found that DNA ligase has great practicality in molecular biology and diagnostic applications, including restriction enzyme cloning, adapter connection for cloning / sequencing, SNP or sequence analysis, and assembling DNA fragments from multiple smaller fragments.

[0007] The prototype DNA ligase comes from bacteriophage T4, which is the most commonly used ligase in molecular biology and diagnostic applications. T4 DNA ligase can connect the cohesive or "sticky" ends of DNA, oligonucleotides, and some RNA and RNA-DNA hybrids. It can also efficiently connect blunt-ended DNA. T4 DNA ligase uses ATP as a cofactor. T4 DNA ligase is usually active between 4°C and 37°C, but loses activity at higher temperatures. T4 DNA ligase is also sensitive to buffer additives such as monovalent salts, which inhibit activity, particularly end-joining activity. In addition, T4 DNA ligase prefers the sequences of the last and penultimate bases of the ligation site. Therefore, although T4 DNA ligase has become an important tool for connecting DNA molecules in research and diagnostic applications, it is ideal to provide a ligase that is easier and more efficient for connecting DNA substrates. Summary of the Invention

[0008] The present disclosure provides engineered DNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered DNA ligase polypeptides. The present disclosure also provides methods for ligating polynucleotides using the engineered DNA ligase polypeptides and compositions thereof.

[0009] In one aspect, the present disclosure provides an engineered DNA ligase or a functional fragment thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2 and an even numbered SEQ ID NO among SEQ ID NO: 40 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO among SEQ ID NO: 2 and 40 to 1184, wherein the amino acid sequence comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to residues One or more substitutions of the reference sequence of NO: 2, 62, 138, 318, 722 or 938.

[0010] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 2, 62, 138, 318, 722 or 938, wherein the amino acid sequence comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: One or more substitutions of the reference sequence of NO: 2, 62, 138, 318, 722 or 938.

[0011] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0012] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0013] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0014] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the amino acid sequence at amino acid positions 11, 12, 13, 14, 18, 30, 31, 33, 34, 36, 37, 44, 50, 56, 59, 60, 61, 63, 67, 68, 69, 71, 73, 74, 76, 77, 82, 88, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 3, 117, 125, 128, 130, 132, 138, 139, 148, 149, 150, 155, 156, 159, 161, 162, 164, 165, 177, 186, 188, 189, 190, 191, 195, 196, 197, 198, 201, 205, 207, 208, 212, 220, 226, 228, 230, 231, 232, 233, 235, 237, 238 9, 240, 242, 251, 254, 258, 263, 264, 266, 267, 269, 271, 273, 277, 278, 282, 283, 284, 286, 288, 289, 290, 294, 295, 297, 300, 301, 305, 306, 308, 309, 317, 323, 328, 334, 337, 339, 349, 355, 356, 357, 358, 359 9, 360, 362, 364, 367, 370, 372, 374, 375, 378, 379, 380, 381, 382, ​​384, 386, 387, 388, 389, 390, 392, 396, 397, 404, 405, 408, 414, 415, 416, 417, 418, 419, 421, 422, 423 or 428, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0015] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or amino acid residue 11D, 12A / I, 13G / R, 14G / S / T / V, 18D / N / S, 30C / H / S, 31R, 33M / R / V, 34L / R, 36T / Y, 37G / L / N / S, 44S, 50G / I / S / T, 56P, 59E, 60Y, 61T / V, 63F / R, 67R, 68A / M / S / V / Y, 69T, 71G / L / P / R, 73C / K / P / T / V / W, 74S, 76F / G / H / L / N / R, 77D, 82R, 88V, 95A / L / R / V, 96A / G / T / V, 97G, 99 G / I, 100V, 101R, 102G / K / L / S, 103V, 104K, 105K / S / T, 106L / S / V, 110R, 112M , 113A / T, 117G / S / V / Y, 125R / T, 128C, 130T, 132R, 138L / R, 139T, 148P, 149P, 150C / F / T, 155R, 156C, 159Q, 161R / V, 162W, 164A / R, 165K, 177G, 186A / C / E / H / L / M / R / T / V, 188A, 189C / T, 190R, 191T, 195R, 196E / V, 197R, 198A / D / K / L / N / R / V / W, 201L / S, 205E / G / K, 207L, 208D / F / H, 212F / G / M / S / W, 220V, 226D / E / Q / S / V, 228E / I / M / S, 230L / M, 231P, 232R, 233G / T / W, 235W, 237L / M / R / S / V / Y , 239M / N / P / Q / S / T / V / W, 240E / G / K / Q / R / S / Y, 242P / Q / T, 251L, 254G / S, 258L / S / V, 263G / L / Q / T, 264A / C, 266M / T, 267D / W / Y, 269L, 271A / G / N / S, 273A / G / S , 277Q / R, 278E, 282G / L / M / T / V / Y, 283A / G / K / L / M / R / S / V, 284D, 286F / L / S, 2 88I, 289A / L / S / V, 290L, 294L, 295K, 297W, 300G / T, 301F / L, 305K, 306I / K / S / V, 308K / L / S, 309G / R, 317Q, 323S, 328R, 334L / R, 337G / L / M / P / R / S, 339Y, 34 9E, 355S, 356A / V / W, 357H / K / P / R / S / V, 358C, 359N / R, 360H / M / P, 362G, 363R,364R, 367C / L, 370C / G, 372N / Q, 374A / S, 375W, 378T, 379A / G / P, 380T, 381K / R, 382V, 384C / V, 386F, 387G, 388K / Y, 389K / L / Q / R, 390E, 392C / I / K / L / R / S, 396C / H, 397K / L / M, 404S, 405I, 408C / V, 414A / L / Q / R / T / V, 415A / C / E / H / I / K / L / V, 416K, 417D / G / L, 418A / G / I / L / M / P / S / T, 419G, 421R, 422N, 423R / T or 428F / R / S, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0016] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises a substitution or set of substitutions at least at amino acid position 233, 317, 191, 288, 207, 149, 251, 205, 269, 164, 36, 428, 105 / 132, or 105, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0017] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least one substitution listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of the engineered DNA ligase variants listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to SEQ ID NO: 2.

[0019] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising at least a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0020] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

[0021] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184.

[0022] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

[0023] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

[0024] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the amino acid sequence at amino acid positions 11, 12, 13, 14, 18, 30, 31, 33, 34, 36, 37, 44, 50, 56, 59, 60, 61, 63, 67, 68, 69, 71, 73, 74, 76, 77, 82, 88, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 3, 117, 125, 128, 130, 132, 138, 139, 148, 149, 150, 155, 156, 159, 161, 162, 164, 165, 177, 186, 188, 189, 190, 191, 195, 196, 197, 198, 201, 205, 207, 208, 212, 220, 226, 228, 230, 231, 232, 233, 235, 237, 238 9, 240, 242, 251, 254, 258, 263, 264, 266, 267, 269, 271, 273, 277, 278, 282, 283, 284, 286, 288, 289, 290, 294, 295, 297, 300, 301, 305, 306, 308, 309, 317, 323, 328, 334, 337, 339, 349, 355, 356, 357, 358, 359 8, 318, 722, or 938, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0025] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or amino acid residue 11D, 12A / I, 13G / R, 14G / S / T / V, 18D / N / S, 30C / H / S, 31R, 33M / R / V, 34L / R, 36T / Y, 37G / L / N / S, 44S, 50G / I / S / T, 56P, 59E, 60Y, 61T / V, 63F / R, 67R, 68A / M / S / V / Y, 69T, 71G / L / P / R, 73C / K / P / T / V / W, 74S, 76F / G / H / L / N / R, 77D, 82R, 88V, 95A / L / R / V, 96A / G / T / V, 97G, 99 G / I, 100V, 101R, 102G / K / L / S, 103V, 104K, 105K / S / T, 106L / S / V, 110R, 112M , 113A / T, 117G / S / V / Y, 125R / T, 128C, 130T, 132R, 138L / R, 139T, 148P, 149P, 150C / F / T, 155R, 156C, 159Q, 161R / V, 162W, 164A / R, 165K, 177G, 186A / C / E / H / L / M / R / T / V, 188A, 189C / T, 190R, 191T, 195R, 196E / V, 197R, 198A / D / K / L / N / R / V / W, 201L / S, 205E / G / K, 207L, 208D / F / H, 212F / G / M / S / W, 220V, 226D / E / Q / S / V, 228E / I / M / S, 230L / M, 231P, 232R, 233G / T / W, 235W, 237L / M / R / S / V / Y, 239M / N / P / Q / S / T / V / W, 240E / G / K / Q / R / S / Y, 242P / Q / T / V, 251L, 254G / S, 258 L / S / V, 263G / L / Q / T, 264A / C, 266M / T, 267D / W / Y, 269L, 271A / G / N / S, 273A / G / S, 277Q / R, 278E, 282G / L / M / T / V / Y, 283A / F / G / K / L / M / R / S / V, 284D, 286F / L / S / W, 288I, 289A / L / S / V, 290L, 294L, 295K, 297W, 300G / T, 301F / L, 305K, 306I / K / S / V, 308K / L / S, 309G, 317T / Q, 323S, 328R, 334L / R, 337G / L / M / P / R / S, 33 9Y, 349E, 355S, 356A / V / W, 357H / K / P / R / S / V, 358C, 359N / R, 360H / M / P, 362G,364R, 367C / L, 370C / G, 372N / Q, 374A / S, 375W, 378T, 379A / G / P, 380T, 381K / R, 382V, 3 84C / V, 386F, 387G, 388K / Y, 389K / L / Q / R, 390E, 392C / I / K / L / R / S, 396C / H, 397K / L / M, 4 04S, 405I, 408C / V, 414A / L / Q / R / S / T / V, 415A / C / E / H / I / K / L / V, 416K, 417D / G / L, 418A / G / I / K / L / M / P / S / T, 419G, 421R, 422N, 423R / T, or 428E / F / R / S, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0026] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 63, 242, 283, 286, 317, 414, 418, or 428, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0027] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or amino acid residue 63R, 242Q, 283L, 286S, 317Q, 414Q, 418S, or 428R, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0028] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to a reference sequence corresponding to SEQ ID NO: 62, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to the reference sequence corresponding to SEQ ID NO: 62.

[0029] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 68 to 312, or to a reference sequence corresponding to an even numbered SEQ ID NO: 68 to 312, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to the reference sequence corresponding to SEQ ID NO: 62.

[0030] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 196, 242, 337, 33, 277, 30, 359, 283, 415, 387, 379, 205, 186, 389, 102, 164, 301, 375, 267, 380, 254, 317, 77 / 139 / 317 / 417, 105 / 317 / 417, 317 / 349 / : 62, 144, 73, 89, 125, 136, 182, 226, 362, 386, 105, 230, 418, 370, 297, 237, 428, 362, 233, 235, 148, 100, 97, 382, ​​or 358, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or relative to the reference sequence corresponding to SEQ ID NO: 62.

[0031] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to a reference sequence corresponding to SEQ ID NO: 138, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to a reference sequence corresponding to SEQ ID NO: 138.

[0032] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 314 to 458, or to a reference sequence corresponding to an even numbered SEQ ID NO: 314 to 458, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to the reference sequence corresponding to SEQ ID NO: 138.

[0033] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 242 / 283 / 286 / 359 / 418, 283 / 286, 283 / 286 / 418, 186 / 242 / 283 / 286 / 418, 205 / 286 / 359, 283, 242 / 286 / 418, 186 / 205 / 242, 283 / 286 / 359, 277 / 286 / 359 / 418, 186 / 205 / 283 / 286 / 359, 242 / 277 / 418, 242 / 283 / 286 / 418, 112 / 196 / 389、286、283 / 286 / 359 / 418、242 / 359 / 418、186 / 283、186 / 359 / 418、205 / 242 / 418、186 / 205 / 242 / 283 / 286 / 359 / 418、205 / 359 / 418、186 / 205 / 283 / 286 / 418、186 / 283 / 359、186 / 242、186 / 242 / 359、283 / 359 / 418、277 / 418、186 / 188 / 283、186 / 286 / 4 18, 186 / 242 / 286 / 359 / 418, 418, 186 / 242 / 283 / 286 / 359 / 418, 186 / 277 / 359 / 418, 242 / 283 / 286, 205 / 418, 30 / 297, 205 / 242 / 286 / 359 / 418, 186 / 205 / 359 / 418, 359 / 418, 186, 230, 33 / 297, 186 / 205, 186 / 283 / 359 / 418, 186 / 418, 205 / 242 / 283 / 359 / 418, 33 / 3 96 / 242 / 283 / 286 / 359 / 418, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or relative to the reference sequence corresponding to SEQ ID NO: 138.

[0034] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to a reference sequence corresponding to SEQ ID NO: 318, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to the reference sequence corresponding to SEQ ID NO: 318.

[0035] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 460 to 936, or to a reference sequence corresponding to an even numbered SEQ ID NO: 460 to 936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to the reference sequence corresponding to SEQ ID NO: 318.

[0036] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 363, 63, 389, 381, 197, 359, 102, 165, 388, 414, 337, 164, 416, 101, 415, 423, 364, 73, 50, 71, 388 / 419, 357, 396, 68, 76, 14, 271, 360, 266, 208, 74, 263, 264, 13, 378, 372, 300, 294, 290, 397, 95, 258, 161, 21 2, 198, 138, 18, 404, 273, 117, 240, 69, 278, 289, 82, 328, 61 / 186 / 417, 186 / 370 / 417, 267, 61 / 370, 186 / 267 / 370 / 417, 61 / 186, 370 / 417, 417, 61 / 370 / 382, 61 / 186 / 267 / 370 / 417, 61, 267 / 370 / 417, 267 / 370, 61 / 417, 61 / 186 / 237 / 267 / 370, 61 / 237 / 370 / 417, 370, 186 / 370, 61 / 186 / 267 / 417, 61 / 186 / 370 / 382, 370 / 382 / 417, 61 / 186 / 370, 237 / 267 / 370 / 417, 61 / 186 / 382, 61 / 267, 61 / 267 / 417, 61 / 237 / 267 / 382, 186 / 370 / 382, 237 / 267 / 370, 61 / 186 / 267 / 370, 186 / 237 / 267 / 370, 61 / 186 / 267, 390, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or relative to the reference sequence corresponding to SEQ ID NO: 318.

[0037] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to a reference sequence corresponding to SEQ ID NO: 722, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to the reference sequence corresponding to SEQ ID NO: 722.

[0038] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 938 to 1098, or to a reference sequence corresponding to an even numbered SEQ ID NO: 938 to 1098, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to the reference sequence corresponding to SEQ ID NO: 722.

[0039] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 63, 63 / 96 / 370, 389, 13 / 267 / 363 / 389, 13 / 186 / 389, 50 / 267 / 363 / 370 / 389, 363 / 370, 96 / 370, 61 / 63 / 212, 11 / 305, 11, 242 / 283 / 286 / 317 / 414 / 418, 323, 334, 3 : 39, 356, 384, 408, 67, 392, 104, 355, 159, 155, 367, 31, 231, 36, 150, 239, 103, 125, 228, 37, 189, 177, 422, 128, 220, 130, 56, 190, 156, 232, 423, 34, 99, 59, 60, 421 or 195, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or relative to the reference sequence corresponding to SEQ ID NO: 722.

[0040] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to a reference sequence corresponding to SEQ ID NO: 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to the reference sequence corresponding to SEQ ID NO: 938.

[0041] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 1100 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO: 1100 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to the reference sequence corresponding to SEQ ID NO: 938.

[0042] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 308 / 357 / 390, 74 / 76 / 201 / 308 / 357, 61 / 74 / 76 / 186 / 201 / 308 / 309 / 357 / 390, 14 / 201 / 240 / 289 / 357, 308 / 415, 76 / 357 / 396, 263 / 308 / 396 、61 / 76 / 96 / 240 / 308 / 309、14 / 306 / 415、14 / 73 / 106 / 415、12 / 14 / 258 / 263 / 289 / 308 / 309 / 396、74 / 76 / 117 / 309 / 357、14 / 258 / 263 / 357 / 396、14 / 96 / 106 / 306、14 / 106、12 / 14 / 308 / 3 09, 14 / 357 / 390, 14 / 117 / 258 / 309 / 357, 390, 240 / 273 / 357 / 390, 61 / 76 / 186 / 201 / 308 / 309, 14 / 396, 309, 14, 106 / 306 / 308, 14 / 240 / 306 / 308, 12 / 14 / 186 / 357, 309 / 390, 14 / 306, or a substitution at or set of substitutions at 14 / 76 / 308, 117 / 208 / 258 / 263 / 289 / 308 / 309, 14 / 73 / 106, 76 / 208 / 263, 357, 14 / 308, 263, 76, 14 / 300 / 308 / 415, 240, 33 / 357 / 390, 14 / 76 / 273 or 74, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or relative to the reference sequence corresponding to SEQ ID NO: 938.

[0043] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least one substitution listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0044] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0045] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising at least a substitution or set of substitutions provided in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0046] In some embodiments, the engineered DNA ligase comprises an amino acid sequence comprising residues 12 to 437 of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, or a sequence comprising an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2.

[0047] In some embodiments, the engineered DNA ligase comprises an amino acid sequence comprising residues 12 to 437 of an even numbered SEQ ID NO among SEQ ID NOs: 40 to 1184, or an amino acid sequence comprising an even numbered SEQ ID NO among SEQ ID NOs: 40 to 1184, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0048] In some embodiments, the engineered DNA ligase comprises an amino acid sequence comprising residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, 938, or 1108, or an amino acid sequence comprising SEQ ID NO: 62, 138, 318, 722, 938, or 1108, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.

[0049] In some embodiments, the engineered DNA ligase has DNA ligase activity. In some embodiments, the engineered DNA ligase has DNA ligase activity and is characterized in that it has at least one improved property compared to a reference DNA ligase. In some embodiments, the improved property of the engineered DNA ligase is selected from i) increased activity, ii) increased stability, iii) increased thermal stability, iv) increased product yield, v) increased solubility, vi) reduced sequence preference, and vii) insensitivity or reduced sensitivity to input DNA concentration, or any combination of i), ii), iii), iv), v), vi), and vii). In some embodiments, the improved property of the engineered DNA ligase is compared to a reference DNA ligase having a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722, or 938, or a sequence corresponding to SEQ ID NO: 2, 62, 138, 318, 722, or 938. In some embodiments, the improved properties of the engineered DNA ligase are compared to a reference DNA ligase having a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2. In some embodiments, the reference DNA ligase is wild-type T4 DNA ligase.

[0050] In some further embodiments, the engineered DNA ligase is purified.In some embodiments, the engineered DNA ligase is provided in solution, as a lyophilizate, or immobilized on a substrate such as a solid substrate, a porous substrate, a membrane, or a particle.

[0051] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding an engineered DNA ligase disclosed herein.

[0052] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1311 of SEQ ID NO: 1, 61, 137, 317, 721 or 937, or a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 61, 137, 317, 721 or 937, wherein the recombinant polynucleotide encodes an engineered DNA ligase.

[0053] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1311 of an odd-numbered SEQ ID NO: 39 to 1183, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 39 to 1183, wherein the recombinant polynucleotide encodes an engineered DNA ligase.

[0054] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide encoding the engineered DNA ligase is codon-optimized for expression in an organism or a cell type thereof, such as a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

[0055] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1311 of SEQ ID NO. 1, 61, 137, 317, 721 or 937, or a polynucleotide sequence comprising SEQ ID NO: 1, 61, 137, 317, 721 or 937.

[0056] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1311 of an odd-numbered SEQ ID NO among SEQ ID NOs: 39 to 1183, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO among SEQ ID NOs: 39 to 1183.

[0057] In a further aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding an engineered DNA ligase as provided herein. In some embodiments, the recombinant polynucleotide of the expression vector is operably linked to a control sequence. In some embodiments, the control sequence includes a promoter, particularly a heterologous promoter.

[0058] On the other hand, the present disclosure also provides a host cell comprising the recombinant polynucleotide or expression vector provided herein. In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

[0059] In a further aspect, the present disclosure provides a method for producing an engineered DNA ligase polypeptide, the method comprising culturing a host cell as described herein under suitable culture conditions such that at least one engineered DNA ligase is produced. In some embodiments, the method further comprises recovering or isolating the engineered DNA ligase from the culture medium and / or host cell. In some embodiments, the method further comprises purifying the engineered DNA ligase.

[0060] On the other hand, the present disclosure provides a composition comprising at least one engineered DNA ligase disclosed herein. In some embodiments, the composition comprises at least a buffer. In some embodiments, the composition further comprises a nucleotide substrate (e.g., ATP) and / or one or more DNA ligase substrates. In some embodiments, the DNA ligase substrate comprises an adapter or a joint.

[0061] In a further aspect, the present disclosure provides a method for ligating at least a first DNA strand and a second DNA strand, the method comprising contacting the first DNA strand and the second DNA strand with an engineered DNA ligase as described herein in the presence of a nucleotide substrate under conditions suitable for ligating the first DNA strand to the second DNA strand, wherein the first DNA strand comprises a ligatable 5' end and the second DNA strand comprises a 3' end ligatable to the 5' end of the first DNA strand. In some embodiments, the 3' end of the second DNA strand is a 3'-hydroxyl group and the 5' end of the first DNA strand is a 5'-phosphate group.

[0062] In some embodiments, the method further comprises a third DNA or polynucleotide chain, wherein the first DNA chain and the second DNA chain are hybridized adjacent to each other on the third DNA or polynucleotide chain to position the 5' end of the first DNA chain adjacent to the 3' end of the second DNA chain. In some embodiments, the third DNA or polynucleotide chain is continuous with the first DNA chain or the second DNA chain. In some embodiments, the third DNA chain is continuous with the first DNA chain and the second DNA chain to form a single continuous DNA ligase substrate.

[0063] In some embodiments of the method, the first DNA strand hybridizes with the third DNA strand to form a first dsDNA substrate, and the second DNA strand hybridizes with the fourth DNA strand to form a second dsDNA substrate. In some embodiments, the first dsDNA substrate comprises a 5' end of the blunt end of the first DNA strand, and the second dsDNA substrate comprises a 3' end of the blunt end of the second DNA strand. In some embodiments of the method, the first dsDNA substrate comprises an overhang on at least one end of the first dsDNA substrate, and the second dsDNA substrate comprises an overhang on at least one end of the second dsDNA substrate, wherein the overhang on the first dsDNA substrate and the overhang on the second dsDNA substrate are complementary and capable of hybridizing to each other and forming one or more nicks that can be ligated.

[0064] In a further aspect, the present disclosure also provides a kit comprising at least one engineered DNA ligase disclosed herein. In some embodiments, the kit further comprises one or more of a buffer, a nucleotide substrate, a reducing agent, one or more DNA ligase substrates, and / or a ligation enhancer. DETAILED DESCRIPTION

[0065] The present disclosure provides engineered DNA ligase polypeptides and compositions thereof, as well as polynucleotides encoding engineered DNA ligase polypeptides. The present disclosure also provides methods for using engineered DNA ligase polypeptides and compositions thereof for molecular biology, diagnostics, and other purposes. In some embodiments, the engineered DNA ligase polypeptides exhibit, among other things, increased activity, increased stability, increased thermostability, increased solubility, and / or decreased sequence preference.

[0066] Abbreviations and definitions

[0067] Unless otherwise defined, 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 the invention belongs. Generally, the nomenclature used herein and the experimental procedures in 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.

[0068] Although any suitable method and material similar to or equal to those described herein can be used in the practice of the present invention, exemplary methods and materials are described herein. It should be understood that the present invention is not limited to the described ad hoc methods, protocols and reagents, as these can be changed according to the circumstances in which they are used by those skilled in the art. Therefore, the terms to be defined hereinafter are more fully described by reference to the application as a whole.

[0069] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0070] As used herein, the term "comprising" and its cognates are used in their inclusive sense (ie, equivalent to the term "including" and its corresponding cognates).

[0071] It will also be understood that where the description of an embodiment uses the term "comprising" and its cognates, the embodiment may also be described using the language "consisting essentially of" or "consisting of.

[0072] In addition, numerical ranges are inclusive of the numbers defining the range. Thus, each numerical range disclosed herein is intended to encompass each narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein. It is also intended that each maximum (or minimum) numerical limitation disclosed herein includes each lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0073] As used herein, the term "about" means an acceptable error for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some cases, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0074] Additionally, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the application as a whole.Accordingly, the terms defined below are more fully defined by reference to the application as a whole.

[0075] The "EC" number refers to the enzyme nomenclature of the International Joint Nomenclature Committee on Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0076] "ATCC" refers to the American Type Culture Collection, whose biological collection includes genes and strains.

[0077] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0078] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (eg, glycosylation or phosphorylation).

[0079] In this article, amino acids are represented by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations for the amino acids used in the genetic code are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). When three-letter abbreviations are used, unless specifically preceded by "L" or "D" or clear from the context in which the abbreviation is used, the amino acid may be referred to with respect to the α-carbon (C α ) is in the L-configuration or the D-configuration. For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When single-letter abbreviations are used, uppercase letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is conventionally presented in an amino (N) to carboxyl (C) direction.

[0080] "Fusion protein" and "chimeric protein" and "chimera" refer to a hybrid protein produced by joining two or more polynucleotides that originally encoded separate proteins. In some embodiments, the fusion protein is produced by recombinant technology.

[0081] "DNA ligase" refers to an enzyme that covalently joins the 5' phosphate terminus ("donor") and the 3' hydroxyl terminus ("acceptor") of DNA to each other. DNA ligases can be divided into two families based on cofactor requirements: ATP-dependent ligases and NAD+-dependent ligases. DNA ligases of eukaryotic and archaeal organisms are generally ATP-dependent. DNA ligases of eubacterial origin are generally NAD+-dependent. DNA ligases include enzymes in the general class of EC 6.5.1.

[0082] "Polynucleotide," "nucleic acid," or "oligonucleotide" is used herein to refer to a polymer comprising at least two nucleotides, wherein the nucleotide is a deoxyribonucleotide or a ribonucleotide or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise specified, abbreviated nucleosides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated individually or as a whole as ribonucleosides or 2'-deoxyribonucleosides. When a polynucleotide, nucleic acid, or oligonucleotide sequence is presented as a string of single-letter abbreviations, the sequence is conventionally presented in a 5' to 3' direction and phosphates are not indicated. The term "DNA" refers to deoxyribonucleic acid. The term "RNA" refers to ribonucleic acid. A polynucleotide or nucleic acid may be single-stranded or double-stranded, or may include single-stranded and double-stranded regions.

[0083] "Duplex" and "ds" refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule composed of two single-stranded polynucleotide molecules that are complementary in their sequence (A to T or U, C to G), arranged in an antiparallel 5' to 3' orientation, and held together by hydrogen bonds between the nucleobases (i.e., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]).

[0084] "Complementary" is used herein to describe the structural relationship between nucleotide bases that can form base pairs with each other. For example, purine nucleotide bases on a polynucleotide that are complementary to pyrimidine nucleotide bases on a polynucleotide can base pair by forming hydrogen bonds with each other. Complementary nucleotide bases can base pair by Watson / Crick base pairing or in any other manner except forming a stable duplex or other nucleic acid structure.

[0085] "Watson / Crick base pairing" refers to the pattern of specific nucleobase pairs and analogs that are held together by sequence-specific hydrogen bonds, eg, A pairs with T or U and G pairs with C.

[0086] "Annealing" or "hybridization" refers to the base pairing interaction of one nucleobase polymer (e.g., polynucleotides and oligonucleotides) with another nucleobase polymer, which results in the formation of a duplex structure, a triplex structure, or a quaternary structure. Annealing or hybridization can occur through Watson / Crick base pairing interactions, but can be mediated by other hydrogen bonding interactions, such as Hoostein base pairing. In some embodiments, the nucleobase polymer that anneals or hybridizes to another nucleobase polymer is a single nucleobase polymer, while in other embodiments, the nucleobase polymer is a separate nucleobase polymer.

[0087] "Engineered," "recombinant," and "non-naturally occurring" when used with respect to cells, polynucleotides, or polypeptides refer to materials that have been modified in a way that does not originally exist in nature or that correspond to a natural or native form of the material, or are identical to naturally occurring cells, nucleic acids, or polypeptides, but are produced or derived from synthetic materials and / or through manipulation using recombinant techniques.

[0088] "Wild-type" and "naturally occurring" refer to a form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and has not been intentionally modified by human manipulation.

[0089] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0090] " sequence identity percentage " refers to the comparison between polynucleotide or polypeptide, and is determined by comparing the sequences of two best alignments on a comparison window, wherein compared with the reference sequence for the best alignment of two sequences, the part of the polynucleotide or polypeptide sequence in the comparison window can include addition or deletion (that is, room). Percentage can be calculated by determining the number of positions of the identical nucleic acid base or amino acid residue present in the two sequences to obtain the number of matching positions, the number of matching positions divided by the total number of positions in the comparison window and multiplying the result by 100 to obtain sequence identity percentage. Alternatively, percentage can be calculated as follows: determine the number of positions in which identical nucleic acid base or amino acid residue or nucleic acid base or amino acid residue aligns with the room to produce the number of matching positions, the number of matching positions divided by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used for comparing two sequences. Optimal alignment of sequences for comparison can be performed, for example, 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 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 suitable for determining percentages of sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms (see, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short fields of length "W" in the query sequence that match or meet a certain positive threshold score "T" when aligned with the same length field of the database sequence. T is referred to as the neighboring field score threshold (Altschul et al., supra). These initial neighboring field hits serve as seeds for initiating searches to find longer HSPs containing them. Subsequently, field hits are extended bidirectionally along each sequence as long as the cumulative alignment score can be increased.For nucleotide sequences, the cumulative score is calculated using the parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hit in each direction stops when the cumulative alignment score decreases by an amount X from its maximum achieved value; when the cumulative score reaches or falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when 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 the default word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and compares both chains. For amino acid sequences, the BLASTP program uses the default word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:109-15). Exemplary determinations of sequence alignments and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0091] "Reference sequence" refers to a sequence of a definition used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) comprise a similar sequence (i.e., a portion of a complete sequence) between the two sequences, and (2) can further comprise a different sequence between the two sequences, the sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides on a "comparison window" to identify and compare local regions of sequence similarity. In certain embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more variations in the primary sequence. For example, the phrase “the reference sequence corresponding to SEQ ID NO:2 with aspartic acid at the residue corresponding to X11” (or “the reference sequence corresponding to SEQ ID NO:2 with aspartic acid at the residue corresponding to position 11”) refers to the reference sequence in which the corresponding residue at position X11 in SEQ ID NO:2 (e.g., glycine) has been changed to aspartic acid.

[0092] A "comparison window" refers to a conceptual segment of consecutive nucleotide positions or amino acid residues in which a sequence can be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window may contain 20% or fewer additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15 to 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100, or longer.

[0093] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference," and "relative to," refer to the numbering of the residues of a given amino acid or polynucleotide sequence when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbers or residue positions of a given polymer are designated with respect to a reference sequence, rather than being designated by the actual numerical position of the residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered DNA ligase, can be aligned with a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, the residues in the given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned, despite the presence of gaps.

[0094] "Mutation" refers to a change in a nucleic acid sequence. In certain embodiments, a mutation results in a change in the encoded polypeptide sequence (i.e., compared to the original sequence without the mutation). In certain embodiments, a mutation comprises a substitution so that a different amino acid is produced. In some alternative embodiments, a mutation comprises an addition so that an amino acid is added (e.g., inserted) into the original polypeptide sequence. In some further embodiments, a mutation comprises a deletion so that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may exist in a given sequence. In certain embodiments, a "substitution" comprises an amino acid deletion and, if present, may be represented by a "-" symbol.

[0095] "Amino acid difference" or "residue difference" refers to the change of the amino acid residue at one position of a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X14 compared to SEQ ID NO:2" (or "residue difference at position 14 compared to SEQ ID NO:2") refers to the change of the amino acid residue at the polypeptide position corresponding to SEQ ID NO:2, position 14. Therefore, if the reference polypeptide SEQ ID NO:2 has lysine at position 14, "residue difference at position X14 compared to SEQ ID NO:2" refers to the amino acid substitution of any residue except lysine at the position of the polypeptide corresponding to SEQ ID NO:2, position 14. In some examples herein, the specific amino acid residue difference at a position is indicated as "XnY," where "Xn" specifies the corresponding residue and position (as described above) of the reference polypeptide, and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue different from the reference polypeptide). In some cases (e.g., in the tables of the examples), the present invention also provides specific amino acid differences represented by the conventional notation "AnB," where A is the single-letter identifier for 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 for the residue substitution in the sequence of the engineered polypeptide. In some embodiments, an amino acid difference, e.g., a substitution, is represented by the abbreviation "nB" without the identifier for the residue in the reference sequence. In some embodiments, the phrase "amino acid residue nB" indicates the presence of an amino acid residue in the engineered polypeptide that may or may not be a substitution in the context of a reference polypeptide or amino acid sequence.

[0096] In some cases, the polypeptides of the present disclosure may include one or more amino acid residue differences relative to a reference sequence, as indicated by a list of designated positions at which changes have been made relative to the reference sequence. In some embodiments, where more than one amino acid can be used at a particular residue position in a polypeptide, the various amino acid residues that can be used are separated by " / " (e.g., X12A / X12I, X12A / I, or 129A / I).

[0097] "Amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, the substitution set comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, the substitution set refers to a collection of amino acid substitutions present in any of the variant DNA ligase polypeptides listed in any of the tables in the Examples. In these substitution sets, individual substitutions are separated by semicolons (";"; e.g., L105K; T317Q) or slashes (" / "; e.g., L105K / T317Q or L105K / 317Q).

[0098] "Conservative amino acid substitutions" refer to the replacement of a residue with a different residue having a similar side chain, and thus generally include the replacement of an amino acid in a polypeptide with an amino acid from the same or similar defined class of amino acids. By way of example and not limitation, an amino acid having an aliphatic side chain can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain can be substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain can be substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain can be substituted with another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain can be substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic amino acid or a hydrophilic amino acid can be substituted with another hydrophobic amino acid or a hydrophilic amino acid, respectively.

[0099] A "non-conservative substitution" refers to a substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, defined groups and affect: (a) the structure of the peptide backbone in the region of the substitution (e.g., substitution of glycine with proline), (b) the charge or hydrophobicity, and / or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0100] "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include the removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids making up the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or retaining the improved properties of the engineered DNA ligase. Deletion can involve internal portions and / or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous. In some embodiments, deletion is indicated by a "-" and can be present in a substitution set.

[0101] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids to a reference polypeptide. The insertion may be internal to the polypeptide or at the carboxyl or amino terminus. As used herein, insertion includes fusion proteins as are known in the art. The insertion may be a continuous stretch of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0102] "Functional fragment" and "biologically active fragment" are used interchangeably herein and refer to polypeptides having amino-terminal and / or carboxyl-terminal deletions and / or internal deletions, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., the full-length engineered DNA ligase of the present disclosure) and retains substantially all of the activity of the full-length polypeptide.

[0103] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). Engineered DNA ligase polypeptides can be present intracellularly, in cell culture medium, or prepared in various forms, such as lysates or isolated preparations.

[0104] "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 typically a substantially pure composition when the target species constitutes at least about 50% of the macromolecular species present, by mole or % weight. Typically, a substantially pure DNA ligase composition will contain about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more, by mole or % weight, of all macromolecular species present in the composition. In some embodiments, the target species is purified to substantial homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant DNA ligase polypeptide is a substantially pure polypeptide composition.

[0105] "Improved enzymatic properties" refers to an engineered DNA ligase polypeptide that exhibits improvements in any enzymatic property compared to a reference DNA ligase polypeptide (such as a wild-type DNA ligase polypeptide or another engineered DNA ligase polypeptide). Improved properties may include, but are not limited to, properties such as increased protein expression, increased thermal activity, increased thermal stability, increased stability, increased enzymatic activity, increased substrate specificity and / or affinity, increased substrate range, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved solvent stability, increased solubility, and increased inhibitor resistance / tolerance.

[0106] "Increased enzymatic activity" and "enhanced catalytic activity" refer to improved properties of an engineered DNA ligase polypeptide, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) and / or an increase in the percent conversion of substrate to product (e.g., percent conversion of a starting amount of substrate to product over a specified time period using a specified amount of DNA ligase) compared to a reference DNA ligase (e.g., wild-type DNA ligase and / or another engineered DNA ligase). Exemplary methods for determining enzymatic activity are provided in the Examples. Any property related to enzymatic activity may be affected, including classical enzyme properties such as kinase activity and kinase activity. m 、V max or k cat , which changes can result in increased enzymatic activity. The improvement in enzymatic activity can be from about 1.1 times the enzymatic activity of the corresponding wild-type enzyme to about 1.5 times, 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times or more enzymatic activity than a naturally occurring DNA ligase or another engineered DNA ligase from which the DNA ligase polypeptide is derived.

[0107] "Hybridization stringency" refers to hybridization conditions in nucleic acid hybridization, such as washing conditions. Typically, hybridization reactions are performed under conditions of lower stringency, followed by different but higher stringency washes (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term "moderate stringency hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids having about 60% identity, preferably about 75% identity, about 85% identity to target DNA, and greater than about 90% identity to target polynucleotides. Exemplary moderate stringency conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5× Denhart solution, 5× SSPE, 0.2% SDS, followed by washing at 42°C in 0.2× SSPE, 0.2% SDS. "High stringency hybridization" generally refers to a hybridization with a polynucleotide sequence as defined, determined under solution conditions at a thermal melting temperature, T m Conditions that differ by about 10°C or less. In some embodiments, high stringency conditions refer to conditions that only allow hybridization of those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if the hybrid is unstable in 0.018M NaCl at 65°C, it is unstable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridizing in conditions equivalent to 42°C, 50% formamide, 5× Denhart solution, 5× SSPE, 0.2% SDS, followed by washing in 0.1× SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridizing in 5× SSC containing 0.1% (w:v) SDS at 65°C and washing in 0.1× SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderate stringency conditions, are described in the references cited above.

[0108] " Codon optimization " refers to that the codons of the polynucleotides encoding proteins are changed to those codons preferentially used in a particular organism so that the encoded protein is effectively expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that the codon usage of a particular organism is non-random and has a preference for specific codon triplets. With respect to the aggregated protein coding region of a given gene, a gene with a common function or ancestral origin, a highly expressed protein contrast low copy number protein, and the genome of an organism, this codon usage preference may be higher. In certain embodiments, the polynucleotide encoding DNA ligase is codon optimized to obtain optimal production from the host organism selected for expression.

[0109] "Control sequences" herein refer to all components that are necessary or advantageous for the expression of the polynucleotides and / or polypeptides 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 sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an initiation sequence, and a transcription terminator. At a minimum, control sequences include promoters and transcription and translation termination signals. In some embodiments, control sequences may be provided with linkers for the purpose of introducing specific restriction sites that facilitate connection of the control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0110] "Operably linked" refers to 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, and in some embodiments, regulates the expression of the encoded polypeptide of interest.

[0111] "Promoter" or "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 comprises transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in the selected host cell, including mutants, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0112] "Suitable reaction conditions" refer to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvent, cofactors, etc.) under which the DNA ligase polypeptides of the present disclosure are able to convert a polynucleotide substrate into the desired ligated product polynucleotide. Exemplary "suitable reaction conditions" are provided herein (see Examples).

[0113] "Product" in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of a DNA ligase polypeptide on a substrate.

[0114] "Culturing" refers to growing a population of cells under suitable conditions using any suitable medium (eg, liquid, gel, or solid).

[0115] "Vector" refers to a recombinant construct for introducing a polynucleotide of interest into a cell. In some embodiments, a vector is an expression vector operably linked to a suitable control sequence capable of affecting expression of the polynucleotide or a polypeptide encoded therein in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a polynucleotide (e.g., a transgene) to drive expression in a host cell, and in some embodiments, further comprises a transcription terminator sequence.

[0116] "Expression" includes any step involved in the production of the polypeptide of interest, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from the cell.

[0117] "Produce" refers to the production of proteins and / or other compounds by a cell. The term is intended to include any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from the cell.

[0118] "Heterologous" or "recombinant" refers to the relationship between two or more nucleic acid or polypeptide sequences (eg, promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and not related in nature.

[0119] "Host cell" and "host strain" refer to a suitable host for an expression vector comprising a polynucleotide as provided herein (e.g., a polynucleotide sequence encoding at least one DNA ligase variant). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0120] Engineered DNA ligase polypeptide

[0121] In one aspect, the present disclosure provides DNA ligases, including engineered DNA ligase polypeptide variants, having DNA ligase activity and characterized by improved properties compared to naturally occurring wild-type DNA ligases. In some embodiments, DNA ligases and engineered DNA ligase polypeptide variants can be used to ligate polynucleotide substrates, particularly DNA substrates. In some embodiments, engineered DNA ligases can be prepared and used as non-fusion polypeptides or fusion polypeptides.

[0122] In some embodiments, the engineered DNA ligase or a functional fragment thereof comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NOs: 2 and even numbered SEQ ID NOs among SEQ ID NOs: 2 and 40 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NOs among SEQ ID NOs: 2 and 40 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NOs: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NOs: 2, 62, 138, 318, 722 or 938.

[0123] In some embodiments, the engineered DNA ligase or a functional fragment thereof comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 2, 62, 138, 318, 722 or 938, wherein the amino acid sequence comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: One or more substitutions of the reference sequence of NO: 2, 62, 138, 318, 722 or 938.

[0124] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0125] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0126] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0127] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the amino acid sequence at amino acid positions 11, 12, 13, 14, 18, 30, 31, 33, 34, 36, 37, 44, 50, 56, 59, 60, 61, 63, 67, 68, 69, 71, 73, 74, 76, 77, 82, 88, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 3, 117, 125, 128, 130, 132, 138, 139, 148, 149, 150, 155, 156, 159, 161, 162, 164, 165, 177, 186, 188, 189, 190, 191, 195, 196, 197, 198, 201, 205, 207, 208, 212, 220, 226, 228, 230, 231, 232, 233, 235, 237, 238 9, 240, 242, 251, 254, 258, 263, 264, 266, 267, 269, 271, 273, 277, 278, 282, 283, 284, 286, 288, 289, 290, 294, 295, 297, 300, 301, 305, 306, 308, 309, 317, 323, 328, 334, 337, 339, 349, 355, 356, 357, 358, 359 9, 360, 362, 364, 367, 370, 372, 374, 375, 378, 379, 380, 381, 382, ​​384, 386, 387, 388, 389, 390, 392, 396, 397, 404, 405, 408, 414, 415, 416, 417, 418, 419, 421, 422, 423 or 428, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0128] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or amino acid residue 11D, 12A / I, 13G / R, 14G / S / T / V, 18D / N / S, 30C / H / S, 31R, 33M / R / V, 34L / R, 36T / Y, 37G / L / N / S, 44S, 50G / I / S / T, 56P, 59E, 60Y, 61T / V, 63F / R, 67R, 68A / M / S / V / Y, 69T, 71G / L / P / R, 73C / K / P / T / V / W, 74S, 76F / G / H / L / N / R, 77D, 82R, 88V, 95A / L / R / V, 96A / G / T / V, 97G, 99 G / I, 100V, 101R, 102G / K / L / S, 103V, 104K, 105K / S / T, 106L / S / V, 110R, 112M , 113A / T, 117G / S / V / Y, 125R / T, 128C, 130T, 132R, 138L / R, 139T, 148P, 149P, 150C / F / T, 155R, 156C, 159Q, 161R / V, 162W, 164A / R, 165K, 177G, 186A / C / E / H / L / M / R / T / V, 188A, 189C / T, 190R, 191T, 195R, 196E / V, 197R, 198A / D / K / L / N / R / V / W, 201L / S, 205E / G / K, 207L, 208D / F / H, 212F / G / M / S / W, 220V, 226D / E / Q / S / V, 228E / I / M / S, 230L / M, 231P, 232R, 233G / T / W, 235W, 237L / M / R / S / V / Y , 239M / N / P / Q / S / T / V / W, 240E / G / K / Q / R / S / Y, 242P / Q / T, 251L, 254G / S, 258L / S / V, 263G / L / Q / T, 264A / C, 266M / T, 267D / W / Y, 269L, 271A / G / N / S, 273A / G / S , 277Q / R, 278E, 282G / L / M / T / V / Y, 283A / G / K / L / M / R / S / V, 284D, 286F / L / S, 2 88I, 289A / L / S / V, 290L, 294L, 295K, 297W, 300G / T, 301F / L, 305K, 306I / K / S / V, 308K / L / S, 309G / R, 317Q, 323S, 328R, 334L / R, 337G / L / M / P / R / S, 339Y, 34 9E, 355S, 356A / V / W, 357H / K / P / R / S / V, 358C, 359N / R, 360H / M / P, 362G, 363R,364R, 367C / L, 370C / G, 372N / Q, 374A / S, 375W, 378T, 379A / G / P, 380T, 381K / R, 382V, 384C / V, 386F, 387G, 388K / Y, 389K / L / Q / R, 390E, 392C / I / K / L / R / S, 396C / H, 397K / L / M, 404S, 405I, 408C / V, 414A / L / Q / R / T / V, 415A / C / E / H / I / K / L / V, 416K, 417D / G / L, 418A / G / I / L / M / P / S / T, 419G, 421R, 422N, 423R / T or 428F / R / S, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0129] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitutions G11D, M12A / I, T13G / R, K14G / S / T / V, Q18D / N / S, T30C / H / S, S31R, T33M / R / V, A34L / R, E36T / Y, D37G / L / N / S, D44S, E50G / I / S / T, Y56P, D59E, L60Y, I61T / V, G63F / R, K67R, I68A / M / S / V / Y, K69T, K71G / L / P / R, L73C / K / P / T / V / W, P74S, D76F / G / H / L / N / R, Y77D, D82R, L88V, Y95A / L / R / V, N96A / G / T / V, R97G, L99G / I, T100V, G101R, N102G / K / L / S, A103V, A1 04K, L105K / S / T, D106L / S / V, V110R, L112M, S113A / T, Q117G / S / V / Y, K125R / T , Q128C, D130T, K132R, K138L / R, S139T, I148P, A149P, E150C / F / T, L155R, A 156C, L159Q, K161R / V, Y162W, K164A / R, R165K, C177G, K186A / C / E / H / L / M / R / T / V, V188A, L189C / T, K190R, S191T, K195R, I196E / V, I197R, T198A / D / K / L / N / R / V / W, T201L / S, Q205E / G / K, I207L, A208D / F / H, V212F / G / M / S / W, I220V, K 226D / E / Q / S / V, A228E / I / M / S, V230L / M, Q231P, K232R, S233G / T / W, F235W, K 237L / M / R / S / V / Y, D239M / N / P / Q / S / T / V / W, D240E / G / K / Q / R / S / Y, V242P / Q / T, V251L, H254G / S, A258L / S / V, R263G / L / Q / T, I264A / C, E266M / T, Q267D / W / Y, I269L, F271A / G / N / S, R273A / G / S, E277Q / R, Q278E, E282G / L / M / T / V / Y, F283A / G / K / L / M / R / S / V, P284D, W286F / L / S, L288I, E289A / L / S / V, W290L, D294L, G 295K, F297W, S300G / T, E301F / L, Q305K, E306I / K / S / V, F308K / L / S, H309G / R,T317Q, M323S, D328R, K334L / R, F337G / L / M / P / R / S, I339Y, D349E, F355S, E356A / V / W, E357H / K / P / R / S / V, G358C, K359N / R, E360H / M / P, T362G, K363R, N364R, V367C / L, A370C / G, V372N / Q, E374A / S, Y375W, N378T, E379A / G / P, V380T, S381K / R, I382V, G384C / V, Y386F , T387G, D388K / Y, E389K / L / Q / R, M390E, V392C / I / K / L / R / S, A396C / H, R397K / L / M, K404S, V405I, I408C / V, S414A / L / Q / R / T / V, T415A / C / E / H / I / K / L / V, S416K, S417D / G / L, K418A / G / I / L / M / P / S / T, T419G, K421R, K422N, S423R / T, or E428F / R / S, or a combination thereof, wherein amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0130] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 63, 242, 283, 286, 317, 414, 418, or 428, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitutions 63F / R, 242P / Q / T, 283A / G / K / L / M / R / S / V, 286F / L / S, 317Q, 414A / L / Q / R / T / V, 418A / G / I / L / M / P / S / T, or 428F / R / S, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least substitutions 63R, 242Q, 283L, 286S, 317Q, 414Q, 418S, or 428R, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0131] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 428, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution 428R, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution E428R, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0132] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 317, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution 317Q, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution T317Q, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0133] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 283, 286, or 418, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution 283L, 286S, or 418S, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution F283L, W286S, or K418S, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0134] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 242 or 414, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 242Q or 414Q, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position V242Q or S414Q, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0135] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 63, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution 63R, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution G63R, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0136] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises a substitution or set of substitutions at least at amino acid position 233, 317, 191, 288, 207, 149, 251, 205, 269, 164, 36, 428, 105 / 132, or 105, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0137] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least substitution or set of substitutions 233T, 317Q, 191T, 288I, 207L, 149P, 251L, 205E, 269L, 164A, 36T, 428R, 105K / 132R, or 105K, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0138] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or set of substitutions S233T, T317Q, S191T, L288I, I207L, A149P, V251L, Q205E, I269L, K164A, E36T, E428R, L105K / K132R, or L105K, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0139] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 196 / 428, 242 / 428, 337 / 428, 33 / 428, 277 / 428, 30 / 428, 359 / 428, 283 / 428, 415 / 428, 387 / 428, 379 / 428, 205 / 428, 186 / 428, 389 / 428, 102 / 428, 164 / 428, 301 / 428, 375 / 428, 267 / 428, 380 / 428, 254 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428, 317 / 428. 428, 233 / 428, 235 / 428, 148 / 428, 100 / 428, 97 / 428, 382 / 428, or 358 / 428, wherein the amino acid position is relative to the amino acid corresponding to SEQ ID NO: 428, wherein the amino acid position is relative to the amino acid corresponding to SEQ ID NO: 428, 236 / 428, 237 / 428, 428, 362 / 428, 233 / 428, 235 / 428, 148 / 428, 100 / 428, 97 / 428, 382 / 428, or 358 / 428, wherein the amino acid position is relative to the amino acid corresponding to SEQ ID NO: 428, wherein the amino acid position is relative to the amino acid corresponding to SEQ ID NO: 428, wherein the amino acid position is relative to the amino acid corresponding to SEQ ID NO: 428, SEQ ID NO: 2.

[0140] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or set of substitutions 196E / 428R, 242P / 428R, 337L / 428R, 33R / 428R, 33V / 428R, 277Q / 428R, 337P / 428R, 30C / 428R, 359N / 428R, 337G / 428R, 283S / 428R, 242Q / 428R, 415A / 428R, 387G / 428R, 415H / 428R, 379A / 428R, 205G / 428R, 186M / 428R, 337M / 428R, 389Q / 428R, 102L / 428R, 389L / 428R, R, 359R / 428R, 205K / 428R, 164A / 428R, 415E / 428R, 301F / 428R, 30H / 428R, 3 79G / 428R, 375W / 428R, 267W / 428R, 380T / 428R, 415C / 428R, 254G / 428R, 186 T / 428R, 317Q / 428R, 77D / 139T / 317Q / 417G / 428R, 105K / 317Q / 417G / 428R, 3 17Q / 349E / 362G / 386F / 428R, 105K / 317Q / 428R, 139T / 317Q / 362G / 428R, 233T / 317Q / 405I / 428R, 139T / 317Q / 428R, 162W / 428R, 283V / 428R, 286S / 428R, 4 14L / 428R, 417L / 428R, 226S / 428R, 61T / 428R, 226Q / 428R, 105S / 428R, 186E / 428R, 230M / 428R, 61V / 428R, 186V / 428R, 186L / 428R, 379P / 428R, 415V / 42 8R, 415I / 428R, 186A / 428R, 283R / 428R, 226V / 428R, 418L / 428R, 418S / 428R, 370G / 428R, 283A / 428R, 337S / 428R, 297W / 428R, 237S / 428R, 428S, 186C / 42 8R, 362G / 428R, 283G / 428R, 196V / 428R, 237L / 428R, 415L / 428R, 233W / 428R , 242T / 428R, 277R / 428R, 235W / 428R, 148P / 428R, 254S / 428R, 102G / 428R, 1 05T / 428R, 301L / 428R, 286F / 428R, 100V / 428R, 237Y / 428R, 30S / 428R, 428F,414Q / 428R, 414V / 428R, 283L / 428R, 414A / 428R, 414R / 428R, 370C / 428R, 283K / 428R, 286L / 428R, 186H / 428R, 417D / 428R, 226D / 428R, 233G / 428R, 267Y / 428R, 97G / 428R, 226E / 428R, 418T / 428R, 230L / 428R , 414T / 428R, 418A / 428R, 237V / 428R, 418G / 428R, 382V / 428R, 267D / 428R, 283M / 428R, 418P / 428R, 237R / 428R, 237M / 428R, 418I / 428R, 358C / 428R, 186R / 428R, 418M / 428R, or 102S / 428R, wherein amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0141] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of the engineered DNA ligase variants listed in Tables 11.2 and 12.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0142] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 242 / 283 / 286 / 317 / 359 / 418 / 428, 283 / 286 / 317 / 428, 283 / 286 / 317 / 418 / 428, 186 / 242 / 283 / 286 / 317 / 418 / 428, 205 / 286 / 317 / 359 / 428, 283 / 317 / 428, 242 / 286 / 317 / 418 / 428, 186 / 205 / 242 / 317 / 428, 283 / 286 / 317 / 359 / 428、242 / 277 / 317 / 418 / 428、242 / 283 / 286 / 317 / 418 / 428、112 / 196 / 317 / 389 / 428、286 / 317 / 428、283 / 286 / 317 / 359 / 418 / 428、242 / 3 17 / 359 / 418 / 428、186 / 283 / 317 / 428、186 / 317 / 359 / 418 / 428、205 / 242 / 317 / 418 / 428、186 / 205 / 242 / 283 / 286 / 317 / 359 / 418 / 428、205 / 317 / 359 / 418 / 42 8. 186 / 205 / 283 / 286 / 317 / 418 / 428, 186 / 283 / 317 / 359 / 428, 186 / 242 / 317 / 428, 186 / 242 / 317 / 359 / 428, 283 / 317 / 359 / 418 / 428, 277 / 317 / 418 / 428, 186 / 188 / 283 / 317 / 428、186 / 286 / 317 / 418 / 428、186 / 242 / 286 / 317 / 359 / 418 / 428、317 / 418 / 428、186 / 242 / 283 / 286 / 317 / 359 / 418 / 428、186 / 277 / 317 / 359 / 418 / 428, 242 / 283 / 286 / 317 / 428, 205 / 317 / 418 / 428, 30 / 297 / 317 / 428, 205 / 242 / 286 / 317 / 359 / 418 / 428, 186 / 205 / 317 / 359 / 418 / 428, 317 / 359 / 418 / 428 8. 186 / 317 / 428, 230 / 317 / 428, 33 / 297 / 317 / 428, 186 / 205 / 317 / 428, 186 / 283 / 317 / 359 / 418 / 428, 186 / 317 / 418 / 428, 205 / 242 / 283 / 317 / 359 / 418 / 428,33 / 317 / 375 / 389 / 428, 33 / 230 / 317 / 428, 196 / 242 / 283 / 286 / 317 / 359 / 418 / 428, 186 / 242 / 283 / 317 / 359 / 418 / 428, 186 / 317 / 359 / 428, 33 / 196 / 317 / 428, 186 / 277 / 317 / 418 / 428, 242 / 317 / 428, 3 3 / 196 / 297 / 301 / 317 / 428, 205 / 237 / 242 / 283 / 286 / 317 / 359 / 428, 186 / 205 / 283 / 317 / 359 / 418 / 428, 33 / 317 / 389 / 428, or 186 / 196 / 242 / 283 / 286 / 317 / 359 / 418 / 428, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0143] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or set of substitutions 242Q / 283V / 286S / 317Q / 359R / 418L / 428R, 283L / 286S / 317Q / 428R, 283L / 286S / 317Q / 418S / 428R, 186E / 242Q / 283V / 286S / 317Q / 418L / 428R, 205K / 286S / 317Q / 359R / 428R, 283V / 317Q / 428R, 242P / 286S / 317Q / 418T / 428R, 186E / 242Q / 283V / 286S / 317Q / 418L / 428R 3L / 286S / 317Q / 359R / 428R, 277Q / 286S / 317Q / 359N / 418T / 428R, 186A / 20 5K / 283V / 286S / 317Q / 359R / 428R, 242Q / 277Q / 317Q / 418L / 428R, 242Q / 28 3S / 286S / 317Q / 418T / 428R, 112M / 196V / 317Q / 389Q / 428R, 286S / 317Q / 42 8R、242P / 283R / 286S / 317Q / 359R / 418L / 428R、283L / 286S / 317Q / 359R / 418 S / 428R, 242Q / 317Q / 359N / 418S / 428R, 186E / 283L / 317Q / 428R, 186E / 317 Q / 359R / 418S / 428R, 205K / 242T / 317Q / 418S / 428R, 186E / 205K / 242Q / 283 S / 286S / 317Q / 359R / 418S / 428R, 205K / 317Q / 359N / 418L / 428R, 186A / 205 K / 283L / 286S / 317Q / 418L / 428R, 242T / 283V / 286S / 317Q / 359R / 418S / 428R , 186E / 283L / 317Q / 359R / 428R, 186E / 242P / 317Q / 428R, 283V / 286S / 317Q / 418S / 428R、186E / 242T / 317Q / 359R / 428R、283L / 317Q / 359R / 418L / 428R ,277Q / 317Q / 418S / 428R, 186E / 188A / 283V / 317Q / 428R, 186E / 286S / 317Q / 418S / 428R、186A / 242T / 286S / 317Q / 359N / 418L / 428R、317Q / 418L / 428R、186A / 242P / 283V / 286S / 317Q / 359R / 418S / 428R、186A / 277Q / 317Q / 359R / 418S / 428R、242Q / 283R / 286S / 317Q / 428R、205K / 317Q / 418S / 428R、 30C / 297W / 317Q / 428R、283L / 317Q / 359R / 418S / 428R、186E / 286S / 317Q / 418L / 428R、317Q / 418T / 428R、205K / 242T / 286S / 317Q / 359R / 418S / 4 28R, 186E / 205K / 317Q / 359N / 418T / 428R, 317Q / 418S / 428R, 186E / 242T / 283S / 286S / 317Q / 418S / 428R, 317Q / 359R / 418S / 428R, 242T / 286S / 317Q / 418S / 428R, 186E / 317Q / 428R, 230L / 317Q / 428R, 33V / 297W / 317Q / 428R, 186E / 205K / 317Q / 428R, 186A / 283L / 317Q / 359R / 418S / 428R, 186 E / 317Q / 418L / 428R, 186E / 277Q / 317Q / 359R / 418S / 428R, 205K / 242T / 283R / 317Q / 359R / 418L / 428R, 186E / 317Q / 418T / 428R, 33V / 317Q / 375W / 389Q / 428R, 33R / 230L / 317Q / 428R, 196E / 242T / 283V / 286S / 317Q / 359R / 418T / 428R, 186E / 242T / 283L / 317Q / 359R / 418S / 428R, 186E / 317Q / 3 59R / 428R、33V / 196V / 317Q / 428R、33R / 317Q / 375W / 389Q / 428R、186A / 277Q / 317Q / 418S / 428R、186A / 242T / 283L / 286S / 317Q / 418L / 428R、242T / 317Q / 428R、33V / 196V / 297W / 301F / 317Q / 428R、205K / 237Y / 242P / 283L / 286S / 317Q / 359R / 428R、186A / 205K / 283L / 317Q / 359R / 418T / 428R、33V / 317Q / 389Q / 428R or 186A / 196E / 242T / 283L / 286S / 317Q / 359N / 418T / 428R, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0144] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of the engineered DNA ligase variants listed in Table 13.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0145] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 283 / 286 / 317 / 363 / 418 / 428, 63 / 283 / 286 / 317 / 418 / 428, 283 / 286 / 317 / 389 / 418 / 428, 283 / 286 / 317 / 381 / 418 / 428, 197 / 283 / 286 / 317 / 418 / 428, 283 / 286 / 317 / 359 / 418 / 428, 102 / 283 / 286 / 317 / 418 / 428, 165 / 283 / 286 / 317 / 418 / 428, 283 / 286 / 317 / 389 / 418 / 428. 、283 / 286 / 317 / 414 / 418 / 428、283 / 286 / 317 / 337 / 418 / 428、164 / 283 / 286 / 317 / 418 / 428、283 / 286 / 317 / 416 / 418 / 428、101 / 283 / 286 / 317 / 418 / 428、28 3 / 286 / 317 / 415 / 418 / 428, 283 / 286 / 317 / 418 / 423 / 428, 283 / 286 / 317 / 364 / 418 / 428, 73 / 283 / 286 / 317 / 418 / 428, 50 / 283 / 286 / 317 / 418 / 428, 71 / 283 / 2 86 / 317 / 418 / 428, 283 / 286 / 317 / 388 / 418 / 419 / 428, 283 / 286 / 317 / 357 / 418 / 428, 283 / 286 / 317 / 396 / 418 / 428, 68 / 283 / 286 / 317 / 418 / 428, 76 / 283 / 286 / 317 / 418 / 428 6 / 317 / 418 / 428, 14 / 283 / 286 / 317 / 418 / 428, 271 / 283 / 286 / 317 / 418 / 428, 283 / 286 / 317 / 360 / 418 / 428, 266 / 283 / 286 / 317 / 418 / 428, 208 / 283 / 286 / 317 / 418 / 428、74 / 283 / 286 / 317 / 418 / 428、263 / 283 / 286 / 317 / 418 / 428、264 / 283 / 286 / 317 / 418 / 428、13 / 283 / 286 / 317 / 418 / 428、283 / 286 / 317 / 378 / 418 / 428, 283 / 286 / 317 / 372 / 418 / 428, 283 / 286 / 300 / 317 / 418 / 428, 283 / 286 / 294 / 317 / 418 / 428, 283 / 286 / 290 / 317 / 418 / 428, 283 / 286 / 317 / 397 / 418 / 428,95 / 283 / 286 / 317 / 418 / 428、258 / 283 / 286 / 317 / 418 / 428、161 / 283 / 286 / 317 / 418 / 428、212 / 283 / 286 / 317 / 418 / 428、198 / 283 / 286 / 317 / 418 / 428、138 / 283 / 286 / 317 / 418 / 428、18 / 283 / 286 / 317 / 418 / 428、283 / 286 / 317 / 404 / 418 / 428、273 / 283 / 286 / 317 / 418 / 428、117 / 283 / 286 / 317 / 418 / 428、240 / 283 / 286 / 317 / 418 / 428、69 / 283 / 286 / 317 / 418 / 428、278 / 283 / 286 / 317 / 418 / 428、283 / 286 / 289 / 317 / 418 / 428、82 / 283 / 286 / 317 / 418 / 428、283 / 286 / 317 / 328 / 418 / 428、61 / 186 / 283 / 286 / 317 / 417 / 418 / 428、186 / 283 / 286 / 317 / 370 / 417 / 418 / 428、267 / 283 / 286 / 317 / 418 / 428、61 / 283 / 286 / 317 / 370 / 418 / 428、186 / 267 / 283 / 286 / 317 / 370 / 417 / 418 / 428、61 / 186 / 283 / 286 / 317 / 418 / 428、283 / 286 / 317 / 370 / 417 / 418 / 428、283 / 286 / 317 / 417 / 418 / 428、61 / 283 / 286 / 317 / 370 / 382 / 418 / 428、61 / 186 / 267 / 283 / 286 / 317 / 370 / 417 / 418 / 428、61 / 283 / 286 / 317 / 418 / 428、267 / 283 / 286 / 317 / 370 / 417 / 418 / 428、267 / 283 / 286 / 317 / 370 / 418 / 428、61 / 283 / 286 / 317 / 417 / 418 / 428、61 / 186 / 237 / 267 / 283 / 286 / 317 / 370 / 418 / 428、61 / 237 / 283 / 286 / 317 / 370 / 417 / 418 / 428、283 / 286 / 317 / 370 / 418 / 428、186 / 283 / 286 / 317 / 370 / 418 / 428、61 / 186 / 267 / 283 / 286 / 317 / 417 / 418 / 428、61 / 186 / 283 / 286 / 317 / 370 / 382 / 418 / 428、283 / 286 / 317 / 370 / 382 / 417 / 418 / 428、61 / 186 / 283 / 286 / 317 / 370 / 418 / 428、237 / 267 / 283 / 286 / 317 / 370 / 417 / 418 / 428、61 / 186 / 283 / 286 / 317 / 382 / 418 / 428、61 / 267 / 283 / 286 / 317 / 418 / 428、61 / 267 / 283 / 286 / 317 / 417 / 418 / 428、61 / 237 / 267 / 283 / 286 / 317 / 382 / 418 / 428、186 / 283 / 286 / 317 / 370 / 382 / 418 / 428、237 / 267 / 283 / 286 / 317 / 370 / 418 / 428、61 / 186 / 267 / 283 / 286 / 317 / 370 / 418 / 428、186 / 237 / 267 / 283 / 286 / 317 / 370 / 418 / 428、61 / 186 / 267 / 283 / 286 / 317 / 418 / 428、61 / 186 / 237 / 283 / 286 / 317 / 418 / 428、186 / 283 / 286 / 317 / 418 / 428、186 / 267 / 283 / 286 / 317 / 418 / 428、237 / 283 / 286 / 317 / 370 / 417 / 418 / 428、242 / 283 / 286 / 317 / 414 / 418 / 428、162 / 283 / 286 / 317 / 414 / 418 / 428、267 / 283 / 286 / 317 / 414 / 418 / 428、105 / 283 / 286 / 317 / 414 / 418 / 428、162 / 242 / 283 / 286 / 317 / 414 / 418 / 428、97 / 162 / 283 / 286 / 317 / 414 / 418 / 428、105 / 162 / 267 / 283 / 286 / 317 / 414 / 418 / 428、283 / 286 / 317 / 356 / 418 / 428、283 / 286 / 317 / 392 / 418 / 428、106 / 283 / 286 / 317 / 418 / 428、283 / 286 / 308 / 317 / 418 / 428、283 / 286 / 306 / 317 / 418 / 428、96 / 283 / 286 / 317 / 418 / 428、282 / 283 / 286 / 317 / 418 / 428、113 / 283 / 286 / 317 / 418 / 428、283 / 286 / 309 / 317 / 418 / 428、110 / 283 / 286 / 317 / 418 / 428、37 / 283 / 286 / 317 / 418 / 428、201 / 283 / 286 / 317 / 418 / 428、283 / 286 / 317 / 418 / 428, 283 / 286 / 317 / 374 / 418 / 428, 283 / 286 / 295 / 317 / 418 / 428, 88 / 283 / 286 / 317 / 418 / 428, 44 / 283 / 286 / 317 / 418 / 428, 12 / 283 / 286 / 317 / 418 / 428, or 283 / 286 / 317 / 390 / 418 / 428, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0146] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or set of substitutions 283L / 286S / 317Q / 363R / 418S / 428R, 63R / 283L / 286S / 317Q / 418S / 428R, 283L / 286S / 317Q / 389K / 418S / 428R, 283L / 286S / 317Q / 381R / 418S / 428R, 283L / 286S / 317Q / 389R / 418S / 428R, 197R / 283L / 286S / 317Q / 418S / 428R, 283L / 286S / 317Q / 389R / 418S / 428R, 286S / 317Q / 381K / 418S / 428R, 102K / 283L / 286S / 317Q / 418S / 428R, 165K / 2 83L / 286S / 317Q / 418S / 428R, 283L / 286S / 317Q / 388K / 418S / 428R, 283L / 286 S / 317Q / 414R / 418S / 428R, 283L / 286S / 317Q / 337R / 418S / 428R, 164R / 283L / 286S / 317Q / 418S / 428R, 283L / 286S / 317Q / 416K / 418S / 428R, 101R / 283L / 28 6S / 317Q / 418S / 428R, 283L / 286S / 317Q / 415K / 418S / 428R, 283L / 286S / 317 Q / 418S / 423R / 428R, 283L / 286S / 317Q / 364R / 418S / 428R, 73P / 283L / 286S / 3 17Q / 418S / 428R, 50I / 283L / 286S / 317Q / 418S / 428R, 71L / 283L / 286S / 317Q / 418S / 428R, 283L / 286S / 317Q / 388Y / 418S / 419G / 428R, 50G / 283L / 286S / 317 Q / 418S / 428R, 283L / 286S / 317Q / 357K / 418S / 428R, 283L / 286S / 317Q / 396H / 418S / 428R、68Y / 283L / 286S / 317Q / 418S / 428R、76F / 283L / 286S / 317Q / 418 S / 428R, 71P / 283L / 286S / 317Q / 418S / 428R, 14V / 283L / 286S / 317Q / 418S / 42 8R、271G / 283L / 286S / 317Q / 418S / 428R、68A / 283L / 286S / 317Q / 418S / 428R、68M / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 360P / 418S / 428R、266M / 283L / 286S / 317Q / 418S / 428R、208D / 283L / 286S / 317Q / 418S / 428R、74S / 283L / 286S / 317Q / 418S / 428R、263T / 283L / 286S / 317Q / 418S / 428R、264C / 283L / 286S / 317Q / 418S / 428R、13R / 283L / 286S / 317Q / 418S / 428R、263L / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 360M / 418S / 428R、283L / 286S / 317Q / 378T / 418S / 428R、76H / 283L / 286S / 317Q / 418S / 428R、13G / 283L / 286S / 317Q / 418S / 428R、76R / 283L / 286S / 317Q / 418S / 428R、50T / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 372Q / 418S / 428R、50S / 283L / 286S / 317Q / 418S / 428R、283L / 286S / S300G / 317Q / 418S / 428R、73T / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 294L / 317Q / 418S / 428R、283L / 286S / 317Q / 372N / 418S / 428R、76N / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 290L / 317Q / 418S / 428R、283L / 286S / 317Q / 397L / 418S / 428R、76G / 283L / 286S / 317Q / 418S / 428R、95L / 283L / 286S / 317Q / 418S / 428R、258L / 283L / 286S / 317Q / 418S / 428R、161V / 283L / 286S / 317Q / 418S / 428R、212M / 283L / 286S / 317Q / 418S / 428R、198V / 283L / 286S / 317Q / 418S / 428R、138L / 283L / 286S / 317Q / 418S / 428R、18D / 283L / 286S / 317Q / 418S / 428R、18S / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 404S / 418S / 428R、273G / 283L / 286S / 317Q / 418S / 428R、117G / 283L / 286S / 317Q / 418S / 428R、240E / 283L / 286S / 317Q / 418S / 428R、68S / 283L / 286S / 317Q / 418S / 428R、73C / 283L / 286S / 317Q / 418S / 428R、69T / 283L / 286S / 317Q / 418S / 428R、278E / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 360H / 418S / 428R、198D / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 300T / 317Q / 418S / 428R、161R / 283L / 286S / 317Q / 418S / 428R、73K / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 289V / 317Q / 418S / 428R、82R / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 328R / 418S / 428R、61T / 186A / 283L / 286S / 317Q / 417D / 418S / 428R、186A / 283L / 286S / 317Q / 370C / 417L / 418S / 428R、267Y / 283L / 286S / 317Q / 418S / 428R、61T / 283L / 286S / 317Q / 370C / 418S / 428R、186A / 267Y / 283L / 286S / 317Q / 370C / 417L / 418S / 428R、61T / 186C / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 370C / 417D / 418S / 428R、283L / 286S / 317Q / 417D / 418S / 428R、61T / 283L / 286S / 317Q / 370C / 382V / 418S / 428R、61T / 186A / 267Y / 283L / 286S / 317Q / 370C / 417D / 418S / 428R、61T / 283L / 286S / 317Q / 418S / 428R、267Y / 283L / 286S / 317Q / 370C / 417D / 418S / 428R、267Y / 283L / 286S / 317Q / 370C / 418S / 428R、61T / 283L / 286S / 317Q / 417L / 418S / 428R、61T / 186H / 237R / 267Y / 283L / 286S / 317Q / 370C / 418S / 428R、61T / 237R / 283L / 286S / 317Q / 370C / 417L / 418S / 428R、283L / 286S / 317Q / 370C / 417L / 418S / 428R、283L / 286S / 317Q / 370C / 418S / 428R、186E / 283L / 286S / 317Q / 370C / 418S / 428R、61T / 186E / 267Y / 283L / 286S / 317Q / 417D / 418S / 428R、61T / 186C / 283L / 286S / 317Q / 370C / 382V / 418S / 428R、283L / 286S / 317Q / 370C / 382V / 417D / 418S / 428R、61T / 186C / 267Y / 283L / 286S / 317Q / 417D / 418S / 428R、61T / 186C / 283L / 286S / 317Q / 370C / 418S / 428R、237R / 267Y / 283L / 286S / 317Q / 370C / 417D / 418S / 428R、61T / 186E / 283L / 286S / 317Q / 417D / 418S / 428R、61T / 186H / 283L / 286S / 317Q / 418S / 428R、61T / 186C / 283L / 286S / 317Q / 382V / 418S / 428R、61T / 186H / 283L / 286S / 317Q / 370C / 418S / 428R、61T / 267Y / 283L / 286S / 317Q / 418S / 428R、61T / 267Y / 283L / 286S / 317Q / 417L / 418S / 428R、61T / 237R / 267Y / 283L / 286S / 317Q / 382V / 418S / 428R、186H / 283L / 286S / 317Q / 370C / 382V / 418S / 428R、237R / 267Y / 283L / 286S / 317Q / 370C / 418S / 428R、186H / 283L / 286S / 317Q / 370C / 417D / 418S / 428R、61T / 186V / 267Y / 283L / 286S / 317Q / 370C / 418S / 428R、267Y / 283L / 286S / 317Q / 370C / 417L / 418S / 428R、61T / 186V / 283L / 286S / 317Q / 370C / 418S / 428R、186H / 237R / 267Y / 283L / 286S / 317Q / 370C / 418S / 428R、61T / 186E / 267Y / 283L / 286S / 317Q / 418S / 428R、61T / 186C / 237R / 283L / 286S / 317Q / 418S / 428R、61T / 186H / 237R / 283L / 286S / 317Q / 418S / 428R、61T / 186C / 283L / 286S / 317Q / 417L / 418S / 428R、186H / 283L / 286S / 317Q / 418S / 428R、186A / 267Y / 283L / 286S / 317Q / 418S / 428R、186C / 283L / 286S / 317Q / 370C / 418S / 428R、61T / 186C / 237R / 267Y / 283L / 286S / 317Q / 370C / 418S / 428R、186E / 267Y / 283L / 286S / 317Q / 418S / 428R、237R / 283L / 286S / 317Q / 370C / 417L / 418S / 428R、186H / 283L / 286S / 317Q / 370C / 418S / 428R、242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、283L / 286S / 317Q / 414T / 418S / 428R、283L / 286S / 317Q / 414Q / 418S / 428R、162W / 283L / 286S / 317Q / 414Q / 418S / 428R、162W / 283L / 286S / 317Q / 414V / 418S / 428R、267D / 283L / 286S / 317Q / 414T / 418S / 428R、283L / 286S / 317Q / 414V / 418S / 428R、267D / 283L / 286S / 317Q / 414Q / 418S / 428R、283L / 286S / 317Q / 414A / 418S / 428R、162W / 283L / 286S / 317Q / 414L / 418S / 428R、105S / 283L / 286S / 317Q / 414T / 418S / 428R、162W / 242Q / 283L / 286S / 317Q / 414T / 418S / 428R、97G / 162W / 283L / 286S / 317Q / 414Q / 418S / 428R、105S / 162W / 267D / 283L / 286S / 317Q / 414V / 418S / 428R、283L / 286S / 317Q / 356V / 418S / 428R、273A / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 357P / 418S / 428R、14G / 283L / 286S / 317Q / 418S / 428R、14S / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 396C / 418S / 428R、240R / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 392K / 418S / 428R、273S / 283L / 286S / 317Q / 418S / 428R、106V / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 308S / 317Q / 418S / 428R、283L / 286S / 308L / 317Q / 418S / 428R、283L / 286S / 306I / 317Q / 418S / 428R、96A / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 397K / 418S / 428R、263Q / 283L / 286S / 317Q / 418S / 428R、282T / 283L / 286S / 317Q / 418S / 428R、138R / 283L / 286S / 317Q / 418S / 428R、258S / 283L / 286S / 317Q / 418S / 428R、76L / 283L / 286S / 317Q / 418S / 428R、14T / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 289S / 317Q / 418S / 428R、240G / 283L / 286S / 317Q / 418S / 428R、106S / 283L / 286S / 317Q / 418S / 428R、117S / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 357S / 418S / 428R、96G / 283L / 286S / 317Q / 418S / 428R、113T / 283L / 286S / 317Q / 418S / 428R、71G / 283L / 286S / 317Q / 418S / 428R、282V / 283L / 286S / 317Q / 418S / 428R、117V / 283L / 286S / 317Q / 418S / 428R、282M / 283L / 286S / 317Q / 418S / 428R、258V / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 309G / 317Q / 418S / 428R、283L / 286S / 306K / 317Q / 418S / 428R、283L / 286S / 308K / 317Q / 418S / 428R、283L / 286S / 317Q / 357R / 418S / 428R、212S / 283L / 286S / 317Q / 418S / 428R、18N / 283L / 286S / 317Q / 418S / 428R、113A / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 306S / 317Q / 418S / 428R、212F / 283L / 286S / 317Q / 418S / 428R、198N / 283L / 286S / 317Q / 418S / 428R、110R / 283L / 286S / 317Q / 418S / 428R、240K / 283L / 286S / 317Q / 418S / 428R、198L / 283L / 286S / 317Q / 418S / 428R、71R / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 357V / 418S / 428R、198R / 283L / 286S / 317Q / 418S / 428R、271A / 283L / 286S / 317Q / 418S / 428R、282Y / 283L / 286S / 317Q / 418S / 428R、212G / 283L / 286S / 317Q / 418S / 428R、95V / 283L / 286S / 317Q / 418S / 428R、37S / 283L / 286S / 317Q / 418S / 428R、68V / 283L / 286S / 317Q / 418S / 428R、240S / 283L / 286S / 317Q / 418S / 428R、117Y / 283L / 286S / 317Q / 418S / 428R、201S / 283L / 286S / 317Q / 418S / 428R、271N / 283L / 286S / 317Q / 418S / 428R、240Q / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 289L / 317Q / 418S / 428R、212W / 283L / 286S / 317Q / 418S / 428R、198W / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 317Q / 357H / 418S / 428R、208F / 283L / 286S / 317Q / 418S / 428R、282L / 283L / 286S / 317Q / 418S / 428R、283L / 286S / 306V / 317Q / 418S / 428R、283L / 284D / 286S / 317Q / 418S / 428R, 96T / 283L / 286S / 317Q / 418S / 428R, 266T / 283L / 286S / 317Q / 418S / 428R, 283L / 286S / 317 Q / 374S / 418S / 428R, 95R / 283L / 286S / 317Q / 418S / 428R, 283L / 286S / 309R / 317Q / 418S / 428R, 73W / 283L / 286S / 317Q / 418S / 428 R、283L / 286S / 317Q / R397M / 418S / 428R、283L / 286S / 289A / 317Q / 418S / 428R、283L / 286S / 295K / 317Q / 418S / 428R、106L / 283L / 286S / 317Q / 418S / 428R, 95A / 283L / 286S / 317Q / 418S / 428R, 283L / 286S / 317Q / 374A / 418S / 428R, 88V / 283L / 286S / 317Q / 418S / 428R、96V / 283L / 286S / 317Q / 418S / 428R、271S / 283L / 286S / 317Q / 418S / 428R、44S / 283L / 286S / 317Q / 418S / 428R、208H / 283 L / 286S / 317Q / 418S / 428R, 263G / 283L / 286S / 317Q / 418S / 428R, 12A / 283L / 286S / 317Q / 418S / 428R, 201L / 283L / 286S / 317Q / 41 8S / 428R, 198A / 283L / 286S / 317Q / 418S / 428R, 198K / 283L / 286S / 317Q / 418S / 428R, 282G / 283L / 286S / 317Q / 418S / 428R, 283L / 286S / 317Q / 390E / 418S / 428R, 264A / 283L / 286S / 317Q / 418S / 428R, or 73V / 283L / 286S / 317Q / 418S / 428R, wherein amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0147] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitutions listed in Tables 14.2, 15.2, and 16.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0148] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 63 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 63 / 96 / 242 / 283 / 286 / 317 / 370 / 414 / 418 / 428, 242 / 283 / 286 / 317 / 389 / 414 / 418 / 428, 13 / 242 / 267 / 283 / 286 / 317 / 363 / 389 / 414 / 418 / 428, 13 / 186 / 242 / 283 / 286 / 317 / 389 / 414 / 418 / 428, and 50 / 242 / 267 / 283 / 286 / 317 / 370 / 414 / 418 / 428. 0 / 389 / 414 / 418 / 428, 242 / 283 / 286 / 317 / 363 / 370 / 414 / 418 / 428, 96 / 242 / 283 / 286 / 317 / 370 / 414 / 418 / 428, 61 / 63 / 212 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 11 / 242 / 283 / 286 / 305 / 317 / 414 / 418 / 428, 11 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 428, 242 / 283 / 286 / 317 / 323 / 414 / 418 / 428, 242 / 283 / 286 / 317 / 3 34 / 414 / 418 / 428、242 / 283 / 286 / 317 / 339 / 414 / 418 / 428、242 / 283 / 286 / 317 / 356 / 414 / 418 / 428、242 / 283 / 286 / 317 / 384 / 414 / 418 / 428、242 / 283 / 286 / 317 / 408 / 414 / 418 / 428、67 / 242 / 283 / 286 / 317 / 414 / 418 / 428、242 / 283 / 286 / 317 / 392 / 414 / 418 / 428、104 / 242 / 283 / 286 / 317 / 414 / 418 / 428、242 / 283 / 286 / 317 / 355 / 414 / 418 / 428、159 / 242 / 283 / 286 / 317 / 414 / 418 / 428、155 / 242 / 283 / 286 / 317 / 414 / 418 / 428、242 / 283 / 286 / 317 / 367 / 414 / 418 / 428、31 / 242 / 283 / 286 / 317 / 414 / 418 / 428、231 / 242 / 283 / 286 / 317 / 414 / 418 / 428、36 / 242 / 283 / 286 / 317 / 414 / 418 / 428、150 / 242 / 283 / 286 / 317 / 414 / 418 / 428、239 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 103 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 125 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 228 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 37 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 189 / 242 / 283 / 286 / 317 / 414 14 / 418 / 428、177 / 242 / 283 / 286 / 317 / 414 / 418 / 428、242 / 283 / 286 / 317 / 414 / 418 / 422 / 428、128 / 242 / 283 / 286 / 317 / 414 / 418 / 428、220 / 242 / 283 / 286 / 317 / 414 / 418 / 428、130 / 242 / 283 / 286 / 317 / 414 / 418 / 428、56 / 242 / 283 / 286 / 317 / 414 / 418 / 428、190 / 242 / 283 / 286 / 317 / 414 / 418 / 428、156 / 242 / 283 / 286 / 317 / 414 / 418 / 428、232 / 242 / 283 / 286 / 317 / 414 / 418 / 428、242 / 283 / 286 / 317 / 414 / 418 / 423 / 428、34 / 242 / 283 / 286 / 317 / 414 / 418 / 428、9 9 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 59 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 60 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 242 / 283 / 286 / 317 / 414 / 418 / 421 / 428, or 195 / 242 / 283 / 286 / 317 / 414 / 418 / 428, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0149] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution set 63R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 63R / 96T / 242Q / 283L / 286S / 317Q / 370C / 414Q / 418S / 428R, 242Q / 283L / 286S / 317Q / 389K / 414Q / 418S / 428R, 13R / 242Q / 267Y / 283L / 286S / 317Q / 363R / 389K / 414Q / 418S / 428R, K / 414Q / 418S / 428R, 50T / 242Q / 267Y / 283L / 286S / 317Q / 363R / 370C / 389K / 414Q / 418S / 428R, 242Q / 283L / 286S / 317Q / 363R / 370C / 414Q / 418S / 428R, 96 T / 242Q / 283L / 286S / 317Q / 370C / 414Q / 418S / 428R, 61T / 63R / 212W / 242Q / 28 3L / 286S / 317Q / 414Q / 418S / 428R, 11D / 242Q / 283L / 286S / Q305K / 317Q / 414Q / 418S / 428R, 11D / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 428R, 242Q / 2 83L / 286S / 317Q / 323S / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 334L / 414 Q / 418S / 428R, 242Q / 283L / 286S / 317Q / 339Y / 414Q / 418S / 428R, 242Q / 283L / 286S / 317Q / 356V / 414Q / 418S / 428R, 242Q / 283L / 286S / 317Q / 384V / 414Q / 41 8S / 428R, 242Q / 283L / 286S / 317Q / 408C / 414Q / 418S / 428R, 67R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 392R / 414Q / 418S / 4 28R、104K / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317 Q / 355S / 414Q / 418S / 428R, 159Q / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R,155R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 367L / 414Q / 418S / 428R、31R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、231P / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、36Y / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、150T / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、239V / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、103V / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 356A / 414Q / 418S / 428R、63F / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、125T / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 367C / 414Q / 418S / 428R、228I / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、37S / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、37G / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、239M / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、239W / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、239Q / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、189C / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、239S / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、239T / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、177G / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、239P / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、37N / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 414Q / 418S / 422N / 428R、228S / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、125R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、128C / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、189T / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 356W / 414Q / 418S / 428R、220V / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 408V / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 392S / 414Q / 418S / 428R、130T / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、228M / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、56P / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、228E / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、190R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 392I / 414Q / 418S / 428R、156C / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、232R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、150F / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、150C / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、239N / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 392L / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 414Q / 418S / 423T / 428R、34L / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、99I / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 384C / 414Q / 418S / 428R、59E / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、242Q / 283L / 286S / 317Q / 334R / 414Q / 418S / 428R, 60Y / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 99G / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 34R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 37L / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 242Q / 283L / 286S / 317Q / 414Q / 418S / 421R / 428R, or 195R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, wherein amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0150] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of the engineered DNA ligase variants listed in Table 17.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0151] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 63 / 242 / 283 / 286 / 308 / 317 / 357 / 390 / 414 / 418 / 428, 63 / 74 / 76 / 201 / 242 / 283 / 286 / 308 / 317 / 357 / 414 / 418 / 428, 61 / 63 / 74 / 76 / 186 / 201 / 242 / 283 / 286 / 308 / 317 / 357 / 414 / 418 / 428, / 242 / 283 / 286 / 308 / 317 / 414 / 415 / 418 / 428、63 / 76 / 242 / 283 / 286 / 317 / 357 / 396 / 414 / 418 / 428、63 / 242 / 263 / 283 / 286 / 308 / 317 / 396 / 414 / 418 / 428、61 / 63 / 76 / 96 / 240 / 242 / 283 / 286 / 308 / 309 / 317 / 414 / 418 / 428、14 / 63 / 242 / 283 / 286 / 306 / 317 / 414 / 415 / 418 / 428、14 / 63 / 73 / 106 / 242 / 283 / 286 / 317 / 414 / 415 / 418 / 428、12 / 14 / 63 / 242 / 258 / 263 / 283 / 286 / 289 / 308 / 309 / 317 / 396 / 414 / 418 / 428、63 / 74 / 76 / 117 / 242 / 283 / 286 / 309 / 317 / 357 / 414 / 418 / 428、14 / 63 / 242 / 258 / 263 / 283 / 286 / 317 / 357 / 396 / 414 / 418 / 428、14 / 63 / 96 / 106 / 242 / 283 / 286 / 306 / 317 / 414 / 418 / 428、14 / 63 / 106 / 242 / 28 3 / 286 / 317 / 414 / 418 / 428、12 / 14 / 63 / 242 / 283 / 286 / 308 / 309 / 317 / 414 / 418 / 428、14 / 63 / 242 / 283 / 286 / 317 / 357 / 390 / 414 / 418 / 428、14 / 63 / 117 / 242 / 258 / 283 / 286 / 309 / 317 / 357 / 414 / 418 / 428、63 / 242 / 283 / 286 / 317 / 390 / 414 / 418 / 428、63 / 240 / 242 / 273 / 283 / 286 / 317 / 357 / 390 / 414 / 418 / 428、61 / 63 / 76 / 186 / 201 / 242 / 283 / 286 / 308 / 309 / 317 / 414 / 418 / 428、14 / 63 / 242 / 283 / 286 / 317 / 396 / 414 / 418 / 428、63 / 242 / 283 / 286 / 309 / 317 / 414 / 418 / 428、14 / 63 / 242 / 283 / 286 / 317 / 414 / 418 / 428、63 / 106 / 242 / 283 / 286 / 306 / 308 / 317 / 414 / 418 / 428、14 / 63 / 240 / 242 / 283 / 286 / 306 / 30 8 / 317 / 414 / 418 / 428、12 / 14 / 63 / 186 / 242 / 283 / 286 / 317 / 357 / 414 / 418 / 428、63 / 242 / 283 / 286 / 309 / 317 / 390 / 414 / 418 / 428、14 / 63 / 242 / 283 / 286 / 306 / 317 / 414 / 418 / 428、14 / 63 / 76 / 242 / 283 / 286 / 308 / 317 / 414 / 418 / 428、63 / 117 / 208 / 242 / 258 / 263 / 283 / 286 / 289 / 308 / 309 / 317 / 414 / 418 / 428, 14 / 63 / 73 / 106 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 63 / 76 / 208 / 242 / 263 / 283 / 286 / 317 / 414 / 418 / 428, 63 / 242 / 283 / 286 / 317 / 357 / 414 / 418 / 428, 14 / 63 / 242 / 283 / 286 / 308 / 317 / 414 / 418 / 428, 63 / 242 / 263 / 283 / 286 / 317 / 414 / 418 / 428 414 / 418 / 428, 63 / 240 / 242 / 283 / 286 / 317 / 414 / 418 / 428, 33 / 63 / 242 / 283 / 286 / 317 / 357 / 390 / 414 / 418 / 428, 14 / 63 / 76 / 242 / 273 / 283 / 286 / 317 / 414 / 418 / 428, or 63 / 74 / 242 / 283 / 286 / 317 / 414 / 418 / 428, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2. 、

[0152] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution set 63R / 242Q / 283L / 286S / 308L / 317Q / 357S / 390E / 414Q / 418S / 428R, 63R / 74S / 76L / 201S / 242Q / 283L / 286S / 308L / 317Q / 357P / 414Q / 418S / 428R, 61T / 63R / 74S / 76L / 186A / 201S / 242Q / 283L / 286S / 308L / 309R / 317Q / 357P / 390E / 414Q / 418S / 428R, / 242Q / 283L / 286S / 289S / 317Q / 357P / 414Q / 418S / 428R、63R / 242Q / 283L / 28 6S / 308S / 317Q / 414Q / 415I / 418S / 428R, 63R / 76L / 242Q / 283L / 286S / 317Q / 3 57S / 396C / 414Q / 418S / 428R, 63R / 242Q / 263L / 283L / 286S / 308L / 317Q / 396C / 414Q / 418S / 428R、61T / 63R / 76L / 96A / 240R / 242Q / 283L / 286S / 308L / 309R / 317Q / 414Q / 418S / 428R, 14S / 63R / 242Q / 283L / 286S / 306I / 317Q / 414Q / 415 I / 418S / 428R, 14S / 63R / 73W / 106S / 242Q / 283L / 286S / 317Q / 414Q / 415I / 418 S / 428R, 12A / 14G / 63R / 242Q / 258S / 263Q / 283L / 286S / 289S / 308L / 309R / 317 Q / 396C / 414Q / 418S / 428R, 63R / 74S / 76L / Q117V / 242Q / 283L / 286S / 309R / 31 7Q / 357S / 414Q / 418S / 428R, 14T / 63R / 242Q / 258S / 263Q / 283L / 286S / 317Q / 3 57S / 396C / 414Q / 418S / 428R, 14S / 63R / 96G / 106S / 242Q / 283L / 286S / 306I / 3 17Q / 414Q / 418S / 428R, 14S / 63R / 106S / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、12A / 14G / 63R / 242Q / 283L / 286S / 308L / 309R / 317Q / 414Q / 418S / 428R、14G / 63R / 242Q / 283L / 286S / 317Q / 357S / 390E / 414Q / 418S / 428R、14G / 63R / 117V / 242Q / 258S / 283L / 286S / 309R / 317Q / 357S / 414Q / 418S / 428R、63R / 242Q / 283L / 286S / 317Q / 390E / 414Q / 418S / 428R、63R / 240R / 242Q / 273S / 283L / 286S / 317Q / 357P / 390E / 414Q / 418S / 428R、61T / 63R / 76L / 186V / 201S / 242Q / 283L / 286S / 308L / 309R / 317Q / 414Q / 418S / 428R、14G / 63R / 242Q / 283L / 286S / 317Q / 396C / 414Q / 418S / 428R、63R / 242Q / 283L / 286S / 309R / 317Q / 414Q / 418S / 428R、14S / 63R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、63R / 106V / 242Q / 283L / 286S / 306I / 308S / 317Q / 414Q / 418S / 428R、14S / 63R / 240S / 242Q / 283L / 286S / 306I / 308S / 317Q / 414Q / 418S / 428R、12I / 14G / 63R / 186V / 242Q / 283L / 286S / 317Q / 357S / 414Q / 418S / 428R、63R / 242Q / 283L / 286S / 309R / 317Q / 390E / 414Q / 418S / 428R、14S / 63R / 242Q / 283L / 286S / 306I / 317Q / 414Q / 418S / 428R、14T / 63R / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、14S / 63R / 76R / 242Q / 283L / 286S / 308S / 317Q / 414Q / 418S / 428R、63R / 117V / 208D / 242Q / 258S / 263Q / 283L / 286S / 289S / 308L / 309R / 317Q / 414Q / 418S / 428R、14S / 63R / 73W / 106S / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R、63R / 76L / 208D / 242Q / 263Q / 283L / 286S / 317Q / 414Q / 418S / 428R、63R / 242Q / 283L / 286S / 317Q / 357S / 414Q / 418S / 428R, 14S / 63R / 242Q / 283L / 286S / 308S / 317Q / 414Q / 418S / 428R, 63R / 242Q / 263L / 283L / 286S / 317Q / 414Q / 418S / 428R, 63R / 76L / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 14S / 63R / 242Q / 283L / 286S / 300G / 308S / 317Q / 414Q / 4 418S / 428R, 15I / 418S / 428R, 63R / 240Y / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, 33M / 63R / 242Q / 283L / 286S / 317Q / 357S / 390E / 414Q / 418S / 428R, 14G / 63R / 76L / 242Q / 273S / 283L / 286S / 317Q / 414Q / 418S / 428R, or 63R / 74S / 242Q / 283L / 286S / 317Q / 414Q / 418S / 428R, wherein amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0153] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of the engineered DNA ligase variants listed in Table 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0154] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at an amino acid position listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acids are positions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0155] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least one substitution listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0156] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions at an amino acid position listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0157] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of the engineered DNA ligase variants listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0158] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0159] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

[0160] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184.

[0161] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

[0162] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

[0163] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the amino acid sequence at amino acid positions 11, 12, 13, 14, 18, 30, 31, 33, 34, 36, 37, 44, 50, 56, 59, 60, 61, 63, 67, 68, 69, 71, 73, 74, 76, 77, 82, 88, 95, 96, 97, 99, 100, 101, 102, 103, 104, 105, 106, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 3, 117, 125, 128, 130, 132, 138, 139, 148, 149, 150, 155, 156, 159, 161, 162, 164, 165, 177, 186, 188, 189, 190, 191, 195, 196, 197, 198, 201, 205, 207, 208, 212, 220, 226, 228, 230, 231, 232, 233, 235, 237, 238 9, 240, 242, 251, 254, 258, 263, 264, 266, 267, 269, 271, 273, 277, 278, 282, 283, 284, 286, 288, 289, 290, 294, 295, 297, 300, 301, 305, 306, 308, 309, 317, 323, 328, 334, 337, 339, 349, 355, 356, 357, 358, 360 8, 318, 722, or 938, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0164] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or amino acid residue 11D, 12A / I, 13G / R, 14G / S / T / V, 18D / N / S, 30C / H / S, 31R, 33M / R / V, 34L / R, 36T / Y, 37G / L / N / S, 44S, 50G / I / S / T, 56P, 59E, 60Y, 61T / V, 63F / R, 67R, 68A / M / S / V / Y, 69T, 71G / L / P / R, 73C / K / P / T / V / W, 74S, 76F / G / H / L / N / R, 77D, 82R, 88V, 95A / L / R / V, 96A / G / T / V, 97G, 99 G / I, 100V, 101R, 102G / K / L / S, 103V, 104K, 105K / S / T, 106L / S / V, 110R, 112M , 113A / T, 117G / S / V / Y, 125R / T, 128C, 130T, 132R, 138L / R, 139T, 148P, 149P, 150C / F / T, 155R, 156C, 159Q, 161R / V, 162W, 164A / R, 165K, 177G, 186A / C / E / H / L / M / R / T / V, 188A, 189C / T, 190R, 191T, 195R, 196E / V, 197R, 198A / D / K / L / N / R / V / W, 201L / S, 205E / G / K, 207L, 208D / F / H, 212F / G / M / S / W, 220V, 226D / E / Q / S / V, 228E / I / M / S, 230L / M, 231P, 232R, 233G / T / W, 235W, 237L / M / R / S / V / Y, 239M / N / P / Q / S / T / V / W, 240E / G / K / Q / R / S / Y, 242P / Q / T / V, 251L, 254G / S, 258 L / S / V, 263G / L / Q / T, 264A / C, 266M / T, 267D / W / Y, 269L, 271A / G / N / S, 273A / G / S, 277Q / R, 278E, 282G / L / M / T / V / Y, 283A / F / G / K / L / M / R / S / V, 284D, 286F / L / S / W, 288I, 289A / L / S / V, 290L, 294L, 295K, 297W, 300G / T, 301F / L, 305K, 306I / K / S / V, 308K / L / S, 309G, 317T / Q, 323S, 328R, 334L / R, 337G / L / M / P / R / S, 33 9Y, 349E, 355S, 356A / V / W, 357H / K / P / R / S / V, 358C, 359N / R, 360H / M / P, 362G,364R, 367C / L, 370C / G, 372N / Q, 374A / S, 375W, 378T, 379A / G / P, 380T, 381K / R, 382V, 3 84C / V, 386F, 387G, 388K / Y, 389K / L / Q / R, 390E, 392C / I / K / L / R / S, 396C / H, 397K / L / M, 4 04S, 405I, 408C / V, 414A / L / Q / R / S / T / V, 415A / C / E / H / I / K / L / V, 416K, 417D / G / L, 418A / G / I / K / L / M / P / S / T, 419G, 421R, 422N, 423R / T, or 428E / F / R / S, or a combination thereof, wherein amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0165] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 63, 242, 283, 286, 317, 414, 418, or 428, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or amino acid residues 63F / R, 242P / Q / T / V, 283A / F / G / K / L / M / R / S / V, 286F / L / S / W, 317T / Q, 414A / L / Q / R / S / T / V, 418A / G / I / K / L / M / P / S / T, or 428E / F / R / S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938. In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or amino acid residue 63R, 242Q, 283L, 286S, 317Q, 414Q, 418S, or 428R, or a combination thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0166] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to a reference sequence corresponding to SEQ ID NO: 62, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to the reference sequence corresponding to SEQ ID NO: 62.

[0167] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 68 to 312, or to a reference sequence corresponding to an even numbered SEQ ID NO: 68 to 312, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to the reference sequence corresponding to SEQ ID NO: 62.

[0168] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the amino acid positions 196, 242, 337, 33, 277, 30, 359, 283, 415, 387, 379, 205, 186, 389, 102, 164, 301, 375, 267, 380, 254, 317, 77 / 139 / 317 / 417, 105 / 317 / 417, 317 / 349 / 36 62, 2 / 386, 105 / 317, 139 / 317 / 362, 233 / 317 / 405, 139 / 317, 162, 286, 414, 417, 226, 61, 105, 230, 418, 370, 297, 237, 428, 362, 233, 235, 148, 100, 97, 382 or 358, wherein the amino acid position is a substitution or set of substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or relative to the reference sequence corresponding to SEQ ID NO: 62 comprising one or more substitutions.

[0169] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least one substitution or set of substitutions 196E, 242P, 337L, 33R, 33V, 277Q, 337P, 30C, 359N, 337G, 283S, 242Q, 415A, 387G, 415H, 379A, 205G, 186M, 337M, 389Q, 102L, 389L, 359R, 205K, 164A, 415E, 301F, 30H, 379G, 375W, 26 7W, 380T, 415C, 254G, 186T, 317Q, 77D / 139T / 317Q / 417G, 105K / 317Q / 417G, 317Q / 349E / 362G / 386F, 105K / 317Q , 139T / 317Q / 362G, 233T / 317Q / 405I, 139T / 317Q, 162W, 283V, 286S, 414L, 417L, 226S, 61T, 226Q, 105S, 186E, 2 30M, 61V, 186V, 186L, 379P, 415V, 415I, 186A, 283R, 226V, 418L, 418S, 370G, 283A, 337S, 297W, 237S, 428S, 186 C, 362G, 283G, 196V, 237L, 415L, 233W, 242T, 277R, 235W, 148P, 254S, 102G, 105T, 301L, 286F, 100V, 237Y, 30S, 86L, 186H, 417D, 226D, 233G, 267Y, 97G, 226E, 418T, 230L, 414T, 418A, 237V, 418G, 382V, 267D, 283M, 418P, 237R, 237M, 418I, 358C, 186R, 418M or 102S, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or relative to the reference sequence corresponding to SEQ ID NO: 62.

[0170] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least one substitution or set of substitutions: I196E, V242P, F337L, T33R, T33V, E277Q, F337P, T30C, K359N, F337G, F283S, V242Q, T415A, T387G, T415H, E379A, Q205G, K186M, F337M, E389Q, N102L, E389L, K359R, Q205K, K164A, T415E, E301F, T30H, E379G, Y375W, Q267W, V 380T, T415C, H254G, K186T, T317Q, Y77D / S139T / T317Q / S417G, L105K / T317Q / S417G, T317Q / D349E / T362G / Y386F, L105K / T317Q, S1 39T / T317Q / T362G, S233T / T317Q / V405I, S139T / T317Q, Y162W, F283V, W286S, S414L, S417L, K226S, I61T, K226Q, L105S, K186E, V23 0M, I61V, K186V, K186L, E379P, T415V, T415I, K186A, F283R, K226V, K418L, K418S, A370G, F283A, F337S, F297W, K237S, R428S, K186 C. T362G, F283G, I196V, K237L, T415L, S233W, V242T, E277R, F235W, I148P, H254S, N102G, L105T, E301L, W286F, T100V, K237Y, T30S , R428F, S414Q, S414V, F283L, S414A, S414R, A370C, F283K, W286L, K186H, S417D, K226D, S233G, Q267Y, R97G, K226E, K418T, V230L, S414T, K418A, K237V, K418G, I382V, Q267D, F283M, K418P, K237R, K237M, K418I, G358C, K186R, K418M or N102S, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or relative to the reference sequence corresponding to SEQ ID NO: 62.

[0171] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to a reference sequence corresponding to SEQ ID NO: 138, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to a reference sequence corresponding to SEQ ID NO: 138.

[0172] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 314 to 458, or to a reference sequence corresponding to an even numbered SEQ ID NO: 314 to 458, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to the reference sequence corresponding to SEQ ID NO: 138.

[0173] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 242 / 283 / 286 / 359 / 418, 283 / 286, 283 / 286 / 418, 186 / 242 / 283 / 286 / 418, 205 / 286 / 359, 283, 242 / 286 / 418, 186 / 205 / 242, 283 / 286 / 359, 277 / 286 / 359 / 418, 186 / 205 / 283 / 286 / 359, 242 / 277 / 418, 242 / 283 / 286 / 418, 112 / 196 / 389、286、283 / 286 / 359 / 418、242 / 359 / 418、186 / 283、186 / 359 / 418、205 / 242 / 418、186 / 205 / 242 / 283 / 286 / 359 / 418、205 / 359 / 418、186 / 205 / 283 / 286 / 418、186 / 283 / 359、186 / 242、186 / 242 / 359、283 / 359 / 418、277 / 418、186 / 188 / 283、186 / 286 / 4 18, 186 / 242 / 286 / 359 / 418, 418, 186 / 242 / 283 / 286 / 359 / 418, 186 / 277 / 359 / 418, 242 / 283 / 286, 205 / 418, 30 / 297, 205 / 242 / 286 / 359 / 418, 186 / 205 / 359 / 418, 359 / 418, 186, 230, 33 / 297, 186 / 205, 186 / 283 / 359 / 418, 186 / 418, 205 / 242 / 283 / 359 / 418, 33 / 3 96 / 242 / 283 / 286 / 359 / 418, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or relative to the reference sequence corresponding to SEQ ID NO: 138.

[0174] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or set of substitutions 242Q / 283V / 286S / 359R / 418L, 283L / 286S, 283L / 286S / 418S, 186E / 242Q / 283V / 286S / 418L, 205K / 286S / 359R, 283V, 242P / 286S / 418T, 186E / 205K / 242T, 283L / 286S / 359R, 277Q / 286S / 359N / 418T,

[0175] 186A / 205K / 283V / 286S / 359R, 242Q / 277Q / 418L, 242Q / 283S / 286S / 418T,

[0176] 112M / 196V / 389Q, 286S, 242P / 283R / 286S / 359R / 418L,

[0177] 283L / 286S / 359R / 418S, 242Q / 359N / 418S, 186E / 283L, 186E / 359R / 418S,

[0178] 205K / 242T / 418S, 186E / 205K / 242Q / 283S / 286S / 359R / 418S, 205K / 359N / 418L, 186A / 205K / 283L / 286S / 418L, 242T / 283V / 286S / 359R / 418S, 186E / 283L / 359R, 186E / 242P, 283V / 286S / 418S, 186E / 242T / 359R, 283L / 359R / 418L, 277Q / 418S, 186E / 188A / 283V, 186E / 286S / 418S, 186A / 242T / 286S / 359N / 418L, 418L, 186A / 242P / 283V / 286S / 359R / 418S, 186A / 277Q / 359R / 418S, 242Q / 283R / 286S, 205K / 418S , 30C / 297W, 283L / 359R / 418S, 186E / 286S / 418L, 418T, 205K / 242T / 286S / 359R / 418S, 186E / 205K / 359N / 418T, 418S, 186E / 242T / 283S / 286S / 418S, 359R / 418S, 242T / 286S / 418S, 186E, 230L, 33V / 297W, 186E / 205K, 186A / 283L / 359R / 418S, 186E / 418L, 186E / 277Q / 359R / 4 18S, 205K / 242T / 283R / 359R / 418L, 186E / 418T, 33V / 375W / 389Q, 33R / 230L, 196E / 242T / 283V / 286S / 359R / 418T, 186E / 242T / 283L / 359 96S / 359N / 418T, wherein amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or relative to the reference sequence corresponding to SEQ ID NO: 138.

[0179] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or set of substitutions V242Q / F283V / W286S / K359R / K418L, F283L / W286S, F283L / W286S / K418S, K186E / V242Q / F283V / W286S / K418L, Q205K / W286S / K359R, F283V, V242P / W286S / K418T, K186E / Q205K / V242T, F283L / W286S / K359R, E277Q / W286S / K359N / K418T, K186A / Q205K / F283V / W286S / K359R, V242Q / E277Q / K418L, V242Q / F283S / W286S / K418T, L112M / I196V / E3 89Q, W286S, V242P / F283R / W286S / K359R / K418L, F283L / W286S / K359R / K418S , V242Q / K359N / K418S, K186E / F283L, K186E / K359R / K418S, Q205K / V242T / K 418S, K186E / Q205K / V242Q / F283S / W286S / K359R / K418S, Q205K / K359N / K418 L, K186A / Q205K / F283L / W286S / K418L, V242T / F283V / W286S / K359R / K418S, K186E / F283L / K359R, K186E / V242P, F283V / W286S / K418S, K186E / V242T / K3 59R, F283L / K359R / K418L, E277Q / K418S, K186E / V188A / F283V, K186E / W286 S / K418S, K186A / V242T / W286S / K359N / K418L, K418L, K186A / V242P / F283V / W 286S / K359R / K418S, K186A / E277Q / K359R / K418S, V242Q / F283R / W286S, Q20 5K / K418S, T30C / F297W, F283L / K359R / K418S, K186E / W286S / K418L, K418T, Q 205K / V242T / W286S / K359R / K418S, K186E / Q205K / K359N / K418T, K418S, K18 6E / V242T / F283S / W286S / K418S, K359R / K418S, V242T / W286S / K418S, K186E,V230L, T33V / F297W, K186E / Q205K, K186A / F283L / K359R / K418S, K186E / K418L, K186E / E277Q / K359R / K418S, Q205K / V242T / F283R / K359R / K418L, K186E / K418T, T33V / Y375W / E389Q, T33R / V230L, I196E / V242T / F283V / W2 86S / K359R / K418T, K186E / V242T / F283L / K359R / K418S, K186E / K359R, T3 3V / I196V, T33R / Y375W / E389Q, K186A / E277Q / K418S, K186A / V242T / F283L / W286S / K418L, V242T, T33V / I196V / F297W / E301F, Q205K / K237Y / V242P / F283L / W286S / K359R, K186A / Q205K / F283L / K359R / K418T, T33V / E389Q or K186A / I196E / V242T / F283L / W286S / K359N / K418T, wherein amino acid positions are relative to the amino acid positions corresponding to SEQ ID NO: 138, or relative to a reference sequence corresponding to SEQ ID NO: 138.

[0180] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to a reference sequence corresponding to SEQ ID NO: 318, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to the reference sequence corresponding to SEQ ID NO: 318.

[0181] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 460 to 936, or to a reference sequence corresponding to an even numbered SEQ ID NO: 460 to 936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to the reference sequence corresponding to SEQ ID NO: 318.

[0182] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 363, 63, 389, 381, 197, 359, 102, 165, 388, 414, 337, 164, 416, 101, 415, 423, 364, 73, 50, 71, 388 / 419, 357, 396, 68, 76, 14, 271, 360, 266, 208, 74, 263, 264, 13, 378, 372, 300, 294, 290, 397, 95, 258, 161, 21 2, 198, 138, 18, 404, 273, 117, 240, 69, 278, 289, 82, 328, 61 / 186 / 417, 186 / 370 / 417, 267, 61 / 370, 186 / 267 / 370 / 417, 61 / 186, 370 / 417, 417, 61 / 370 / 382, 61 / 186 / 267 / 370 / 417, 61, 267 / 370 / 417, 267 / 370, 61 / 417, 61 / 186 / 237 / 267 / 370, 61 / 237 / 370 / 417, 370, 186 / 370, 61 / 186 / 267 / 417, 61 / 186 / 370 / 382, 370 / 382 / 417, 61 / 186 / 370, 237 / 267 / 370 / 417, 61 / 186 / 382, 61 / 267, 61 / 267 / 417, 61 / 237 / 267 / 382, 186 / 370 / 382, 237 / 267 / 370, 61 / 186 / 267 / 370, 186 / 237 / 267 / 370, 61 / 186 / 267, 390, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or relative to the reference sequence corresponding to SEQ ID NO: 318.

[0183] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least one substitution or set of substitutions 363R, 63R, 389K, 381R, 389R, 197R, 359R, 381K, 102K,

[0184] 165K、388K、414R、337R、164R、416K、101R、415K、423R、364R、73P、50I、71L、388Y / 419G、50G、357K、396H、68Y、76F、71P、14V、271G、68A、68M、360P、266M、208D、74S、263T、264C、13R、263L、360M、378T、76H、13G、76R、50T、372Q、50S、300G、73T、294L、372N、76N、290L、397L、76G、95L、258L、161V、212M、198V、138L、18D、18S、404S、273G、117G、240E、68S、73C、69T、278E、360H、198D、300T、161R、73K、289V、82R、328R、61T / 186A / 417D、186A / 370C / 417L、267Y、61T / 370C、186A / 267Y / 370C / 417L、61T / 186C、370C / 417D、417D、61T / 370C / 382V、61T / 186A / 267Y / 370C / 417D、61T、267Y / 370C / 417D、267Y / 370C、61T / 417L、61T / 186H / 237R / 267Y / 370C、61T / 237R / 370C / 417L、370C / 417L、370C、186E / 370C、61T / 186E / 267Y / 417D、61T / 186C / 370C / 382V、370C / 382V / 417D、61T / 186C / 267Y / 417D、61T / 186C / 370C、237R / 267Y / 370C / 417D、61T / 186E / 417D、61T / 186H、61T / 186C / 382V、61T / 186H / 370C、61T / 267Y、61T / 267Y / 417L、61T / 237R / 267Y / 382V、186H / 370C / 382V、237R / 267Y / 370C、186H / 370C / 417D、61T / 186V / 267Y / 370C、267Y / 370C / 417L、61T / 186V / 370C、186H / 237R / 267Y / 370C、61T / 186E / 267Y、61T / 186C / 237R、61T / 186H / 237R、61T / 186C / 417L、186H、186A / 267Y、186C / 370C、61T / 186C / 237R / 267Y / 370C、186E / 267Y、237R / 370C / 417L, 186H / 370C, 242Q / 414Q, 414T, 414Q, 162W / 414Q, 162W / 414V, 267D / 414T, 414 V, 267D / 414Q, 414A, 162W / 414L, 105S / 414T, 162W / 242Q / 414T, 97G / 162W / 414Q, 105S / 162W / 267 D / 414V, 356V, 273A, 357P, 14G, 14S, 396C, 240R, 392K, 273S, 106V, 308S, 308L, 306I, 96A, 397K, 263Q, 282T, 138R, 258S, 76L, 14T, 289S, 240G, 106S, 117S, 357S, 96G, 113T, 71G, 282V, 117V, 282 M, 258V, 309G, 306K, 308K, 357R, 212S, 18N, 113A, 306S, 212F, 198N, 110R, 240K, 198L, 71R, 357 V, 198R, 271A, 282Y, 212G, 95V, 37S, 68V, 240S, 117Y, 201S, 271N, 240Q, 289L, 212W, 198W, 357H, : 208F, 282L, 306V, 284D, 96T, 266T, 374S, 95R, 309R, 73W, 397M, 289A, 295K, 106L, 95A, 374A, 88V, 96V, 271S, 44S, 208H, 263G, 12A, 201L, 198A, 198K, 282G, 390E, 264A or 73V, wherein amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or relative to a reference sequence corresponding to SEQ ID NO: 318.

[0185] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or set of substitutions K363R, G63R, E389K, S381R, E389R, I197R, K359R, S381K, N102K, R165K, D388K, S414R, F337R, K164R, S416K, G101R, T415K, S423R, N364R, L73P, E50I, K71L, D388Y / T419G, E50G, E357K, A396H, I68Y, D76F, K71P, K14V, F271G, I68A, I68M, E360P, E266M, A208D, P74S , R263T, I264C, T13R, R263L, E360M, N378T, D76H, T13G, D76R, E50T, V372Q, E50S, S300G, L73T, D294L, V372N, D76N, W290L, R397L, D76G, Y95L, A258L, K 161V, V212M, T198V, K138L, Q18D, Q18S, K404S, R273G, Q117G, D240E, I68S, L73C, K69T, Q278E, E360H, T198D, S300T, K161R, L73K, E289V, D82R, D328R, I61T / K186A / S417D, K186A / A370C / S417L, Q267Y, I61T / A370C, K186A / Q267 Y / A370C / S417L, I61T / K186C, A370C / S417D, S417D, I61T / A370C / I382V, I6 1T / K186A / Q267Y / A370C / S417D, I61T, Q267Y / A370C / S417D, Q267Y / A370C, I61T / S417L, I61T / K186H / K237R / Q267Y / A370C, I61T / K237R / A370C / S417L , A370C / S417L, A370C, K186E / A370C, I61T / K186E / Q267Y / S417D, I61T / K18 6C / A370C / I382V, A370C / I382V / S417D, I61T / K186C / Q267Y / S417D, I61T / K 186C / A370C, K237R / Q267Y / A370C / S417D, I61T / K186E / S417D, I61T / K186H , I61T / K186C / I382V, I61T / K186H / A370C, I61T / Q267Y, I61T / Q267Y / S417L,I61T / K237R / Q267Y / I382V、K186H / A370C / I382V、K237R / Q267Y / A370C、K186H / A370C / S417D、I61T / K186V / Q267Y / A370C、Q267Y / A370C / S417L、I61T / K186V / A370C、K186H / K237R / Q267Y / A370C、I61T / K186E / Q267Y、I61T / K186C / K237R、I61T / K186H / K237R、I61T / K186C / S417L、K186H、K186A / Q267Y、K186C / A370C、I61T / K186C / K237R / Q267Y / A370C、K186E / Q267Y、K237R / A370C / S417L、K186H / A370C、V242Q / S414Q、S414T、S414Q、Y162W / S414Q、Y162W / S414V、Q267D / S414T、S414V、Q267D / S414Q、S414A、Y162W / S414L、L105S / S414T、Y162W / V242Q / S414T、R97G / Y162W / S414Q、L105S / Y162W / Q267D / S414V、E356V、R273A、E357P、K14G、K14S、A396C、D240R、V392K、R273S、D106V、F308S、F308L、E306I、N96A、R397K、R263Q、E282T、K138R、A258S、D76L、K14T、E289S、D240G、D106S、Q117S、E357S、N96G、S113T、K71G、E282V、Q117V、E282M、A258V、H309G、E306K、F308K、E357R、V212S、Q18N、S113A、E306S、V212F、T198N、V110R、D240K、T198L、K71R、E357V、T198R、F271A、E282Y、V212G、Y95V、D37S、I68V、D240S、Q117Y、T201S、F271N、D240Q、E289L、V212W、T198W、E357H、A208F、E282L、E306V、P284D、N96T、E266T、E374S、Y95R、H309R、L73W、R397M、E289A、G295K、D106L、Y95A、E374A、L88V、N96V、F271S、D44S、A208H、R263G、M12A、T201L, T198A, T198K, E282G, M390E, I264A or L73V, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or relative to a reference sequence corresponding to SEQ ID NO: 318.

[0186] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to a reference sequence corresponding to SEQ ID NO: 722, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to the reference sequence corresponding to SEQ ID NO: 722.

[0187] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 938 to 1098, or to a reference sequence corresponding to an even numbered SEQ ID NO: 938 to 1098, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to the reference sequence corresponding to SEQ ID NO: 722.

[0188] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 63, 63 / 96 / 370, 389, 13 / 267 / 363 / 389, 13 / 186 / 389, 50 / 267 / 363 / 370 / 389, 363 / 370, 96 / 370, 61 / 63 / 212, 11 / 305, 11, 242 / 283 / 286 / 317 / 414 / 418, 323, 334, 3 : 722, 356, 384, 408, 67, 392, 104, 355, 159, 155, 367, 31, 231, 36, 150, 239, 103, 125, 228, 37, 189, 177, 422, 128, 220, 130, 56, 190, 156, 232, 423, 34, 99, 59, 60, 421 or 195, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or relative to the reference sequence corresponding to SEQ ID NO: 722.

[0189] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least one substitution or set of substitutions 63R, 63R / 96T / 370C, 389K, 13R / 267Y / 363R / 389K, 13R / 186H / 389K, 50T / 267Y / 363R / 370C / 389K, 363R / 370C, 96T / 370C, 61 T / 63R / 212W, 11D / 305K, 11D, 242V / 283F / 286W / 317T / 414S / 418K, 323S, 334L, 339 Y, 356V, 384V, 408C, 67R, 392R, 104K, 355S, 159Q, 155R, 367L, 31R, 231P, 36Y, 150 T, 239V, 103V, 356A, 63F, 125T, 367C, 228I, 37S, 37G, 239M, 239W, 239Q, 189C, 23 9S, 239T, 177G, 239P, 37N, 422N, 228S, 125R, 128C, 189T, 356W, 220V, 408V, 392S, 722, 130T, 228M, 56P, 228E, 190R, 392I, 156C, 232R, 150F, 150C, 239N, 392L, 423T, 34L, 99I, 384C, 59E, 334R, 60Y, 99G, 34R, 37L, 392C, 421R or 195R, wherein amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or relative to a reference sequence corresponding to SEQ ID NO: 722.

[0190] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution set G63R, G63R / N96T / A370C, E389K, T13R / Q267Y / K363R / E389K, T13R / K186H / E389K, E50T / Q267Y / K363R / A370C / E389K, K363R / A370C, N96T / A370C, I61T / G63R 3R / V212W, G11D / Q305K, G11D, Q242V / L283F / S286W / Q317T / Q414S / S418K, M323S, K334L, I339Y, E356V, G384V, I408C, K67R, V392R, A104K, F355S, L159Q, L155R, V367L, S31R, Q231P, E36Y, E150T , D239V, A103V, E356A, G63F, K125T, V367C, A228I, D37S, D37G, D239M, D239W, D239Q, L189C, D23 9S, D239T, C177G, D239P, D37N, K422N, A228S, K125R, Q128C, L189T, E356W, I220V, I408V, V392S, : D130T, A228M, Y56P, A228E, K190R, V392I, A156C, K232R, E150F, E150C, D239N, V392L, S423T, A34L, L99I, G384C, D59E, K334R, L60Y, L99G, A34R, D37L, V392C, K421R or K195R, wherein amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or relative to the reference sequence corresponding to SEQ ID NO: 722.

[0191] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to a reference sequence corresponding to SEQ ID NO: 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to the reference sequence corresponding to SEQ ID NO: 938.

[0192] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 1100 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO: 1100 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to the reference sequence corresponding to SEQ ID NO: 938.

[0193] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 308 / 357 / 390, 74 / 76 / 201 / 308 / 357, 61 / 74 / 76 / 186 / 201 / 308 / 309 / 357 / 390, 14 / 201 / 240 / 289 / 357, 308 / 415, 76 / 357 / 396, 263 / 308 / 396 、61 / 76 / 96 / 240 / 308 / 309、14 / 306 / 415、14 / 73 / 106 / 415、12 / 14 / 258 / 263 / 289 / 308 / 309 / 396、74 / 76 / 117 / 309 / 357、14 / 258 / 263 / 357 / 396、14 / 96 / 106 / 306、14 / 106、12 / 14 / 308 / 309, 14 / 357 / 390, 14 / 117 / 258 / 309 / 357, 390, 240 / 273 / 357 / 390, 61 / 76 / 186 / 201 / 308 / 309, 14 / 396, 309, 14, 106 / 306 / 308, 14 / 240 / 306 / 308, 12 / 14 / 186 / 357, 309 / 390, 14 / 306 , 14 / 76 / 308, 117 / 208 / 258 / 263 / 289 / 308 / 309, 14 / 73 / 106, 76 / 208 / 263, 357, 14 / 308, 263, 76, 14 / 300 / 308 / 415, 240, 33 / 357 / 390, 14 / 76 / 273 or 74, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or relative to the reference sequence corresponding to SEQ ID NO: 938.

[0194] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution set 308L / 357S / 390E, 74S / 76L / 201S / 308L / 357P, 61T / 74S / 76L / 186A / 201S / 308L / 309R / 357P / 390E, 14G / 201S / 240R / 289S / 357P, 308S / 415I, 76L / 357S / 396C, 263L / 308L / 396C, 61T / 76L / 96A / 240R / 308L / 309R, 14S / 306I / 415I, 14S / 73W / 106S / 415I, 12A / 14G / 258S / 263Q / 289S / 308L / 309R / 3 96C, 74S / 76L / 117V / 309R / 357S, 14T / 258S / 263Q / 357S / 396C, 14S / 96G / 106S / 306I, 14S / 106S, 12A / 14G / 308L / 309R、14G / 357S / 390E、14G / 117V / 258S / 309R / 357S、390E、240R / 273S / 357P / 390E、61T / 76L / 186V / 201S / 308 L / 309R, 14G / 396C, 309R, 14S, 106V / 306I / 308S, 14S / 240S / 306I / 308S, 12I / 14G / 186V / 357S, 309R / 390E, 14S / 306I, 14T, 14S / 76R / 308S, 117V / 208D / 258S / 263Q / 289S / 308L / 309R, 14S / 73W / 106S, 76L / 208D / 263Q, 357S, 14S / 308S, 263L, 76L, 14S / 300G / 308S / 415I, 240Y, 33M / 357S / 390E, 14G / 76L / 273S or 74S, wherein amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or relative to the reference sequence corresponding to SEQ ID NO: 938.

[0195] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution set F308L / E357S / M390E, P74S / D76L / T201S / F308L / E357P, I61T / P74S / D76L / K186A / T201S / F308L / H309R / E357P / M390E, K14G / T201S / D240R / E289S / E357P, F308S / T415I, D76L / E357S / A396C, R263L / F308L / A396C, I61T / P74S / D76L / K186A / T201S / F308L / H309R / E357P / M390E / N96A / D240R / F308L / H309R, K14S / E306I / T415I, K14S / L73W / D106S / T415I, M12A / K14G / A258S / R263Q / E289S / F308L / H309R / A396C , P74S / D76L / Q117V / H309R / E357S, K14T / A258S / R263Q / E357S / A396C, K14S / N96G / D106S / E306I, K14S / D106S, M12A / K14G / F308L / H 309R, K14G / E357S / M390E, K14G / Q117V / A258S / H309R / E357S, M390E, D240R / R273S / E357P / M390E, I61T / D76L / K186V / T201S / F308 L / H309R, K14G / A396C, H309R, K14S, D106V / E306I / F308S, K14S / D240S / E306I / F308S, M12I / K14G / K186V / E357S, H309R / M390E, K14 S / E306I, K14T, K14S / D76R / F308S, Q117V / A208D / A258S / R263Q / E289S / F308L / H309R, K14S / L73W / D106S, D76L / A208D / R263Q, E357S, K14S / F308S, R263L, D76L, K14S / S300G / F308S / T415I, D240Y, T33M / E357S / M390E, K14G / D76L / R273S or P74S, wherein amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or relative to the reference sequence corresponding to SEQ ID NO: 938.

[0196] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution at an amino acid position listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0197] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least one substitution listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0198] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions at an amino acid position listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0199] In some embodiments, the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0200] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0201] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of residues 12 to 437 of the corresponding engineered DNA ligase variants listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2.

[0202] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to residues 12 to 437 of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236 , 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 58, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478,480、482、484、486、488、490、492、494、496、498、500、502、504、506、508、510、512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、700、702、704、706、708、710、712、714、716、718、720、722、724、728、730、732。734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, or 1184.

[0203] In some embodiments, the engineered DNA ligase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 119, 200, 201 20, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240 , 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 3 62, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482,484、486、488、490、492、494、496、498、500、502、504、506、508、510、512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、700、702、704、706、708、710、712、714、716、718、720、722、724、728、730、732。734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998、1000、1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, or 1184.

[0204] In some embodiments, the engineered DNA ligase comprises an amino acid sequence comprising residues 12 to 437 of an even-numbered SEQ ID NO among SEQ ID NOs: 40 to 1184, or an amino acid sequence comprising an even-numbered SEQ ID NO among SEQ ID NOs: 40 to 1184. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions.

[0205] In some embodiments, the engineered DNA ligase comprises a DNA ligase comprising the following SEQ ID The amino acid sequence of residues 12 to 437 of NO is: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 6, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407 8, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528,530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、700、702、704、706、708、710、712、714、716、718、720、722、724、728、730、732。734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998、1000、1002、1004、1006、1008、1010、1012、1014、1016、1018、1020、1022、1024、1028、1030、1032、1034、1036、1038、1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1174, 1176, 1178, 1180, 1182, or 1184. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0206] In some embodiments, the engineered DNA ligase comprises a DNA ligase comprising the following SEQ ID Amino acid sequence of NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 , 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 29 2, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 413 14, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534,536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、700、702、704、706、708、710、712、714、716、718、720、722、724、728、730、732。734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998、1000、1002、1004、1006、1008、1010、1012、1014、1016、1018、1020、1022、1024、1028、1030、1032、1034、1036、1038、1040、1042、1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1174, 1176, 1178, 1180, 1182, or 1184. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0207] In some embodiments, the engineered DNA ligase comprises an amino acid sequence comprising residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or an amino acid sequence comprising SEQ ID NO: 62, 138, 318, 722, or 938. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions, insertions, and / or deletions. In some embodiments, the amino acid sequence of the engineered DNA ligase optionally comprises 1, 2, 3, 4, or 5 substitutions.

[0208] In some of the foregoing embodiments, the engineered DNA ligase polypeptide has 1, 2, 3, 4, or up to 5 substitutions in the amino acid sequence. In some embodiments, the engineered DNA ligase polypeptide has 1, 2, 3, or 4 substitutions in the amino acid sequence. In some embodiments, the substitutions comprise non-conservative substitutions or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions. In some embodiments, the substitutions comprise non-conservative substitutions. In some embodiments, the variants disclosed herein provide guidance on non-conservative substitutions and conservative substitutions.

[0209] In some embodiments, the engineered DNA ligases of the present disclosure have DNA ligase activity. In some embodiments, the engineered DNA ligases have DNA ligase activity and are characterized by or exhibit one or more improved or enhanced properties described herein compared to a reference DNA ligase.

[0210] In some embodiments, the engineered DNA ligase has increased activity compared to a reference DNA ligase. In some embodiments, the increased activity is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold or more compared to a reference DNA ligase. Exemplary improvements in activity are provided in the Examples.

[0211] In some embodiments, the engineered DNA ligase has increased stability compared to a reference DNA ligase. In some embodiments, the engineered DNA ligase has increased thermostability compared to a reference DNA ligase. In some embodiments, the increased thermostability is at a temperature of about 25°C to 55°C, about 30°C to about 45°C, 35°C to about 40°C, particularly about 40°C to about 50°C. In some embodiments, the increased thermostability is at 25°C, 30°C, 35°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. In some embodiments, the increased thermostability is at a specified temperature and treated for 15 minutes, 30 minutes, 45 minutes or 1 hour.

[0212] In some embodiments, the engineered DNA ligase has an increased product yield compared to a reference DNA ligase. In some embodiments, the increase in product yield is under the substrates and reaction conditions provided in the examples.

[0213] In some embodiments, the engineered DNA ligase has increased solubility compared to a reference DNA ligase. In some embodiments, the engineered DNA ligase has reduced sequence preference compared to a reference DNA ligase. In some embodiments, the engineered DNA ligase is insensitive or has reduced sensitivity to input DNA substrate concentration.

[0214] In some embodiments, the reference DNA ligase has a sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, 938, or 1108, or a sequence corresponding to SEQ ID NO: 62, 138, 318, 722, 938, or 1108. In some embodiments, the reference DNA ligase has a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2. In some embodiments, the reference DNA ligase is wild-type T4 DNA ligase.

[0215] In some embodiments, the engineered DNA ligase has an improved property selected from the group consisting of: i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased product yield, v) increased solubility, vi) reduced sequence preference, and vii) insensitivity or reduced sensitivity to input DNA concentration, or any combination of i), ii), iii), iv), v), vi), and vii). In some embodiments, the reference DNA ligase has a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722, or 938, or a sequence corresponding to SEQ ID NO: 2, 62, 138, 318, 722, or 938. In some embodiments, the reference DNA ligase has a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2. In some embodiments, the reference DNA ligase is wild-type T4 DNA ligase.

[0216] In some embodiments, the present disclosure further provides an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to

[0217] (a) a sequence corresponding to residues 12 to 437 of SEQ ID NO:2; a sequence corresponding to residues 12 to 613 of SEQ ID NO:4; residues 12 to 614 of SEQ ID NO:6; residues 12 to 610 of SEQ ID NO:8; residues 12 to 606 of SEQ ID NO:10; residues 12 to 615 of SEQ ID NO:12; residues 12 to 594 of SEQ ID NO:14; residues 12 to 620 of SEQ ID NO:16; residues 12 to 608 of SEQ ID NO:18; residues 12 to 611 of SEQ ID NO:20; residues 12 to 614 of SEQ ID NO:22; residues 12 to 611 of SEQ ID NO:24; residues 12 to 609 of SEQ ID NO:26; residues 12 to 422 of SEQ ID NO:28; residues 12 to 518 of SEQ ID NO:30; residues 12 to 438 of SEQ ID NO:32; residues 12 to 381 of SEQ ID NO:34; residues 12 to 424 of SEQ ID NO:36; or residues 12 to 390 of SEQ ID NO:38, or

[0218] (b) a sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.

[0219] In some embodiments, the engineered DNA ligase comprises the amino acid sequence

[0220] (a) comprises residues 12 to 437 of SEQ ID NO:2; residues 12 to 613 of SEQ ID NO:4; residues 12 to 614 of SEQ ID NO:6; residues 12 to 610 of SEQ ID NO:8; residues 12 to 606 of SEQ ID NO:10; residues 12 to 615 of SEQ ID NO:12; residues 12 to 594 of SEQ ID NO:14; residues 12 to 620 of SEQ ID NO:16; residues 12 to 608 of SEQ ID NO:18; residues 12 to 611 of SEQ ID NO:20; residues 12 to 614 of SEQ ID NO:22; residues 12 to 611 of SEQ ID NO:24; residues 12 to 609 of SEQ ID NO:26; residues 12 to 422 of SEQ ID NO:28; residues 12 to 518 of SEQ ID NO:30; residues 12 to 438 of SEQ ID NO:32; residues 12 to 381 of SEQ ID NO:34; residues 12 to 424 of SEQ ID NO:36; or residues 12 to 390 of SEQ ID NO:38, or

[0221] (b) comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.

[0222] In certain embodiments, the engineered DNA ligase is expressed as a fusion protein. In certain embodiments, the engineered DNA ligase as described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, a polypeptide tag that can be used for detection and / or purification. In certain embodiments, the fusion protein of the engineered DNA ligase comprises a glycine-histidine or histidine tag (His tag). In certain embodiments, the fusion protein of the engineered DNA ligase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In certain embodiments, the fusion protein of the engineered DNA ligase comprises a GST, SUMO, Strep, MBP, or GFP tag. In certain embodiments, the fusion is to the amino (N-) end of the engineered DNA ligase polypeptide. In certain embodiments, the fusion is to the carboxyl (C-) end of the engineered DNA ligase polypeptide.

[0223] In some embodiments, the engineered DNA ligase polypeptides described herein are isolated compositions. In some embodiments, the engineered DNA ligase polypeptides are purified or are purified preparations, as further discussed herein.

[0224] In some embodiments, the present disclosure also provides functional fragments or biologically active fragments of the engineered DNA ligase polypeptides described herein. Therefore, for each embodiment of the engineered DNA ligase, functional fragments or biologically active fragments of the engineered DNA ligase are provided herein. In some embodiments, the functional fragments or biologically active fragments of the engineered DNA ligase comprise at least about 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the DNA ligase polypeptide (i.e., parent DNA ligase) from which it originates. In some embodiments, the functional fragments or biologically active fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the parent sequence of the DNA ligase. In some embodiments, the functional fragments will be truncated to less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50 amino acids, less than 55 amino acids, less than 60 amino acids, less than 65 amino acids or less than 70 amino acids.

[0225] In some embodiments, the functional fragment of the engineered DNA ligase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the parent sequence of the engineered DNA ligase. In some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65 or less than 70 amino acids.

[0226] In some embodiments, the functional or biologically active fragments of the engineered DNA ligase polypeptides described herein comprise at least one mutation or set of mutations in the amino acid sequence of the engineered DNA ligase described herein. Thus, in some embodiments, the functional or biologically active fragments of the engineered DNA ligase exhibit enhanced or improved properties relative to the mutation or set of mutations in the parent DNA ligase.

[0227] Polynucleotides encoding engineered polypeptides, expression vectors, and host cells

[0228] In another aspect, the present disclosure provides recombinant polynucleotides encoding the engineered DNA ligases described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide construct capable of expressing the engineered DNA ligase. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding an engineered DNA ligase polypeptide is introduced into an appropriate host cell to express the corresponding DNA ligase polypeptide.

[0229] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to the various amino acids provides a description of all polynucleotides capable of encoding the polypeptides of the present invention. The degeneracy of the genetic code allows for the preparation of a vast array of nucleic acids, all of which encode the engineered DNA ligases of the present invention, when the same amino acid is encoded by alternative or synonymous codons. Thus, the present disclosure provides methods and compositions for generating each possible polynucleotide variant encoding the engineered DNA ligase polypeptides described herein by selecting combinations based on possible codon selection, and all such polynucleotide sequence variants will be considered to be specifically disclosed for any polypeptide described herein, including the examples (e.g., Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2) and the amino acid sequences presented in the sequence listing.

[0230] In some embodiments, codons are preferably optimized for utilization by the selected host cell for protein production. In some embodiments, preferred codons in bacteria are used for expression in bacteria. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, the codon-optimized polynucleotide encoding the engineered DNA ligase polypeptides described herein contains a preferred codon at about 40%, 50%, 60%, 70%, 80%, 90% or more of the codon positions in the full-length coding region.

[0231] Thus, in some embodiments, the recombinant polynucleotides of the present disclosure encode the engineered DNA ligase polypeptides described herein.In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon-optimized.

[0232] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NOs: 2 and an even numbered SEQ ID NO among SEQ ID NOs: 2 and 40 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO among SEQ ID NOs: 2 and 40 to 1184, wherein the amino acid sequence comprises residues 12 to 437 of the reference sequence corresponding to SEQ ID NOs: 2, 62, 138, 318, 722, or 938, or to a reference sequence corresponding to SEQ ID NOs: One or more substitutions of the reference sequence of NO: 2, 62, 138, 318, 722 or 938.

[0233] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722, or 938, or to a reference sequence corresponding to SEQ ID NO: 2, 62, 138, 318, 722, or 938, wherein the amino acid sequence comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722, or 938, or to a reference sequence corresponding to SEQ ID NO: One or more substitutions of the reference sequence of NO: 2, 62, 138, 318, 722 or 938.

[0234] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0235] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0236] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to the reference sequence corresponding to SEQ ID NO: 2.

[0237] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least one of the following: , 106, 110, 112, 113, 117, 125, 128, 130, 132, 138, 139, 148, 149, 150, 155, 156, 159, 161, 162, 164, 165, 177, 186, 188, 189, 190, 191, 195, 196, 197, 198, 201, 205, 207, 208, 212, 220, 226, 228, 230, 231, 232, 233, 234 5, 237, 239, 240, 242, 251, 254, 258, 263, 264, 266, 267, 269, 271, 273, 277, 278, 282, 283, 284, 286, 288, 289, 290, 294, 295, 297, 300, 301, 305, 306, 308, 309, 317, 323, 328, 334, 337, 339, 349, 355, 356, 357, 358, : 14, 17, 18, 20, 25, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 59, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 51, 52, 53, 54, 55, 56, 57, 58, 59, 5

[0238] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least an amino acid sequence comprising a substitution at amino acid position 63, 242, 283, 286, 317, 414, 418, or 428, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0239] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least an amino acid sequence comprising a substitution or set of substitutions at amino acid position 233, 317, 191, 288, 207, 149, 251, 205, 269, 164, 36, 428, 105 / 132, or 105, wherein the amino acid position is relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0240] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least a substituted amino acid sequence at an amino acid position listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0241] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence comprising at least one substitution listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0242] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least an amino acid sequence comprising a substitution or set of substitutions at the amino acid positions listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0243] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence comprising at least a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 2.

[0244] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0245] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

[0246] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938, wherein the amino acid sequence comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: One or more substitutions of a reference sequence of ID NO: 62, 138, 318, 722 or 938.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184, wherein the amino acid sequence comprises residues 12 to 437 relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938, or to a reference sequence corresponding to an even numbered SEQ ID NO: One or more substitutions of the reference sequence of NO: 62, 138, 318, 722 or 938.

[0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least one of the following: , 106, 110, 112, 113, 117, 125, 128, 130, 132, 138, 139, 148, 149, 150, 155, 156, 159, 161, 162, 164, 165, 177, 186, 188, 189, 190, 191, 195, 196, 197, 198, 201, 205, 207, 208, 212, 220, 226, 228, 230, 231, 232, 233, 234 5, 237, 239, 240, 242, 251, 254, 258, 263, 264, 266, 267, 269, 271, 273, 277, 278, 282, 283, 284, 286, 288, 289, 290, 294, 295, 297, 300, 301, 305, 306, 308, 309, 317, 323, 328, 334, 337, 339, 349, 355, 356, 357, 358, 8, 359, 360, 362, 364, 367, 370, 372, 374, 375, 378, 379, 380, 381, 382, ​​384, 386, 387, 388, 389, 390, 392, 396, 397, 404, 405, 408, 414, 415, 416, 417, 418, 419, 421, 422, 423 or 428, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 63, 242, 283, 286, 317, 414, 418, or 428, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0251] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to a reference sequence corresponding to SEQ ID NO: 62, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to the reference sequence corresponding to SEQ ID NO: 62.

[0252] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least one of amino acid positions 196, 242, 337, 33, 277, 30, 359, 283, 415, 387, 379, 205, 186, 389, 102, 164, 301, 375, 267, 380, 254, 317, 77 / 139 / 317 / 417, 105 / 317 / 417, 82, 358, 148, 100, 97, 382, ​​or 358, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or relative to the reference sequence corresponding to SEQ ID NO: 62.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 68 to 312, or to a reference sequence corresponding to an even numbered SEQ ID NO: 68 to 312, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to the reference sequence corresponding to SEQ ID NO: 62.

[0254] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to a reference sequence corresponding to SEQ ID NO: 138, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to the reference sequence corresponding to SEQ ID NO: 138.

[0255] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 314 to 458, or to a reference sequence corresponding to an even numbered SEQ ID NO: 314 to 458, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to the reference sequence corresponding to SEQ ID NO: 138.

[0256] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least one of amino acid positions 242 / 283 / 286 / 359 / 418, 283 / 286, 283 / 286 / 418, 186 / 242 / 283 / 286 / 418, 205 / 286 / 359, 283, 242 / 286 / 418, 186 / 205 / 242, 283 / 286 / 359, 277 / 286 / 359 / 418, 186 / 205 / 283 / 286 / 359, 242 / 277 / 418, 242 / 283 / 286 / 418、112 / 196 / 389、286、283 / 286 / 359 / 418、242 / 359 / 418、186 / 283、186 / 359 / 418、205 / 242 / 418、186 / 205 / 242 / 283 / 286 / 359 / 418、205 / 359 / 418、186 / 205 / 283 / 286 / 418、186 / 283 / 359、186 / 242、186 / 242 / 359、283 / 359 / 418、277 / 418、186 / 188 / 283、 186 / 286 / 418, 186 / 242 / 286 / 359 / 418, 418, 186 / 242 / 283 / 286 / 359 / 418, 186 / 277 / 359 / 418, 242 / 283 / 286, 205 / 418, 30 / 297, 205 / 242 / 286 / 359 / 418, 186 / 205 / 359 / 418, 359 / 418, 186, 230, 33 / 297, 186 / 205, 186 / 283 / 359 / 418, 186 / 418, 205 / 242 / 283 / 359 / 418, 33 186 / 242 / 283 / 286 / 359 / 418, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or relative to the reference sequence corresponding to SEQ ID NO: 138.

[0257] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to a reference sequence corresponding to SEQ ID NO: 318, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to the reference sequence corresponding to SEQ ID NO: 318.

[0258] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 460 to 936, or to a reference sequence corresponding to an even numbered SEQ ID NO: 460 to 936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to the reference sequence corresponding to SEQ ID NO: 318.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least amino acid positions 363, 63, 389, 381, 197, 359, 102, 165, 388, 414, 337, 164, 416, 101, 415, 423, 364, 73, 50, 71, 388 / 419, 357, 396, 68, 76, 14, 271, 360, 266, 208, 74, 263, 264, 13, 378, 372, 300, 294, 290, 396. 7, 95, 258, 161, 212, 198, 138, 18, 404, 273, 117, 240, 69, 278, 289, 82, 328, 61 / 186 / 417, 186 / 370 / 417, 267, 61 / 370, 186 / 267 / 370 / 417, 61 / 186, 370 / 417, 417, 61 / 370 / 382, 61 / 186 / 267 / 370 / 417, 61, 267 / 370 / 417, 267 / 370, 61 / 417, 61 / 186 / 237 / 267 / 37 0, 61 / 237 / 370 / 417, 370, 186 / 370, 61 / 186 / 267 / 417, 61 / 186 / 370 / 382, 370 / 382 / 417, 61 / 186 / 370, 237 / 267 / 370 / 417, 61 / 186 / 382, 61 / 267, 61 / 267 / 417, 61 / 237 / 267 / 382, 186 / 370 / 382, 237 / 267 / 370, 61 / 186 / 267 / 370, 186 / 237 / 267 / 370, 61 / 186 / 267, 390, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or relative to the reference sequence corresponding to SEQ ID NO: 318.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to a reference sequence corresponding to SEQ ID NO: 722, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to the reference sequence corresponding to SEQ ID NO: 722.

[0261] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 938 to 1098, or to a reference sequence corresponding to an even numbered SEQ ID NO: 938 to 1098, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to the reference sequence corresponding to SEQ ID NO: 722.

[0262] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least one of amino acid positions 63, 63 / 96 / 370, 389, 13 / 267 / 363 / 389, 13 / 186 / 389, 50 / 267 / 363 / 370 / 389, 363 / 370, 96 / 370, 61 / 63 / 212, 11 / 305, 11, 242 / 283 / 286 / 317 / 414 / 418, 3 722, 354, 336, 357, 369, 421, 437, 442, 461, 473, 489, 501, 512, 513, 514, 520, 516, 521, 517, 522, 518, 523, 524, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 541, 542, 543, 544, 529, 545, 546, 547, 548, 551, 552, 553, 554, 528, 529, 530, 531, 532, 533, 539, 541, 542, 543, 544, 529

[0263] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to a reference sequence corresponding to SEQ ID NO: 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to the reference sequence corresponding to SEQ ID NO: 938.

[0264] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 1100 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO: 1100 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to the reference sequence corresponding to SEQ ID NO: 938.

[0265] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least amino acid positions 308 / 357 / 390, 74 / 76 / 201 / 308 / 357, 61 / 74 / 76 / 186 / 201 / 308 / 309 / 357 / 390, 14 / 201 / 240 / 289 / 357, 308 / 415, 76 / 357 / 396, 263 / 308 / 396, 61 / 76 / 96 / 240 / 308 / 309, 14 / 306 / 415, 14 / 73 / 106 / 415, 12 / 14 / 258 / 263 / 289 / 308 / 309 / 396, 74 / 76 / 117 / 309 / 357, 14 / 258 / 263 / 357 / 396, 14 / 96 / 106 / 306, 14 / 106, 12 / 14 / 308 / 309, 14 / 357 / 390, 14 / 117 / 258 / 309 / 357, 390, 240 / 273 / 357 / 390, 61 / 76 / 186 / 201 / 308 / 309, 14 / 396, 309, 14, 106 / 306 / 308, 14 / 240 / 306 / 308, 12 / 14 / 186 / 357, 309 / 390, 14 / 30 6, 14 / 76 / 308, 117 / 208 / 258 / 263 / 289 / 308 / 309, 14 / 73 / 106, 76 / 208 / 263, 357, 14 / 308, 263, 76, 14 / 300 / 308 / 415, 240, 33 / 357 / 390, 14 / 76 / 273 or 74, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or relative to the reference sequence corresponding to SEQ ID NO: 938.

[0266] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least a substituted amino acid sequence at an amino acid position listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0267] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence comprising at least one substitution listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0268] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least an amino acid sequence comprising a substitution or set of substitutions at an amino acid position listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0269] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising at least a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

[0270] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising at least a substitution or set of substitutions provided in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 and 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or relative to the reference sequence corresponding to SEQ ID NO: Reference sequence of NO:62, 138, 318, 722 or 938.

[0271] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising a sequence corresponding to residues 12 to 437 of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, or a sequence corresponding to residues 12 to 437 of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2. The sequences of the engineered DNA ligase variants listed in 3.2, 14.2, 15.2, 16.2, 17.2 and 18.2 have amino acid sequences of at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0272] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence comprising residues 12 to 437 of an even numbered SEQ ID NO among SEQ ID NOs: 40 to 1184, or an amino acid sequence comprising an even numbered SEQ ID NO among SEQ ID NOs: 40 to 1184, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0273] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, 938, or 1108, or comprising the amino acid sequence of SEQ ID NO: 62, 138, 318, 722, 938, or 1108, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.

[0274] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1311 of SEQ ID NO: 1, 61, 137, 317, 721 or 937, or a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 61, 137, 317, 721 or 937, wherein the recombinant polynucleotide encodes an engineered DNA ligase.

[0275] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1311 of an odd-numbered SEQ ID NO: 39 to 1183, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 39 to 1183, wherein the recombinant polynucleotide encodes an engineered DNA ligase.

[0276] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to nucleotide residues 34 to 1311 of the following SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233 , 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 3 55, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475,477、479、481、483、485、487、489、491、493、495、497、499、501、503、505、507、509、511、513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、923、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955、957、959、961、963、965、967、969、971、973、975、977、979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1 035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1 087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1 139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181 or 1183, wherein the recombinant polynucleotide encodes a DNA ligase.

[0277] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence corresponding to SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 118 9, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 41, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483,485、487、489、491、493、495、497、499、501、503、505、507、509、511、513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、923、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955、957、959、961、963、965、967、969、971、973、975、977、979、981、983、985、987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 103 9, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089 、1091、1093、1095、1097、1099、1101、1103、1105、1107、1109、1111、1113、1115、1117、1119、1121、1123、1125、1127、1129、1131、1133、1135、1137、1139、 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181 or 1183, wherein the recombinant polynucleotide encodes a DNA ligase.

[0278] In some embodiments, as described above, the recombinant polynucleotide comprises a codon-optimized polynucleotide sequence for expressing the encoded engineered DNA ligase. In some embodiments, the polynucleotide sequence is codon-optimized for expression in prokaryotes. In some embodiments, the polynucleotide sequence is codon-optimized for expression in eukaryotic cells. In some embodiments, the polynucleotide sequence is codon-optimized for expression in bacterial cells.

[0279] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1311 of an odd-numbered SEQ ID NO among SEQ ID NOs: 39 to 1183, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO among SEQ ID NOs: 39 to 1183.

[0280] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1311 of the following SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163 , 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 284 5, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 4 07, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527,529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、923、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955、957、959、961、963、965、967、969、971、973、975、977、979、981、983、985、987、989、991、993、995、997、999、1001、1003、1005、1007、1009、1011、1013、1015、1017、1019、1021、1023、1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, or 1183.

[0281] In some embodiments, the recombinant polynucleotide comprises the following SEQ ID Polynucleotide sequence of NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 , 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 29 1, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 4 13, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533,535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、923、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955、957、959、961、963、965、967、969、971、973、975、977、979、981、983、985、987、989、991、993、995、997、999、1001、1003、1005、1007、1009、1011、1013、1015、1017、1019、1021、1023、1025、1027、1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, or 1183.

[0282] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1311 of SEQ ID NO. 1, 61, 137, 317, 721, 937, or 1107, or a polynucleotide sequence comprising SEQ ID NO: 1, 61, 137, 317, 721, 937, or 1107.

[0283] In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions to a reference polynucleotide sequence encoding an engineered DNA ligase described herein, such as the recombinant polynucleotides provided in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, or their reverse complements. In some embodiments, the reference polynucleotide sequence corresponds to nucleotide residues 34 to 1311 of SEQ ID NO: 1, 61, 137, 317, 721, or 937, or to a sequence of SEQ ID NO: 1, 61, 137, 317, 721, or 937, or its reverse complement, or a polynucleotide sequence encoding one of the other engineered DNA ligases provided herein. In some embodiments, the recombinant polynucleotide encodes a DNA ligase and hybridizes under high stringency conditions to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1311 of an odd-numbered SEQ ID NO among SEQ ID NOs: 39 to 1183, or the reverse complement of a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO among SEQ ID NOs: 39 to 1183.

[0284] In some embodiments, the recombinant polynucleotide hybridizes under high stringency conditions to the reverse complement of a reference polynucleotide sequence encoding an engineered DNA ligase, wherein the engineered DNA ligase comprises an amino acid sequence with one or more amino acid differences compared to SEQ ID NO: 2, 62, 138, 318, 722 or 938 at a residue position selected from any of the positions listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2. In some embodiments, the polynucleotide that hybridizes under high stringency conditions comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 39 to 1183 of SEQ ID NO: 1, 61, 137, 317, 721 or 937, or a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 61, 137, 317, 721 or 937. In some other embodiments, the polynucleotide that hybridizes under high stringency conditions comprises a reference polynucleotide sequence within nucleotide residues 39 to 1183 of the corresponding polynucleotide sequences listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, or a polynucleotide sequence listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2 having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0285] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to

[0286] (a) a sequence corresponding to residues 12 to 437 of SEQ ID NO:2; a sequence corresponding to residues 12 to 613 of SEQ ID NO:4; residues 12 to 614 of SEQ ID NO:6; residues 12 to 610 of SEQ ID NO:8; residues 12 to 606 of SEQ ID NO:10; residues 12 to 615 of SEQ ID NO:12; residues 12 to 594 of SEQ ID NO:14; residues 12 to 620 of SEQ ID NO:16; residues 12 to 608 of SEQ ID NO:18; residues 12 to 611 of SEQ ID NO:20; residues 12 to 614 of SEQ ID NO:22; residues 12 to 611 of SEQ ID NO:24; residues 12 to 609 of SEQ ID NO:26; residues 12 to 422 of SEQ ID NO:28; residues 12 to 518 of SEQ ID NO:30; residues 12 to 438 of SEQ ID NO:32; residues 12 to 381 of SEQ ID NO:34; residues 12 to 424 of SEQ ID NO:36; or residues 12 to 390 of SEQ ID NO:38, or

[0287] (b) a sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.

[0288] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered DNA ligase comprising the amino acid sequence

[0289] (a) comprises residues 12 to 437 of SEQ ID NO:2; residues 12 to 613 of SEQ ID NO:4; residues 12 to 614 of SEQ ID NO:6; residues 12 to 610 of SEQ ID NO:8; residues 12 to 606 of SEQ ID NO:10; residues 12 to 615 of SEQ ID NO:12; residues 12 to 594 of SEQ ID NO:14; residues 12 to 620 of SEQ ID NO:16; residues 12 to 608 of SEQ ID NO:18; residues 12 to 611 of SEQ ID NO:20; residues 12 to 614 of SEQ ID NO:22; residues 12 to 611 of SEQ ID NO:24; residues 12 to 609 of SEQ ID NO:26; residues 12 to 422 of SEQ ID NO:28; residues 12 to 518 of SEQ ID NO:30; residues 12 to 438 of SEQ ID NO:32; residues 12 to 381 of SEQ ID NO:34; residues 12 to 424 of SEQ ID NO:36; or residues 12 to 390 of SEQ ID NO:38, or

[0290] (b) comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.

[0291] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to

[0292] (a) a polynucleotide sequence corresponding to residues 34 to 1311 of SEQ ID NO: 1; residues 34 to 1839 of SEQ ID NO: 3; residues 34 to 1842 of SEQ ID NO: 5; residues 34 to 1830 of SEQ ID NO: 7; residues 34 to 1818 of SEQ ID NO: 9; residues 34 to 1845 of SEQ ID NO: 11; residues 34 to 1782 of SEQ ID NO: 13; residues 34 to 1860 of SEQ ID NO: 15; residues 34 to 1824 of SEQ ID NO: 17; residues 34 to 1833 of SEQ ID NO: 19; residues 34 to 1842 of SEQ ID NO: 21; residues 34 to 1833 of SEQ ID NO: 23; residues 34 to 1827 of SEQ ID NO: 25; residues 34 to 1266 of SEQ ID NO: 27; residues 34 to 1554 of SEQ ID NO: 29; residues 34 to 1314 of SEQ ID NO:31; residues 34 to 1143 of SEQ ID NO:33; residues 34 to 1272 of SEQ ID NO:35; or residues 34 to 1170 of SEQ ID NO:37; or

[0293] (b) a polynucleotide sequence corresponding to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37; wherein the polynucleotide sequence encodes a DNA ligase.

[0294] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence (a) comprising residues 34 to 1311 of SEQ ID NO: 1; residues 34 to 1839 of SEQ ID NO: 3; residues 34 to 1842 of SEQ ID NO: 5; residues 34 to 1830 of SEQ ID NO: 7; residues 34 to 1818 of SEQ ID NO: 9; residues 34 to 1845 of SEQ ID NO: 11; residues 34 to 1782 of SEQ ID NO: 13; residues 34 to 1860 of SEQ ID NO: 15; residues 34 to 1824 of SEQ ID NO: 17; residues 34 to 1833 of SEQ ID NO: 19; residues 34 to 1842 of SEQ ID NO: 21; residues 34 to 1833 of SEQ ID NO: 23; residues 34 to 1827 of SEQ ID NO: 25; residues 34 to 1266 of SEQ ID NO: 27; residues 34 to 1554 of SEQ ID NO:29; residues 34 to 1314 of SEQ ID NO:31; residues 34 to 1143 of SEQ ID NO:33; residues 34 to 1272 of SEQ ID NO:35; or residues 34 to 1170 of SEQ ID NO:37, or

[0295] (b) comprises SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37.

[0296] In some embodiments, the recombinant polynucleotide encoding any of the DNA ligases herein is manipulated in a variety of ways to promote expression of the DNA ligase polypeptide. In some embodiments, the recombinant polynucleotide encoding the DNA ligase comprises an expression vector in which one or more control sequences are present to regulate the expression of the DNA ligase polynucleotide and / or polypeptide. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, control sequences include, among others, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator.

[0297] In some embodiments, a suitable promoter is selected based on the host cell selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters obtained from the Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis dagA gene, the Streptomyces coelicolor ... subtilis levansucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes and prokaryotic β-lactase gene (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), and the tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase), and mutants, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in insect cells include, but are not limited to, polyhedrin, p10, ELT, OpIE2, and hr5 / ie1 promoters. Exemplary promoters for use in mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), chicken β-actin promoter fused to the CMV enhancer, simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, β-actin, elongation factor-1a, or 3-glyceraldehyde-3-phosphate dehydrogenase, and from chicken β-actin.

[0298] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence that is recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the DNA ligase polypeptide. Any suitable terminator that works in the selected host cell can be used in the present invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that depends on the Rho transcription factor, or a Rho-independent or intrinsic terminator that does not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5): 793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae 3-phosphoglyceraldehyde dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., supra). Exemplary terminators for insect and mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), simian virus 40 (SV40), Homo sapiens growth hormone hGH, bovine growth hormone BGH, and human or rabbit beta globulin.

[0299] In certain embodiments, the control sequence is a suitable leader sequence, i.e., the non-translated region of an mRNA that is important for translation by the host cell. In certain embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the DNA ligase polypeptide. Any suitable leader sequence that works in the host cell of selection can be used in the present invention. Exemplary leader sequences for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leader sequences for yeast host cells are obtained from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / 3-glyceraldehyde phosphate dehydrogenase (ADH2 / GAP). Suitable leader sequences for mammalian host cells include, but are not limited to, the 5'-UTR elements present in orthopoxvirus mRNA.

[0300] In certain embodiments, control sequences are polyadenylation sequences (i.e., sequences that are operably linked to the 3' end of the nucleotide sequence and that are recognized by the host cell as signals for adding polyadenosine residues to the mRNA of transcription when transcribed). Any suitable polyadenylation sequence that works in the host cell of selection can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3'UTR sequences for insect and mammalian host cells include, but are not limited to, the OpIE2 polyA sequence, the D. melanogaster metallothionein (Mt) polyA signal sequence, D. melanogaster alcohol dehydrogenase (adh), the SV40 polyA signal sequence, and 3'-UTRs of α- and β-globin mRNAs that carry sequence elements that increase mRNA stability and translation.

[0301] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of the cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in the translation reading frame to the segment of the coding region encoding the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is exogenous to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell can be used for expression of the engineered polypeptide. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the genes for Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57: 109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for insect and mammalian host cells include, but are not limited to, those from immunoglobulin gamma (IgG) genes and human secretory proteins, such as the signal peptides in human β-galactosidase polypeptides.

[0302] In some embodiments, the control sequence is a propeptide coding region encoding an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." Propolypeptide can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source, including but not limited to genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease, and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). In the case where both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned near the amino terminus of the polypeptide, and the signal peptide region is positioned near the amino terminus of the propeptide region.

[0303] In some embodiments, regulatory sequences are also utilized. These sequences contribute to the expression of the polypeptide relative to host cell growth. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). In prokaryotic host cells, suitable regulatory sequences include but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include but are not limited to the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include but are not limited to the TAKA α-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0304] In another aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide encoding an engineered DNA ligase polypeptide and one or more expression control regions, such as a promoter and terminator, an origin of replication, etc., depending on the type of host into which they are to be introduced. In some embodiments, the various nucleic acids and control sequences described herein are joined together (i.e., operably linked) to produce a recombinant expression vector. Alternatively, in some embodiments, the nucleic acid sequences of the present disclosure are expressed by inserting a nucleic acid sequence or nucleic acid construct comprising the sequence into a suitable expression vector.

[0305] The recombinant expression vector can be any suitable vector (e.g., a plasmid or virus) that can readily undergo recombinant DNA procedures and result in expression of the DNA ligase polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0306] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector existing as an extrachromosomal entity that replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may comprise any means for ensuring self-replication. In some alternative embodiments, the vector is a vector that is integrated into the genome when introduced into the host cell and replicates together with the chromosome into which it has been integrated. In addition, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids, are utilized that together contain the total DNA to be introduced into the host cell genome, and / or a transposon.

[0307] In certain embodiments, recombinant polynucleotides can be provided on non-replicating expression vectors or plasmids. In certain embodiments, non-replicating expression vectors or plasmids can be based on replication-defective viral vectors (see, e.g., Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0308] In some embodiments, the expression vector comprises one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene whose product confers biocide or viral resistance, resistance to heavy metals, native nutrition to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis or markers that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from Aspergillus niger or Aspergillus oryzae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from Aspergillus niger or Aspergillus oryzae), sC (sulfate adenyltransferase), and trpC (anthranilate synthetase), and equivalents thereof.

[0309] On the other hand, the present disclosure provides a host cell comprising a polynucleotide encoding at least one engineered DNA ligase polypeptide of the present disclosure, the polynucleotide being operably linked to one or more control sequences for expressing the engineered DNA ligase in the host cell. In some embodiments, the host cell comprises an expression vector comprising a polynucleotide encoding an engineered DNA ligase polypeptide as described herein, wherein the polynucleotide is operably linked to one or more control sequences. Host cells suitable for use in expressing polypeptides encoded by the expression vectors of the present invention are known in the art and include, but are not limited to, bacterial cells such as Escherichia coli, Bacillus subtilis, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC accession number 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowman's melanoma cells; and plant cells. Exemplary host cells also include various E. coli strains (e.g., W3110 (ΔfhuA) and BL21).

[0310] In another aspect, the present disclosure provides a method for producing an engineered DNA ligase polypeptide, wherein the method comprises culturing a host cell capable of expressing a polynucleotide encoding the engineered DNA ligase polypeptide under conditions suitable for expressing the polypeptide, such that the engineered DNA ligase is produced. In some embodiments, the method further comprises a step of isolating and / or purifying the DNA ligase polypeptide described herein.

[0311] Suitable culture media and culture conditions for host cells are known in the art. Any suitable method for introducing a polynucleotide for expressing a DNA ligase polypeptide into a cell is contemplated for use in the present invention. Suitable techniques include, but are not limited to, electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0312] In certain embodiments, any suitable method known in the art can be used to produce recombinant polypeptide (such as DNA ligase variants).For example, a variety of different mutagenesis techniques are well known to those skilled in the art. In addition, mutagenesis kits also can be obtained from many commercial molecular biology suppliers. It is available to carry out specific replacement (site-directed) at the amino acid place of definition, carry out specific or random mutation (region-specific) or carry out random mutagenesis (such as, saturation mutagenesis) on the whole gene in the local area of ​​gene. Many suitable methods known to those skilled in the art generate enzyme variants, including but not limited to the site-directed mutagenesis of single-stranded DNA or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, fallibility PCR, reorganization and chemical saturation mutagenesis or any other suitable method known in the art. Non-limiting examples of methods for DNA and protein engineering are provided in the following patents: U.S. Patent No. 6,117,679; U.S. Patent No. 6,420,175; U.S. Patent No. 6,376,246; U.S. Patent No. 6,586,182; U.S. Patent No. 7,747,391; U.S. Patent No. 7,747,393; U.S. Patent No. 7,783,428; and U.S. Patent No. 8,383,346. After the variants are generated, they can be screened for any desired property (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased stability, increased substrate range, increased inhibitor resistance / tolerance, increased solubility, pH stability, etc.).

[0313] In some embodiments, by subjecting polynucleotides encoding naturally occurring or engineered DNA ligase polypeptides to appropriate mutagenesis and / or directed evolution methods known in the art, e.g., as described herein, engineered DNA ligase polypeptides having properties disclosed herein can be obtained. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91: 10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529).

[0314] Mutagenesis and directed evolution methods can be applied to polynucleotides encoding DNA ligase to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods can be used in the present disclosure and are known in the art (see, e.g., U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,303,345, 6,304,350, 6,306,351,360, 6,307,361, 6,308,362, 6,309,371, 6,313,374, 6,314,375, 6,315,376, 6,316,377,638, 6,317,376, 6,318,377,639, 6,320,331, 6,321,333, 6,323,334, 6,324,335, 6,326,336, 6,327,337, 6,372,497,6,337,186,6,376,246,6,379,964,6,387,702,6,391,552,6,391,640,6,395,547,6,406,855, 6,406,910、6,413,745、6,413,774、6,420,175、6,423,542、6,426,224、6,436,675、6,444,468、6,455,253、6,479,652、6,482,647、6,483,011、6,484,105、6,489,146、6,500,617、6,500,639、6,506,602、6,506,603、6,518,065、6,519,065、6,521,453、6,528,311、6,537,746、6,573,09 8、6,576,467、6,579,678、6,586,182、6,602,986、6,605,430、6,613,514、6,653,072、6,686,515、6,703,240、6,716,631、6,825,001、6,902,922、6,917,882、6,946,296、6,961,664、6,995,017、7,024,312、7,058,515、7,105,297、7,148,054、7,220,566、7,288,375、7,384,387、7,421,347、7,430,477、7,462,469、7,534,564、7,620,500、7,620,502、7,629,170、7,702,464、7,747,391、7,747,393、7,751,986、7,776,598、7,783,428、7,795,030、7,853,410、7,868,138、7,783,428、7,873,477、7,873,499、7,904,249、7,957,912、7,981,614、8,014,961、8,029,988、8,048,674、 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,665,694, 9,684,771, and all related PCT and non-U.S. counterpart applications; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74 ; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J., 1986, 237:1-7; Kramer et al., Cell, 1984, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. USA, 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature, 1994, 366:389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994,91: 10747-10751; EP 3 049 973; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; WO 2009 / 152336 and WO 2015 / 048573, all of which are incorporated herein by reference).

[0315] In some embodiments, clones obtained after mutagenesis are screened by subjecting the enzyme preparation to defined treatment conditions or assay conditions (e.g., temperature, pH conditions, ligase substrate type, input substrate concentration, etc.) and measuring DNA ligase activity after treatment or other suitable assay conditions. Clones containing polynucleotides encoding the polypeptide of interest are subsequently isolated from the gene, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in a host cell. Measurement of enzyme activity from the expression library can be performed using any suitable method known in the art and as described in the Examples.

[0316] For engineered polypeptides of known sequences, the polynucleotides encoding the polypeptides can be prepared by standard solid phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then joined (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired continuous sequence (see, e.g., Hughes et al., Cold Spring Harb Perspect Biol. 2017 Jan; 9 (1): a023812). For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the phosphoramidite method (see, e.g., Beaucage et al., Tet. Lett., 1981, 22: 1859-69; and Matthes et al., EMBO J., 1984, 3: 801-05), because it is typically implemented with automated synthetic methods.

[0317] In some embodiments, a method for preparing an engineered DNA ligase polypeptide may comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant as described herein, and (b) expressing the DNA ligase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide may optionally have one or more (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions are conservative substitutions or non-conservative substitutions.

[0318] In some embodiments, any engineered DNA ligase polypeptide expressed in host cells is recovered and / or purified from the cells and / or culture medium using any one or more known protein purification techniques, including, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0319] Chromatographic techniques for isolating / purifying the DNA ligase polypeptide include, among others, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, hydrophobic-interaction chromatography, size exclusion chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying the DNA ligase depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and will be apparent to those skilled in the art. In certain embodiments, affinity techniques can be used to isolate the DNA ligase. For affinity chromatography purification, antibodies that specifically bind to the DNA ligase polypeptide can be used. In certain embodiments, for isolation / purification purposes, affinity tags, such as His tags, can be introduced into the DNA ligase polypeptide.

[0320] Composition

[0321] In a further aspect, the present disclosure provides compositions of the DNA ligase disclosed herein. In some embodiments, the compositions comprise at least one engineered DNA ligase polypeptide described herein. In some embodiments, the engineered DNA ligase polypeptide in the composition is isolated or purified. In some embodiments, the DNA ligase is combined with other components and compounds to provide compositions and formulations comprising engineered DNA ligase polypeptides suitable for various applications and uses.

[0322] In some embodiments, the composition comprises at least one engineered DNA ligase described herein, e.g., an engineered DNA ligase provided in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2. The composition comprises an engineered DNA ligase comprising an amino acid sequence of residues 12 to 437 of an even-numbered SEQ ID NO: 40 to 1184, or an amino acid sequence of an even-numbered SEQ ID NO: 40 to 1184.

[0323] In some embodiments, the composition comprises a DNA ligase provided in Table 8.1. In some embodiments, the composition comprises a DNA ligase comprising an amino acid sequence

[0324] (a) comprises residues 12 to 437 of SEQ ID NO:2; residues 12 to 613 of SEQ ID NO:4; residues 12 to 614 of SEQ ID NO:6; residues 12 to 610 of SEQ ID NO:8; residues 12 to 606 of SEQ ID NO:10; residues 12 to 615 of SEQ ID NO:12; residues 12 to 594 of SEQ ID NO:14; residues 12 to 620 of SEQ ID NO:16; residues 12 to 608 of SEQ ID NO:18; residues 12 to 611 of SEQ ID NO:20; residues 12 to 614 of SEQ ID NO:22; residues 12 to 611 of SEQ ID NO:24; residues 12 to 609 of SEQ ID NO:26; residues 12 to 422 of SEQ ID NO:28; residues 12 to 518 of SEQ ID NO:30; residues 12 to 438 of SEQ ID NO:32; residues 12 to 381 of SEQ ID NO:34; residues 12 to 424 of SEQ ID NO:36; or residues 12 to 390 of SEQ ID NO:38; or

[0325] (b) comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.

[0326] In some embodiments, the composition further comprises one or more of a buffer, a nucleotide substrate (e.g., ATP), and / or at least one or more DNA ligase substrates (such as synthetic oligonucleotides or recombinant polynucleotides). In some embodiments, the DNA ligase substrate comprises a DNA adapter or linker. In some embodiments, the composition further comprises a divalent cation (e.g., Mg +2 or Mn +2 ), one or more of a reducing agent (e.g., DTT) and / or a salt (e.g., KCl and / or NaCl).

[0327] In some embodiments, the composition further comprises an additive or attachment enhancer including, among others, one or more of DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., dextran 6000). In some embodiments, the composition comprises 1% to 40% v / v DMSO. In some embodiments, the composition comprises 0.1M to 3M betaine. In some embodiments, the composition comprises 0.5% to 20% w / v PEG (e.g., PEG 6000 or PEG 8000).

[0328] In some embodiments, the composition further comprises a second nucleic acid modification enzyme, including, among others, a DNA polymerase (e.g., thermal DNA polymerase, isothermal polymerase, reverse transcriptase, RNA polymerase, etc.), a restriction enzyme, a polynucleotide kinase, or a terminal transferase.

[0329] In certain embodiments, engineered DNA ligase as herein described is provided in solution, as lyophilized material or fixed on substrate.In certain embodiments, substrate is solid substrate, porous substrate, film or particle.Enzyme can be trapped in matrix or film.In certain embodiments, matrix comprises polymeric material, such as calcium alginate, agar, kappa carrageenan, polyacrylamide, agarose or its derivative (for example cross-linked agarose) and collagen, or solid matrix, such as activated carbon, porous ceramic and diatomaceous earth.In certain embodiments, matrix is ​​particle, film or fiber.The type of film includes, among others, nylon, cellulose, polysulfone or polyacrylate.

[0330] In certain embodiments, the enzyme is immobilized on the surface of a support material. In certain embodiments, the enzyme is adsorbed on a support material. In certain embodiments, the enzyme is immobilized on a support material by covalent bonding. The support material includes, among others, inorganic materials such as aluminum oxide, silicon dioxide, porous glass, ceramics, diatomaceous earth, clay and bentonite, or organic materials such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polystyrene and ion exchange resins such as Amberlite, Sephadex and Dowex.

[0331] Uses and kits of engineered DNA ligase polypeptides

[0332] In another aspect, the present disclosure provides uses of engineered DNA ligases for ligating or joining DNA molecules, such as for polynucleotide synthesis, DNA repair, or other molecular biology and diagnostic uses.

[0333] In certain embodiments, engineered DNA ligase is used to connect polynucleotide or oligonucleotide.In certain embodiments, engineered DNA ligase is used to synthesize polynucleotide from shorter oligonucleotide or polynucleotide.In certain embodiments, the method for connecting at least the first polynucleotide chain and the second polynucleotide chain is included in being suitable for being connected to the first polynucleotide chain under the conditions of the second polynucleotide chain, in the presence of nucleotide substrate, the first polynucleotide chain and the second polynucleotide chain are contacted with engineered DNA ligase as herein described, wherein the first polynucleotide chain comprises connectable 5 ' end and the second polynucleotide chain comprises 3 ' end that can be connected to the 5 ' end of the first polynucleotide chain.In certain embodiments, the 5 ' end of the first polynucleotide chain has 5 '-phosphoric acid (for example, -PO4) and the 3 ' end of the second polynucleotide chain has 3 ' hydroxyl (OH).In certain embodiments, the first polynucleotide chain and the second polynucleotide chain comprise a mixture of DNA or DNA and RNA.

[0334] In certain embodiments, the method further includes a third polynucleotide chain, wherein the first polynucleotide chain and the second polynucleotide chain hybridize adjacent to each other on the third polynucleotide chain so that the 5' end of the first polynucleotide chain is positioned near the 3' end of the second polynucleotide chain. In some embodiments of the method, the third polynucleotide chain is continuous with the first polynucleotide chain or the second polynucleotide chain. In some embodiments of the method, the third polynucleotide chain is continuous with the first polynucleotide chain and the second polynucleotide chain to form a single continuous polynucleotide ligase substrate. In certain embodiments, the first polynucleotide chain, the second polynucleotide chain and the third polynucleotide chain comprise independent polynucleotide. In certain embodiments, the third polynucleotide chain is a mixture of RNA, DNA or RNA and DNA, preferably DNA.

[0335] In some embodiments of the method, the third polynucleotide comprises a splint or bridge polynucleotide, wherein the 5' terminal sequence of the first polynucleotide chain and the 3' terminal sequence of the second polynucleotide chain hybridize adjacent to each other on the splint or bridge polynucleotide to position the 5' end of the first polynucleotide chain adjacent to the 3' end of the second polynucleotide chain. In some embodiments, the splint or bridge polynucleotide is RNA, DNA, or a mixture of RNA and DNA, preferably DNA.

[0336] In some embodiments of the method, the first polynucleotide strand is hybridized to the third polynucleotide strand to form a first double-stranded polynucleotide substrate, and the second polynucleotide strand is hybridized to the fourth polynucleotide strand to form a second double-stranded polynucleotide substrate.

[0337] In some embodiments, the first double-stranded polynucleotide substrate comprises a blunt 5' end of the first polynucleotide strand and the second double-stranded polynucleotide substrate comprises a blunt 3' end of the second polynucleotide strand.

[0338] In some embodiments, the first double-stranded polynucleotide substrate comprises an overhang on at least one end of the first double-stranded polynucleotide substrate, and the second double-stranded polynucleotide substrate comprises an overhang on at least one end of the second double-stranded polynucleotide substrate, wherein the overhang on the first double-stranded polynucleotide substrate and the overhang on the second double-stranded polynucleotide substrate are complementary and thus capable of hybridizing to each other and forming one or more ligatable nicks. In some embodiments, the nick is between the 5' end of the first polynucleotide chain and the 3' end of the second polynucleotide chain.

[0339] In some embodiments, the first double-stranded polynucleotide substrate and the second double-stranded polynucleotide substrate comprise DNA, eg, a first dsDNA substrate and a second dsDNA substrate.

[0340] In some embodiments, the DNA ligase substrate comprises an adapter or joint. In some embodiments, the adapter or joint has a blunt end or a cohesive (sticky) end. In some embodiments, the joint substrate has two blunt ends. In some embodiments, the adapter has one blunt end and one cohesive or sticky end.

[0341] In some embodiments, the method includes a nucleotide substrate used by the DNA ligase to catalyze the ligation reaction. In some embodiments, the nucleotide substrate is ATP. In some embodiments of the method, the reaction conditions of the ligation reaction include additional components, such as a divalent metal (e.g., Mg +2 ), buffer, reducing agent (e.g., DTT) and / or salt (KCl or NaCl).

[0342] In some embodiments, the reaction conditions further include a ligation enhancing agent, which includes, among others, DMSO, betaine, polyethylene glycol (e.g., PEG 6000 and PEG 8000) or other molecular crowding agents, such as, but not limited to, bovine serum albumin (BSA), dextran, 1,2-propylene glycol, and Ficoll.

[0343] In some embodiments, the ligation reaction is carried out at a suitable temperature and reaction time period. In some embodiments, the ligation reaction temperature is about 2 ° C to about 50 ° C. In some embodiments, the ligation reaction temperature is 2 ° C to 45 ° C, 4 ° C to 40 ° C, 4 ° C to 35 ° C, 4 ° C to 30 ° C or 10 ° C to 25 ° C. In some embodiments, the ligation reaction temperature is 2 ° C, 3 ° C, 4 ° C, 5 ° C, 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 37 ° C, 40 ° C, 45 ° C or 50 ° C. In some embodiments, the ligation reaction temperature can use different temperatures, such as a temperature that forms a stable hybrid between the polynucleotide substrates, followed by a higher temperature to promote the completion of the ligation reaction.

[0344] In some embodiments, the ligation reaction time can be sufficient time for connecting the polynucleotide substrate. In some embodiments, the ligation reaction time is 0.5 to 72 hours or longer. In some embodiments, the ligation reaction time is 1 to 72 hours, 2 to 48 hours or 2 to 24 hours. In some embodiments, the ligation reaction time is 0.5, 1, 2, 4, 5, 12, 24, 48 or 72 hours or longer.

[0345] In some embodiments, the DNA ligase described herein is used in a ligase chain reaction (LCR) or a ligase detection reaction (LDR) (see, e.g., Gibriel et al., Mutat Res Rev Mutat Res. 2017, 773: 66–90). In some embodiments, the DNA ligase described herein is used to generate a template for rolling circle amplification. In some embodiments, the DNA ligase is used for DNA sequencing or single nucleotide polymorphism (SNP) detection, e.g., by supported oligonucleotide ligation and detection (SOLiD).

[0346] In a further aspect, the present disclosure provides a kit comprising a DNA ligase as described herein. In some embodiments, the kit further comprises a buffer, a nucleotide substrate (e.g., ATP) and / or one or more polynucleotide ligase substrates, particularly one or more of a synthetic oligonucleotide or a recombinant polynucleotide. In some embodiments, the kit further comprises an additive or ligation enhancer, including one or more of DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., dextran 6000).

[0347] Examples

[0348] Following example, comprises the result of experiment and acquisition, only provides and should not be construed as limiting the present invention for the purpose of illustration.For many reagents and equipment described below, there are various suitable sources.The present invention is not intended to be limited to any particular source of any reagent or equipment project.

[0349] In the experimental disclosure below, the following abbreviations apply: M (mole / liter); mM (millimol / liter); uM and μM (micromol / liter); nM (nanomole / liter); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); ul, uL, μl and μL (microliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min (s) ( minutes); h (s) and hr (hours); U (unit); MW (molecular weight); rpm (revolutions per minute); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); IPTG (isopropyl β-Dl-thiogalactopyranose); LB (lysogeny broth); TB (terrific broth); SFP (shake flask powder); CDS (coding sequence); E. coli W3110 (a commonly used laboratory E. coli strain available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); FIOPC and FIOP (fold improvement over the positive control or fold improvement over the parent, respectively).

[0350] Example 1

[0351] DNA ligase gene acquisition and expression vector construction

[0352] Genes encoding various N-terminal 6-histidine-tagged variants of wild-type (WT) dsDNA ligase were designed using codon optimization for expression in E. coli, synthesized, and subcloned into the E. coli expression vector pCK100900i (see, e.g., U.S. Patent No. 7,629,157 and U.S. Patent Application Publication No. 2016 / 0244787, both of which are incorporated herein by reference). These plasmid constructs were used to transform an E. coli strain derived from W3110. Directed evolution techniques commonly known to those skilled in the art were used to generate libraries of gene variants from these plasmids (see, e.g., U.S. Patent No. 8,383,346 and WO2010 / 144103, both of which are incorporated herein by reference). Substitutions in the enzyme variants described herein are indicated with reference to an N-terminal 6-histidine-tagged form of the reference WT dsDNA ligase (i.e., SEQ ID NO: 2) or variants thereof, as indicated.

[0353] Example 2

[0354] High-throughput (HTP) DNA ligase expression and thermal cleavage

[0355] High-throughput (HTP) growth of dsDNA ligase and variants

[0356] Transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol. After incubation at 37°C overnight, colonies were plated in 96-well shallow-bottom NUNC TM The wells of the (Thermo-Scientific) plate were filled with 180 μl / well LB culture medium supplemented with 1% glucose and 30 μg / ml chloramphenicol. The culture was grown overnight for 18 to 20 hours in a shaker (200 rpm, 30°C and 85% relative humidity; Kuhner). The overnight grown sample (20 μL) was transferred to a Costar 96-well deep plate filled with 380 μL of premium broth supplemented with 30 μg / ml chloramphenicol. The plate was incubated for 120 minutes in a shaker (250 rpm, 30°C and 85% relative humidity; Kuhner) until the OD 600 The p-value was between 0.4 and 0.8. The cells were then induced with 10 mM IPTG in 40 μL sterile water and incubated overnight for 18 to 20 hours in a shaker (250 rpm, 30°C and 85% relative humidity; Kuhner). The cells were pelleted (4000 rpm x 20 min), the supernatant was discarded, and the cells were frozen at -80°C before analysis.

[0357] Heat lysis of HTP cell pellets with lysozyme

[0358] 200 μl of buffer containing 50mM Tris-HCl buffer (pH 7.5) is added to the cell pellet in each well. The cells are completely resuspended at room temperature for 15 to 20 minutes and shaken on a benchtop shaker. At the same time, 60uL of lysis buffer containing 50mM Tris-HCl buffer (pH 7.5) and 0.2g / L lysozyme is transferred to the BioRad plate. The 60 μL fully resuspended cell pellet is then transferred to the BioRad plate. The plate is completely mixed and heat-challenged at a specific temperature for 1 hour. The plate is then centrifuged at 4,000rpm and 4°C for 15 minutes. The clarified supernatant is then used for biocatalytic reactions to determine its activity level.

[0359] Example 3

[0360] High-throughput (HTP) DNA ligase expression and purification

[0361] Transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol. After incubation at 37°C overnight, colonies were plated in 96-well shallow-bottom NUNC TMThe wells of the (Thermo-Scientific) plate were filled with 180 μl / well LB culture medium supplemented with 1% glucose and 30 μg / ml chloramphenicol. The culture was grown overnight for 18 to 20 hours in a shaker (200 rpm, 30°C and 85% relative humidity; Kuhner). The overnight grown sample (20 μL) was transferred to a Costar 96-well deep plate filled with 380 μL of premium broth supplemented with 30 μg / ml chloramphenicol. The plate was incubated for 120 minutes in a shaker (250 rpm, 30°C and 85% relative humidity; Kuhner) until the OD 600 The p-value was between 0.4 and 0.8. The cells were then induced with 10 mM IPTG in 40 μL sterile water and incubated overnight for 18 to 20 hours in a shaker (250 rpm, 30°C and 85% relative humidity; Kuhner). The cells were pelleted (4000 rpm x 20 min), the supernatant was discarded, and the cells were frozen at -80°C before analysis.

[0362] Lysis of HTP cell pellets for HTP purification of dsDNA ligase from crude lysate

[0363] The cell pellet was resuspended in 400 μl / well lysis mixture [50 mM Tris-HCl buffer (pH 7.5), 75% (v / v) B-Per reagent (Thermo Fisher), 300 mM NaCl, 10 mM imidazole, and 0.2% (v / v) Triton X-100] and added to the cell suspension. The mixture was stirred at room temperature for 2 hours, precipitated (4000 rpm x 20 min), and the supernatant was retained for purification.

[0364] According to the manufacturer's instructions, use HIS- High Capacity (HC) Nickel Coated Plates (Sigma) are purified from thick Escherichia coli extract by metal affinity chromatography. Use 800 μ l wash buffer (50mM Tris-HCl (pH 7.5), 500mM NaCl, 20mM imidazoles, 0.02% v / v Triton X-100 reagent) / well balance HIS-Select plate. Then, 200 μ l HTP lysates containing dsDNA ligase in each well are loaded onto the plate, and at 2115 relative centrifugal forces (rcf) and 4 ℃ of centrifugal 1 minute. Plate is washed twice with 400 μ l wash buffer / well, each washing at 2115rcf and 4 ℃ of centrifugal 2 to 3 minutes. The dsDNA ligase sample was eluted by adding 120 μl / well elution buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 250 mM imidazole, 0.02% v / v Triton X-100 reagent) by centrifugation at 2115 rcf and 4°C for 1 minute.

[0365] Using Zeba TM Spin desalting plate (Thermo Fisher) carries out buffer exchange to eluent.In brief, plate is balanced twice with 375 μ l 2x dsDNA ligase storage buffer (80mM Tris-HCl pH7.5, 200mM KCl and 0.2mMEDTA) / hole, and is centrifuged 1 minute at 2115 relative centrifugal forces (rcf) and 4 DEG C. Desalting plate is loaded with 90 μ l HIS-Select sample eluent and is centrifuged 2 minutes at 2115rcf and 4 DEG C. Retention eluent from desalting plate and mix with equal-volume glycerol, form the final storage buffer concentration of 40mM Tris-HCl pH 7.5, 100mM KCl, 0.1mM EDTA and 50% glycerol.

[0366] Example 4

[0367] Shake flask expression and DNA ligase purification

[0368] Shake flask expression

[0369] Selected HTP cultures grown as described above were plated onto LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol and grown overnight at 37°C. A single colony from each culture was transferred to 5 ml of LB broth containing 1% glucose and 30 μg / ml chloramphenicol. The cultures were grown at 30°C, 250 rpm for 20 hours and subcultured in 250 ml of premium broth containing 30 μg / ml chloramphenicol at a dilution of approximately 1:50 to a final OD of600 The culture was incubated at 30°C, 250 rpm for about 195 minutes until the OD 600 The cell suspension was cooled in an ice bath and lysed using a microfluidizer (Microfluidics M-110L). The crude lysate was precipitated by centrifugation (4 °C, 11,000 rpm, 60 minutes) and filtered through a 0.2 μm PES membrane to further clarify the lysate.

[0370] Purification of DNA ligase from shake flask lysates

[0371] Additional NaCl and imidazole are added to the clarified dsDNA ligase lysate to adjust their concentrations to 500 mM NaCl and 20 mM imidazole, respectively. Lysate was subsequently purified using an AKTA Pure purification system and a 5 ml HisTrap FF column (GE Healthcare) using an AC Step HiF setup (run parameters are provided below). SF wash buffer comprises 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 0.02% v / v Triton X-100 reagent.

[0372]

[0373] The three most concentrated 1 ml fractions were identified by UV absorption (280 nm) and analyzed on a 3.5K Slide-A-Lyzer TM Buffer exchange was performed by dialyzing overnight into dialysis buffer (40 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA and 50% glycerol) in a dialysis cassette (Thermo Fisher).The concentration of the purified and dialyzed dsDNA ligase samples was measured by absorbance at 280 nm.

[0374] Example 5

[0375] Preparation of dsDNA 50-mer inserts for HTP screening

[0376] A 6-carboxyfluorescein-labeled double-stranded 50-mer DNA fragment containing two single-stranded HPLC-purified synthetic oligonucleotides (Integrated DNA Technologies) was prepared by annealing them in 1x annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM EDTA). The resulting double-stranded "50-mer fluorescently labeled insert" has a single-base deoxyadenine 3' overhang and a 5' monophosphate end at both ends of the molecule and is internally labeled with a fluorescent dye (6-carboxyfluorescein attached to the 5-position of the thymine ring via a 6-carbon spacer arm).

[0377] Example 6

[0378] Preparation of dsDNA 20-mer adapters for HTP screening

[0379] Double-stranded "20-mer adapter" molecules containing two single-stranded HPLC-purified oligonucleotides (Integrated DNA Technologies) (SEQ ID NO: 1217 and SEQ ID NO: 1218) were prepared by annealing in 1x annealing buffer (10 mM Tris pH 7.5, 50 mM NaCl, 10 mM EDTA). The resulting 20-mer adapter duplex had phosphorothioate-protected 5' deoxythymidine overhangs and 5' phosphates at the ligation-compatible ends.

[0380] Example 7

[0381] Capillary electrophoresis (CE) analysis of oligonucleotides

[0382] Sample preparation for reaction analysis using CE

[0383] The reaction samples were analyzed by capillary electrophoresis using an ABI 3500xl Genetic Analyzer (Thermo Fisher). The reaction (25 μL) was quenched by adding 5 μL of 50 mM aqueous EDTA. 2 μL of this quenched solution was transferred to a new 96-well MicroAmp optical PCR plate or a plate containing 18 μL of Hi-Di TMThe PCR was performed in a 384-well MicroAmp optical PCR plate in formamide (Thermo Fisher) with appropriate size standards. The ABI 3500xl was configured with POP6 polymer, 50 cm capillaries, and a 45°C oven temperature. The pre-run setting was 18 kV for 180 seconds. The injection was 5 kV for 5 seconds, and the run setting was 19.5 kV for 1500 seconds. The FAM-labeled oligonucleotide substrate and product were identified by their size relative to the size ladder, with the substrate oligonucleotide peak at approximately 49 bp and the ligation product appearing in the region of approximately 85 bp.

[0384] Example 8

[0385] Expression, purification and activity verification of DNA ligase gene in Escherichia coli shake flasks

[0386] Twenty genes (SEQ ID NO: 1 / 2 to SEQ ID NO: 37 / 38) were cloned for recombinant expression in E. coli as described in Example 1 and expressed and purified as described in Example 4.

[0387] Ligation reaction is carried out in 200 μ L BioRad PCR plates of 96-well format.Reaction includes 1 nM insert (SEQ ID:1185 / SEQ ID:1186), 200 nM adapter (SEQ ID:1217 / SEQ ID:1218) and 1X connection buffer, and described connection buffer comprises 66 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP.Reaction is set up as follows: (i) all reaction components except the dsDNA ligase of purification are premixed in a single solution, and 80 μ L of this solution are aliquoted into each well of 96-well plates, (ii) 20 μ L of dsDNA ligase solution (1.28 uM is final concentration) of purification are subsequently added into well to initiate reaction.Reaction plate is heat-sealed with peelable aluminum seal and incubated 30 minutes at 23 DEG C in thermal cycler, then maintained at 4 DEG C until reaction is quenched.

[0388] The reaction was quenched by adding 20 μL of 90 mM EDTA, followed by 2.8 g / L proteinase K in 20 μL of water (0.2 g / L proteinase K final concentration). The quenched reaction was incubated at 50° C. in a thermal cycler for 2 hours and then cleaned with ZR-96 DNA Cleaner and Concentrator-5 (Catalog # D4024, cleaning solution included in the kit). The cleaned mixture was analyzed using a Caliper (Perkin Elmer) Labchip GX capillary electrophoresis instrument using a DNA high sensitivity assay according to the manufacturer's instructions.

[0389] The conversion rate was calculated as the peak area of ​​the double ligation product relative to the sum of the peak areas of the unligated insert, single ligation, and double ligation products. The results are shown in Table 8.1. The amino acid sequence identity (excluding the His tag) between each DNA ligase examined is shown in Table 8.2.

[0390]

[0391]

[0392] Example 9

[0393] Improvement in DNA ligase activity relative to SEQ ID NO: 2

[0394] HTP screening for improved dsDNA ligase variants

[0395] SEQ ID NO: 2 was selected as the parent DNA ligase. A library of engineered genes was generated from the parent gene using recognized techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1.

[0396] Ligation reactions were performed in 200 μL BioRad PCR plates in a 96-well format. The reactions included inserts, adapters, and reaction buffer as described in Table 9.1. The reactions were set up as follows: (i) all reaction components except dsDNA ligase were premixed in a single solution, and 20 μL of this solution was aliquoted into each well of the 96-well plate, (ii) 5 μL of dsDNA ligase solution was then added to the wells to initiate the reaction. The reaction plates were heat-sealed with peelable aluminum seals and incubated in a thermal cycler at the specified temperature and reaction time, and then maintained at 4°C until the reaction was quenched. Reaction and quenching details are detailed in Table 9.1. After quenching the reaction, capillary electrophoresis (CE) sample preparation was performed as described in Table 9.1.

[0397]

[0398] The activity relative to SEQ ID NO: 2 (active FIOP) was calculated as the fold improvement in conversion of the variant divided by the conversion observed in the reaction of SEQ ID NO: 2 (where the conversion can be set as the average of the replicates or, as appropriate, the highest single sample). The conversion was calculated as the peak area of ​​the double ligated product relative to the sum of the peak areas of the unligated insert, single ligated, and double ligated products. The results are shown in Table 9.2.

[0399]

[0400]

[0401] Example 10

[0402] Improvement in DNA ligase activity relative to SEQ ID NO: 2

[0403] HTP screening for improved dsDNA ligase variants

[0404] SEQ ID NO: 2 was selected as the parent DNA ligase. A library of engineered genes was generated from the parent gene using recognized techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in an HTP and prepared as described in Table 10.1.

[0405] Ligation reactions were performed in 200 μL BioRad PCR plates in a 96-well format. The reactions included inserts, adapters, and reaction buffer as described in Table 10.1. The reactions were set up as follows: (i) all reaction components except dsDNA ligase were premixed in a single solution and 20 μL of this solution was aliquoted into each well of the 96-well plate, (ii) 5 μL of dsDNA ligase solution was then added to the wells to initiate the reaction. The reaction plates were heat-sealed with peelable aluminum seals and incubated in a thermal cycler at the specified temperature and reaction time, and then maintained at 4°C until the reaction was quenched. Reaction and quenching details are detailed in Table 10.1. After quenching the reaction, capillary electrophoresis (CE) sample preparation was performed as described in Table 10.1.

[0406]

[0407]

[0408] The activity relative to SEQ ID NO: 2 (active FIOP) was calculated as the improvement in intermediate product conversion of the variant divided by the intermediate product conversion observed in the reaction of SEQ ID NO: 2 (wherein the intermediate product conversion can be set as the average of the replicates or, as appropriate, as the highest single sample). Intermediate product conversion was calculated as the peak area of ​​the single ligation product relative to the sum of the peak areas of the unligated insert and the single ligation product. The results are shown in Table 10.2.

[0409]

[0410] Example 11

[0411] Improvement in DNA ligase activity relative to SEQ ID NO: 62 HTP screening for improved dsDNA ligase variants select

[0412] SEQ ID NO: 62 was selected as the parent DNA ligase. Libraries of engineered genes were generated from the parent gene using recognized techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTP and prepared as described in Table 11.1.

[0413] Ligation reactions were performed in 200 μL BioRad PCR plates in a 96-well format. The reactions included inserts, adapters, and reaction buffer as described in Table 11.1. The reactions were set up as follows: (i) all reaction components except dsDNA ligase were premixed in a single solution and 20 μL of this solution was aliquoted into each well of the 96-well plate, (ii) 5 μL of dsDNA ligase solution was then added to the wells to initiate the reaction. The reaction plates were heat-sealed with peelable aluminum seals and incubated in a thermal cycler at the specified temperature and reaction time, and then maintained at 4°C until the reaction was quenched. Reaction and quenching details are detailed in Table 11.1. After quenching the reaction, capillary electrophoresis (CE) sample preparation was performed as described in Table 11.1.

[0414]

[0415]

[0416] The activity relative to SEQ ID NO: 62 (active FIOP) was calculated as the fold improvement in conversion of the variant divided by the conversion observed in the reaction of SEQ ID NO: 62 (where the conversion can be set as the average of the replicates or, as appropriate, as the highest single sample). The conversion was calculated as the peak area of ​​the double ligated product relative to the sum of the peak areas of the unligated insert, single ligated, and double ligated products. The results are shown in Table 11.2.

[0417]

[0418]

[0419]

[0420] Example 12

[0421] Improvement in DNA ligase activity relative to SEQ ID NO: 62 HTP screening for improved dsDNA ligase variants select

[0422] SEQ ID NO: 62 was selected as the parent DNA ligase. A library of engineered genes was generated from the parent gene using recognized techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in an HTP and prepared as described in Table 12.1.

[0423] Ligation reactions were performed in 200 μL BioRad PCR plates in a 96-well format. The reactions included inserts, adapters, and reaction buffer as described in Table 12.1. The reactions were set up as follows: (i) all reaction components except dsDNA ligase were premixed in a single solution and 20 μL of this solution was aliquoted into each well of the 96-well plate, (ii) 5 μL of dsDNA ligase solution was then added to the wells to initiate the reaction. The reaction plates were heat-sealed with peelable aluminum seals and incubated in a thermal cycler at the specified temperature and reaction time, and then maintained at 4°C until the reaction was quenched. Reaction and quenching details are detailed in Table 12.1. After quenching the reaction, capillary electrophoresis (CE) sample preparation was performed as described in Table 12.1.

[0424]

[0425] The activity relative to SEQ ID NO: 62 (active FIOP) was calculated as the fold improvement in conversion of the variant divided by the conversion observed in the reaction of SEQ ID NO: 62 (where the conversion can be set as the average of the replicates or, as appropriate, the highest single sample). The conversion was calculated as the peak area of ​​the double ligated product relative to the sum of the peak areas of the unligated insert, single ligated, and double ligated products. The results are shown in Table 12.2.

[0426]

[0427]

[0428] Example 13

[0429] Improvements in DNA ligase activity and thermostability relative to SEQ ID NO: 138 Targeting improved dsDNA ligase HTP screening of variants

[0430] SEQ ID NO: 138 was selected as the parent DNA ligase. Libraries of engineered genes were generated from the parent gene using recognized techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTP and prepared as described in Table 13.1.

[0431] Ligation reactions were performed in 200 μL BioRad PCR plates in a 96-well format. The reactions included inserts, adapters, and reaction buffer as described in Table 13.1. The reactions were set up as follows: (i) all reaction components except dsDNA ligase were premixed in a single solution and 20 μL of this solution was aliquoted into each well of the 96-well plate, (ii) 5 μL of dsDNA ligase solution was then added to the wells to initiate the reaction. The reaction plates were heat-sealed with peelable aluminum seals and incubated in a thermal cycler at the specified temperature and reaction time, and then maintained at 4°C until the reaction was quenched. Reaction and quenching details are detailed in Table 13.1. After quenching the reaction, capillary electrophoresis (CE) sample preparation was performed as described in Table 13.1.

[0432]

[0433] The activity relative to SEQ ID NO: 138 (active FIOP) was calculated as the fold improvement in conversion of the variant divided by the conversion observed in the reaction of SEQ ID NO: 138 (where the conversion can be set as the average of the replicates or, as appropriate, as the highest single sample). The conversion was calculated as the peak area of ​​the double ligated product relative to the sum of the peak areas of the unligated insert, single ligated, and double ligated products. The results are shown in Table 13.2.

[0434]

[0435]

[0436]

[0437]

[0438] Example 14

[0439] Improvement in DNA ligase activity relative to SEQ ID NO: 318 HTPs targeting improved dsDNA ligase variants filter

[0440] SEQ ID NO: 318 was selected as the parent DNA ligase. Libraries of engineered genes were generated from the parent gene using recognized techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). Polypeptides encoded by each gene were produced in HTP and prepared as described in Table 14.1.

[0441] Ligation reactions were performed in 200 μL BioRad PCR plates in a 96-well format. The reactions included inserts, adapters, and reaction buffer as described in Table 14.1. The reactions were set up as follows: (i) all reaction components except dsDNA ligase were premixed in a single solution and 20 μL of this solution was aliquoted into each well of the 96-well plate, (ii) 5 μL of dsDNA ligase solution was then added to the wells to initiate the reaction. The reaction plates were heat-sealed with peelable aluminum seals and incubated in a thermal cycler at the specified temperature and reaction time, and then maintained at 4°C until the reaction was quenched. Reaction and quenching details are detailed in Table 14.1. After quenching the reaction, capillary electrophoresis (CE) sample preparation was performed as described in Table 14.1.

[0442]

[0443] The activity relative to SEQ ID NO: 318 (active FIOP) was calculated as the fold improvement in conversion of the variant divided by the conversion observed in the reaction of SEQ ID NO: 318 (where the conversion can be set as the average of the replicates or, as appropriate, as the highest single sample). The conversion was calculated as the peak area of ​​the double ligation product relative to the sum of the peak areas of the unligated insert, single ligation, and double ligation products. The results are shown in Table 14.2.

[0444]

[0445]

[0446]

[0447] Example 15

[0448] Improvements in DNA ligase activity, thermostability, and solubility relative to SEQ ID NO: 318, as determined by % conversion of double ligation products

[0449] HTP screening for imp...

Claims

1. An engineered DNA ligase or a functional fragment thereof comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2 and an even-numbered SEQ ID NO: 40 to 1184, or to a reference sequence corresponding to an even-numbered SEQ ID NO: 2 and 40 to 1184, wherein the amino acid sequence comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to an even-numbered SEQ ID NO: One or more substitutions of the reference sequence of NO: 2, 62, 138, 318, 722 or 938.

2. The engineered DNA ligase of claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 2, 62, 138, 318, 722 or 938. One or more substitutions of the reference sequence of NO: 2, 62, 138, 318, 722 or 938.

3. The engineered DNA ligase of claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO:

2.

4. The engineered DNA ligase or functional fragment thereof according to claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

5. The engineered DNA ligase or functional fragment thereof according to claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even-numbered SEQ ID NO: 40 to 1184, or a reference sequence corresponding to an even-numbered SEQ ID NO: 40 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

6. The engineered DNA ligase according to any one of claims 1 to 5, wherein the amino acid sequence of the engineered DNA ligase comprises at least one of amino acid positions 11, 12, 13, 14, 18, 30, 31, 33, 34, 36, 37, 44, 50, 56, 59, 60, 61, 63, 67, 68, 69, 71, 73, 74, 76, 77, 82, 88, 95, 96, 97, 99, 100, 101, 102, 103, 104 , 105, 106, 110, 112, 113, 117, 125, 128, 130, 132, 138, 139, 148, 149, 150, 155, 156, 159, 161, 162, 164, 165, 177, 186, 188, 189, 190, 191, 195, 196, 197, 198, 201, 205, 207, 208, 212, 220, 226, 228, 230, 231, 232, 233 3, 235, 237, 239, 240, 242, 251, 254, 258, 263, 264, 266, 267, 269, 271, 273, 277, 278, 282, 283, 284, 286, 288, 289, 290, 294, 295, 297, 300, 301, 305, 306, 308, 309, 317, 323, 328, 334, 337, 339, 349, 355, 356, 357, : 358, 359, 360, 362, 364, 367, 370, 372, 374, 375, 378, 379, 380, 381, 382, ​​384, 386, 387, 388, 389, 390, 392, 396, 397, 404, 405, 408, 414, 415, 416, 417, 418, 419, 421, 422, 423 or 428, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

7. The engineered DNA ligase according to any one of claims 1 to 6, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or amino acid residues 11D, 12A / I, 13G / R, 14G / S / T / V, 18D / N / S, 30C / H / S, 31R, 33M / R / V, 34L / R, 36T / Y, 37G / L / N / S, 44S, 50G / I / S / T, 56P, 59E, 60Y, 61T / V, 63F / R, 67R, 68A / M / S / V / Y, 69T, 71G / L / P / R, 73C / K / P / T / V / W, 74S, 76F / G / H / L / N / R, 77D, 82R, 88V, 95A / L / R / V, 96A / G / T / V, 97G, 99G / I, 100V, 101R, 102G / K / L / S, 103V, 104K, 1 05K / S / T, 106L / S / V, 110R, 112M, 113A / T, 117G / S / V / Y, 125R / T, 128C, 130T, 1 32R, 138L / R, 139T, 148P, 149P, 150C / F / T, 155R, 156C, 159Q, 161R / V, 162W, 164A / R, 165K, 177G, 186A / C / E / H / L / M / R / T / V, 188A, 189C / T, 190R, 191T, 195 R, 196E / V, 197R, 198A / D / K / L / N / R / V / W, 201L / S, 205E / G / K, 207L, 208D / F / H , 212F / G / M / S / W, 220V, 226D / E / Q / S / V, 228E / I / M / S, 230L / M, 231P, 232R, 23 3G / T / W, 235W, 237L / M / R / S / V / Y, 239M / N / P / Q / S / T / V / W, 240E / G / K / Q / R / S / Y , 242P / Q / T, 251L, 254G / S, 258L / S / V, 263G / L / Q / T, 264A / C, 266M / T, 267D / W / Y, 269L, 271A / G / N / S, 273A / G / S, 277Q / R, 278E, 282G / L / M / T / V / Y, 283A / G / K / L / M / R / S / V, 284D, 286F / L / S, 288I, 289A / L / S / V, 290L, 294L, 295K, 297W, 30 0G / T, 301F / L, 305K, 306I / K / S / V, 308K / L / S, 309G / R, 317Q, 323S, 328R, 334 L / R, 337G / L / M / P / R / S, 339Y, 349E, 355S, 356A / V / W, 357H / K / P / R / S / V, 358C,359N / R, 360H / M / P, 362G, 363R, 364R, 367C / L, 370C / G, 372N / Q, 374A / S, 375W, 378T, 379A / G / P, 380T, 381K / R, 382V, 384C / V, 386F, 387G, 388K / Y, 389K / L / Q / R, 390E, 392C / I / K / L / R / S, 39 6C / H, 397K / L / M, 404S, 405I, 408C / V, 414A / L / Q / R / T / V, 415A / C / E / H / I / K / L / V, 416K, 417D / G / L, 418A / G / I / L / M / P / S / T, 419G, 421R, 422N, 423R / T, or 428F / R / S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

8. The engineered DNA ligase of any one of claims 1 to 6, wherein the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 63, 242, 283, 286, 317, 414, 418, or 428, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

9. The engineered DNA ligase according to any one of claims 1 to 6 and 8, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitutions 63R, 242Q, 283L, 286S, 317Q, 414Q, 418S or 428R or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

10. The engineered DNA ligase of any one of claims 1 to 6, wherein the amino acid sequence of the engineered DNA ligase comprises at least a substitution or group of substitutions at amino acid position 233, 317, 191, 288, 207, 149, 251, 205, 269, 164, 36, 428, 105 / 132, or 105, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

11. The engineered DNA ligase of any one of claims 1 to 6 and 10, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution group 233T, 317Q, 191T, 288I, 207L, 149P, 251L, 205E, 269L, 164A, 36T, 428R, 105K / 132R, or 105K, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

12. The engineered DNA ligase of claim 1 , wherein the amino acid sequence of the engineered DNA ligase comprises at least one substitution listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

13. The engineered DNA ligase of claim 1 , wherein the amino acid sequence of the engineered DNA ligase comprises at least a substitution or substitution group of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to a reference sequence corresponding to SEQ ID NO:

2.

14. The engineered DNA ligase of claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising at least a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2 and 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO:

2.

15. The engineered DNA ligase of claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

16. The engineered DNA ligase of claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even-numbered SEQ ID NO: 40 to 1184, or a reference sequence corresponding to an even-numbered SEQ ID NO: 40 to 1184.

17. The engineered DNA ligase of claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

18. The engineered DNA ligase of claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 40 to 1184, or a reference sequence corresponding to an even numbered SEQ ID NO: 40 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722 or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722 or 938.

19. The engineered DNA ligase of claim 17 or 18, wherein the amino acid sequence of the engineered DNA ligase comprises at least one of the following: 105, 106, 110, 112, 113, 117, 125, 128, 130, 132, 138, 139, 148, 149, 150, 155, 156, 159, 161, 162, 164, 165, 177, 186, 188, 189, 190, 191, 195, 196, 197, 198, 201, 205, 207, 208, 212, 220, 226, 228, 230, 231, 232, 233 3, 235, 237, 239, 240, 242, 251, 254, 258, 263, 264, 266, 267, 269, 271, 273, 277, 278, 282, 283, 284, 286, 288, 289, 290, 294, 295, 297, 300, 301, 305, 306, 308, 309, 317, 323, 328, 334, 337, 339, 349, 355, 356, 357, 8, 372, 374, 375, 378, 379, 380, 381, 382, ​​384, 386, 387, 388, 389, 390, 392, 396, 397, 404, 405, 408, 414, 415, 416, 417, 418, 419, 421, 422, 423, or 428, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

20. The engineered DNA ligase of any one of claims 17 to 19, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or amino acid residues 11D, 12A / I, 13G / R, 14G / S / T / V, 18D / N / S, 30C / H / S, 31R, 33M / R / V, 34L / R, 36T / Y, 37G / L / N / S, 44S, 50G / I / S / T, 56P, 59E, 60Y, 61T / V, 63F / R, 67R, 68A / M / S / V / Y, 69T, 71G / L / P / R, 73C / K / P / T / V / W, 74S, 76F / G / H / L / N / R, 77D, 82 R, 88V, 95A / L / R / V, 96A / G / T / V, 97G, 99G / I, 100V, 101R, 102G / K / L / S, 103V , 104K, 105K / S / T, 106L / S / V, 110R, 112M, 113A / T, 117G / S / V / Y, 125R / T, 128 C, 130T, 132R, 138L / R, 139T, 148P, 149P, 150C / F / T, 155R, 156C, 159Q, 161R / V, 162W, 164A / R, 165K, 177G, 186A / C / E / H / L / M / R / T / V, 188A, 189C / T, 190R , 191T, 195R, 196E / V, 197R, 198A / D / K / L / N / R / V / W, 201L / S, 205E / G / K, 207 L, 208D / F / H, 212F / G / M / S / W, 220V, 226D / E / Q / S / V, 228E / I / M / S, 230L / M, 23 1P, 232R, 233G / T / W, 235W, 237L / M / R / S / V / Y, 239M / N / P / Q / S / T / V / W, 240E / G / K / Q / R / S / Y, 242P / Q / T / V, 251L, 254G / S, 258L / S / V, 263G / L / Q / T, 264A / C, 2 66M / T, 267D / W / Y, 269L, 271A / G / N / S, 273A / G / S, 277Q / R, 278E, 282G / L / M / T / V / Y, 283A / F / G / K / L / M / R / S / V, 284D, 286F / L / S / W, 288I, 289A / L / S / V, 290 L, 294L, 295K, 297W, 300G / T, 301F / L, 305K, 306I / K / S / V, 308K / L / S, 309G, 3 17T / Q, 323S, 328R, 334L / R, 337G / L / M / P / R / S, 339Y, 349E, 355S, 356A / V / W,357H / K / P / R / S / V, 358C, 359N / R, 360H / M / P, 362G, 364R, 367C / L, 370C / G, 372N / Q, 374A / S, 375W, 37 8T, 379A / G / P, 380T, 381K / R, 382V, 384C / V, 386F, 387G, 388K / Y, 389K / L / Q / R, 390E, 392C / I / K / L / R / S, 396C / H, 397K / L / M, 404S, 405I, 408C / V, 414A / L / Q / R / S / T / V, 415A / C / E / H / I / K / L / V, 416K, 417D / G / L, 418A / G / I / K / L / M / P / S / T, 419G, 421R, 422N, 423R / T, or 428E / F / R / S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

21. The engineered DNA ligase of any one of claims 17 to 19, wherein the amino acid sequence of the engineered DNA ligase comprises at least a substitution at amino acid position 63, 242, 283, 286, 317, 414, 418, or 428, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

22. The engineered DNA ligase of any one of claims 17 to 19, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or amino acid residues 63F / R, 242P / Q / T / V, 283A / F / G / K / L / M / R / S / V, 286F / L / S / W, 317T / Q, 414A / L / Q / R / S / T / V, 418A / G / I / K / L / M / P / S / T, or 428E / F / R / S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, or 938, or relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

23. The engineered DNA ligase of claim 17, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to a reference sequence corresponding to SEQ ID NO: 62, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to a reference sequence corresponding to SEQ ID NO:

62.

24. The engineered DNA ligase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 68 to 312, or to a reference sequence corresponding to an even numbered SEQ ID NO: 68 to 312, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or to the reference sequence corresponding to SEQ ID NO:

62.

25. The engineered DNA ligase of claim 23 or 24, wherein the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 196, 242, 337, 33, 277, 30, 359, 283, 415, 387, 379, 205, 186, 389, 102, 164, 301, 375, 267, 380, 254, 317, 77 / 139 / 317 / 417, 105 / 317 / 4 82, 233, 235, 148, 100, 97, 382, ​​or 358, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or relative to the reference sequence corresponding to SEQ ID NO:

62.

26. The engineered DNA ligase of any one of claims 23 to 25, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution group 196E, 242P, 337L, 33R, 33V, 277Q, 337P, 30C, 359N, 337G, 283S, 242Q, 415A, 387G, 415H, 379A, 205G, 186M, 337M, 389Q, 102L, 389L, 359R, 205K, 164A, 415E, 301F, 30H, 379G, 375W, 267W, 380T, 415C, 254G, 186T, 317Q, 77D / 139T / 317Q / 417G, 105K / 317Q / 417G, 317Q / 349E / 3 62G / 386F, 105K / 317Q, 139T / 317Q / 362G, 233T / 317Q / 405I, 139T / 317Q, 162W, 283V, 286S, 414L, 417L, 226S, 61T, 22 6Q, 105S, 186E, 230M, 61V, 186V, 186L, 379P, 415V, 415I, 186A, 283R, 226V, 418L, 418S, 370G, 283A, 337S, 297W, 23 7S, 428S, 186C, 362G, 283G, 196V, 237L, 415L, 233W, 242T, 277R, 235W, 148P, 254S, 102G, 105T, 301L, 286F, 100V, 23 7Y, 30S, 428F, 414Q, 414V, 283L, 414A, 414R, 370C, 283K, 286L, 186H, 417D, 226D, 233G, 267Y, 97G, 226E, 418T, 230L, 414T, 418A, 237V, 418G, 382V, 267D, 283M, 418P, 237R, 237M, 418I, 358C, 186R, 418M or 102S, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 62, or relative to the reference sequence corresponding to SEQ ID NO:

62.

27. The engineered DNA ligase of claim 17, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to a reference sequence corresponding to SEQ ID NO: 138, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to a reference sequence corresponding to SEQ ID NO:

138.

28. The engineered DNA ligase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 314 to 458, or to a reference sequence corresponding to an even numbered SEQ ID NO: 314 to 458, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or to the reference sequence corresponding to SEQ ID NO:

138.

29. The engineered DNA ligase of claim 27 or 28, wherein the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 242 / 283 / 286 / 359 / 418, 283 / 286, 283 / 286 / 418, 186 / 242 / 283 / 286 / 418, 205 / 286 / 359, 283, 242 / 286 / 418, 186 / 205 / 242, 283 / 286 / 359, 277 / 286 / 359 / 418, 186 / 205 / 283 / 286 / 359, 242 / 277 / 4 18, 242 / 283 / 286 / 418, 112 / 196 / 389, 286, 283 / 286 / 359 / 418, 242 / 359 / 418, 186 / 283, 186 / 359 / 418, 205 / 242 / 418, 186 / 205 / 242 / 283 / 286 / 359 / 418, 205 / 359 / 418, 186 / 205 / 283 / 286 / 418, 186 / 283 / 359, 186 / 242, 186 / 242 / 359, 283 / 359 / 418, 277 / 418, 186 / 188 / 2 83, 186 / 286 / 418, 186 / 242 / 286 / 359 / 418, 418, 186 / 242 / 283 / 286 / 359 / 418, 186 / 277 / 359 / 418, 242 / 283 / 286, 205 / 418, 30 / 297, 205 / 242 / 286 / 359 / 418, 186 / 205 / 359 / 418, 359 / 418, 186, 230, 33 / 297, 186 / 205, 186 / 283 / 359 / 418, 186 / 418, 205 / 242 / 283 / 359 / 41 8, 33 / 375 / 389, 33 / 230, 196 / 242 / 283 / 286 / 359 / 418, 186 / 242 / 283 / 359 / 418, 186 / 359, 33 / 196, 186 / 277 / 418, 242, 33 / 196 / 297 / 301, 205 / 237 / 242 / 283 / 286 / 359, 186 / 205 / 283 / 359 / 418, 33 / 389 or 186 / 196 / 242 / 283 / 286 / 359 / 418, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or relative to the reference sequence corresponding to SEQ ID NO:

138.

30. The engineered DNA ligase of claim 27 or 28, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution group 242Q / 283V / 286S / 359R / 418L, 283L / 286S, 283L / 286S / 418S, 186E / 242Q / 283V / 286S / 418L, 205K / 286S / 359R, 283V, 242P / 286S / 418T, 186E / 205K / 242T, 283L / 286S / 359R, 277Q / 286S / 359N / 418T, 277Q / 418L, 242Q / 283S / 286S / 418T, 112M / 196V / 389Q, 286S, 242P / 283R / 28 6S / 359R / 418L, 283L / 286S / 359R / 418S, 242Q / 359N / 418S, 186E / 283L, 186E / 359R / 418S, 205K / 242T / 418S, 186E / 205K / 242Q / 283S / 286S / 359R / 418S, 2 05K / 359N / 418L, 186A / 205K / 283L / 286S / 418L, 242T / 283V / 286S / 359R / 418S , 186E / 283L / 359R, 186E / 242P, 283V / 286S / 418S, 186E / 242T / 359R, 283L / 3 59R / 418L, 277Q / 418S, 186E / 188A / 283V, 186E / 286S / 418S, 186A / 242T / 286 S / 359N / 418L, 418L, 186A / 242P / 283V / 286S / 359R / 418S, 186A / 277Q / 359R / 418S, 242Q / 283R / 286S, 205K / 418S, 30C / 297W, 283L / 359R / 418S, 186E / 286 S / 418L, 418T, 205K / 242T / 286S / 359R / 418S, 186E / 205K / 359N / 418T, 418S, 186E / 242T / 283S / 286S / 418S, 359R / 418S, 242T / 286S / 418S, 186E, 230L, 33 V / 297W, 186E / 205K, 186A / 283L / 359R / 418S, 186E / 418L, 186E / 277Q / 359R / 418S, 205K / 242T / 283R / 359R / 418L, 186E / 418T, 33V / 375W / 389Q, 33R / 230L,196E / 242T / 283V / 286S / 359R / 418T, 186E / 242T / 283L / 359R / 418S, 186E / 359R, 33 V / 196V, 33R / 375W / 389Q, 186A / 277Q / 418S, 186A / 242T / 283L / 286S / 418L, 242T, 33 V / 196V / 297W / 301F, 205K / 237Y / 242P / 283L / 286S / 359R, 186A / 205K / 283L / 359R / 418T, 33V / 389Q, or 186A / 196E / 242T / 283L / 286S / 359N / 418T, wherein the amino acid positions are relative to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 138, or relative to a reference sequence corresponding to SEQ ID NO:

138.

31. The engineered DNA ligase of claim 17, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to a reference sequence corresponding to SEQ ID NO: 318, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to a reference sequence corresponding to SEQ ID NO:

318.

32. The engineered DNA ligase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 460 to 936, or to a reference sequence corresponding to an even numbered SEQ ID NO: 460 to 936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or to the reference sequence corresponding to SEQ ID NO:

318.

33. The engineered DNA ligase of claim 31 or 32, wherein the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 363, 63, 389, 381, 197, 359, 102, 165, 388, 414, 337, 164, 416, 101, 415, 423, 364, 73, 50, 71, 388 / 419, 357, 396, 68, 76, 14, 271, 360, 266, 208, 74, 263, 264, 13, 378, 372, 300, 294, 290 、397、95、258、161、212、198、138、18、404、273、117、240、69、278、289、82、328、61 / 186 / 417、186 / 370 / 417、267、61 / 370、186 / 267 / 370 / 417、61 / 186、370 / 417、417、61 / 370 / 382、61 / 186 / 267 / 370 / 417、61、267 / 370 / 417、267 / 370、61 / 417、61 / 186 / 237 / 267 / 370, 61 / 237 / 370 / 417, 370, 186 / 370, 61 / 186 / 267 / 417, 61 / 186 / 370 / 382, 370 / 382 / 417, 61 / 186 / 370, 237 / 267 / 370 / 417, 61 / 186 / 382, 61 / 267, 61 / 267 / 417, 61 / 237 / 267 / 382, 186 / 370 / 382, 237 / 267 / 370, 61 / 186 / 267 / 370, 186 / 237 / 267 / 370, 61 / 186 / 2 37, 201, 284, 374, 295, 88, 44, 12, or 390, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or relative to the reference sequence corresponding to SEQ ID NO:

318.

34. The engineered DNA ligase of claim 31 or 32, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution group 363R, 63R, 389K, 381R, 389R, 197R, 359R, 381K, 102K, 165K, 388K, 414R, 337R, 164R, 416K, 101R, 415K, 423R, 364R, 73P, 501, 71L, 388Y / 419G, 50G, 357K, 396H, 68Y, 76F, 71P, 14V, 271G, 68A, 68M, 360P, 266M, 208D, 74S, 263T, 264C, 13R , 263L, 360M, 378T, 76H, 13G, 76R, 50T, 372Q, 50S, 300G, 73T, 294L, 372N, 76 N, 290L, 397L, 76G, 95L, 258L, 161V, 212M, 198V, 138L, 18D, 18S, 404S, 273G , 117G, 240E, 68S, 73C, 69T, 278E, 360H, 198D, 300T, 161R, 73K, 289V, 82R, 3 28R、61T / 186A / 417D、186A / 370C / 417L、267Y、61T / 370C、186A / 267Y / 370C / 4 17L, 61T / 186C, 370C / 417D, 417D, 61T / 370C / 382V, 61T / 186A / 267Y / 370C / 4 17D, 61T, 267Y / 370C / 417D, 267Y / 370C, 61T / 417L, 61T / 186H / 237R / 267Y / 3 70C, 61T / 237R / 370C / 417L, 370C / 417L, 370C, 186E / 370C, 61T / 186E / 267Y / 417D, 61T / 186C / 370C / 382V, 370C / 382V / 417D, 61T / 186C / 267Y / 417D, 61T / 1 86C / 370C, 237R / 267Y / 370C / 417D, 61T / 186E / 417D, 61T / 186H, 61T / 186C / 3 82V, 61T / 186H / 370C, 61T / 267Y, 61T / 267Y / 417L, 61T / 237R / 267Y / 382V, 18 6H / 370C / 382V, 237R / 267Y / 370C, 186H / 370C / 417D, 61T / 186V / 267Y / 370C, 267Y / 370C / 417L, 61T / 186V / 370C, 186H / 237R / 267Y / 370C, 61T / 186E / 267Y,61T / 186C / 237R, 61T / 186H / 237R, 61T / 186C / 417L, 186H, 186A / 267Y, 186C / 370C, 61T / 186C / 237R / 267Y / 370C, 186E / 267Y, 237R / 370C / 417L, 186H / 370C, 242Q / 414Q, 414T, 414Q, 162W / 414Q, 162W / 414V, 267D / 414T, 414V, 26 7D / 414Q, 414A, 162W / 414L, 105S / 414T, 162W / 242Q / 414T, 97G / 162W / 414Q, 105S / 162W / 267D / 414V, 356V, 273A , 357P, 14G, 14S, 396C, 240R, 392K, 273S, 106V, 308S, 308L, 306I, 96A, 397K, 263Q, 282T, 138R, 258S, 76L, 14T, 2 89S, 240G, 106S, 117S, 357S, 96G, 113T, 71G, 282V, 117V, 282M, 258V, 309G, 306K, 308K, 357R, 212S, 18N, 113A, 306S, 212F, 198N, 110R, 240K, 198L, 71R, 357V, 198R, 271A, 282Y, 212G, 95V, 37S, 68V, 240S, 117Y, 201S, 271N, 2 40Q, 289L, 212W, 198W, 357H, 208F, 282L, 306V, 284D, 96T, 266T, 374S, 95R, 309R, 73W, 397M, 289A, 295K, 106L, 95A, 374A, 88V, 96V, 271S, 44S, 208H, 263G, 12A, 201L, 198A, 198K, 282G, 390E, 264A or 73V, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 318, or relative to the reference sequence corresponding to SEQ ID NO:

318.

35. The engineered DNA ligase of claim 17, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to a reference sequence corresponding to SEQ ID NO: 722, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to a reference sequence corresponding to SEQ ID NO:

722.

36. The engineered DNA ligase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 938 to 1098, or to a reference sequence corresponding to an even numbered SEQ ID NO: 938 to 1098, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or to the reference sequence corresponding to SEQ ID NO:

722.

37. The engineered DNA ligase of claim 35 or 36, wherein the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 63, 63 / 96 / 370, 389, 13 / 267 / 363 / 389, 13 / 186 / 389, 50 / 267 / 363 / 370 / 389, 363 / 370, 96 / 370, 61 / 63 / 212, 11 / 305, 11, 242 / 283 / 286 / 317 / 414 / 4 722, 354, 372, 384, 423, 392, 54, 472, 56, 214, 256, 232, 215, 220, 130, 56, 190, 156, 232, 215, 220, 130, 56, 214, 2 ...190, 156, 232, 215, 220, 130, 56, 190, 156, 232, 215, 220, 1 38. The engineered DNA ligase of claim 35 or 36, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution groups 63R, 63R / 96T / 370C, 389K, 13R / 267Y / 363R / 389K, 13R / 186H / 389K, 50T / 267Y / 363R / 370C / 389K, 363 R / 370C, 96T / 370C, 61T / 63R / 212W, 11D / 305K, 11D, 242V / 283F / 286W / 317T / 414S / 418 K, 323S, 334L, 339Y, 356V, 384V, 408C, 67R, 392R, 104K, 355S, 159Q, 155R, 367L, 31R, 231P, 36Y, 150T, 239V, 103V, 356A, 63F, 125T, 367C, 228I, 37S, 37G, 239M, 239W, 239Q , 189C, 239S, 239T, 177G, 239P, 37N, 422N, 228S, 125R, 128C, 189T, 356W, 220V, 408V, 392S, 130T, 228M, 56P, 228E, 190R, 392I, 156C, 232R, 150F, 150C, 239N, 392L, 423T, 34L, 99I, 384C, 59E, 334R, 60Y, 99G, 34R, 37L, 392C, 421R or 195R, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 722, or relative to the reference sequence corresponding to SEQ ID NO:

722.

39. The engineered DNA ligase of claim 17, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to a reference sequence corresponding to SEQ ID NO: 938, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to a reference sequence corresponding to SEQ ID NO:

938.

40. The engineered DNA ligase of claim 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 12 to 437 of an even numbered SEQ ID NO: 1100 to 1184, or to a reference sequence corresponding to an even numbered SEQ ID NO: 1100 to 1184, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or to the reference sequence corresponding to SEQ ID NO:

938.

41. The engineered DNA ligase of claim 39 or 40, wherein the amino acid sequence of the engineered DNA ligase comprises at least amino acid positions 308 / 357 / 390, 74 / 76 / 201 / 308 / 357, 61 / 74 / 76 / 186 / 201 / 308 / 309 / 357 / 390, 14 / 201 / 240 / 289 / 357, 308 / 415, 76 / 3 57 / 396、263 / 308 / 396、61 / 76 / 96 / 240 / 308 / 309、14 / 306 / 415、14 / 73 / 106 / 415、12 / 14 / 258 / 263 / 289 / 308 / 309 / 396、74 / 76 / 117 / 309 / 357、14 / 258 / 263 / 357 / 396、14 / 96 / 106 / 306、14 / 106 、12 / 14 / 308 / 309、14 / 357 / 390、14 / 117 / 258 / 309 / 357、390、240 / 273 / 357 / 390、61 / 76 / 186 / 201 / 308 / 309、14 / 396、309、14、106 / 306 / 308、14 / 240 / 306 / 308、12 / 14 / 186 / 357、309 / 390、14 93, 14 / 76 / 308, 117 / 208 / 258 / 263 / 289 / 308 / 309, 14 / 73 / 106, 76 / 208 / 263, 357, 14 / 308, 263, 76, 14 / 300 / 308 / 415, 240, 33 / 357 / 390, 14 / 76 / 273 or 74, wherein the amino acid position is relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or relative to the reference sequence corresponding to SEQ ID NO:

938.

42. The engineered DNA ligase of claim 39 or 40, wherein the amino acid sequence of the engineered DNA ligase comprises at least the substitution or substitution groups 308L / 357S / 390E, 74S / 76L / 201S / 308L / 357P, 61T / 74S / 76L / 186A / 201S / 308L / 309R / 357P / 390E, 14G / 201S / 240R / 289S / 357P, 308S / 415I, 76L / 357S / 396C, 263L / 308L / 396C, 61T / 76L / 96A / 240R / 308L / 309R, 14S / 306I / 415I, 14S / 73W / 106S / 415I, 12A / 14G / 258S / 263Q / 2 89S / 308L / 309R / 396C, 74S / 76L / 117V / 309R / 357S, 14T / 258S / 263Q / 357S / 396C, 14S / 96G / 106S / 306I, 14S / 106S, 1 2A / 14G / 308L / 309R, 14G / 357S / 390E, 14G / 117V / 258S / 309R / 357S, 390E, 240R / 273S / 357P / 390E, 61T / 76L / 186V / 201S / 308L / 309R, 14G / 396C, 309R, 14S, 106V / 306I / 308S, 14S / 240S / 306I / 308S, 12I / 14G / 186V / 357S, 309R / 390 E, 14S / 306I, 14T, 14S / 76R / 308S, 117V / 208D / 258S / 263Q / 289S / 308L / 309R, 14S / 73W / 106S, 76L / 208D / 263Q, 357S, 14S / 308S, 263L, 76L, 14S / 300G / 308S / 415I, 240Y, 33M / 357S / 390E, 14G / 76L / 273S or 74S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to 437 of SEQ ID NO: 938, or relative to the reference sequence corresponding to SEQ ID NO:

938.

43. The engineered DNA ligase of claim 1, wherein the amino acid sequence of the engineered DNA ligase comprises at least one substitution listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

44. The engineered DNA ligase of claim 1, wherein the amino acid sequence of the engineered DNA ligase comprises at least a substitution or set of substitutions of an engineered DNA ligase variant listed in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

45. The engineered DNA ligase of claim 1 , comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence comprising at least a substitution or set of substitutions provided in Tables 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, and 18.2, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 62, 138, 318, 722, or 938.

46. ​​The engineered DNA ligase of claim 1, wherein the amino acid sequence of the DNA ligase comprises residues 12 to 437 of an even-numbered SEQ ID NO. among SEQ ID NOs: 40 to 1184, or comprises an even-numbered SEQ ID NO. among SEQ ID NOs: 40 to 1184, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9 or at most 10 substitutions.

47. The engineered DNA ligase of claim 1, wherein the amino acid sequence of the DNA ligase comprises residues 12 to 437 of SEQ ID NO: 62, 138, 318, 722, 938, or 1108, or comprises SEQ ID NO: 62, 138, 318, 722, 938, or 1108, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.

48. The engineered DNA ligase of any one of claims 1 to 47, wherein the engineered DNA ligase has DNA ligase activity and improved properties compared to a reference DNA ligase having a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722, or 938, or a sequence corresponding to SEQ ID NO: 2, 62, 138, 318, 722, or 938.

49. The engineered DNA ligase according to any one of claims 1 to 48, characterized in that An improved property selected from the group consisting of i) increased activity, ii) increased stability, iii) increased thermostability, iv) increased product yield, v) increased solubility, vi) reduced sequence preference, and vii) insensitivity or reduced sensitivity to input DNA substrate concentration, or any combination of i), ii), iii), iv), v), and vi) compared to a reference DNA ligase having a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, 62, 138, 318, 722, or 938, or a sequence corresponding to SEQ ID NO: 2, 62, 138, 318, 722, or 938.

50. The engineered DNA ligase of claim 48 or 49, wherein the reference DNA ligase has a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO:

2.

51. An engineered DNA ligase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to: (a) a sequence corresponding to residues 12 to 437 of SEQ ID NO: 2; residues 12 to 613 of SEQ ID NO: 4; residues 12 to 614 of SEQ ID NO: 6; residues 12 to 610 of SEQ ID NO: 8; and residues 12 to 606 of SEQ ID NO: 10; residues 12 to 615 of SEQ ID NO: 12; residues 12 to 594 of SEQ ID NO: 14; residues 12 to 620 of SEQ ID NO: 16; residues 12 to 608 of SEQ ID NO: 18; residues 12 to 611 of SEQ ID NO: 20; residues 12 to 614 of SEQ ID NO: 22; residues 12 to 611 of SEQ ID NO: 24; residues 12 to 609 of SEQ ID NO: 26; residues 12 to 422 of SEQ ID NO: 28; residues 12 to 518 of SEQ ID NO: 30; residues 12 to 438 of SEQ ID NO: 32; residues 12 to 381 of SEQ ID NO: 34; residues 12 to 424 of SEQ ID NO: 36; or residues 12 to 390 of SEQ ID NO: 38, or (b) corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, sequence of 22, 24, 26, 28, 30, 32, 34, 36 or 38.

52. The engineered DNA ligase of claim 51 , wherein the amino acid sequence of the engineered DNA ligase comprises: (a) residues 12 to 437 of SEQ ID NO:2; residues 12 to 613 of SEQ ID NO:4; residues 12 to 614 of SEQ ID NO:6; residues 12 to 610 of SEQ ID NO:8; residues 12 to 606 of SEQ ID NO:10; residues 12 to 615 of SEQ ID NO:12; residues 12 to 594 of SEQ ID NO:14; residues 12 to 620 of SEQ ID NO:16; residues 12 to 608 of SEQ ID NO:18; residues 12 to 611 of SEQ ID NO:20; and residues 12 to 614 of SEQ ID NO:22; residues 12 to 611 of SEQ ID NO:24; residues 12 to 609 of SEQ ID NO:26; residues 12 to 422 of SEQ ID NO:28; residues 12 to 518 of SEQ ID NO:30; residues 12 to 438 of SEQ ID NO:32; residues 12 to 381 of SEQ ID NO:34; residues 12 to 424 of SEQ ID NO:36; or residues 12 to 390 of SEQ ID NO:38, or (b) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.

53. The engineered DNA ligase of any one of claims 1 to 52, wherein the engineered DNA ligase is purified.

54. A recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered DNA ligase of claim 51 or 52.

55. A recombinant polynucleotide comprising a polynucleotide sequence encoding the engineered DNA ligase of any one of claims 1 to 50.

56. The recombinant polynucleotide of claim 55, comprising a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1311 of SEQ ID NO: 1, 61, 137, 317, 721 or 937, or a reference polynucleotide sequence corresponding to SEQ ID NO: 1, 61, 137, 317, 721 or 937, wherein the recombinant polynucleotide encodes an engineered DNA ligase.

57. The recombinant polynucleotide of claim 55, comprising a polynucleotide sequence having at least 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 polynucleotide sequence corresponding to nucleotide residues 34 to 1311 of an odd-numbered SEQ ID NO: 39 to 1183, or a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 39 to 1183, wherein the recombinant polynucleotide encodes an engineered DNA ligase.

58. The recombinant polynucleotide of any one of claims 55 to 57, wherein the polynucleotide sequence is codon-optimized for expression of the encoded engineered DNA ligase.

59. The recombinant polynucleotide of claim 55, comprising a polynucleotide sequence comprising nucleotide residues 34 to 1311 of an odd-numbered SEQ ID NO: 39 to 1183, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO: 39 to 1183.

60. The recombinant polynucleotide of claim 55, comprising a polynucleotide sequence comprising nucleotide residues 34 to 1311 of SEQ ID NO. 1, 61, 137, 317, 721, 937, or 1107, or a polynucleotide sequence comprising SEQ ID NO: 1, 61, 137, 317, 721, 937, or 1107.

61. An expression vector comprising at least one recombinant polynucleotide according to any one of claims 54 to 61.

62. The expression vector of claim 61, wherein the polynucleotide is operably linked to a control sequence.

63. The expression vector of claim 62, wherein the control sequence comprises at least a promoter.

64. A host cell comprising the expression vector of any one of claims 61 to 63.

65. The host cell of claim 64, which comprises a prokaryotic cell or a eukaryotic cell.

66. The host cell of claim 65, which comprises a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

67. A method of producing an engineered DNA ligase polypeptide in a host cell, the method comprising culturing the host cell of any one of claims 64 to 66 under suitable culture conditions such that at least one engineered DNA ligase is produced.

68. The method of claim 67, further comprising recovering the engineered DNA ligase polypeptide from the culture and / or the host cell.

69. The method of claim 67 or 68, further comprising purifying the engineered DNA ligase.

70. A composition comprising the engineered DNA ligase of any one of claims 1 to 53.

71. The composition of claim 70, further comprising one or more of a buffer, ATP, a reducing agent, and / or one or more DNA ligase substrates.

72. The composition of claim 70 or 71 further comprising a linkage enhancer.

73. A method of ligating at least a first DNA strand and a second DNA strand, the method comprising contacting the first DNA strand and the second DNA strand with the engineered DNA ligase of any one of claims 1 to 53 in the presence of a nucleotide substrate under conditions suitable for ligating the first DNA strand to the second DNA strand, wherein the first DNA strand comprises a ligatable 5' end and the second DNA strand comprises a 3' end ligatable to the 5' end of the first DNA strand.

74. The method of claim 73, further comprising a third DNA strand, wherein the first DNA strand and the second DNA strand are hybridized adjacent to each other on the third DNA strand to position the 5' end of the first DNA strand adjacent to the 3' end of the second DNA strand.

75. The method of claim 74, wherein the third DNA strand is continuous with the first DNA strand or the second DNA strand.

76. The method of claim 74, wherein the third DNA strand is continuous with the first DNA strand and the second DNA strand to form a single continuous DNA ligase substrate.

77. The method of claim 73, wherein the first DNA strand is hybridized to a third DNA strand to form a first dsDNA substrate, and the second DNA strand is hybridized to a fourth DNA strand to form a second dsDNA substrate.

78. The method of claim 77, wherein the first dsDNA substrate comprises a blunt-ended 5' end of the first DNA strand, and the second dsDNA substrate comprises a blunt-ended 3' end of the second DNA strand.

79. The method of claim 77, wherein the first dsDNA substrate comprises an overhang on at least one end of the first dsDNA substrate, and the second dsDNA substrate comprises an overhang on at least one end of the second dsDNA substrate, wherein the overhang on the first dsDNA substrate and the overhang on the second dsDNA substrate are complementary and capable of hybridizing to each other and forming one or more nicks that can be ligated.

80. The method of any one of claims 73 to 79, wherein the 3' end of the second DNA strand is a 3'-OH and wherein the 5' end of the first DNA strand is a 5'-phosphate.

81. The method of any one of claims 73 to 80, wherein the nucleotide substrate is ATP.

82. A kit comprising at least the DNA ligase according to any one of claims 1 to 53.

83. The kit of claim 82, further comprising one or more of a buffer, a nucleotide substrate, a DNA substrate for the ligase, and / or a ligation enhancer.

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