Method for synthesizing a non-natural base deoxynucleotide triphosphate

Abstract

The application specifically relates to a method for synthesizing unnatural base deoxynucleotide triphosphates by using enzymes. The application is based on the amino acid modification of wild-type deoxynucleotide kinases from Drosophila melanogaster and wild-type polyphosphate kinases from Thermus rubens, and mutants with improved activities for unnatural base deoxynucleotides and unnatural base deoxynucleotide monophosphates are obtained. The obtained nucleotide kinase mutants can efficiently convert unnatural base deoxynucleotides dCNMO and dTPT3 into corresponding unnatural base deoxynucleotide monophosphates, and then the polyphosphate kinase mutants can further efficiently convert the unnatural base deoxynucleotide monophosphates into corresponding unnatural base deoxynucleotide triphosphates. The present research provides a new strategy for the synthesis of unnatural base deoxynucleotide triphosphates, which is efficient, economical and green, and overcomes the limitations of the existing chemical synthesis method, such as complicated steps, harmful reagents and high cost, and lays an important foundation for the low-cost and green efficient production of nucleotide derivatives.

Description

Technical Field

[0001] This invention relates to the fields of biochemistry and bioengineering, specifically to a method for synthesizing non-natural deoxyribonucleoside triphosphates. Background Technology

[0002] The four bases in DNA form 64 triplet codons, encoding 20 common amino acids. These bases, through Watson-Crick pairing, ensure the precise transmission of genetic information during replication, transcription, and translation. However, relying solely on the four bases for storing and transmitting genetic information limits the information capacity and function of DNA and RNA. By introducing non-natural base pairs, the genetic alphabet can be expanded, providing DNA and RNA with greater information capacity and new functions.

[0003] In recent years, several non-natural base pairs have been developed and successfully replicated, transcribed, and translated in vitro and in vivo. Among them, dCNMO-dTPT3 and its derivatives have been used to construct hexa-base *E. coli*. The application of non-natural base pairs in vitro and in vivo relies on the use of non-natural deoxynucleotides. Currently, the synthesis of non-natural deoxynucleotides depends on chemical synthesis, which is cumbersome, has extremely low yields, and is costly. Therefore, it is necessary to develop a green and efficient method for synthesizing non-natural deoxynucleotides. Summary of the Invention

[0004] The first objective of this invention is to provide a nucleoside kinase mutant that efficiently phosphorylates non-natural deoxynucleosides to generate non-natural deoxynucleosides monophosphate and a polyphosphate kinase mutant that efficiently phosphorylates non-natural deoxynucleosides monophosphate to generate non-natural deoxynucleosides triphosphate.

[0005] A second objective of this invention is to provide applications of deoxynucleoside kinase mutants and polyphosphate kinase mutants.

[0006] The third objective of this invention is to provide a method for the enzymatic synthesis of non-natural nucleoside triphosphates.

[0007] The objective of this invention is achieved through the following technical solution: Deoxynucleoside kinase mutant Dm dNK-Q81H, whose amino acid sequence is shown in SEQ ID No. 7; Furthermore, the aforementioned Dm The nucleotide sequence of the dNK-Q81H encoding gene is shown in SEQ ID No. 2.

[0008] Polyphosphate kinase mutant Mr PPK-M1, whose amino acid sequence is shown in SEQ ID No. 9; Furthermore, the aforementioned Mr The nucleotide sequence of the gene encoding PPK-M1 is shown in SEQ ID No. 4.

[0009] Polyphosphate kinase mutant Mr PPK-M2, the amino acid sequence of which is shown in SEQ ID No. 10; Furthermore, the aforementioned Mr The nucleotide sequence of the PPK-M2 encoding gene is shown in SEQ ID No. 5.

[0010] Deoxynucleoside kinase mutant Dm Application of dNK-Q81H in the synthesis of non-natural deoxynucleoside monophosphate and / or non-natural deoxynucleoside triphosphate.

[0011] Furthermore, in the application, non-natural deoxyribonucleosides are used as raw materials to synthesize non-natural deoxyribonucleosides monophosphate and / or non-natural deoxyribonucleosides triphosphate; the non-natural deoxyribonucleosides include at least one of dCNMO or dTPT3, the structure of dCNMO is shown in Formula 1, and the structure of dTPT3 is shown in Formula 2. .

[0012] Polyphosphate kinase mutant Mr Application of PPK-M1 in the synthesis of dCNMOTP.

[0013] Furthermore, in the aforementioned application, non-natural deoxynucleoside monophosphate dCNMOMP is used as a raw material to synthesize non-natural deoxynucleoside triphosphate dCNMOTP, the structure of which is shown in Formula 5. .

[0014] Polyphosphate kinase mutant Mr Application of PPK-M2 in the synthesis of dTPT3TP.

[0015] Furthermore, in the aforementioned application, non-natural deoxynucleoside monophosphate dTPT3MP is used as a raw material to synthesize non-natural deoxynucleoside triphosphate dTPT3TP, the structure of which is shown in Formula 6. .

[0016] A method for the enzymatic synthesis of non-natural deoxyribonucleoside triphosphates includes the following steps: S1. Non-natural deoxynucleosides containing adenosine triphosphate (ATP), MgCl2, and deoxynucleoside kinase mutants. Dm The reaction was carried out in the dNK-Q81H system, and the non-natural deoxyribonucleoside monophosphate was isolated and purified. S2. The non-natural base deoxyribonucleoside monophosphate obtained in step S1 is reacted in a system containing polyphosphate, MnCl2 and polyphosphate kinase mutant to obtain non-natural base deoxyribonucleoside triphosphate; The deoxynucleoside kinase mutant described in step S1 Dm The amino acid sequence of dNK-Q81H is shown in SEQ ID No. 7.

[0017] The polyphosphate kinase mutants mentioned in step S2 include Mr PPK-M1 and Mr At least one of PPK-M2.

[0018] Further, the non-natural deoxyribonucleoside mentioned in step S1 includes at least one of dCNMO or dTPT3; even further, the corresponding non-natural deoxyribonucleoside monophosphate is dCNMOMP or dTPT3MP; the structure of dCNMO is shown in Formula 1, and the structure of dTPT3 is shown in Formula 2. ; The structure of dCNMOMP is shown in Equation 3, and the structure of dTPT3MP is shown in Equation 4. .

[0019] Furthermore, the concentration of ATP in the system described in step S1 is 1–20 mM; even further, it is 12 mM.

[0020] Furthermore, the concentration of the non-natural deoxyribonucleoside mentioned in step S1 in the system is 1–20 mM; even further, it is 10 mM.

[0021] Furthermore, the divalent metal ion mentioned in step S1 is a magnesium ion, provided by a magnesium salt; even further, it is provided by MgCl2.

[0022] Furthermore, the concentration of the divalent metal ions in the system described in step S1 is 5–100 mM; even further, it is 20 mM.

[0023] Furthermore, the aforementioned Dm The nucleotide sequence of the gene encoding dNK-Q81H is shown in SEQ ID No. 2.

[0024] Furthermore, the deoxynucleoside kinase described in step S1 Dm The concentration of dNK-Q81H in the system ranges from 1 to 10 μM; further, it is 6 μM.

[0025] Furthermore, the system described in step S1 further includes 10–60 mM Tris-HCl with pH 5.0–10.0 and 1–150 mM KCl; even further, it includes 50 mM Tris-HCl with pH 8.0 and 100 mM KCl.

[0026] Furthermore, the reaction described in step S1 is carried out at a temperature of 30-40°C for 1-10 hours; even further, it is carried out at a temperature of 37°C for 3 hours.

[0027] Furthermore, the polyphosphoric acid mentioned in step S2 is ammonium polyphosphate, and the degree of polymerization of ammonium polyphosphate is 6 to 100; even further, the degree of polymerization is 30 to 50.

[0028] Furthermore, the concentration of the polyphosphoric acid in the system described in step S2 is 0.1-2 mg / mL; even more specifically, it is 0.776 mg / mL.

[0029] Furthermore, the non-natural deoxynucleotide monophosphate mentioned in step S2 includes at least one of dCNMOMP and dTPT3MP; When the non-natural deoxyribonucleotide monophosphate mentioned in step S2 is dCNMOMP, the polyphosphate kinase mutant mentioned in step S2 is... Mr PPK-M1, whose amino acid sequence is shown in SEQ ID No. 9, and the non-natural base deoxynucleoside triphosphate obtained by the reaction is dCNMOTP, whose structure is shown in Formula 5; When the non-natural deoxyribonucleotide monophosphate mentioned in step S2 is dTPT3MP, the polyphosphate kinase mutant mentioned in step S2 is Mr PPK-M2, whose amino acid sequence is shown in SEQ ID No. 10, and the non-natural base deoxynucleoside triphosphate obtained by the reaction is dTPT3TP, whose structure is shown in Formula 6; .

[0030] Furthermore, the aforementioned Mr The nucleotide sequence of the gene encoding PPK-M1 is shown in SEQ ID No. 4; Furthermore, the aforementioned Mr The nucleotide sequence of the gene encoding PPK-M2 is shown in SEQ ID No. 5.

[0031] Furthermore, the concentration of the non-natural deoxyribonucleoside monophosphate mentioned in step S2 in the system is 0.1–5 mM; even further, it is 1 mM.

[0032] Furthermore, the divalent metal ion mentioned in step S2 is a manganese ion, provided by a manganese salt; even further, it is provided by MnCl2.

[0033] Furthermore, the concentration of the divalent metal ions in the system described in step S2 is 5–50 mM; even further, it is 15 mM.

[0034] Further, the concentration of the polyphosphate kinase mutant in step S2 is 0.1–5 μM; even further, the non-natural deoxyribonucleotide monophosphate in step S2 is dCNMOMP, and the polyphosphate kinase mutant is… Mr When PPK-M1 is used, Mr The concentration of PPK-M1 in the system was 0.5 μM; the non-natural deoxyribonucleotide monophosphate mentioned in step S2 was dTPT3MP, and the polyphosphate kinase mutant was... Mr When PPK-M2, Mr The concentration of PPK-M2 in the system was 0.3 μM.

[0035] Furthermore, the system described in step S2 further includes 10–100 mM Tris-HCl with pH 5.0–10.0; and even further includes 20 mM Tris-HCl with pH 8.0.

[0036] Furthermore, the reaction described in step S2 is carried out at a temperature of 30–80 °C for 0.5–24 h; even further, it is carried out at 37 °C for 2 h.

[0037] The present invention has the following advantages and effects compared with the prior art: This invention proposes a method for synthesizing non-natural deoxyribonucleotide triphosphates dCNMOTP and dTPT3TP using deoxyribonucleotide kinase mutants and polyphosphate kinase mutants. This method achieves the enzymatic green synthesis of dCNMOTP and dTPT3TP under mild conditions, providing a new technical route for the synthesis of non-natural deoxyribonucleotides. Specifically, the synthesis of dCNMOTP and dTPT3TP utilizes Drosophila melanogaster deoxyribonucleotide kinase. Dm dNK mutant ( Dm The synthesis of dNK-Q81H); dCNMOTP was performed using a polyphosphate kinase mutant of *Thermus rubrum*. Mr The synthesis of PPK-M1, dTPT3TP was performed using a polyphosphate kinase mutant of *Thermophyton floccosum*. Mr PPK-M2. Deoxynucleoside kinase mutant compared to wild type. Dm dNK-Q81H significantly enhances the activity of synthesizing non-natural deoxynucleotide monophosphates, and the polyphosphate kinase mutant... Mr PPK-M1 andMr PPK-M2 significantly enhances the activity of synthesizing non-natural deoxynucleoside triphosphates. Attached Figure Description

[0038] Figure 1 It is to utilize Dm dNK mutants and Mr Technical roadmap for the enzymatic synthesis of non-natural deoxyribonucleoside triphosphates using PPK mutants.

[0039] Figure 2 It is an expression of purification Dm dNK and Dm SDS-PAGE analysis results of dNK-Q81H protein.

[0040] Figure 3 It is an expression of purification Mr PPK-WT, Mr PPK-M1 and Mr SDS-PAGE analysis results of PPK-M1 protein.

[0041] Figure 4 It is Dm HPLC analysis results of the products synthesized from non-natural deoxynucleoside monophosphates catalyzed by dNK-Q81H. Among them, Figure 4 -A is used Dm HPLC analysis results of the product dCNMO phosphorylation to dCNMOMP catalyzed by dNK-Q81H; Figure 4 -B is used Dm HPLC analysis results of the product dTPT3MP generated by phosphorylation of dTPT3 catalyzed by dNK-Q81H.

[0042] Figure 5 It is Mr PPK-WT and Mr PPK-M1 or Mr HPLC analysis results of the products synthesized from non-natural deoxyribonucleoside triphosphates catalyzed by PPK-M2. Among them, Figure 5 -A is Mr PPK-WT and mutants Mr HPLC analysis results of the product of dCNMOTP generated by phosphorylation of dCNMOMP catalyzed by PPK-M1; Figure 5 -B is used Mr PPK-WT and mutants Mr HPLC analysis results of the product dTPT3TP generated by PPK-M2 catalytic phosphorylation of dTPT3MP. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the biological materials used in the embodiments were prepared or obtained by conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0044] The chemical structures of dCNMO, dTPT3, and the corresponding synthesized dCNMOMP, dTPT3MP, dCNMOTP, and dTPT3TP used in the examples are as follows: Figure 1 As shown.

[0045] Example 1. Dm dNK mutants and Mr Construction of PPK mutant recombinant expression strains Deoxynucleoside kinase mutants, specifically pET- from our laboratory Dm Using the dNK-WT plasmid as a template, conventional overlap extension PCR was performed to mutate glutamine at position 81 to histidine, thus constructing a plasmid containing... Dm dNK mutant Dm Plasmid of the dNK-Q81H gene; in which pET- Dm In the dNK-WT plasmid, Dm The sequence of the dNK-WT gene is shown in SEQ ID NO. 1, and the corresponding enzyme... Dm The amino acid sequence of dNK-WT is shown in SEQ ID NO. 6; the mutated... Dm The sequence of the dNK-Q81H gene is shown in SEQ ID NO. 2, and the corresponding enzyme... Dm The amino acid sequence of dNK-Q81H is shown in SEQ ID NO. 7. Polyphosphate kinase mutants are expressed as pET- Mr Using the PPK-WT plasmid as a template, conventional overlap extension PCR was performed to mutate glycine at position 92 to lysine, aspartic acid at position 127 to asparagine, and lysine at position 134 to arginine, thus constructing a plasmid containing... Mr PPK mutants Mr PPK-M1 and Mr The plasmid of the PPK-M2 gene. Mr In the PPK-M1 mutant, glycine at position 92, aspartic acid at position 127, and lysine at position 134 are mutated to lysine, asparagine, and arginine, respectively. Mr In the PPK-M2 mutant, glycine at position 92 and lysine at position 134 are mutated to lysine and arginine, respectively. pET- Mr PPK-WT plasmid Mr The sequence of the PPK gene is shown in SEQ ID NO. 3, and the corresponding enzyme... MrThe amino acid sequence of PPK-WT is shown in SEQ ID NO. 8; the mutated... Mr PPK-M1 and Mr The sequences of the PPK-M2 gene are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the corresponding enzymes are... Mr PPK-M1 and Mr The amino acid sequences of PPK-M2 are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0046] Table 1. Primer sequences used in overlap extension PCR site-directed mutagenesis

[0047] Table 2. PCR reaction system for site-directed mutagenesis amplification of each gene fragment

[0048] The specific steps for constructing the mutant recombinant expression strain are as follows: (1) pET-28a(+)- Dm Using dNK-WT plasmid as a template, pET-T7-F / Q81-R and Q81H-F / pET-T7-R primers were used for amplification according to the system in Table 2. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 30 cycles; 68 ℃ for 5 min; extension times t were 20 s and 36 s respectively. The amplified plasmid was used to construct pET-28a(+)- Dm The two gene fragments required for the dNK-Q81H plasmid. (The pET-28a(+)-) Dm Using dNK-WT as a template and T7F-rev / T7R-rev as primers, amplification was performed according to the system in Table 2. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 310 s, 30 cycles; 68 ℃ for 5 min; the amplified product was used to construct pET-28a(+)- Dm The linear vector fragment required for the dNK-Q81H plasmid.

[0049] After the PCR reaction, gene fragments and vector fragments were recovered by gel extraction using an agarose gel DNA recovery kit (Guangzhou Meiji Biotechnology Co., Ltd.). Using the two gene fragments as templates, the high-fidelity enzyme PrimeSTAR was employed. ®The full-length gene was amplified by overlap extension PCR using HS DNA polymerase. The reaction system is shown in Table 3. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 1 min, 30 cycles; 68 ℃ for 5 min. The amplified products were recovered by gel excision using an agarose gel DNA recovery kit to obtain the full-length gene containing the Q81H mutation.

[0050] Table 3. Overlap extension PCR system for site-directed mutagenesis assembly and amplification of full-length genes

[0051] Homologous recombination was performed between the full-length gene containing the Q81H mutation and the linearized vector: the homologous recombination system was prepared according to Table 4 and incubated at 50 °C for 20 min. 5 µL of the homologous recombination product was transformed into 100 µL of *E. coli* DH5α competent cells via electroporation. After recovery, the cells were plated on kanamycin-resistant plates and cultured overnight. Positive clones were identified by colony PCR, and selected for sequencing. After verifying the sequence correctness, the corresponding plasmid was extracted and transformed into *E. coli* expression strain BL21(DE3) pLysS competent cells via electroporation.

[0052] Table 4. Homologous recombination system

[0053] (2) pET-28a(+)- Mr Using PPK2-WT as a template, pET-T7-F / MrG92-R, MrG92K-F / MrK134-R, and MrK134R-F / pET-T7-R were used as primers, respectively. Amplification was performed according to the system in Table 2. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 30 cycles; 68 ℃ for 5 min; extension times t were 18 s, 10 s, and 36 s, respectively. The amplified primers were used to construct pET-28a(+)- Mr The PPK-M2 plasmid requires three gene fragments. (The fragment is incomplete and requires more context.) Mr Using PPK2-WT as a template and T7F-rev / T7R-rev as primers, amplification was performed according to the system in Table 2. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 310 s, 30 cycles; 68 ℃ for 5 min; the amplified product was used to construct pET-28a(+)- Mr The linear vector fragment required for PPK2-M2 plasmid.

[0054] After the PCR reaction, the DNA fragments were recovered by gel extraction using an agarose gel DNA recovery kit (Guangzhou Meiji Biotechnology Co., Ltd.). Using the three gene fragments as templates, the high-fidelity enzyme PrimeSTAR was employed for extraction. ® The full-length gene was amplified by overlap extension PCR using HS DNA polymerase. The reaction system is shown in Table 3. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 75 s, 30 cycles; 68 ℃ for 5 min. The amplified products were recovered from the gel using an agarose gel DNA recovery kit, yielding the full-length gene containing the G92K and K134R mutations.

[0055] Homologous recombination was performed between the full-length gene containing G92K and K134R mutations and a linearized vector: the homologous recombination system was prepared according to Table 4 and incubated at 50 °C for 20 min. 5 µL of the homologous recombination product was transformed into 100 µL of *E. coli* DH5α competent cells via electroporation. After recovery, the cells were plated on kanamycin-resistant plates and cultured overnight. Positive clones were identified by colony PCR, and selected for sequencing. After verifying the sequence correctness, the corresponding plasmid was extracted and transformed into *E. coli* expression strain BL21(DE3) pLysS competent cells via electroporation.

[0056] (3) pET-28a(+)- Mr Using PPK2-M2 plasmid as a template, pET-T7-F / MrD127-R and MrD127N-F / pET-T7-R primers were used, respectively, and amplification was performed according to the system in Table 2. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 30 cycles; 68 ℃ for 5 min; extension times t were 30 s and 40 s, respectively. The amplified plasmid was used to construct pET- Mr The two gene fragments required for the PPK-M1 plasmid. pET-28a(+)- Mr Using PPK2-WT as a template and T7F-rev / T7R-rev as primers, amplification was performed according to the system in Table 2. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 310 s, 30 cycles; 68 ℃ for 5 min. The amplified product was used to construct pET-28a(+)- Mr The linear vector fragment required for PPK2-M1 plasmid.

[0057] After the PCR reaction, DNA fragments were recovered by gel extraction using an agarose gel DNA recovery kit (Guangzhou Meiji Biotechnology Co., Ltd.). Using two gene fragments as templates, the high-fidelity enzyme PrimeSTAR was employed. ®The full-length gene was amplified by overlap extension PCR using HS DNA polymerase. The reaction system is shown in Table 3. The amplification program was: 98 ℃ for 2 min; 98 ℃ for 10 s, 68 ℃ for 75 s, 30 cycles; 68 ℃ for 5 min. The amplified products were recovered from the gel using an agarose gel DNA recovery kit, yielding the full-length gene containing the G92K, D127N, and K134R mutations.

[0058] The full-length gene containing the G92K, D127N, and K134R mutations was homologously recombinated with a linearized vector. The homologous recombination system was prepared according to Table 4 and incubated at 50 °C for 20 min. 5 µL of the homologous recombination product was transformed into 100 µL of *E. coli* DH5α competent cells via electroporation. After recovery, the cells were plated on kanamycin-resistant plates and cultured overnight. Positive clones were identified by colony PCR, and selected clones were sequenced. After verifying the sequence correctness, the corresponding plasmid was extracted and transformed into *E. coli* expression strain BL21(DE3) pLysS competent cells via electroporation.

[0059] Example 2. Dm dNK - WT Dm dNK-Q81H, Mr PPK-WT, Mr PPK-M1, Mr Expression and purification of PPK-M2 protein Select those containing pET-28a(+)- Dm dNK - WT, pET-28a(+)- Dm dNK-Q81H, pET-28a(+)- Mr PPK-WT, pET-28a(+) Mr PPK-M1 and pET-28a(+) Mr A single colony of BL21(DE3) pLysS cells containing the PPK-M2 plasmid was inoculated into 10 mL of 2× YT medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured overnight at 37 °C and 220 rpm. 10 mL of the overnight culture was then transferred to 1 L of 2× YT medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37 °C until OD500. 600The pH was set to 0.6–0.8, and 0.5 mM IPTG was added, followed by induction at 18 °C for 18 h. After centrifugation to collect the bacterial cells, they were resuspended in buffer A (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM imidazole) and homogenized using a high-pressure homogenizer. After centrifugation at 4 °C and 10,000 rpm for 1 h, the supernatant was filtered through a 0.45 μm filter to remove cell debris. The filtered supernatant was purified by affinity chromatography using a nickel column. The target protein was eluted using 1×elution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10–500 mM imidazole) containing different concentrations of imidazole.

[0060] After SDS-PAGE analysis to verify the results, the eluent fractions containing the target protein were collected. The target protein was then dialyzed and concentrated using a 10 kDa Amicon-Ultra centrifuge ultrafiltration tube and 1× dialysis buffer (100 mM Tris-HCl, 150 mM NaCl, pH 8.0). Finally, an equal volume of 100% glycerol was added to the purified protein, and the mixture was stored at -20 °C for use in subsequent experiments. Figure 2 For expression purification Dm dNK - WT and Dm SDS-PAGE analysis of dNK-Q81H protein showed that the purified protein was of the theoretical size. Figure 3 For expression purification Mr PPK-WT, Mr PPK-M1, Mr SDS-PAGE analysis of PPK-M2 protein showed that the purified protein was of the theoretical size.

[0061] Example 3. Dm dNK-WT and Dm Determination of kinetic parameters of dCNMO or dTPT3 by dNK-Q81H The reaction system was prepared in a UV-STAR® microplate (Griener) and contained: 50 mM Tris-HCl (pH 8.0), 150 mM KCl, 20 mM MgCl2, 0.3 mM NADH, 0.5 mM PEP, 0.5 mM ATP, 30 U / mL pyruvate kinase, 30 U / mL lactate dehydrogenase, different concentrations of nucleoside substrate, and 30-50 nM... Dm dNK or DmdNK-Q81H. The reaction system was placed in a Spectra Max iD3 multi-functional microplate reader, and the absorbance change at 340 nm was monitored in real time at 37 ℃. This change corresponds to the NADH consumption rate. The NADH consumption rate corresponds to the ADP generation rate, reflecting the phosphorylation reaction rate (V). (V) was fitted with the corresponding nucleoside substrate concentration [S] using a nonlinear curve to obtain the kinetic curve and parameters conforming to the Michaelis-Menten equation. The results are shown in Table 5. Compared with the wild type, Dm The catalytic efficiency of dNK-Q81H for dCNMO and dTPT3 was increased by 2.39 and 1.31 times, respectively.

[0062] Table 5. Dm dNK-WT and Dm dNK-Q81H for kinetic parameters of dCNMO or dTPT3

[0063] Example 4. Dm dNK-Q81H-catalyzed synthesis of non-natural deoxynucleotide monophosphate Non-natural deoxyribonucleosides dCNMO and dTPT3 via Dm The dNK-Q81H-catalyzed reaction yielded the corresponding monophosphate, and the reaction was analyzed by HPLC. The reaction system contained 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 12 mM ATP, 10 mM nucleoside, 20 mM MgCl2, and 6 μM... Dm dNK-Q81H. After incubating the reaction solution at 37 °C for 3 h, the protein was removed by centrifugation at 6000 rpm using a 3 kDa ultrafiltration column. 20 μL of the filtrate was then analyzed by HPLC. The mobile phase A for HPLC was 10 mM ammonium acetate (pH 6.0), and the mobile phase B was 75% acetonitrile. The column temperature was 35 °C, the mobile phase flow rate was 1 mL / min, and the detection wavelength of the HPLC UV detector was 254 nm. Different gradient elution programs were used to analyze the various products, and the results are as follows: Figure 4 As shown. Figure 4 -A consists of the following from top to bottom: Dm Chromatograms of the reaction solution for dCNMOMP synthesis catalyzed by dNK-Q81H, dCNMO standard, ATP standard, and ADP standard; Figure 4 The following are the characters from top to bottom in -B: DmChromatograms of the reaction solution for dNK-Q81H-catalyzed dTPT3MP synthesis, dTPT3 standard, ATP standard, and ADP standard. Compared with the chromatograms of ATP, ADP, and non-natural deoxynucleoside standards, Dm In the chromatogram of the catalytic reaction solution of dNK-Q81H, the peak intensity of non-natural nucleoside base deoxynucleosides was significantly reduced, while a new peak of non-natural nucleoside base deoxynucleosides monophosphate appeared. Furthermore, based on the peak area integral, the yields of dCNMOMP (dCNMO nucleoside monophosphate) and dTPT3MP (dTPT3 nucleoside monophosphate) both exceeded 90%.

[0064] Example 5. Mr PPK-WT and Mr Comparison of the effects of PPK mutants on the synthesis of non-natural base deoxynucleoside triphosphates Non-natural deoxynucleoside monophosphate dCNMOMP via Mr PPK-WT or Mr The PPK-M1-catalyzed reaction produces triphosphoric acid. The reaction system contains 50 mM Tris-HCl (pH 8.0), 0.776 mg / mL polyphosphoric acid, 1 mM dCNMOMP, 15 mM MnCl2, and 0.5 μM... Mr PPK-WT or Mr PPK-M1. After reacting at 37 °C for 2 h, the reaction solution was diluted with 4 times its volume of water, then centrifuged at 6000 rpm using a 3 kDa ultrafiltration column to remove proteins, and the composition of the reaction product was analyzed by HPLC. Non-natural deoxynucleoside monophosphate dTPT3MP was subjected to... Mr PPK-WT or Mr The PPK-M2 catalyzed reaction produces triphosphoric acid. The reaction system contains 50 mM Tris-HCl (pH 8.0), 0.776 mg / mL polyphosphoric acid, 1 mM dTPT3MP, 15 mM MnCl2, and 0.3 μM... Mr PPKK-WT or Mr PPK-M2 was reacted at 37 °C for 2 h. The reaction solution was then diluted with 4 times its volume of water, followed by centrifugation at 6000 rpm using a 3 kDa ultrafiltration column to remove proteins. The composition of the reaction products was analyzed by HPLC. The mobile phase A for HPLC was a 5% methanol solution containing 10 mM dipotassium hydrogen phosphate and 5 mM tetrabutylammonium hydrogen sulfate (pH 5.2), and the mobile phase B was a 75% acetonitrile solution. The injection volume for HPLC detection was 15 μL, the detection temperature was 35 °C, the mobile phase flow rate was 0.8 mL / min, and the detection wavelength was 254 nm using a UV detector. Different gradient elution programs were used to analyze each product. The results are as follows: Figure 5 As shown, where, Figure 5 -A is Mr PPK-WT and mutants Mr Comparison of HPLC analysis results of products from the phosphorylation of dCNMOMP to dCNMOTP catalyzed by PPK-M1; Figure 5 -B is Mr PPK-WT and mutants Mr Comparison of HPLC analyses of the products from the phosphorylation of dTPT3MP to dTPT3TP catalyzed by PPK-M2. When dCNMOMP is used as the substrate... Mr PPK-WT can only generate a small amount of dCNMODP (dCNMO nucleoside diphosphate), while Mr The yield of dCNMOTP synthesized by PPK-M1 can reach 69.3%; when dTPT3MP is used as the substrate, Mr The yield of PPK-WT synthesis of dTPT3TP was only 9.5%, while Mr The yield of PPK-M2 to synthesize dTPT3TP reached 76.5%.

[0065] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention patent.

Claims

1. A deoxynucleoside kinase mutant, DmdNK-Q81H, characterized in that, Its amino acid sequence is shown in SEQ ID No.

7.

2. The polyphosphate kinase mutant MrPPK-M1, characterized in that, Its amino acid sequence is shown in SEQ ID No.

9.

3. The polyphosphate kinase mutant MrPPK-M2, characterized in that, Its amino acid sequence is shown in SEQ ID No.

10.

4. The use of the deoxynucleoside kinase mutant DmdNK-Q81H according to claim 1 in the synthesis of non-natural deoxynucleoside monophosphate and / or non-natural deoxynucleoside triphosphate; In the application, non-natural deoxynucleosides are used as raw materials to synthesize non-natural deoxynucleosides monophosphate and / or non-natural deoxynucleosides triphosphate; the non-natural deoxynucleosides include at least one of dCNMO and dTPT3, the structure of dCNMO is shown in Formula 1, and the structure of dTPT3 is shown in Formula 2. 。 5. The application of the polyphosphate kinase mutant MrPPK-M1 according to claim 2 in the synthesis of dCNMOTP, characterized in that, The structure of dCNMOTP is shown in Equation 5; 。 6. The application of the polyphosphate kinase mutant MrPPK-M2 according to claim 3 in the synthesis of dTPT3TP, characterized in that, The structure of dTPT3TP is shown in Equation 6; 。 7. A method for the enzymatic synthesis of non-natural deoxyribonucleoside triphosphates, characterized in that, Includes the following steps: S1. Non-natural deoxyribonucleosides are reacted in a system containing adenosine triphosphate, MgCl2 and the deoxyribonucleoside kinase mutant DmdNK-Q81H as described in claim 1, and the reaction product is separated and purified to obtain non-natural deoxyribonucleosides monophosphate. S2. The non-natural deoxynucleotide monophosphate obtained in step S1 is reacted in a system containing polyphosphate, MnCl2 and a polyphosphate kinase mutant to obtain non-natural deoxynucleotide triphosphate. The polyphosphate kinase mutants mentioned in step S2 include at least one of MrPPK-M1 as described in claim 2 and MrPPK-M2 as described in claim 3.

8. The method according to claim 7, characterized in that, The non-natural deoxyribonucleoside mentioned in step S1 includes at least one of dCNMO and dTPT3, the structure of dCNMO is shown in Formula 1, and the structure of dTPT3 is shown in Formula 2. ； The non-natural deoxynucleotide monophosphate mentioned in step S2 includes at least one of dCNMOMP and dTPT3MP, the structure of dCNMOMP is shown in Formula 3, and the structure of dTPT3MP is shown in Formula 4. 。 9. The method according to claim 8, characterized in that, When the non-natural deoxyribonucleoside monophosphate mentioned in step S2 is dCNMOMP, the polyphosphate kinase mutant mentioned in step S2 is MrPPK-M1 as described in claim 2, and the non-natural deoxyribonucleoside triphosphate obtained by the reaction is dCNMOTP, the structure of which is shown in Formula 5. When the non-natural deoxyribonucleoside monophosphate mentioned in step S2 is dTPT3MP, the polyphosphate kinase mutant mentioned in step S2 is MrPPK-M2 as described in claim 3, and the non-natural deoxyribonucleoside triphosphate obtained by the reaction is dTPT3TP, the structure of which is shown in Formula 6. 。 10. The method according to claim 7, characterized in that, The concentration of adenosine triphosphate in the system mentioned in step S1 is 1–20 mM; The concentration of the non-natural deoxyribonucleoside mentioned in step S1 in the system is 1–20 mM; The divalent metal ion mentioned in step S1 is a magnesium ion; The concentration of the divalent metal ions in the system mentioned in step S1 is 5–100 mM; The concentration of the deoxynucleoside kinase mutant DmdNK-Q81H described in step S1 in the system is 1–10 μM; The system described in step S1 further includes 10–60 mM Tris-HCl with pH 5.0–10.0 and 1–150 mM KCl; The reaction described in step S1 is carried out at a temperature of 30–40 °C for 1–10 h. The polyphosphoric acid mentioned in step S2 is ammonium polyphosphate, and the degree of polymerization of ammonium polyphosphate is 6 to 100. The concentration of the polyphosphoric acid mentioned in step S2 in the system is 0.1-2 mg / mL; The concentration of the non-natural deoxyribonucleoside monophosphate mentioned in step S2 in the system is 0.1–5 mM; The divalent metal ion mentioned in step S2 is a manganese ion; The concentration of the divalent metal ions in the system mentioned in step S2 is 5–50 mM; The concentration of the polyphosphokinase mentioned in step S2 in the system is 0.1–5 μM; The system described in step S2 also includes 10–100 mM Tris-HCl with a pH of 5.0–10.0; The reaction described in step S2 is carried out at a temperature of 30–80 °C for 0.5–24 h.

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