A method for synthesizing deoxynucleoside monophosphate by biological enzyme

Abstract

This invention discloses a method for the bioenzymatic synthesis of deoxynucleoside monophosphate. The invention provides a mutant protein of dNK, comprising mutating amino acid residues at positions 84, 88, 110, and 172 of the amino acid sequence shown in SEQ ID NO:1, while leaving other amino acid residues unchanged, to obtain a protein with deoxynucleoside kinase activity. This invention obtains a deoxynucleoside kinase mutant capable of stably and efficiently catalyzing the synthesis of 3'-O-NH2-deoxynucleoside monophosphate using 3'-O-NH2-deoxynucleoside as a substrate, providing a new approach and pathway for nucleotide production. The bioenzymatic synthesis of (deoxy)nucleoside monophosphate of this invention has advantages such as high conversion rate, mild reaction conditions, simple production process, environmental friendliness, and ease of large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a method for the bioenzymatic synthesis of deoxynucleoside monophosphate. Background Technology

[0002] Nucleosides are compounds formed by the linkage of a base and ribose sugar via glycosidic bonds. Based on the different bases they contain, they can be classified into several types, including adenosine, guanosine, cytidine, thymidine in DNA, and uridine in RNA. Nucleosides derived from D-ribose are called ribonucleosides, which participate in the formation of ribonucleic acid (RNA); nucleosides derived from D-2-deoxyribose are called deoxyribonucleosides, which participate in the formation of deoxyribonucleic acid (DNA).

[0003] Nucleotides are formed by adding phosphate groups to nucleosides. They are the basic building blocks of nucleic acids, including ribonucleotides and deoxyribonucleotides. Nucleic acids are long-chain polymers composed of multiple nucleotides linked by phosphodiester bonds, mainly including DNA and RNA.

[0004] Based on the number of phosphate groups, ribonucleotides can be classified into nucleoside monophosphates (NMP), nucleoside diphosphates (NDP), and nucleoside triphosphates (NTP). Similarly, deoxyribonucleotides can be classified into deoxyribonucleoside monophosphates (dNMP), deoxyribonucleoside diphosphates (dNDP), and deoxyribonucleoside triphosphates (dNTP).

[0005] Nucleotides, as an important class of small biological molecules, not only participate in various biochemical reactions in organisms, including genetic metabolism, but also have wide applications in fine chemistry, pharmaceuticals, food science, and life science research. In the pharmaceutical field, many important antiviral and antitumor drugs are chemically modified from nucleotides and nucleosides. In scientific research, PCR technology has made unprecedented contributions to life sciences since its inception, rapidly driving the development of biotechnology, and deoxynucleoside triphosphates are the material basis of PCR technology. Deoxynucleoside monophosphates are one of the essential raw materials for the synthesis of deoxynucleoside triphosphates.

[0006] Modified nucleotides are molecules with similar structures formed by replacing groups or atoms in natural nucleotides. They possess similar physical and chemical properties to nucleotides and can replace natural nucleotide molecules to be incorporated into DNA and RNA, participating in or interfering with the enzymatic synthesis of polynucleotides. In the field of enzymatic DNA synthesis, most methods employ template-free polymerases to add 3'-O-blocked nucleoside triphosphates to the primer chain, then unblock to obtain open 3'-OH groups for the next synthesis cycle. This "synthesis-unblocking" cycle is repeated to obtain the target sequence of polynucleotide. Currently, various 3'-O-blocking groups are available, among which the 3'-O-NH2 blocking group has gained widespread attention both domestically and internationally due to its small size and ease of cleavage.

[0007] Typically, the synthesis of deoxynucleosides into deoxynucleosides triphosphates requires three catalytic steps. The synthesis of deoxynucleosides into deoxynucleosides monophosphates, as the first and rate-limiting step, plays a crucial role. Therefore, utilizing deoxynucleoside kinases to catalyze the synthesis of the corresponding 3'-O-NH2-deoxynucleosides from 3'-O-NH2-deoxynucleosides has significant research value.

[0008] The synthesis of deoxynucleoside monophosphates can be divided into two types: chemical and biological methods. Chemical synthesis is currently the most commonly used method, as illustrated by patents CN200910048035.1 and CN202211177402.X. This method typically utilizes organic reagents such as phosphorus oxychloride to phosphorylate the substrate (deoxy)nucleoside, followed by a series of separation and purification steps to obtain the pure (deoxy)nucleoside monophosphate. However, most of the chemical reagents used in chemical synthesis are toxic, and the process is also cumbersome, produces numerous byproducts, is difficult to separate, has high costs, and carries certain operational risks. Compared to chemical synthesis, biological enzymatic methods offer numerous advantages, including milder reaction conditions, simpler operation, faster reaction times, and environmental friendliness.

[0009] However, previous literature reports mainly focused on Drosophila melanogaster (Drosophila dimerans). Drosophila melanogasterThe functional studies of deoxynucleoside kinase (dNK) and its homologous mutants catalyzing natural deoxynucleoside substrates have not been conducted. There are no studies using 3'-O-NH2-deoxynucleoside as a substrate, and the reported effects of deoxynucleoside kinase and its homologous mutants catalyzing the synthesis of 3'-O-NH2-dAMP and 3'-O-NH2-dGMP are not very good.

[0010] Therefore, it is necessary to find enzymes and methods that can efficiently synthesize the four types of 3'-O-NH2-dNMP, especially 3'-O-NH2-dAMP and 3'-O-NH2-dGMP. Summary of the Invention

[0011] The technical problem solved by this invention is how to efficiently synthesize four types of 3'-O-NH2-dNMP, and in particular how to promote the synthesis of 3'-O-NH2-dAMP and 3'-O-NH2-dGMP.

[0012] To address the aforementioned technical problems, the first aspect of this invention provides a mutant protein of dNK, which is any one of the following:

[0013] The mutant protein shown in A1) includes a protein with deoxynucleoside kinase activity obtained by mutating amino acid residues at positions 84, 88, 110 and 172 of the amino acid sequence shown in SEQ ID NO:1, while keeping the amino acid residues at other positions unchanged.

[0014] A2) A protein in which, apart from the mutated amino acid residues, the other amino acid residues have more than 80% identity and have the same function;

[0015] The mutant protein shown in A3) is obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in A1) or A2) to obtain the protein with the sequence shown.

[0016] The mutant protein described above has the following mutation:

[0017] The 84th amino acid residue was mutated from V to A;

[0018] The 88th amino acid residue was mutated from M to R;

[0019] The 110th amino acid residue was mutated from A to D;

[0020] The amino acid residue at position 172 was mutated from E to D.

[0021] The positions indicated by bits 84, 88, 110, and 172 above were determined by comparison with SEQ ID NO.1.

[0022] In the above text, the tag sequence can be MBP tag, HIS tag, GST tag, etc.

[0023] In some embodiments, the mutant may be in the form of purified enzyme, enzyme lysis supernatant, or whole cell.

[0024] In a second aspect, the present invention provides biological materials related to the mutant protein described in the first aspect, which are any one of B1) to B4) below:

[0025] B1) A nucleic acid molecule encoding the mutant protein described in the first aspect;

[0026] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0027] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0028] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0029] In the above text, the recombinant vector is a bacterial plasmid, bacteriophage, or yeast plasmid that carries the nucleic acid molecule.

[0030] In some embodiments, the bacterial plasmid is derived from a plasmid vector suitable for expression in Escherichia coli.

[0031] In one embodiment, the plasmid vector is selected from any one of pET-21a(+), pET-22a(+), pET-23a(+), pET-28a(+), pET-30a(+), pET-32a(+), pET-21b(+), pET-22b(+), pET-23b(+), pET-28b(+), pET-30b(+), and pET-32b(+).

[0032] In the above text, recombinant microorganisms are obtained by introducing the recombinant vector into host cells.

[0033] In some embodiments, the host cell may be a prokaryotic cell such as bacteria, specifically Escherichia coli, or a lower eukaryotic cell such as yeast.

[0034] In one implementation, the host cell can be E. coli BL21(DE3).

[0035] Thirdly, the present invention provides the mutant protein described in the first aspect for any of the following applications:

[0036] C1) acts as a deoxynucleoside kinase;

[0037] C2) Synthesis of deoxynucleoside monophosphate;

[0038] C3) Catalytic synthesis of 3'-O-NH2-dNMP from substrate 3'-O-NH2-dN;

[0039] C4) promotes the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP;

[0040] C5) promotes the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP.

[0041] Fourthly, the present invention provides the biomaterials described in the second aspect for any of the following applications:

[0042] D1) Preparation of deoxynucleoside kinase;

[0043] D2) Synthesis of deoxynucleoside monophosphate;

[0044] D3) Catalytic synthesis of 3'-O-NH2-dNMP from substrate 3'-O-NH2-dN;

[0045] D4) Promotes the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP;

[0046] D5) promotes the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP.

[0047] Fifthly, the present invention provides the mutant protein and polyphosphate kinase SMC described in the first aspect for any of the following applications:

[0048] E1) Synthesizes deoxynucleoside monophosphate;

[0049] E2) Catalytic synthesis of 3'-O-NH2-dNMP from substrate 3'-O-NH2-dN;

[0050] E3) promotes the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP;

[0051] E4) promotes the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP;

[0052] The polyphosphokinase SMC is any one of the following:

[0053] The protein shown in F1) includes the amino acid residues encoded by the nucleotides shown in SEQ ID NO:3;

[0054] F2) Proteins that share more than 80% identity with F1) and have the same function;

[0055] The protein shown in F3) is obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in F1) or F2) to obtain the protein shown in the sequence.

[0056] In a sixth aspect, the present invention provides a method for the bioenzymatic synthesis of deoxynucleoside monophosphate, comprising the following steps: using the mutant protein described in the first aspect and the polyphosphate kinase SMC catalyzing substrate 3'-O-NH2-dN described in the fifth aspect to synthesize 3'-O-NH2-dNMP.

[0057] In a seventh aspect, the present invention provides a method for promoting the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP, comprising the following steps: using the mutant protein described in the first aspect and the polyphosphoric acid kinase SMC described in the fifth aspect to catalyze the substrate 3'-O-NH2-dA to promote the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP.

[0058] Eighthly, the present invention provides a method for promoting the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP, comprising the following steps: using the mutant protein described in the first aspect and the polyphosphoric acid kinase SMC described in the fifth aspect to catalyze the substrate 3'-O-NH2-dG to promote the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP.

[0059] In a ninth aspect, the present invention provides a method for producing or increasing the yield of 3'-O-NH2-modified deoxynucleoside monophosphates, comprising the following steps: synthesizing 3'-O-NH2-dAMP, synthesizing 3'-O-NH2-dGMP, synthesizing 3'-O-NH2-dTMP, and synthesizing 3'-O-NH2-dCMP, thereby achieving the production or increase of the yield of 3'-O-NH2-modified deoxynucleoside monophosphates;

[0060] The method for synthesizing 3'-O-NH2-dAMP includes the following steps: synthesizing 3'-O-NH2-dAMP using the mutant protein described in the first aspect and the polyphosphoric acid kinase SMC catalytic substrate 3'-O-NH2-dA described in the fifth aspect;

[0061] The method for synthesizing 3'-O-NH2-dGMP includes the following steps: synthesizing 3'-O-NH2-dGMP using the mutant protein described in the first aspect and the polyphosphoric acid kinase SMC catalytic substrate 3'-O-NH2-dG described in the fifth aspect;

[0062] The method for synthesizing 3'-O-NH2-dTMP includes the following steps: using dNK protein and the polyphosphoric kinase SMC described in the fifth aspect to catalyze the substrate 3'-O-NH2-dT to synthesize 3'-O-NH2-dT;

[0063] The method for synthesizing 3'-O-NH2-dCMP includes the following steps: using dNK protein and the polyphosphoric kinase SMC described in the fifth aspect to catalyze the substrate 3'-O-NH2-dC to synthesize 3'-O-NH2-dC.

[0064] The dNK protein is any one of the following:

[0065] The protein shown in G1 includes the amino acid residues shown in SEQ ID NO:1;

[0066] Proteins with greater than 80% identity to proteins shown in G2 and G1, and with the same function;

[0067] The protein shown in G3) includes the protein shown in G1) or G2) by attaching a tag to its N-terminus and / or C-terminus to obtain the protein shown in the sequence.

[0068] In the catalysis described above, the catalytic reaction system includes a buffer solution.

[0069] In some embodiments, the buffer solution may be a phosphate buffer; the phosphate buffer solution includes KH2PO4, and the pH value of the buffer solution may be 6-9.

[0070] In some embodiments, the catalytic reaction system further includes metal ions.

[0071] In some embodiments, the metal ion may be Mg. 2+、 Mn 2+ or Co 2+ .

[0072] In some embodiments, the catalytic reaction system further includes a phosphate donor;

[0073] In some embodiments, the phosphate donor may be an inorganic phosphate donor or a nucleoside triphosphate.

[0074] In some implementations, the phosphate donor for SMC-catalyzed ATP cycling can be sodium tripolyphosphate, sodium hexametaphosphate, or sodium polyphosphate.

[0075] In some embodiments, the phosphate donor used for the catalytic reaction of Dm-dNK or its mutants can be ATP, dNTP, or dNTP with a modified group (such as 3'-O-NH2).

[0076] In some embodiments, the catalytic reaction system further includes the substrate 3'-O-NH2-dN.

[0077] In some embodiments, the concentration of the substrate in the reaction system can be 1 to 10 mM.

[0078] In some embodiments, the concentration of the phosphate donor ATP / dNTP in the reaction system can be 1~5 mM.

[0079] In some embodiments, the concentration of the mutant protein in the reaction system can be 0.1 to 1 mg / mL.

[0080] In some embodiments, the catalytic reaction can be carried out at 30-40°C. Specifically, the catalytic reaction temperature can be 37°C.

[0081] In some embodiments, the catalytic reaction can be carried out for 1 to 24 hours.

[0082] In some embodiments, the catalytic reaction can be carried out under oscillating conditions.

[0083] In this invention, amino acids are represented by single-letter or three-letter codes, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Glycine; H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0084] In this invention, mutants are described based on mutations at specific residues, the locations of which are determined by alignment with or reference to the wild-type enzyme sequence. This invention also relates to any variants carrying these same mutations at functionally equivalent residues.

[0085] In this invention, mutation sites and their substitutions are expressed by their position numbers and the amino acid types at those sites. For example, M88R indicates that, compared to SEQ ID NO:1, methionine at position 88 of SEQ ID NO:1 is mutated to arginine; A110D indicates that, compared to SEQ ID NO:1, alanine at position 110 of SEQ ID NO:1 is mutated to aspartic acid. In this invention, " / " is used to represent combinations of mutation sites. For example, "M88 / A110" indicates that both methionine at position 88 and alanine at position 110 are mutated, containing two mutation sites, thus a double mutant. Similarly, "V84 / M88 / A110" indicates that all three corresponding sites are mutated simultaneously, thus a triple mutant; "V84 / M88 / A110 / E172" indicates that all four corresponding sites are mutated simultaneously, thus a quadruple mutant.

[0086] In this invention, the term deoxynucleoside (dN) includes adenine deoxynucleoside, thymine deoxynucleoside, cytosine deoxynucleoside, and guanine deoxynucleoside; the term deoxynucleoside monophosphate (dNMP) includes adenine deoxynucleoside monophosphate, thymine deoxynucleoside monophosphate, cytosine deoxynucleoside monophosphate, and guanine deoxynucleoside monophosphate; and the term deoxynucleoside triphosphate (dNTP) includes adenine deoxynucleoside triphosphate, thymine deoxynucleoside triphosphate, cytosine deoxynucleoside triphosphate, and guanine deoxynucleoside triphosphate.

[0087] Beneficial effects

[0088] This invention yields various deoxynucleoside kinases and their mutants, which can stably and efficiently catalyze the synthesis of 3'-O-NH2-deoxynucleoside monophosphate using 3'-O-NH2-deoxynucleoside as a substrate, providing new ideas and pathways for nucleotide production. The bioenzymatic synthesis of (deoxy)nucleoside monophosphate in this invention has advantages such as high conversion rate, mild reaction conditions, simple production process, environmental friendliness, and ease of large-scale production. Attached Figure Description

[0089] Figure 1 The images show the plasmid maps of pET-28a-dNK and pET-28a-SMC, where a is the plasmid map of pET-28a-dNK and b is the plasmid map of pET-28a-SMC.

[0090] Figure 2 This is the HPLC chromatogram of the 3'-O-NH2-dN standard, which is the substrate of this experiment.

[0091] Figure 3 This is a standard curve showing the HPLC peak area and corresponding concentration of the substrate 3'-O-NH2-dA standard in this experiment.

[0092] Figure 4 This is a standard curve showing the HPLC peak area and corresponding concentration of the substrate 3'-O-NH2-dT standard in this experiment.

[0093] Figure 5 This is a standard curve showing the HPLC peak area and corresponding concentration of the substrate 3'-O-NH2-dC standard in this experiment.

[0094] Figure 6 This is a standard curve showing the HPLC peak area and corresponding concentration of the substrate 3'-O-NH2-dG standard in this experiment.

[0095] Figure 7 The HPLC chromatogram is for the reaction of 5mM 3'-O-NH2-dN catalyzed by wild-type Dm-dNK (the reaction sample was detected after being diluted 1 time).

[0096] Figure 8 HPLC chromatogram of the reaction of 5 mM 3'-O-NH2-dN catalyzed by the Dm-dNK mutant V84A / M88R / A110D / E172D (the reaction sample was not diluted for detection). Detailed Implementation

[0097] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0098] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0099] The sources of materials, reagents, etc. used in the following examples are shown in Table 1.

[0100]

[0101] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0102] Example 1: Gene acquisition and vector construction of deoxynucleoside kinase dNK and polyphosphate kinase SMC

[0103] The gene origin of deoxynucleoside kinase dNK is from the following species. Drosophila melanogasterThe amino acid sequence is shown in SEQ ID NO.1. Without altering the amino acid sequence, the codons of the wild-type gene were replaced with codons preferred (frequently used) by *E. coli*. After codon optimization, the gene sequence contains *E. coli*-preferred codons, and the corresponding gene sequence is shown in SEQ ID NO.2. This gene sequence (SEQ ID NO.2) was directly constructed into the expression vector pET-28a(+) between the restriction enzyme sites BamHI and XhoI. The recombinant plasmid was named pET-28a(+)-dNK (referred to as pET-28a-dNK in the figure, as shown in the diagram). Figure 1 (a) Genes that express deoxynucleoside kinase dNK.

[0104] The gene source of polyphosphate kinase SMC is: Sinorhizobium meliloti The gene sequence is shown in SEQ ID NO.3. This gene sequence (SEQ ID NO.3) was directly constructed into the expression vector pET-28a(+) between the BamHI and Xho I restriction sites. The recombinant plasmid was named pET-28a(+)-SMC (denoted as pET-28a-SMC in the figure, as shown in the diagram). Figure 1 (b) Genes that express polyphosphokinases.

[0105] pET-28a(+) is the expression vector described in the example, but it is not limited to this; all vectors that can be used to express proteins are included. The BamHI and XhoI restriction sites described in the example are not limited to this; all restriction sites in the expression vector that can be used to construct the expressed gene fragment are included.

[0106] Example 2: Obtaining the deoxynucleoside kinase dNK active mutant

[0107] 1. Obtaining active mutants

[0108] This invention relates to the source Drosophila melanogasterHomology modeling was performed on dNK (hereinafter also referred to as Dm-dNK) to obtain its three-dimensional protein structure. By analyzing residues within the 3A range of the active site, several potential sites that may affect its catalytic activity were obtained. These single or multiple mutations were performed using a BVT mixed base pattern, including a total of 9 random mutations (H, P, R, D, A, G, Y, S, C). Combined with high-throughput screening and other methods, deoxynucleoside kinase dNK mutants with high catalytic efficiency and substrate specificity were obtained: I29R, K33C, E52S, V54D, L66Y, Y70P, Q81A, V84A, M88R, R105G, A110D, F114H, E172D, V175C, M88R / A110D, V84A / M88R / A110D, V84A / M88R / A110D / L66Y, V84A / M88R / A110D / E172D, V84A / M88R / A110D / V175C.

[0109] The optimal dNK mutant V84A / M88R / A110D / E172D was determined by comparing response values ​​using an Echo-MS system and peak areas using high-performance liquid chromatography.

[0110] The amino acid sequence of the dNK mutant V84A / M88R / A110D / E172D is obtained by mutating V at position 84 of SEQ ID NO: 1 to A, M at position 88 to R, A at position 110 to D, and E at position 172 to D, while other amino acid residues remain unchanged.

[0111] The nucleotide encoding the dNK mutant V84A / M88R / A110D / E172D is obtained by mutating the T base at position 251 of SEQ ID NO: 2 to a C base, the A base at position 262 to a C base, the T base at position 263 to a G base, the G base at position 264 to an A base, the C base at position 329 to an A base, the A base at position 330 to a C base, and the A base at position 516 to a T base, while other nucleotide residues remain unchanged.

[0112] 2. Construction of vectors expressing dNK mutants

[0113] The recombinant plasmid pET-28a(+)-dNK mutant expressing the dNK mutant was constructed by inserting the coding nucleic acid of the dNK mutant V84A / M88R / A110D / E172D into the expression vector pET-28a(+) between the restriction sites BamHI and XhoI, and expressing the dNK mutant.

[0114] 3. Gene expression

[0115] To detect the activity of deoxynucleoside kinase and its mutants in vitro, the enzyme was expressed and purified exogenously in *E. coli*. The host bacteria described in the examples were... E. coli BL21(DE3), but the host bacteria are not limited to this; all hosts that can be used to express proteins are included.

[0116] 1) The recombinant plasmids pET-28a(+)-dNK and pET-28a(+)-SMC constructed in Example 1 above, as well as the recombinant plasmid pET-28a(+)-dNK mutant constructed above, were respectively transformed into E. coli Recombinant bacteria were obtained from BL21(DE3). Positive clones were selected using kanamycin-resistant plates. + (100 mg / mL), incubate overnight at 37°C;

[0117] 2) Pick a single colony and add it to 8 mL of LB liquid medium (Kan). + (100 mg / mL), cultured at 37°C and 220 r / min until OD 600 The value is 0.6-0.8. Transfer 8 mL of bacterial culture from LB medium to 800 mL of 2YT medium (Kan...). + (100 mg / mL), incubated at 37°C and 220 rpm until OD. 600 When the concentration is 0.6-0.8, cool to 16°C, add IPTG to a final concentration of 0.5 mM, and induce expression for 20 h;

[0118] 3) Collect the above-mentioned bacterial culture into a collection bottle and centrifuge at 5500 r / min for 15 min;

[0119] 4) Discard the supernatant, suspend the obtained bacterial cell pellet with 35 mL of protein buffer (50 mM Tris-HCl, 2 mM EDTA, 0.1% Triton X-100, balance water, pH 7.4), pour into a 50 mL centrifuge tube, and store at -80°C to obtain the individual bacterial cell suspensions.

[0120] 4. Protein purification

[0121] 1) Sterilization: The bacterial suspensions obtained in step 3 above were sterilized twice using a high-pressure cryogenic disruptor at 1200 bar and 4°C. The suspensions were then centrifuged at 10000 r / min for 45 min at 4°C. The precipitate and supernatant were collected for sample preparation.

[0122] 2) Purification: The supernatant was filtered through a 0.45μm microporous membrane and purified by nickel affinity chromatography. The specific steps are as follows:

[0123] a: Column equilibration: Before attaching the supernatant, wash with ddH2O for 2 column volumes, then equilibrate the Ni affinity chromatography column with protein buffer for 1 column volume;

[0124] b: Sample loading: Slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 0.5 mL / min, and repeat once;

[0125] c: Elution of contaminating proteins: Wash with protein buffer for 1 column volume, then use 50 mL of protein buffer containing 50 mM imidazole to elute strongly bound contaminating proteins. Take the first few drops to flow through the sample and prepare the sample.

[0126] d: Elution of target protein: The target protein was eluted with 20 mL of imidazole protein buffer containing 100 mM, 200 mM, and 300 mM, respectively. The first few drops were used to flow through the sample for preparation. The sample was then analyzed by 12% SDS-PAGE (to obtain a 32.6 KD deoxynucleoside kinase dNK or its mutant, and a 37.1 KD polyphosphate kinase SMC).

[0127] 3) Concentration and buffer exchange: The collected target protein was concentrated by centrifugation (4℃, 3400 r / min) using a 50 mL Amicon ultrafiltration tube (10 kDa, Millipore) to a final volume of 1 mL. 10 mL of protein buffer was added, and the concentration was repeated once to obtain purified protein, namely, purified dNK solution, purified dNK mutant V84A / M88R / A110D / E172D solution, and purified SMC solution.

[0128] 4) The concentration of the concentrated protein was detected using a BCA kit and diluted to 10 mg / mL to obtain the purified and concentrated protein.

[0129] Example 3: Protein Function Verification

[0130] 1. Preparation of Standards

[0131] 3'-O-NH2-dA related standard solutions: Different amounts of 3'-O-NH2-dA were dissolved in water to obtain standard solutions, wherein the concentrations of 3'-O-NH2-dA were 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM, respectively.

[0132] 3'-O-NH2-dT related standard solutions: Different amounts of 3'-O-NH2-dT were dissolved in water to obtain standard solutions, wherein the concentrations of 3'-O-NH2-dT were 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM, respectively.

[0133] 3'-O-NH2-dC related standard solutions: Different amounts of 3'-O-NH2-dC were dissolved in water to obtain standard solutions, wherein the concentrations of 3'-O-NH2-dC were 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM, respectively.

[0134] 3'-O-NH2-dG related standard solutions: Different amounts of 3'-O-NH2-dG were dissolved in water to obtain standard solutions, wherein the concentrations of 3'-O-NH2-dG were 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM, respectively.

[0135] The above standard solutions were subjected to HPLC analysis under the following conditions, and the results are as follows. Figure 2 As shown, the peak times of each standard can be obtained. Based on the peak area, a standard curve was plotted comparing the HPLC peak area of ​​the 3'-O-NH2-dN standard with its corresponding molar concentration. The results are shown below. Figures 3-6 As shown.

[0136] HPLC detection conditions: Mobile phase: A: H₂O + TBAH + KH₂PO₄; B: CH₃OH + TBAH. Elution gradient: isocratic gradient. Column: Agilent Polaris 180A C18-A 5μm, 4.6mm × 250mm. Flow rate: 1 mL / min; UV detection wavelength: 254nm; Column oven temperature: 30℃; Injection volume: 10μL.

[0137] 2. Synthesis of deoxynucleoside monophosphate

[0138] The deoxynucleoside monophosphate synthesis reaction system was as follows: 50 mM KH2PO4; 10 mM MgCl2; substrate 5 mM 3'-O-NH2-dN; phosphate donor 1 mM ATP; phosphate donor 3 mM sodium hexametaphosphate; 0.1 mg / mL SMC, 0.3 mg / mL dNK or its mutant, with the balance being water, and the pH value being 7.

[0139] The aforementioned 3'-O-NH2-dN can be 3'-O-NH2-dA, 3'-O-NH2-dT, 3'-O-NH2-dC, or 3'-O-NH2-dG.

[0140] In the above system, the concentration of each substance is the final concentration in the system.

[0141] In the above system, dNK or its mutants and SMC are added in the form of purified dNK solution, purified dNK mutant solution and purified SMC solution.

[0142] Reaction conditions: After reacting overnight with shaking at 37°C, the protein was inactivated at 100°C. Then, the mixture was filtered through a 0.22 μm filter and analyzed by HPLC.

[0143] HPLC detection conditions: Mobile phase: A: H₂O + TBAH + KH₂PO₄; B: CH₃OH + TBAH. Elution gradient: isocratic gradient. Column: Agilent Polaris 180A C18-A 5μm, 4.6mm × 250mm. Flow rate: 1 mL / min; UV detection wavelength: 254nm; Column oven temperature: 30℃; Injection volume: 10μL.

[0144] Conversion rate = 100% - (molar concentration of remaining 3'-O-NH2-dN / initial molar concentration of added 3'-O-NH2-dN)%

[0145] Liquid phase detection results of wild-type Dm-dNK catalyzed 3'-O-NH2-dN are as follows: Figure 7 As shown, by converting the conversion rates, it can be concluded that the conversion rate of 5mM 3'-O-NH2-dT / dC to 3'-O-NH2-dTMP / dCMP by Dm-dNK is 100%; the conversion rate of 5mM 3'-O-NH2-dA / dG to 3'-O-NH2-dAMP / dGMP by Dm-dNK is not good, only about 30% and 1% respectively.

[0146] Liquid phase detection results of 3'-O-NH2-dN catalyzed by the four mutants V84A / M88R / A110D / E172D are as follows: Figure 8 As shown, by converting the conversion rates, it can be concluded that the conversion rate of 5mM 3'-O-NH2-dA to 3'-O-NH2-dAMP catalyzed by the tetra-mutant V84A / M88R / A110D / E172D is 100%; the conversion rate of 5mM 3'-O-NH2-dG to 3'-O-NH2-dGMP catalyzed by the tetra-mutant V84A / M88R / A110D / E172D is about 85%, which improves the conversion rate of 3'-O-NH2-dA / dG to 3'-O-NH2-dAMP / dGMP. However, the conversion rate of 3'-O-NH2-dT / dC to 3'-O-NH2-dTMP / dCMP catalyzed by the tetra-mutant is greatly reduced.

[0147] Therefore, in later experiments, wild-type Dm-dNK was used to catalyze the generation of 3'-O-NH2-dT / dC from 3'-O-NH2-dTMP / dCMP, and the four mutants V84A / M88R / A110D / E172D were used to catalyze the generation of 3'-O-NH2-dA / dG from 3'-O-NH2-dAMP / dGMP, thus achieving efficient synthesis of 3'-O-NH2-dNMP.

[0148] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A mutant protein of dNK, characterized by: The mutant protein of dNK is any one of the following: The mutant protein shown in A1) is obtained by mutating amino acid residues at positions 84, 88, 110 and 172 of the amino acid sequence shown in SEQ ID NO:1, while keeping the amino acid residues at other positions unchanged, resulting in a protein with deoxynucleoside kinase activity. The mutant protein shown in A2) is the protein with the sequence shown by attaching a tag to the N-terminus and / or C-terminus of the protein shown in A1). The mutation is as follows: The 84th amino acid residue was mutated from V to A; The 88th amino acid residue was mutated from M to R; The 110th amino acid residue was mutated from A to D; The amino acid residue at position 172 was mutated from E to D.

2. A biomaterial related to the mutant protein of claim 1, characterized in that: The biomaterial is any one of B1) to B4) below: B1) A nucleic acid molecule encoding the mutant protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The application of the mutant protein according to claim 1, characterized in that: The application is any one of the following: C1) acts as a deoxynucleoside kinase; C2) promotes the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP; C3) promotes the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP.

4. The application of the biomaterial according to claim 2, characterized in that: The application is any one of the following: D1) Preparation of deoxynucleoside kinase; D2) Promotes the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP; D3) promotes the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP.

5. The application of the mutant protein and polyphosphokinase SMC according to claim 1, characterized in that: The application is any one of the following: E1) Promotes the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP; E2) promotes the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP; The polyphosphokinase SMC is any one of the following: The protein shown in F1 represents the amino acid residues encoded by the nucleotide shown in SEQ ID NO:3; The protein shown in F2) is obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in F1) to obtain the sequence shown.

6. A method for synthesizing deoxynucleoside monophosphate via a bioenzymatic process, characterized in that: The method includes the following steps: using the mutant protein of claim 1 and the polyphosphoric acid kinase SMC catalytic substrate 3'-O-NH2-dN of claim 5 to synthesize 3'-O-NH2-dNMP; The 3'-O-NH2-dN is either 3'-O-NH2-dA or 3'-O-NH2-dG; The 3'-O-NH2-dNMP is either 3'-O-NH2-dAMP or 3'-O-NH2-dGMP.

7. A method for promoting the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP, characterized in that: The method includes the following steps: using the mutant protein of claim 1 and the polyphosphoric acid kinase SMC of claim 5 to catalyze the substrate 3'-O-NH2-dA to promote the conversion of substrate 3'-O-NH2-dA to 3'-O-NH2-dAMP.

8. A method for promoting the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP, characterized in that: The method includes the following steps: using the mutant protein of claim 1 and the polyphosphoric acid kinase SMC of claim 5 to catalyze the substrate 3'-O-NH2-dG to promote the conversion of substrate 3'-O-NH2-dG to 3'-O-NH2-dGMP.

9. A method for producing or increasing the yield of 3'-O-NH2-modified deoxynucleoside monophosphates, characterized in that: The method includes the following steps: synthesizing 3'-O-NH2-dAMP, synthesizing 3'-O-NH2-dGMP, synthesizing 3'-O-NH2-dTMP and synthesizing 3'-O-NH2-dCMP, to achieve the production or increase of 3'-O-NH2-modified deoxynucleoside monophosphate yield; The method for synthesizing 3'-O-NH2-dAMP includes the following steps: synthesizing 3'-O-NH2-dAMP using the mutant protein of claim 1 and the polyphosphoric acid kinase SMC catalytic substrate 3'-O-NH2-dA of claim 5; The method for synthesizing 3'-O-NH2-dGMP includes the following steps: synthesizing 3'-O-NH2-dGMP using the mutant protein of claim 1 and the polyphosphoric acid kinase SMC catalytic substrate 3'-O-NH2-dG of claim 5; The method for synthesizing 3'-O-NH2-dTMP includes the following steps: using dNK protein and the polyphosphoric kinase SMC described in claim 5 to catalyze the substrate 3'-O-NH2-dT to synthesize 3'-O-NH2-dT; The method for synthesizing 3'-O-NH2-dCMP includes the following steps: using dNK protein and the polyphosphoric kinase SMC described in claim 5 to catalyze the substrate 3'-O-NH2-dC to synthesize 3'-O-NH2-dC. The dNK protein is any one of the following: The protein shown in G1 represents the amino acid residues shown in SEQ ID NO:1; The protein shown in G2 is obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in G1 to obtain the sequence shown.

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