Terminal deoxynucleotidyl transferase mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a terminal deoxynucleotidyl transferase mutant and application thereof, and the mutant is obtained by performing R189L / K191G mutation on any one of amino acid sequences as shown in SEQ ID NO.1-7. Starting from a truncated terminal deoxynucleotidyl transferase TdT derived from Zonotrichia albicola, 52 key residues located in a substrate binding pocket are fixed to obtain 7 terminal deoxynucleotidyl transferase mutant sequences, the unfolding temperature Tm of five of the 7 terminal deoxynucleotidyl transferase mutant sequences is 10 DEG C or above higher than that of wild type WT / LG, after the obtained mutants are subjected to heat treatment, the mutant sequences are separated from the wild type WT / LG, and the terminal deoxynucleotidyl transferase TdT is separated from the wild type WT / LG to obtain the 7 terminal deoxynucleotidyl transferase mutant sequences. According to activity characterization of the mutant, compared with a wild type, the mutant obtained by the invention can obviously enable a primer to extend, has higher product yield and can play an important role in DNA enzymatic synthesis.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a terminal deoxynucleotidyl transferase mutant and application thereof. Background Art

[0002] Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase that can randomly add deoxynucleotides to the initiator under template-independent conditions. Based on the template-independent characteristics of terminal deoxynucleotidyl transferase, it can be used for DNA synthesis. The use of 3′-ONH2-modified nucleotides can achieve controllable addition of DNA, but when the growing single-stranded DNA molecules reach a certain length, secondary structures will be formed. TdT has poor substrate activity for the formation of secondary structures at the 3′-OH end, resulting in reduced extension efficiency. High temperature can destroy the hairpin structure of the primer chain and restore it to a single chain that is easy to extend, but TdT has poor stability. High temperature will lead to the loss of enzyme activity and catalytic reaction function. Therefore, it is of great significance to improve the thermal stability of TdT by modifying TdT in DNA enzymatic synthesis.

[0003] Traditional protein engineering mainly relies on rational design, semi-rational design and directed evolution. Directed evolution randomly generates a large number of mutant libraries through error-prone PCR, DNA shuffling and other means to screen target mutants. Directed evolution does not need to understand the protein catalytic mechanism and structural information. After multiple rounds of screening, beneficial mutations are accumulated to obtain mutants with improved specific performance. Semi-rational design selects several sites for modification through iterative saturation mutagenesis (ISM) and combinatorial active-site saturation test (CAST). It only needs to screen a smaller mutant library. Rational design relies on the relationship between protein structure and function. Through molecular docking, molecular dynamics simulation and other means, it evaluates the changes in protein structure, binding energy and other aspects. It selects mutants that meet the requirements and combines them with experimental verification, which greatly reduces the number of required libraries. However, traditional protein engineering modification is inefficient, and all of them are modifications to existing proteins in nature, which cannot meet the needs of industrial production.

[0004] In recent years, deep learning has shown great potential in protein design and modification. For example, AlphaFold2 can predict the structure of proteins, which greatly accelerates the research progress of proteins. In addition, for a given protein skeleton, the use of deep learning algorithms can quickly generate candidate amino acid sequences. ProteinMPNN is a protein design algorithm based on deep learning that can quickly and efficiently generate protein sequences with expected functions and structures, and has been successful in increasing protein expression and stability. Summary of the invention

[0005] In view of the defect of poor heat resistance of terminal deoxynucleotidyl transferase in the prior art, the present invention provides a terminal deoxynucleotidyl transferase mutant with high thermal stability and its application, and the specific technical scheme is as follows:

[0006] In a first aspect, the present invention provides a terminal deoxynucleotidyl transferase mutant, wherein the terminal deoxynucleotidyl transferase mutant is one of the following mutants:

[0007] (1) The amino acid sequence shown in SEQ ID NO.1 was subjected to R189L / K191G mutation;

[0008] (2) The amino acid sequence shown in SEQ ID NO. 2 was subjected to R189L / K191G mutation;

[0009] (3) The amino acid sequence shown in SEQ ID NO. 3 was subjected to R189L / K191G mutation;

[0010] (4) obtained by subjecting the amino acid sequence shown in SEQ ID NO.4 to R189L / K191G mutation;

[0011] (5) The amino acid sequence shown in SEQ ID NO.5 was subjected to R189L / K191G mutation.

[0012] The present invention predicts the three-dimensional structure of ZaTdT (sZaTdT, WT) with N-terminal truncation by AlphaFold2 and uses it as the input of ProteinMPNN. Since ProteinMPNN is a structure-based de novo design method, the functional information of the protein is not considered when generating the sequence. Therefore, in order to retain the catalytic activity of TdT, 52 key residues located in the substrate binding pocket are fixed when using ProteinMPNN to design new sequences. Finally, 7 initial terminal deoxynucleotidyl transferase mutant sequences were obtained, and AlphaFold2 was used to predict the structures of these 7 sequences. The results showed that they all reproduced the substrate binding pocket with high confidence (plDDT). The R189L / K191G mutation was combined with 7 sequences respectively to obtain terminal deoxynucleotidyl transferase mutant sequences. Terminal deoxynucleotidyl transferase mutants with improved thermal stability were obtained through expression and purification. It was verified that 5 of the terminal deoxynucleotidyl transferase mutants had a significant improvement in thermal stability, and after heat treatment at 48°C, 51°C, 56°C, 58°C, and 61°C, the yield of DNA synthesis catalyzed by these 5 terminal deoxynucleotidyl transferase mutants was higher than that of the N-terminally truncated terminal deoxynucleotidyl transferase with R189L / K191G mutation.

[0013] In a second aspect, the present invention provides a gene encoding the terminal deoxynucleotidyl transferase mutant.

[0014] In a third aspect, the present invention provides a recombinant vector comprising the encoding gene as described in claim 2.

[0015] In a fourth aspect, the present invention provides a genetically engineered bacterium, comprising a host cell, wherein the host cell contains the encoding gene as claimed in claim 2 or the recombinant vector as claimed in claim 3.

[0016] Furthermore, the host cell is Escherichia coli.

[0017] Furthermore, the host cell is E. coli BL21 (DE3).

[0018] In a fifth aspect, the present invention provides the use of the terminal deoxynucleotidyl transferase mutant in enzymatic DNA synthesis.

[0019] Furthermore, in the enzymatic DNA synthesis, deoxyribonucleotides modified with a reversible blocking group at the 3' end are used as substrates, and DNA is synthesized under the catalysis of the terminal deoxynucleotidyl transferase mutant.

[0020] Furthermore, the reversible blocking group is an oxyamino group.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention starts from a truncated terminal deoxynucleotidyl transferase TdT from Zonotrichia albicollis, fixes 52 key residues located in the substrate binding pocket, and obtains 7 terminal deoxynucleotidyl transferase mutant sequences, among which the unfolding temperatures T of MP1 / LG-MP5 / LG m It was more than 10°C higher than the wild-type WT / LG. After the obtained mutants were heat-treated at 48°C, 51°C, 56°C, 58°C, and 61°C for 5 minutes, their activity was characterized. Compared with the wild-type, the product yield of MP1 / LG-MP5 / LG was higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The gel electrophoresis diagrams of different terminal deoxynucleotidyl transferase mutants after incubation at different temperatures; wherein, WT / LG represents the terminal deoxynucleotidyl transferase with N-terminal truncation, N represents the primer, and N+1 represents the primer extended by 1 base.

[0024] Figure 2 The figure is the gel electrophoresis diagram of the extension reaction of MP6 / LG and MP7 / LG after incubation at 37°C; wherein N+1 indicates the primer extended by 1 base. DETAILED DESCRIPTION

[0025] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0026] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0028] In the following examples, the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0029] In the following examples, the amino acid sequences of the initial terminal deoxynucleotidyl transferase mutants are shown in SEQ ID NOs. 1 to 7, the nucleotide sequences of the genes encoding the initial terminal deoxynucleotidyl transferase mutants are shown in SEQ ID NOs. 8 to 14, the amino acid sequence of the N-terminally truncated terminal deoxynucleotidyl transferase sZaTdT is shown in SEQ ID NO. 15, and the nucleotide sequence of the N-terminally truncated terminal deoxynucleotidyl transferase sZaTdT is shown in SEQ ID NO. 16.

[0030] SEQ ID NO.1:

[0031] MKISQYSCQRRTSLKNLNQKFVDAFNVMADYYEYLEDKEKAEKYRRAAALLRSLPFKVTKMEDIEGLPNIYEECRKIIKEILEKGYSPEVEKIKNDPYYQAMKEFTSVYGFGVKEAKELYKKGYKTVEEVKADKTLKLSATQKAGLKYYDDLVAPVTREEAEAVHRLITETVHAFVPDAVVEI TGGFRRGKKTTHDVDFLISNPGPVEDPDLLDKVLARLEAEGRLLYAERIPSTYDPDRLPSEKLDAMDHFQKCFTIVKLPLPPPPPAAAAAAAPVPPAPPRGWRAVRVDLTYVPYERYPYALLGWTGSRQFVRDLRRYAREERGMLLDNHGLYDRKTGRFLEAASEREIFDHLGLEYIPPEERNA

[0032] SEQ ID NO.2:

[0033] MKISQYSCQRRTSLKDLNKEFTDALKVMAEYYEYKEDKENAEKYRKAAALLRSLPFKVTKLEDIKGLPNISEEVRKIIEEILKNGYSEEVEKIKNDPYYKAMKEFTSVYGFGVKEAKELYAKGYKTVEEVKADKTLKLTEEQKAGLKYYEDLVRPVTREEAEAVYEIISETVHKFVPEAVVEITGGFRRGKKTTHDVDFLISNPGPEEHPDLLQKIIEELKKQGLLLYYELIPSTYDPNRLPSEKLDAMDHFQKCFMIIKLPLPPPPPAAAPEAAPVPPAPPRNYRAVRVDLVYVPKERYPYALLGWTGSRQFVRDLRRYALEERGMLLDNHGLYDRKTGRFLKAESEKEIFDHLGLEYIPPEERNA

[0034] SEQ ID NO.3:

[0035] EVVSQYSCQRRCSLTNLNQKFVDAFDIMAEYYEFKENKKEAEKFRRAAALLRCLPFEVTKMEDIEGLPNIYEEERKIIEEILKNGYSPEVEKIKNDPYFKAMKEFTKIYGFGVKRAKEYYAKGYKTVEEIKADKSLKLSETQKAGLKYYEDLVRPITREEALAVHEIIKSTVHKFRPDAIVEITGGFRRGKETTHDVDFLISNPGPVEDPNLLDKIIEELKEKGILLYYEKIPSTYDPNRLPSTKLDAMDHFQKCFMIIKLYLPPPPPAAAAAAAPVPPAPPQDWRAVRVDLTYVPYSRRPYALLGWTGSRQFVRDLRRYALEERGMILDNHGLYDRKTGKFLEAASEEEIFAHLGLPYIPPEQRNA

[0036] SEQ ID NO.4:

[0037] KKYSQYSCQRRTSLKNLNQKFVDAFEVMARYAEFKGDKETAERYRRAAALLRSLPFEVKSMEDIEGLPNIYPECREIIEEILKYGYSKRVEAIKADPYFKAMSEFVSVYGFGVKRAKELIAKGYTTVEQVKADKTLKLSKTQKAGLKYYEDLVRPVTREEAEVHEIITSTVHKFRPDAVVEVTGGFRRGKETTHDVDFLISNPGPVEDPDLLQKIVDTLKEEGLLLYHEIIPSTYKPDALPSEKLDAMDHFQKCFMIIKLPLPPPPPPAAAAAAPAPPAPPKDWRAVRVDLTLVPAEARPYALLGWTGSRQFVRDLRRYAREERGMLLDNHGLFDRKAGRFVAAASEEEIFAALGLPYIPPEERNA

[0038] SEQ ID NO.5:

[0039] MVYSQYSCQRRTSLKDLNKEFTDAFDIMAEYAEFKEDKETAEKYRDAAALLRSLPFKVTKMEDIEGLPNIYPDCREIIEEILKNGYSSRVEKIKNDPYFKAMKEFTKVLGFGVKRAKEYYAKGYTTVEQIKADKSIKLTPEQKAGLKYYEDLVAPVTREEAEAVAKIIRETVHKFVPNAIVEITGGFRRGKETTHDVDFLISNPGPVDDPLKKVVEELKKEGLLLYAKIIESTYDPDRLPSEKEDAMDHFQKCFMIIKLYLPPPPPPAAGPAPAPVPPAPPRGYRAVRVDLTYVPYERYPFALLGWTGSRQFVRDLRRYAREERGMLLDNHRLYDRRTGRFVEAKSEQEIYDALGLEYIPPEDRNA

[0040] SEQ ID NO.6:

[0041] MKISQYSCQRRTSLKNLNQKFVDAFNVMADYYEYLEDKEKAEKYRRAAALLRSLPFKVTKMEDIEGLPNIYEECRKIIKEILEKGYSPEVEKIKNDPYYQAMKEFTSVYGFGVKEAKELYKKGYHTHHHHHHDKTLKLSATQKAGLKYYDDLVAPVTREEAEAVHRLITETVHAFVPDAVVEITGGFRRGKHHHHDVDFLISNPGPVEDPDLLDKVLARLEAEGRLLYAERIPSTYDPDRLPSEKLDAMDHFQKCFTIVKLPLPPPPPAAAAAAAPVPPAPPRGWRAVRVDLTYVPYERYPYALLGWTGSRQFVRDLRRYAREERGMLLDNHGLYDRKTGRFLEAASEREIFDHLGLEYIPPEERNA

[0042] SEQ ID NO.7:

[0043] MKISQYSCQRRTSLKNLNQKFVDAFNVMADYYEYLEDKEKAEKYRRAAALLRSLPFKPPPPPDPEGLPNIYEECRKIIKEILEKGYSPEVEKIKNDPYYQAMKEFTSVYGFGVKEAKELYKKGYHTHHHHHHDKTLKLSATQKAGLKYYDDLVAPVTREEAEAVHRLITETVHAFVPDAVVEITGGFRRGKHHHHDVDFLISNPGPVEDPDLLDKVLARLEAEGRLLYAERIPSTYDPDRLPSEKLDAMDHFQKCFTIVKLPLPPPPPAAAAAAAPVPPAPPRGWRAVRVDLTYVPYERYPYALLGWTGSRQFVRDLRRYAREERGMLLDNHGLYDRKTGRFLEAASEREIFDHLGLEYIPPEERNA

[0044] SEQ ID NO.8:

[0045]

[0046] SEQ ID NO.9:

[0047]

[0048] SEQ ID NO.10:

[0049]

[0050] SEQ ID NO.11:

[0051]

[0052] SEQ ID NO.12:

[0053]

[0054] SEQ ID NO.13:

[0055]

[0056] SEQ ID NO.14:

[0057]

[0058] SEQ ID NO.15:

[0059] MKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEIIEEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0060] SEQ ID NO.16:

[0061]

[0062] Example 1 Construction of mutants

[0063] (1) In this implementation, the terminal deoxynucleotidyl transferase (ZaTdT) from diphtheria was selected, and the three-dimensional structure of the N-terminally truncated ZaTdT (sZaTdT, WT) was predicted by AlphaFold 2 and used as the input of ProteinMPNN. In order to retain the catalytic activity of terminal deoxynucleotidyl transferase, 52 key residues located in the substrate binding pocket were fixed when using ProteinMPNN to design new sequences. The amino acid residues at positions 4, 5, 8, 9, 10, 114, 184, 185, 186, 187, 188, 189, 190, 191, 195, 196, 198, 235, 243, 245, 249, 250, 251, 252, 253, 254, 255, 256, 289, 291, 303, 304, 305, 306, 307, 308, 309, 310, 311, 315, 318, 319, 330, 331, 332, 337, 355, 356, 357, 365, 366, and 367 of sZaTdT were fixed. ProteinMPNN was used to generate 1000 sequences, from which the sequences of seven initial terminal deoxynucleotidyl transferase mutants with the best global score and lower sequence similarity were selected.

[0064] The initial terminal deoxynucleotidyl transferase mutant gene was synthesized by Qingke Biotechnology Co., Ltd. and inserted into the restriction sites BamH I and Hind III of the vector pET28a to obtain a recombinant plasmid.

[0065] (2) Construction of the superimposed R189L / K191G mutant

[0066] Based on the amino acid sequences of the seven initial terminal deoxynucleotidyl transferase mutants, R189L / K191G mutations were performed to obtain the sequences of seven terminal deoxynucleotidyl transferase mutants (named MP1 / LG, MP2 / LG, MP3 / LG, MP4 / LG, MP5 / LG, MP6 / LG, and MP7 / LG). The terminal deoxynucleotidyl transferase mutant genes were synthesized by Qingke Biotechnology Co., Ltd., and appropriate primers were designed for whole-plasmid PCR. The PCR reaction system and primers are shown in Tables 1 and 2.

[0067] Table 1 PCR reaction system

[0068]

[0069]

[0070] Note: N* refers to 1 to 5, indicating different types of mutants.

[0071] Table 2 Primers used for mutant construction

[0072]

[0073] Example 2 Expression and purification of different terminal deoxynucleotidyl transferase mutants

[0074] The plasmids carrying different terminal deoxynucleotidyl transferase mutant genes were transformed into BL21 (DE3), spread on LB solid medium containing 50 μg / mL kanamycin (Kana), and cultured in a 37°C constant temperature incubator for 12 h; a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL Kana, and cultured at 37°C, 220 rpm for 8-12 h; 2% of the inoculum was transferred to 50 mL of fresh LB liquid medium containing 50 μg / mL Kana, and cultured at 37°C, 220 rpm until OD 600 When the pH value reached about 0.8, IPTG was added at a final concentration of 1 mM and the culture was induced at 18°C ​​and 220 rpm for 16 h.

[0075] After the culture is completed, the bacterial solution is centrifuged at 4000rpm and 4℃ for 15min, the supernatant is discarded, and the collected bacteria are then resuspended in 50mM phosphate buffer with pH 7.5, and ultrasonically disrupted at 400W in an ice water bath, each ultrasonication for 3S, with an interval of 7S. After the disruption is completed, the cell disruption solution is centrifuged at 12000rpm and 4℃ for 30min to remove cell debris, and the obtained supernatant is the crude enzyme solution, which is filtered through a 0.22μm filter membrane for purification, and impurities are washed with a washing buffer (50mM PBS buffer, 50mM imidazole, 100mM NaCl, pH7.5), and the target protein is eluted with an elution buffer (50mM PBS buffer, 250mM imidazole, 100mM NaCl, pH 7.5). The separated target protein is placed in an ultrafiltration centrifuge tube for sufficient desalting and concentration to obtain pure terminal deoxynucleotidyl transferase mutant proteins.

[0076] Example 3 Activity Characterization of Different Terminal Deoxynucleotidyl Transferase Mutants

[0077] The terminal deoxynucleotidyl transferase mutant protein prepared in Example 2 was used to prepare a 0.05 mg / mL enzyme solution. The reaction system was 10 μL, including 1 μM primer, 33 μM 3′-ONH2-dGTP, 0.25 mM CoCl2, 100 mM NaCl and 50 mM phosphate buffer (pH 7.5). The 0.05 mg / mL enzyme solution was reacted at 37°C for 10 min. The reaction was analyzed by 20% urea-polyacrylamide gel electrophoresis. The results are shown in FIG. Figure 1 and Figure 2 shown.

[0078] Example 4 Characterization of thermal stability of different terminal deoxynucleotidyl transferase mutants

[0079] The terminal deoxynucleotidyl transferase mutant protein prepared in Example 2 was used to prepare a 0.05 mg / mL enzyme solution. The 0.5 mg / mL enzyme solution was incubated at 40°C, 45°C, 48°C, 51°C, 56°C, 58°C, and 61°C for 5 min, respectively, and then placed on ice for use. The reaction system was 10 μL, including 1 μM primer, 33 μM 3′-ONH2-dGTP, 0.25 mM CoCl2, 100 mM NaCl, and 50 mM phosphate buffer (pH 7.5). 1 μL of the above enzyme solution was added to start the reaction. The reaction was reacted at 37°C for 10 min. The reaction was analyzed by 20% urea-polyacrylamide gel electrophoresis. The results are as follows: Figure 1 SHST analysis was used to extract the grayscale, measure the grayscale of the extension product and the grayscale of the unextended band, and calculate the yield by the ratio of the grayscale of the extension product to the sum of the grayscales of the extension and unextended bands in the lane. The results are shown in Table 3.

[0080] Table 3 Product yields of terminal deoxynucleotidyl transferase mutants WT / LG, MP1 / LG-MP5 / LG

[0081]

[0082] Example 5 Unfolding Temperature T of Different Terminal Deoxynucleotidyl Transferase Mutants m Determination

[0083] The unfolding temperature T of the mutants was determined using a protein thermal stability analyzer (NanoTemper, USA). m The protein sample with a concentration of 0.5 mg / mL was loaded into a quartz capillary, the power was set to 10%, the scanning rate was 60℃ / h, and the scanning range was 20~90℃. m As shown in Table 4.

[0084] Table 4 Unfolding temperature T of different terminal deoxynucleotidyl transferase mutantsm

[0085] Mutants <![CDATA[T m / ℃]]> WT / LG 46.75±0.05℃ MP1 / LG 74.30±0.10℃ MP2 / LG 64.05±0.25℃ MP3 / LG 82.05±0.15℃ MP4 / LG 81.50±0.30℃ MP5 / LG 64.50±0.90℃ MP6 / LG 40.50±0.20℃ MP7 / LG 42.15±0.15℃

[0086] As can be seen from Table 4, the different terminal deoxynucleotidyl transferase mutants used in this example have higher unfolding temperatures than the wild-type terminal deoxynucleotidyl transferase. The unfolding temperatures T of MP1 / LG, MP2 / LG, MP3 / LG, MP4 / LG, and MP5 / LG are as follows: m More than 10℃ higher than the wild type WT / LG.

Claims

1. A terminal deoxynucleotidyl transferase mutant, characterized in that The terminal deoxynucleotidyl transferase mutant is one of the following mutants: (1) The amino acid sequence shown in SEQ ID NO.1 was subjected to R189L / K191G mutation; (2) The amino acid sequence shown in SEQ ID NO. 2 was subjected to R189L / K191G mutation; (3) The amino acid sequence shown in SEQ ID NO. 3 was subjected to R189L / K191G mutation; (4) The amino acid sequence shown in SEQ ID NO. 4 was subjected to R189L / K191G mutation; (5) The amino acid sequence shown in SEQ ID NO.5 was subjected to R189L / K191G mutation.

2. A gene encoding the terminal deoxynucleotidyl transferase mutant according to claim 1.

3. A recombinant vector, characterized in that: Contains the coding gene as described in claim 2.

4. A genetically engineered bacterium, characterized in that: The invention comprises a host cell, wherein the host cell comprises the encoding gene as claimed in claim 2 or the recombinant vector as claimed in claim 3.

5. The genetically engineered bacterium according to claim 4, characterized in that: The host cell is Escherichia coli.

6. The genetically engineered bacterium according to claim 5, characterized in that: The host cell is E. coli BL21 (DE3).

7. Use of the terminal deoxynucleotidyl transferase mutant according to claim 1 in enzymatic DNA synthesis.

8. The use according to claim 7, characterized in that: In the enzymatic DNA synthesis, deoxyribonucleotides modified with a reversible blocking group at the 3' end are used as substrates, and DNA is synthesized under the catalysis of the terminal deoxynucleotidyl transferase mutant.

9. The use according to claim 8, characterized in that: The reversible blocking group is an oxyamino group.

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