Engineered terminal deoxynucleotidyl transferase mutant and application thereof

Abstract

The invention discloses an engineered terminal deoxynucleotidyl transferase mutant and application thereof. The engineering TdT enzyme variant containing single mutation and multi-site mutation is obtained by screening through a directed evolution technology on the basis of a DNA (Deoxyribonucleic Acid) sequence of wild type ZaTdT from a diphtheria sparrow. The engineered TdT enzyme variant designed by the invention has a unique function of specifically catalyzing an aminooxy modified nucleotide monomer to carry out single base extension, and high-precision controllable synthesis of a single-stranded DNA molecule can be realized under the condition of no template dependence. According to the method, the dependency of traditional enzymatic synthesis on a template chain is broken through, the single-stranded oligonucleotide synthesis efficiency and the sequence control precision are remarkably improved, and a novel tool platform is provided for innovative development in the fields of gene synthesis, molecular diagnosis, biological storage and the like.

Description

Technical Field

[0001] The present invention relates to the technical fields of synthetic biology and molecular enzyme engineering, and particularly relates to engineered terminal deoxynucleotidyl transferase mutants and their applications. Background Art

[0002] DNA enzymatic synthesis technology, with its high fidelity (>99.9%), long fragment synthesis ability (>200 nt), and low-cost production advantages, has become a key development direction for breaking through the bottleneck of chemical synthesis technology. However, in the existing technical system, there is a lack of DNA polymerases with single-base level precision control, which severely restricts its industrial applications in fields such as synthetic biology and DNA data storage.

[0003] Terminal deoxynucleotidyl transferase (TdT), as a non-template-dependent DNA polymerase, has natural advantages in catalyzing the addition of random nucleotides to the 3'-hydroxyl end. However, its wild type has the following key defects: (1) low catalytic efficiency for modified nucleotides (kcat < 0.1 s-1); (2) lack of specific recognition ability for reversible terminators, resulting in insufficient single-base addition precision (error rate > 5%); (3) inability to achieve the stability of multiple-round cyclic extension (active half-life < 30 min). Existing nucleotide polymerization control technologies mainly include: (a) TdT-nucleotide complex technology; (b) apyrase-assisted regulation technology; (c) reversible terminator technology. The first two technologies have technical defects such as large fluctuations in dissociation constants and enzyme activity inhibition caused by by-product accumulation.

[0004] Reversible terminator nucleotides are a class of functionalized dNTP derivatives with a reversible blocking group introduced at the 3'-hydroxyl site. Its core mechanism is that when this nucleotide is incorporated into the 3'-end of single-stranded DNA (ssDNA) through the catalysis of terminal deoxynucleotidyl transferase (TdT), the steric hindrance effect generated by its blocking group can effectively block the non-specific incorporation of subsequent nucleotides, thereby forcibly terminating the current extension reaction. After specifically removing the blocking group through a chemical protection step, the 3'-OH active site is regenerated, and only then can the next round of base addition cycle be initiated. This mechanism enables the template-independent DNA synthesis mediated by TdT to achieve strict single-turnover precision of single-base addition, reducing the synthesis error rate to less than 0.1%.

[0005] However, wild-type TdT cannot effectively incorporate dNTP derivatives containing a 3'-hydroxyl blocking group. Therefore, developing a TdT variant that can efficiently incorporate such dNTP derivatives has important scientific and application values. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides an engineered terminal deoxynucleotidyl transferase mutant and its application. Based on the DNA sequence of the white-throated sparrow ZaTdT, the present invention obtains mutants specifically adapted to 3'-protected nucleotides (aminooxy-dNTPs) by site-directed mutagenesis. The engineered enzyme mutant designed by the present invention has the unique function of specifically catalyzing the single-base extension of aminooxy-modified nucleotide monomers, and can achieve the high-precision and controllable synthesis of single-stranded DNA molecules under template-independent conditions.

[0007] The technical solution of the present invention is as follows:

[0008] An engineered terminal deoxynucleotidyl transferase mutant, wherein the engineered terminal deoxynucleotidyl transferase mutant is obtained by single-site mutation or multi-site mutation of the amino acid sequence shown in SEQ ID No. 2 at positions 8, 9, 12, 18, 21, 27, 46, 56, 129, 138, 178, 186, 189, 193, 196, 203, 292, 460, 474, 478; the single-site mutation does not include the single-site mutations at positions 178, 186, 478; the terminal deoxynucleotidyl transferase mutant has at least 80% identity with the amino acid sequence shown in SEQ ID No. 2 or a functionally equivalent sequence.

[0009] Furthermore, the engineered terminal deoxynucleotidyl transferase mutant is obtained by single-site mutation of the amino acid sequence shown in SEQ ID No. 2 at positions 46 and 56; the engineered terminal deoxynucleotidyl transferase mutant includes mutants that add or delete some amino acids on the basis of SEQ ID NO. 2 and still have the function of synthesizing nucleic acids using 3'-modified nucleotides.

[0010] Furthermore, compared with the amino acid sequence shown in SEQ ID No. 2, the preferred mutation modes of the engineered terminal deoxynucleotidyl transferase mutant are: L46M, G56A; the 3'-modified nucleotide is preferably a 3'-protected nucleotide; the protecting group of the 3'-protected nucleotide is aminooxy.

[0011] Furthermore, the engineered terminal deoxynucleotidyl transferase mutant is obtained by multi-site mutation of the amino acid sequence shown in SEQ ID No. 2 at positions 8, 9, 12, 18, 21, 27, 129, 138, 178, 186, 189, 193, 196, 203, 292, 460, 474, 478; the engineered terminal deoxynucleotidyl transferase mutant includes mutants that add or delete some amino acids on the basis of SEQ ID NO. 2 and still have the function of synthesizing nucleic acids using 3'-modified nucleotides.

[0012] Furthermore, compared with the amino acid sequence shown in SEQ ID No.2, the preferred mutation patterns of the multi-site mutation are as follows:

[0013] Combined mutations at the mutation sites at positions 12, 189, 292, 196 and 203; or combined mutations at the mutation sites at positions 9, 196 and 474; or combined mutations at the mutation sites at positions 21, 196 and 474; or combined mutations at the mutation sites at positions 178, 186, 193, 335, 337, 460 and 478; or combined mutations at the mutation sites at positions 21, 129, 138, 196 and 474; or combined mutations at the mutation sites at positions 18, 21, 27, 196 and 292; or combined mutations at the mutation sites at positions 8, 18, 196 and 203; The nucleotide with 3'-end modification is preferably a 3'-end protected nucleotide; The protecting group of the 3'-end protected nucleotide is an aminooxy group.

[0014] Furthermore, compared with the amino acid sequence shown in SEQ ID No.2, the preferred mutation patterns of the engineered terminal deoxynucleotidyl transferase mutant are: combined mutations at the mutation sites of Q12R, E189A, L292F, L196V and S203T; or combined mutations at the mutation sites of V9L, L196V and I474M; or combined mutations at the mutation sites of S21P, L196V and I474M; or combined mutations at the mutation sites of Y178A, F186R, A193T, R335L, K337H, G460N and H478G; or combined mutations at the mutation sites of S21A, S129C, A138V, L196V and I474M; or combined mutations at the mutation sites of G18A, S21T, S27E, L196V and L292F; or combined mutations at the mutation sites of A8S, G18Q, L196V and S203P.

[0015] Furthermore, compared with the amino acid sequence shown in SEQ ID No.2, the most preferred mutation pattern of the engineered terminal deoxynucleotidyl transferase mutant is: combined mutations at the mutation sites of Y178A, F186R, A193T, R335L, K337H, G460N and H478G.

[0016] A nucleic acid molecule encoding the engineered terminal deoxynucleotidyl transferase mutant.

[0017] An expression vector comprising the nucleic acid molecule.

[0018] A host cell comprising the expression vector.

[0019] A method for producing the engineered terminal deoxynucleotidyl transferase mutant, wherein the host cell is cultured under culture conditions allowing the expression of the nucleic acid encoding the terminal deoxynucleotidyl transferase mutant, and the terminal deoxynucleotidyl transferase mutant is recovered.

[0020] Furthermore, the production method includes the following steps:

[0021] (1) Inducing the host cell to express the recombinant enzyme under suitable culture conditions;

[0022] (2) Lysing the cells and purifying by affinity chromatography (such as His-tag purification);

[0023] (3) Verifying the correctness of the size of the recombinant protein by SDS-PAGE.

[0024] An application of the engineered terminal deoxynucleotidyl transferase mutant, which is used for synthesizing single-stranded DNA with 3'-end modified nucleotides in the absence of a template.

[0025] A method for controllably synthesizing single-stranded DNA in the absence of a template, the method comprising contacting a nucleic acid primer with at least one nucleotide, preferably at least one 3'-end modified nucleotide and the terminal deoxynucleotidyl transferase mutant.

[0026] Furthermore, the method for controllably synthesizing single-stranded DNA includes the following steps:

[0027] (a) Providing a DNA primer with a free 3'-OH;

[0028] (b) Adding to the reaction system:

[0029] i. The ZaTdT mutant,

[0030] and ii. A single type of 3'-end aminooxy-modified nucleotide (concentration range: 0.1 mM - 1 mM),

[0031] and iii. A buffer containing divalent cations (preferably Co 2+ or Mn 2+ , concentration 0.1 mM - 1 mM);

[0032] (c) Incubating at 20 - 40 °C for 1 - 60 min to achieve single-base addition;

[0033] (d) Removing the aminooxy group through a chemical deprotection step (sodium nitrite solution) to regenerate the 3'-OH for the next round of extension.

[0034] The beneficial technical effects of the present invention are as follows:

[0035] By constructing and screening a mutant library, the present invention successfully obtained a set of ZaTdT mutants containing single-site and multi-site combined mutations. Through in vitro functional verification, the TdT mutants of the present invention exhibit specific catalytic activity towards aminooxy-protected nucleotides (3'-ONH2-dNTP), with a significantly improved single-base extension activity compared to the wild type, and achieve the controllable synthesis of single-stranded DNA one by one under template-free conditions. The present invention provides a variety of engineered terminal deoxynucleotidyl transferase mutants, which show significant application potential in the controllable and precise synthesis of single-stranded DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a mechanism diagram for the extension of single-stranded DNA by the engineered terminal deoxynucleotidyl transferase mutant of the present invention.

[0037] Figure 2 It is a chemical structure diagram of 4 reversible terminator deoxynucleotides modified with an aminooxy group at the 3' end.

[0038] Figure 3 It is an SDS-PAGE result diagram of wild-type ZaTdT and its mutants.

[0039] Figure 4 It is a Urea-PAGE result diagram for the incorporation of 1 reversible terminator into the starting strand catalyzed by wild-type ZaTdT and its mutants.

[0040] Figure 5 It is a heat map of the efficiency of incorporating a single reversible terminator by wild-type ZaTdT and its mutants. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The present invention will be specifically described below in conjunction with the drawings and embodiments.

[0042] In this article, amino acids are represented by their single-letter or three-letter codes according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp); and Y: tyrosine (Tyr).

[0043] In this text, the following terms are used to indicate substitutions: A8S means that the amino acid residue (alanine, A) at position 8 of the parental sequence is changed to serine (S); V9L means that the amino acid residue (valine, V) at position 9 of the parental sequence is changed to leucine (L); Q12R means that the amino acid residue (glutamine, Q) at position 12 of the parental sequence is changed to arginine (R); G18Q means that the amino acid residue (glycine, G) at position 18 of the parental sequence is changed to glutamine (Q); S21A / T / P means that the amino acid residue (serine, S) at position 21 of the parental sequence is substituted by one of the following amino acids: alanine (A) or threonine (T) or proline (P); S27E means that the amino acid residue (serine, S) at position 27 of the parental sequence is changed to glutamic acid; L46M means that the amino acid residue (leucine, L) at position 46 of the parental sequence is changed to methionine (M); G56A means that the amino acid residue (glycine, G) at position 56 of the parental sequence is changed to alanine (A); S129C means that the amino acid residue (serine, S) at position 129 of the parental sequence is changed to cysteine (C); A138V means that the amino acid residue (alanine, A) at position 138 of the parental sequence is changed to valine (V); Y178A means that the amino acid residue (tyrosine, Y) at position 178 of the parental sequence is changed to alanine (A); F186R means that the amino acid residue (phenylalanine, F) at position 186 of the parental sequence is changed to arginine (R); E189A means that the amino acid residue (glutamic acid, E) at position 189 of the parental sequence is changed to alanine (A); A193T means that the amino acid residue (alanine, A) at position 193 of the parental sequence is changed to threonine (T); L196V means that the amino acid residue (leucine, L) at position 196 of the parental sequence is changed to valine (V); S203T means that the amino acid residue (serine, S) at position 203 of the parental sequence is changed to threonine (T); L292F means that the amino acid residue (leucine, L) at position 292 of the parental sequence is changed to phenylalanine (F); H341K means that the amino acid residue (histidine, H) at position 341 of the parental sequence is changed to lysine (K); G460N means that the amino acid residue (glycine, G) at position 460 of the parental sequence is changed to asparagine (N); I474M means that the amino acid residue (isoleucine, I) at position 474 of the parental sequence is changed to methionine (M); H478G means that the amino acid residue (histidine, H) at position 478 of the parental sequence is changed to glycine (G).

[0044] In the embodiments of the present invention, the substrates are all constructed based on deoxyribonucleotides (dNTPs), wherein the 3'-OH end is replaced with a reversible terminating group, specifically referring to deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate, and deoxythymidine monophosphate. In a more preferred embodiment, the modifying group at the 3' end adopts an alkoxyamine structure.

[0045] Figure 1 This is a schematic diagram of the extension of single-stranded DNA by the engineered terminal deoxynucleotidyl transferase mutant of the present invention. First, Buffer, ssDNA, metal ions, and reversible terminator nucleotides are sequentially added to the reaction system, and finally the corresponding ZaTdT or its mutant is added; under the catalysis of ZaTdT, the reversible terminator is incorporated into the ssDNA to terminate the reaction. Excess TdT and other impurities are removed by centrifugation; then a deprotection reagent is added to the catalytic system to expose the 3'-OH end for the next round of reaction.

[0046] Example 1 Species source of terminal deoxynucleotidyl transferase (TdT)

[0047] SEQ ID NO.1: According to the records of NCBI, the wild-type ZaTdT gene sequence derived from Zonotrichia albicollis.

[0048] SEQ ID NO.2: The amino acid sequence deduced from the TdT gene sequence of SEQ ID NO.1.

[0049] SEQ ID NO.2:

[0050] MDRFKAPAVISQRKRQKGLHSPKLSCSYEIKFSNFVIFIMQRKMGLTRRMFLMELGRRKGFRVESELSDSVTHIVAENNSYLEVLDWLKGQAVGDSSRFELLDISWFTACMEAGRPVDSEVKYRLMEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0051] The gene sequences corresponding to all the enzymes used in this example were sent to Beijing Tsingke Biosynthesis and inserted into the expression vector pET-28a, and the restriction enzyme sites NdeI and XhoI were retained. After plasmid construction, it was transformed into Escherichia coli BL21 for protein expression and purification. The construction, expression and purification of recombinant TdT enzyme used the vector PET-28a, and the MBP tag was added when needed to promote protein solubility. The host bacterium was Escherichia coli BL21 as an example. The present invention includes but is not limited to this, and all vectors and host bacteria that can successfully express and purify TdT protein should be included.

[0052] Example 2 Obtaining of highly active engineered terminal deoxynucleotidyl transferase (TdT) mutants

[0053] In the present invention, the TdT gene sequence from Zonotrichia albicollis was subjected to homology modeling to obtain its three-dimensional protein structure. Based on the inventors' existing experience, through computer-aided rational design, single-point or multi-point saturation mutagenesis, combined with high-throughput screening and other means, variants with high catalytic efficiency and substrate specificity were obtained. The variants included in the present invention are shown in the following table.

[0054] Table 1 Mutants included in the present invention

[0055]

[0056] Example 3 Obtaining Engineered Terminal Deoxynucleotidyl Transferase (TdT) Mutant Proteins

[0057] To verify the activity of the terminal deoxynucleotidyl transferase mutant in vitro, the enzyme was recombinantly expressed and purified in Escherichia coli. The experimental method is as follows:

[0058] (1) Construction of recombinant plasmids

[0059] The gene sequence SEQ ID NO.1 of wild-type TdT was obtained through NCBI, codon-optimized, synthesized, and ligated to the pET-28a vector to construct a recombinant plasmid. Single-site mutant TdT was generated using the Novizan site-directed mutagenesis kit based on the recombinant wild-type TdT plasmid. For multi-site mutants, the amino acid sequence was codon-optimized, synthesized, and ligated to the pET-28a vector to construct a mutant recombinant plasmid. For some genes, an MBP tag was additionally added to the N-terminus of the TDT sequence and ligated to the pET-28a vector to construct a mutant recombinant plasmid.

[0060] (2) Construction of recombinant Escherichia coli expression strains

[0061] Take 4 μg of the freeze-dried powder of the recombinant plasmid, add 40 μL of Elution, dissolve and mix well. Take 1 μL and dilute it 10 times to make a 40 ng / μL solution. Take 100 μL of competent cells of Escherichia coli BL21(DE3) and place them on ice. After melting, take 1 μL of the above recombinant plasmid solution and add it to the competent cells, mix well, incubate on ice for 30 - 60 min, heat shock in a 42°C water bath for 45 s, transfer to ice and let stand for 2 min; add 300 μL of LB liquid medium, resuscitate at 37°C for 30 - 60 min, take 100 μL and spread it on an LB solid plate containing 50 μg / mL kanamycin, culture overnight at 37°C, and pick single colonies for culture and scale-up culture.

[0062] (3) Inductive expression of recombinant proteins

[0063] Pick a single colony from the LB plate and inoculate it into 10 mL of LB medium containing 50 μg / mL kanamycin. Culture overnight at 37°C and 225 rpm for 12 - 16 h. Then take 5 mL and inoculate it into 1 L of LB liquid medium containing 50 μg / mL kanamycin. Culture at 37°C and 225 rpm until the OD 600nm is between 0.4 and 0.45. Transfer to a shaker at 18°C and 200 rpm for 1 h, add IPTG to a final concentration of 200 μM, and induce expression at 18°C and 200 rpm for 16 - 18 h.

[0064] (4) Protein purification

[0065] Centrifuge the induced bacterial solution at 4 °C and 6000 rpm for 5 min, discard the supernatant, add 30 mL of buffer A (200 mM NaCl, 20 mM Tris, 10% Glycerol, 1 mmol / L TCEP, pH 6.0) to resuspend the bacteria, and sonicate for 1 h; then centrifuge at 12000 rpm for 40 min, collect the supernatant, filter through a 0.22 μM microporous filter membrane, and collect the sample. Purify the protein using AKTA. After loading the sample, perform gradient elution: rinse with 3% Buffer B (200 mM NaCl, 20 mM Tris, 10% Glycerol, 1 mM TCEP, 500 mM Imidazole, pH 6.0) for 5 column volumes; rinse with 7% Buffer B for 5 column volumes; rinse with 10% Buffer B for 3 column volumes, 20% Buffer B for 3 column volumes, 40% Buffer B for 8 column volumes; rinse with 100% Buffer B for 10 column volumes. After elution, according to the UV absorption graph, collect the eluates rinsed with 40% and 100% Buffer B, desalt with a desalting column, concentrate the protein solution using a 30 kDa ultrafiltration tube, and immediately perform enzyme activity tests or quick-freeze in liquid nitrogen and store at -80 °C for later use.

[0066] Figure 3 SDS-PAGE electrophoresis diagram of the terminal deoxynucleotidyl transferase mutant. From Figure 3 It can be seen that the present invention can successfully induce the expression of engineered terminal deoxynucleotidyl transferase mutants S1, S2, Z-3, Z-5, Z-6, Z-7, Z-8, Z-9, Z-10, S3, S11, Z-2, Z-4.

[0067] Example 4 Application of terminal deoxynucleotidyl transferase (ZaTdT) mutant S1 in the controllable synthesis of single-stranded DNA

[0068] (1) Immobilization: According to the instructions of the Novizan kit, immobilize the primer with a biotin label at the 5' end (primer sequence: 5'-TAATACGACTCACTTG-3') on streptavidin magnetic beads (the magnetic beads are purchased from Sangon Biotech, Shanghai, product number D601013-0001) to obtain the immobilized primer.

[0069] (2) Reaction: Add 1 mg / mL terminal deoxynucleotidyl transferase mutant S1 (L46M), 0.5 mmol / L deoxyribonucleotides (dNTPs) modified at the 3'-end with an aminooxy group, and 0.25 mmol / L CoCl2 to a 50 mmol / L Tris-HCl solution at pH = 7.4. Then mix with the immobilized primer obtained in step (1) and react at 30 °C for 20 min.

[0070] (3) Deprotection: Remove the protecting group with a 0.7 mol / L sodium nitrite solution (pH = 5, pH adjusted with nitrous acid).

[0071] (4) Repeat step (2) once to obtain single-stranded DNA with one additional base.

[0072] Example 5 - 17

[0073] Examples 5 - 17 are the applications of terminal deoxynucleotidyl transferase (TdT) mutants S2, Z-3, Z-5, Z-6, Z-7, Z-8, Z-9, Z-10, S3, S11, Z-2, Z-4, and wild-type TdT in the controllable synthesis of single-stranded DNA, respectively. The application process is basically the same as that of Example 4, except that in Examples 5 - 13, terminal deoxynucleotidyl transferase mutants S2, Z-3, Z-5, Z-6, Z-7, Z-8, Z-9, Z-10, S3, S11, Z-2, Z-4, and wild-type TdT are used for the reaction in sequence.

[0074] The single-stranded DNA obtained in Examples 4 - 17 of the present invention was detected by 20% urea-polyacrylamide gel (denaturing PAGE), and the result after staining with 1×SYBR Gold is as Figure 4 shown. As can be seen from Figure 4 the results, compared with wild-type ZaTdT, the ssDNA bands catalyzed by the mutants showed obvious extended migration, indicating that the engineered terminal deoxynucleotidyl transferase mutants of the present invention can catalyze the polymerization of one nucleotide variant. In addition, combined with Figure 5Heat map analysis of the efficiency of wild-type TdT (WT) and its mutants inserted into a single reversible terminator. The darker the color, the higher the catalytic efficiency. Z-3, Z-5, Z-6, Z-7, Z-8, Z-9, Z-10, and S3 all significantly enhanced the extension activity of the four reversible terminators. In addition, the catalytic activity of S1 to the three reversible terminators other than 3'-ONH2-dGTP was greatly improved, and S2 enhanced the catalytic properties of 3'-ONH2-dTTP and 3'-ONH2-dCTP. However, the experimental results also found that the catalytic activity of Z-2 and Z-4 was not optimized. Compared with the ZaTdT mutant R335L-K337L currently reported to have good catalytic activity, the extension activity of the ZaTdT mutant designed in the present invention is also significantly enhanced. In particular, Z-7 can completely incorporate the four reversible terminators into the starting chain. Among all the mutants designed by us, the catalytic activity for 3'-ONH2-dGTP is generally weak, while the enzyme completely incorporates 3'-ONH2-dGTP, indicating that the engineered terminal deoxynucleotidyl transferase mutants of the present invention have significantly improved catalytic ability for the reversible terminator modified with 3'-terminal aminooxy group compared with the wild-type terminal deoxynucleotidyl transferase TdT.

[0075] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. An engineered terminal deoxynucleotidyl transferase mutant, characterized in that, The engineered terminal deoxynucleotidyl transferase mutant is obtained by performing single-site mutations or multi-site mutations at positions 8, 9, 12, 18, 21, 27, 46, 56, 129, 138, 178, 186, 189, 193, 196, 203, 292, 460, 474, and 478 of the amino acid sequence shown in SEQ ID No. 2; the single-site mutations do not include the single-site mutations at positions 178, 186, and 478; the terminal deoxynucleotidyl transferase mutant has at least 80% identity with the amino acid sequence shown in SEQ ID No. 2 or a functionally equivalent sequence.

2. The engineered terminal deoxynucleotidyl transferase mutant according to claim 1, wherein The engineered terminal deoxynucleotidyl transferase mutant is obtained by performing single-site mutations at positions 46 and 56 of the amino acid sequence shown in SEQ ID No. 2; the engineered terminal deoxynucleotidyl transferase mutant includes mutants that have the function of synthesizing nucleic acids using 3'-end modified nucleotides after adding or deleting some amino acids based on SEQ ID NO.

2.

3. The engineered terminal deoxynucleotidyl transferase mutant according to claim 2, wherein, Compared with the amino acid sequence shown in SEQ ID No. 2, the preferred mutation modes of the engineered terminal deoxynucleotidyl transferase mutant are: L46M and G56A; the 3'-end modified nucleotide is preferably a 3'-end protected nucleotide; the protecting group of the 3'-end protected nucleotide is an aminooxy group.

4. The engineered terminal deoxynucleotidyl transferase mutant according to claim 1, wherein The engineered terminal deoxynucleotidyl transferase mutant is obtained by performing multi-site mutations at positions 8, 9, 12, 18, 21, 27, 129, 138, 178, 186, 189, 193, 196, 203, 292, 460, 474, and 478 of the amino acid sequence shown in SEQ ID No. 2; the engineered terminal deoxynucleotidyl transferase mutant includes mutants that have the function of synthesizing nucleic acids using 3'-end modified nucleotides after adding or deleting some amino acids based on SEQ ID NO.

2.

5. The engineered terminal deoxynucleotidyl transferase mutant according to claim 4, wherein Compared with the amino acid sequence shown in SEQ ID No. 2, the preferred mutation modes of the multi-site mutations are as follows: Combined mutations at the mutation sites of positions 12, 189, 292, 196, and 203; or combined mutations at the mutation sites of positions 9, 196, and 474; or combined mutations at the mutation sites of positions 21, 196, and 474; or combined mutations at the mutation sites of positions 178, 186, 193, 335, 337, 460, and 478; or combined mutations at the mutation sites of positions 21, 129, 138, 196, and 474; or combined mutations at the mutation sites of positions 18, 21, 27, 196, and 292; or combined mutations at the mutation sites of positions 8, 18, 196, and 203; the 3'-end modified nucleotide is preferably a 3'-end protected nucleotide; the protecting group of the 3'-end protected nucleotide is an aminooxy group.

6. The engineered terminal deoxynucleotidyl transferase mutant according to claim 4, wherein Compared with the amino acid sequence shown in SEQ ID No. 2, the preferred mutation modes of the engineered terminal deoxynucleotidyl transferase mutant are: combined mutations at the mutation sites of Q12R, E189A, L292F, L196V and S203T; or combined mutations at the mutation sites of V9L, L196V and I474M; or combined mutations at the mutation sites of S21P, L196V and I474M; or combined mutations at the mutation sites of Y178A, F186R, A193T, R335L and K337H, G460N, H478G; or combined mutations at the mutation sites of S21A, S129C, A138V, L196V and I474M; or combined mutations at the mutation sites of G18A, S21T, S27E, L196V and L292F; or combined mutations at the mutation sites of A8S, G18Q, L196V and S203P.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the engineered terminal deoxynucleotidyl transferase mutant according to any one of claims 1-6.

8. An expression vector, wherein the expression vector contains the nucleic acid molecule according to claim 7.

9. A host cell, wherein the host cell contains the expression vector according to claim 8.

10. Use of an engineered terminal deoxynucleotidyl transferase mutant according to any one of claims 1-6, characterized in that, The engineered terminal deoxynucleotidyl transferase mutant is used for synthesizing single-stranded DNA with 3'-end modified nucleotides in the absence of a template.

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