Preparation method and application of natural nucleotide

Abstract

This invention discloses a method for preparing natural nucleotides and their applications. The MrPPK2 mutant protein of this invention is as follows: A1) Mutating amino acid residues 93 and 127 of the amino acid sequence shown in SEQ ID NO:1 to obtain a protein with polyphosphokinase activity; A2) In the protein shown in A1), except for the mutated amino acid residues, the identity of other amino acid residues is greater than 80%, and the protein has the same function; A3) A protein obtained by attaching a tag to the N-terminus and / or C-terminus of the A1) or A2) protein. This invention uses sodium hexametaphosphate as the main phosphate donor, and the MrPPK2 mutant increases the production rate of dGTP and dATP; when the SMC mutant is used in combination with dTMP-K or dCMP-K, sodium hexametaphosphate is the main phosphate donor, and dTTP / dCTP is the secondary donor, increasing the production rate of dTTP and dCTP.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for preparing natural nucleotides and their applications. Background Technology

[0002] dNTPs, or deoxyribonucleotide triphosphates, are essential molecules in DNA replication and repair. Each dNTP molecule consists of a phosphate group, a deoxyribose sugar, and a nitrogenous base. Based on the base composition, dNTPs can be classified into four types: dATP (deoxyadenosine triphosphate), dGTP (deoxyguanine triphosphate), dTTP (deoxythymidine triphosphate), and dCTP (deoxycytosine triphosphate). dATP and dGTP belong to the purine family, while dTTP and dCTP belong to the pyrimidine family. dNTPs play a crucial role in bioscience, primarily serving as raw materials for DNA replication and repair. In molecular biology research, dNTPs are fundamental to experimental techniques such as PCR (polymerase chain reaction) for amplifying specific DNA fragments. In the biotechnology industry, dNTPs are key components in the synthesis of DNA fragments, gene editing, and the production of genetically engineered products.

[0003] Traditional nucleotide synthesis methods are mainly divided into four types: nucleic acid degradation, chemical synthesis, direct microbial fermentation, and enzymatic methods. Nucleic acid degradation: This method originated in the 1960s when Japanese scientists first used 5'-phosphodiesterase to degrade RNA to obtain nucleotides, pioneering the industrial production of nucleotides. The specific process involves extracting RNA from yeast, then using nuclease P1 to degrade it into a mixture of four nucleotides, followed by complex separation and purification steps to obtain the nucleotide monomers. This method has a long production cycle, low product purity, and suffers from low fermentation levels and low enzymatic conversion rates. Chemical synthesis: This method uses phosphorus oxychloride (POCl3) as a phosphate donor. Nucleosides or deoxynucleosides are dissolved in triethyl phosphate (TEP) or trimethyl phosphate (TMP), and then POCl3 is added, causing the phosphate groups to transfer to the nucleosides or deoxynucleosides. Finally, nucleotides are obtained through hydrolysis. Chemical synthesis has a high yield, reaching over 90%, making it more efficient than nucleic acid degradation. However, this method has drawbacks: it accumulates large amounts of hydrochloric acid during production, potentially leading to the degradation of purine nucleotides (such as 5'-dGMP and 5'-dAMP); the chemical reagents used are expensive, increasing production costs; and the TEP, TMP, and POCl3 used in the reaction are all toxic reagents, which can easily cause environmental pollution if not handled properly. Microbial direct fermentation: This method uses screened strains to directly produce nucleotides through fermentation. Currently, its most successful application is the production of inosinic acid (5'-IMP). For example, researchers such as Furuya isolated a strain that... 2+Insensitive ammonia-producing short bacillus mutants, in fermentation media containing sufficient adenine, regulate Mn 2+ Concentration can promote the accumulation of IMP. Nevertheless, direct fermentation still faces the challenge of overcoming the microbial cell membrane barrier, and the natural enzyme systems present within microbial cells may degrade nucleotides, making the production of nucleotides via direct fermentation quite difficult.

[0004] Enzymatic synthesis has mild reaction conditions, fast speed, high conversion rate, and low pollution, making it the most suitable industrial production method for development. However, the high cost of the required energy donor ATP and the large amount of AMP and ADP byproducts make it unsuitable for industrial production. Summary of the Invention

[0005] The technical problem solved by this invention is how to synthesize deoxyribonucleoside triphosphate using biological enzymes.

[0006] To address the aforementioned technical problems, the first aspect of this invention provides a mutant protein of MrPPK2, which is any one of the following:

[0007] The mutant protein shown in A1) involves mutating amino acid residues at positions 93 and 127 of the amino acid sequence shown in SEQ ID NO:1, while keeping other amino acid residues unchanged, to obtain a protein with polyphosphokinase activity;

[0008] 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;

[0009] 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.

[0010] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0011] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0012] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, Myc tag, GST tag, and / or SUMO tag, etc.

[0013] The mutant protein mentioned above originates from the following organisms: Meiothermus ruber .

[0014] Among the mutant proteins mentioned above,

[0015] The mutation is as follows:

[0016] The 93rd amino acid residue was mutated from V to Y;

[0017] The amino acid residue at position 127 is mutated from D to P.

[0018] The mutant protein described above, A1), includes the amino acid residues shown in SEQ ID NO:2.

[0019] In some embodiments, the mutant protein shown in A1) can be the amino acid residue shown in SEQ ID NO:2.

[0020] Among the mutant proteins of MrPPK2 described above, the activity of the mutant protein of MrPPK2 is higher than that of the polyphosphokinase MrPPK2 shown in SEQ ID NO: 1.

[0021] 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:

[0022] B1) A nucleic acid molecule encoding the mutant protein described in the first aspect;

[0023] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0024] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0025] 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).

[0026] In the above text, B1) the nucleic acid molecule encoding the mutant protein described in the first aspect can be SEQ ID NO: 3.

[0027] In the aforementioned biological materials, the expression cassette containing the nucleic acid molecule described in B1) as described in B2) refers to DNA capable of expressing the protein described in the above applications in host cells. This DNA may include not only promoters that initiate transcription of protein-coding genes but also terminators that terminate transcription of protein-coding genes. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0028] In the above text, the recombinant vector is a vector that expresses the mutant protein by inserting the nucleic acid molecule described in B1) into the vector backbone.

[0029] The vector backbone can be derived from pET-28a(+) or other vectors that can be used to express proteins.

[0030] The above insertion site can be the pET-28a(+) carrier. Nde I and Xho The I restriction site can also be any other restriction site on the vector used to construct the expression of gene fragments.

[0031] In the above-mentioned biological materials, the recombinant microorganisms may specifically be yeast, bacteria, algae, or fungi.

[0032] The aforementioned recombinant microorganisms can be recombinant microorganisms obtained by introducing a recombinant vector into a host microorganism.

[0033] The host microorganisms mentioned above can be Escherichia coli, such as E. coli BL21(DE3), or other host microorganisms used for protein expression.

[0034] Thirdly, the present invention provides the use of the mutant protein described in the first aspect in any of the following C1-C6;

[0035] Or the application of the biomaterials described in the second aspect in any of the following C2-C7:

[0036] C1) acts as a polyphosphokinase;

[0037] C2) Catalyzes nucleotide polymerization reactions;

[0038] C3) Bioenzymatic synthesis of dATP or dGTP;

[0039] C4) catalyzes the synthesis of dATP from the substrate dAMP;

[0040] C5) Catalytic synthesis of dGTP from dGMP substrate;

[0041] C6) Increases the yield of deoxyribonucleoside triphosphates;

[0042] C7) to prepare polyphosphokinase.

[0043] In the above text, the catalysis uses sodium hexametaphosphate as the phosphate donor.

[0044] Fourthly, the present invention provides the mutant protein and other polyphosphokinases described in the first aspect for any of the following applications;

[0045] Alternatively, the present invention provides the mutant protein described in the first aspect, other polyphosphokinases, and phosphokinases for any of the following applications:

[0046] D1) Catalyzes nucleotide polymerization reactions;

[0047] D2) Bioenzymatic synthesis of deoxyribonucleoside triphosphates;

[0048] D3) Catalyzes the synthesis of dNTPs from dNMP substrates;

[0049] D4) Increases the yield of deoxyribonucleoside triphosphates.

[0050] In the applications described above, the other polyphosphokinases are SMC or its mutants.

[0051] The SMC or its mutant is any one of the following:

[0052] The nucleotide sequence of the protein shown in E1 includes SEQ ID NO:4;

[0053] The nucleotide sequence of the protein shown in E2 includes SEQ ID NO:5;

[0054] The protein shown in E3) is a protein that shares more than 80% amino acid residue identity with the protein shown in E1) or E2) and has the same function.

[0055] The protein shown in E4 is the protein whose sequence is obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in E1) or E2).

[0056] In some embodiments, the nucleotide sequence of the protein shown in E1) is SEQ ID NO:4;

[0057] In some embodiments, the nucleotide sequence of the protein shown in E2) is SEQ ID NO:5.

[0058] The above applications include using the mutant protein described in the first aspect to catalyze the synthesis of dATP from dAMP or the synthesis of dGTP from dGMP using the mutant protein described in the first aspect; or using the mutant protein described in the first aspect and the other polyphosphokinases and phosphokinases to catalyze the synthesis of dTTP from dTMP; or using the mutant protein described in the first aspect and the other polyphosphokinases and phosphokinases to catalyze the synthesis of dCTP from dCMP.

[0059] In the above text, other polyphosphokinases are used to synthesize dTTP or dCTP.

[0060] In the above text, the phosphokinase may be dTMP-K or dCMP-K.

[0061] The SMC mentioned above may be derived from the following organisms: Sinorhizobium melilot .

[0062] The dTMP-K can be derived from the following organisms: Saccharolobus solfataricus The amino acid sequence of dTMP-K can be SEQ ID NO: 7. dTMP-K catalyzes the conversion of dTMP to dTDP.

[0063] The guanylate kinase dCMP-K is derived from the following organisms: Homo sapiens The amino acid sequence of dCMP-K can be SEQ ID NO: 8. dCMP-K catalyzes the conversion of dCMP to dCDP.

[0064] The SMC catalyzes the conversion of dCDP or dTDP to dCTP or dTTP.

[0065] In the above applications, the catalysis is carried out using sodium hexametaphosphate as the phosphate donor and dTTP or dCTP as the secondary donor, respectively, with the corresponding enzyme catalysis.

[0066] Fifthly, the present invention provides an SMC mutant (which may be SMC-T119R), which is any one of the following:

[0067] The nucleotide sequence of the protein shown in F1 includes SEQ ID NO:5;

[0068] The protein shown in F2) is a protein that shares more than 80% amino acid residue identity with the protein shown in F1) and has the same function.

[0069] The protein shown in F3 is the protein whose sequence is obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in F1) or F2).

[0070] Sixthly, the present invention provides any of the following methods:

[0071] G1. A method for synthesizing dATP by a biological enzymatic method, comprising the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein described in the first aspect to catalyze the substrate dAMP to achieve the synthesis of dATP.

[0072] G2. The present invention provides a method for synthesizing dGTP by a biological enzymatic method, comprising the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein described in the first aspect to catalyze the substrate dGMP to achieve the synthesis of dGTP.

[0073] G3. A method for increasing dATP production, comprising the following steps: using sodium hexametaphosphate as a phosphate donor, using the mutant protein described in the first aspect to catalyze the substrate dAMP, thereby increasing dATP production;

[0074] G4. A method for increasing dGTP yield, comprising the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein described in the first aspect to catalyze the substrate dGMP, thereby increasing dGTP yield.

[0075] The aforementioned increase in dATP or dGTP production is an improvement in dATP or dGTP production compared to the catalysis of the polyphosphokinase MrPPK2 shown in SEQ ID NO: 1.

[0076] In a seventh aspect, the present invention provides any of the following methods:

[0077] H1. A method for synthesizing deoxynucleoside triphosphates by a biological enzymatic process, comprising the following steps: synthesizing dATP, synthesizing dGTP, synthesizing dTTP, and synthesizing dCTP;

[0078] The method for synthesizing dATP includes the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein described in the first aspect to catalyze the substrate dAMP to achieve the synthesis of dATP;

[0079] The steps for synthesizing dGTP include: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein described in the first aspect to catalyze the substrate dGMP to achieve the synthesis of dGTP;

[0080] The steps for synthesizing dTTP include: using sodium hexametaphosphate as a phosphate donor and dTTP as a secondary donor, and using other polyphosphokinases and phosphokinases from the fourth aspect to catalyze the substrate dTMP to achieve the synthesis of dTTP;

[0081] The steps for synthesizing dCTP include: using sodium hexametaphosphate as a phosphate donor and dCTP as a secondary donor, and using other polyphosphokinases and phosphokinases from the fourth aspect to catalyze the substrate dCMP to achieve the synthesis of dCTP.

[0082] H2. This invention provides a method for increasing the yield of deoxynucleoside triphosphates, comprising: increasing dATP yield, increasing dGTP yield, increasing dTTP yield, and increasing dCTP yield, thereby increasing the yield of deoxynucleoside triphosphates.

[0083] The method for increasing dATP production includes the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein described in the first aspect to catalyze the substrate dAMP, thereby increasing dATP production;

[0084] The steps for increasing dGTP production include: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein described in the first aspect to catalyze the substrate dGMP, thereby increasing dGTP production;

[0085] The steps to improve dTTP include: using sodium hexametaphosphate as a phosphate donor and dTTP as a secondary donor, and using other polyphosphokinases and phosphokinases from the fourth aspect to catalyze the substrate dTMP, thereby increasing dTTP yield;

[0086] The steps to improve dCTP include: using sodium hexametaphosphate as a phosphate donor and dCTP as a secondary donor, and using other polyphosphokinases and phosphokinases from the fourth aspect to catalyze the substrate dCMP, thereby increasing dCTP yield.

[0087] The aforementioned increase in dATP or dGTP production is an improvement in dATP or dGTP production compared to the catalysis of the polyphosphokinase MrPPK2 shown in SEQ ID NO: 1.

[0088] The other polyphosphokinases and phosphokinases used in the above synthesis of dTTP were SMC mutant and dTMP-K, respectively.

[0089] The other polyphosphokinases and phosphokinases used in the above synthesis of dCTP were SMC mutant and dCMP-K, respectively.

[0090] In one embodiment of the present invention, sodium hexametaphosphate is used as the phosphate donor, and the mutant protein described in the first aspect catalyzes the reaction of the substrate dAMP in a 200 μL reaction system: sodium hexametaphosphate is used as the phosphate donor at a concentration of 3 mM; dAMP is used as the substrate at a concentration of 5 mM; the salt ion in the reaction solution is selected as 50 mM potassium dihydrogen phosphate, and the pH value is selected as 7.5; the metal ion is MgCl2 at a concentration of 10 mM; MrPPK2 is added to the reaction system at a preferred concentration of 200 μg / mL. The reaction temperature is 37°C, and the reaction time is 3 hours. Under these conditions, the substrate dAMP can be efficiently converted to dATP.

[0091] In one embodiment of the present invention, sodium hexametaphosphate is used as the phosphate donor, and the mutant protein described in the first aspect catalyzes the reaction of the substrate dGMP in a 200 μL reaction system: sodium hexametaphosphate is used as the phosphate donor at a concentration of 3 mM; dGMP is used as the substrate at a concentration of 5 mM; the salt ion in the reaction solution is selected as 50 mM potassium dihydrogen phosphate, and the pH value is selected as 7.5; the metal ion is MgCl2 at a concentration of 10 mM; MrPPK2 is added to the reaction system at a preferred concentration of 200 μg / mL. The reaction temperature is 37°C, and the reaction time is 3 hours. Under these conditions, the substrate dGMP can be efficiently converted to dGTP.

[0092] In one embodiment, sodium hexametaphosphate is used as the primary phosphate donor at a concentration of 3 mM; dTTP is used as the secondary donor at a concentration of 1.5 mM; and dTMP is used as the substrate at a concentration of 5 mM. The salt ion in the reaction solution is selected as 50 mM potassium dihydrogen phosphate, and the pH value is selected as 7.5. The metal ion is MgCl2 at a concentration of 10 mM. SMC-T119R and dTMP-K are added to the reaction system at a concentration of 100 μg / mL and 300 μg / mL, respectively. The reaction temperature is 37°C, and the reaction time is 3 hours. Under these conditions, the substrate dTMP can be efficiently converted to dTTP.

[0093] In one embodiment, sodium hexametaphosphate is used as the primary phosphate donor at a concentration of 3 mM; dCTP is used as the secondary donor at a concentration of 1.5 mM; and dCMP is used as the substrate at a concentration of 5 mM. The salt ion in the reaction solution is selected as 50 mM potassium dihydrogen phosphate, and the pH value is selected as 7.5. The metal ion is MgCl2 at a concentration of 10 mM. SMC-T119R and dCMP-K are added to the reaction system, with SMC-T119R at 100 μg / ml and dCMP-K at 300 μg / ml. The reaction temperature is 37°C, and the reaction time is 3 hours. Under these conditions, the substrate dCMP can be efficiently converted to dCTP.

[0094] This invention uses the most common biologically extracted genomic DNA as raw material, prepares monophosphate nucleotides dNMP through two-step enzymatic hydrolysis, then selects polyphosphokinase as the key enzyme system, uses sodium hexametaphosphate as the main phosphate donor to generate dNTPs, and uses the product for PCR amplification experiments.

[0095] This invention provides a method for producing dNTP products using genomic DNA as raw material, comprising enzymatic hydrolysis of genomic DNA into dNMP, and then using inexpensive sodium hexametaphosphate as a phosphate donor and a polyphosphokinase variant with significantly enhanced catalytic activity to synthesize four types of dNTPs.

[0096] According to the present invention, the selected genomic DNA is DNA extracted from Escherichia coli. The genomic DNA is first broken into short-chain DNA using nuclease I, and then the short-chain DNA is further hydrolyzed into dNMP using nuclease P1 (penicillin). The dNMP mixture is then separated into individual nucleotides by HPLC.

[0097] This invention provides a method for the bioenzymatic synthesis of deoxyribonucleoside triphosphates. Using inexpensive sodium hexametaphosphate (Poly P6) as the primary phosphate donor, a polyphosphokinase MrPPK2 mutant catalyzes the conversion of dA / GMP to dA / GTP. This mutant significantly improves the production rates of dGTP and dATP. Furthermore, by using a polyphosphokinase SMC mutant in combination with dTMP-K or dCMP-K, sodium hexametaphosphate is used as the primary phosphate donor, and dTTP / dCTP as the secondary donor to convert the substrate dT / CMP to dT / CTP. Specifically, dT / CMP→dT / CDP uses dTTP / dCTP as the phosphate donor, and dT / CDP→dT / CTP uses P6 as the phosphate donor. The combined use of the SMC mutant, dTMPK, and dCMPK further enhances the production rates of dTTP and dCTP. Sodium hexametaphosphate is an inorganic compound with the chemical formula (NaPO3)6. It is a white crystalline powder, readily soluble in water but insoluble in organic solvents. It is inexpensive and mainly used in the food industry, causing minimal environmental pollution and thus reducing costs and pollution. The method of this invention can increase the yield of deoxynucleoside triphosphates. Attached Figure Description

[0098] Figure 1 The HPLC separation chromatogram is for dNMP separation.

[0099] Figure 2 This is a schematic diagram of the UPLC for the standard product.

[0100] Figure 3 The diagram shows the UPLC results of the conversion of dAMP / dGMP by MRPPK2-related proteins and the conversion of dCDP / dTDP by SMC-related proteins. A represents the conversion of dAMP by MRPPK2-related proteins; B represents the conversion of dGMP by MRPPK2-related proteins; and C represents the UPLC results of the conversion of 1mM dCDP / dTDP by wild-type SMC and the SMC mutant T119R.

[0101] Figure 4 This is a schematic diagram of the UPLC results for the conversion reactions of four types of dNMP (5mM).

[0102] Figure 5To compare the dNTP products obtained in this invention with those obtained from commercial dNTPs in PCR, A shows the gel images obtained by running agarose gel with the dNTP products obtained in this invention and the PCR products obtained by running commercial dNTPs in PCR; B shows the PCR product accuracy result of A. Detailed Implementation

[0103] 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.

[0104] 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.

[0105] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0106] Definitions and Explanations

[0107] 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: Gly (glycine); H: Histidine; I: Isoleucine (isoleucine); L: Leucine (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).

[0108] In this invention, the mutation site and its substitution are expressed by the position number of the mutation site and the type of amino acid at that site. For example, V93Y indicates that, compared with SEQ ID NO:1, valine at position 93 of SEQ ID NO:1 is mutated to tyrosine; D127P indicates that, compared with SEQ ID NO:1, aspartic acid at position 127 of SEQ ID NO:1 is mutated to proline. In this invention, " / " is used to represent combinations of mutation sites.

[0109] In this invention, the term "transformation" refers to the introduction of DNA into a host cell so that the DNA can be replicated as an extrachromosomal element or through chromosomal integration. That is, transformation refers to the alteration of gene synthesis caused by the introduction of exogenous DNA into a cell. The term "polyphosphokinase produced by the transformant" refers to a product obtained by culturing a transformant according to known methods for culturing microorganisms.

[0110] In this invention

[0111] The term deoxyribonucleic acid monophosphate (dNMP) includes adenine deoxyribonucleic acid monophosphate, guanine deoxyribonucleic acid monophosphate, cytosine deoxyribonucleic acid monophosphate, and thymine deoxyribonucleic acid monophosphate.

[0112] The term deoxyribonucleic acid diphosphate (dNDP) includes adenine deoxyribonucleic acid diphosphate, guanine deoxyribonucleic acid diphosphate, cytosine deoxyribonucleic acid diphosphate, and thymine deoxyribonucleic acid diphosphate.

[0113] The term deoxyribonucleic acid triphosphate (dNTP) includes adenine deoxyribonucleic acid triphosphate, guanine deoxyribonucleic acid triphosphate, cytosine deoxyribonucleic acid triphosphate, and thymine deoxyribonucleic acid triphosphate.

[0114] In this invention, "PPK" refers to polyphosphokinase (PPK), and includes references to purified and recombinant forms of the enzyme.

[0115] The sources of materials used in the following embodiments are shown in Table 1.

[0116]

[0117] Example 1: Hydrolysis of Genomic DNA

[0118] 1. DNA extraction

[0119] Weigh 368.7 g of Escherichia coli BL21 bacterial sludge and dissolve it in 400 mL of ultrapure water. Add the solution to a 1000 mL round-bottom flask and heat in an oil bath at 99 °C for 20 min with stirring. Cool to room temperature. Transfer the solution to a centrifuge cup and centrifuge (9000 rpm, 30 min). Collect the supernatant (supernatant volume: 510 mL), which is the DNA extract.

[0120] 2. Degradation of RNA

[0121] Prepare a RNase (DNase-free) buffer: 10 mM Tris, 15 mM NaCl, balance water, pH 7.5;

[0122] RNA degradation: Take 2 mL of the DNA extract after boiling and alcohol precipitation, add RNase enzyme (final concentration: 100 μg / mL) to it, react at 37℃ for 10 min to obtain the DNA extract with RNA removed.

[0123] Removal of RNase enzyme from the system: Treat the DNA extract (excluding RNA) at 98°C for 10 min to remove the precipitate and collect the supernatant;

[0124] 3. Alcohol precipitation

[0125] Add an equal volume of isopropanol to the supernatant, mix by inversion, and incubate at -20°C for 30 min. Centrifuge (10000 rpm, 10 min) and discard the supernatant. Wash three times with 50 mL of pre-cooled 75% ethanol, then centrifuge to remove the supernatant. Finally, allow to stand at room temperature for 30 min to evaporate, and reconstitute with purified water to a concentration of 100 mg / mL to obtain the DNA solution.

[0126] 4. Breaking long chains

[0127] Prepare a 100 mg / m³ nuclease I solution using a buffer (40 mM Tris + 2.5 mM magnesium sulfate + 1 mM calcium chloride, with the remainder being water). Take 1 mL of DNA solution and 1 mL of nuclease I solution, mix them thoroughly, and react at 30 °C for 0.5 h to obtain the fragmented product.

[0128] 5. Short-fragment hydrolysis

[0129] Dissolve nuclease P1 (Penicillium citrinum, Angel Yeast Co., Ltd.) in water to prepare a 50 mg / mL nuclease P1 solution;

[0130] Add 8 mL of nuclease P1 solution to the fragmented product obtained in step 4, heat to 75 °C, react for 2 h, and then boil to inactivate the enzyme, obtaining a dNMP mixture solution.

[0131] 6. Separation of dNMP

[0132] The dNMP mixture solution was separated using HPLC under the conditions shown in Table 2 below.

[0133]

[0134] The HPLC separation chromatogram is as follows: Figure 1 As shown in the figure, the four peaks from left to right are dCMP, dTMP, dGMP, and dAMP.

[0135] The collected products were recovered by rotary evaporation to obtain dAMP, dTMP, dCMP, and dGMP, respectively.

[0136] dAMP, dTMP, dCMP, and dGMP can also be purchased commercially.

[0137] Example 2: UPLC schematic diagram of standard curve preparation and detection of standards.

[0138] dNTP and dNMP were serially diluted to a maximum concentration of 5 mM. Five concentrations were selected, and UPLC was used to obtain the signal values ​​of each concentration. These values ​​were then used to generate a scatter plot, and trend lines and linear functions were also generated.

[0139] The standard curve function for dATP is y = 7E+06x, where x is the amount of dATP and y is the signal value of the dATP product peak corresponding to UPLC.

[0140] The standard curve function for dTTP is y = 3E+06x, where x is the amount of dTTP and y is the signal value of the dTTP product peak corresponding to UPLC.

[0141] The standard curve function for dCTP is y = 3E+06x, where x is the amount of dCTP and y is the signal value of the dCTP product peak corresponding to UPLC.

[0142] The standard curve function for dGTP is y = 5E+06x, where x is the amount of dGTP and y is the signal value of the dGTP product peak corresponding to UPLC.

[0143] A schematic diagram of the UPLC for the standard product is shown below. Figure 2 As shown, from top to bottom, they are 5MdTM / D / TP, 5mMdCM / D / TP, 5mMdGM / D / TP, and 5MdAM / D / TP.

[0144] Example 3: Obtaining polyphosphate kinase mutants

[0145] 1. Obtaining polyphosphate kinase mutants

[0146] Source Meiothermus ruber The amino acid sequence of the wild-type MrPPK2 protein is SEQ ID NO: 1.

[0147] Homology modeling was performed on MrPPK2 to obtain its three-dimensional protein structure. By analyzing residues within the 3A region of the active site, several potential sites that could affect its catalytic activity were identified. These single- or multi-site saturation mutations, combined with high-throughput screening, yielded a MrPPK2 mutant with high catalytic efficiency and substrate specificity: D127P / V93Y.

[0148] The MrPPK2 mutant D127P / V93Y is formed by mutating V at position 93 of SEQ ID NO: 1 to Y and D at position 127 to P, while other amino acid residues remain unchanged. The amino acid sequence of the MrPPK2 mutant D127P / V93Y is SEQ ID NO: 2, and the nucleotide sequence of the gene encoding this mutant is SEQ ID NO: 3.

[0149] Source Sinorhizobium melilot The amino acid sequence of the wild-type SMC protein is SEQ ID NO: 4.

[0150] Homology modeling of SMC was used to obtain its three-dimensional protein structure. By analyzing residues within the 3A region of the active site, several potential sites that may affect its catalytic activity were identified. These single- or multi-site saturation mutations, combined with high-throughput screening and other methods, yielded an SMC mutant with high catalytic efficiency and substrate specificity: T119R.

[0151] The SMC mutant T119R is formed by mutating the T at position 119 of SEQ ID NO:4 to R, while keeping other amino acid residues unchanged. The amino acid sequence of the SMC mutant T119R is SEQ ID NO:5, and the nucleotide sequence of the gene encoding this mutant is SEQ ID NO:6.

[0152] 2. Construction of the expression carrier

[0153] The recombinant plasmid pET-28a(+)-PPK expressing the wild-type MrPPK2 gene is formed by inserting the gene encoding the MrPPK2 protein into pET-28a(+) (Novagen, Kansas City). + ) Nde I and Xho The recombinant plasmid was obtained by cutting the I restriction site.

[0154] The recombinant plasmid pET-28a(+)-PPKD127P / V93Y expressing the MrPPK2 mutant D127P / V93Y is formed by inserting the coding gene (SEQ ID NO: 3) of the MrPPK2 mutant D127P / V93Y into pET-28a(+). Nde I and Xho The recombinant plasmid was obtained by cutting the I restriction site.

[0155] The recombinant plasmid pET-28a(+)-SMC, expressing the wild-type SMC gene, is created by inserting the SMC protein-coding gene into pET-28a(+) (Novagen, Kansas City). + ) Nde I and Xho The recombinant plasmid was obtained by cutting the I restriction site.

[0156] The recombinant plasmid pET-28a(+)-SMCT119R expressing the SMC mutant T119R is formed by inserting the coding gene (SEQ ID NO: 6) of the SMC mutant T119R into pET-28a(+). Nde I and Xho The recombinant plasmid was obtained by cutting the I restriction site.

[0157] 3. Expression and purification of polyphosphokinase

[0158] To detect polyphosphokinase activity in vitro, the enzyme was expressed and purified exogenously in Escherichia coli.

[0159] 1) Induced expression

[0160] The various recombinant plasmids prepared in step 2 above were transformed into E. coli BL21(DE3), and positive clones were screened using kanamycin resistance plates (Kan+, 100 mg / mL) and cultured overnight at 37°C. Single clones were picked and cultured in 8 mL of LB liquid medium (Kan+, 100 mg / mL) at 37°C and 220 r / min until the OD600 reached 0.6. The bacterial culture in 8 mL of LB medium was transferred to 800 mL of 2YT medium (Kan+, 100 mg / mL), and cultured at 37°C and 220 rpm until the OD600 reached 0.6. The temperature was then lowered to 16°C, and IPTG was added to a final concentration of 0.5 mM. Expression was induced for 20 h to obtain the induced culture product. The induced culture product was collected into a collection bottle, centrifuged at 5500 r / min for 15 min, the supernatant was discarded, and the obtained bacterial cell pellet was resuspended in 35 mL of protein buffer (50 mM TrisHCl, 2 mM EDTA, 0.1% Triton X100, the remainder being water, pH 7.4). The pellet was then poured into a 50 mL centrifuge tube and stored at 80°C to obtain the individual bacterial cell suspensions.

[0161] 2) Protein purification

[0162] (1) Sterilization: The bacterial suspensions obtained in step 1) above were sterilized twice using a high-pressure low-temperature lysing apparatus at a pressure of 1200 bar and a temperature of 4°C. Centrifuged at 10000 r / min for 45 min at 4°C, the precipitate and supernatant were collected and used for sample preparation.

[0163] (2) Purification: The supernatant obtained in (1) above was filtered through a 0.45 μm microporous membrane and purified by nickel affinity chromatography. The specific steps are as follows:

[0164] 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;

[0165] 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 more;

[0166] 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.

[0167] 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 sample preparation and detection by 12% SDS-PAGE (75.8 KD of target protein was obtained).

[0168] (3) Concentration and buffer replacement: The collected target protein was concentrated by centrifugation using a 50 mL Amicon ultrafiltration tube (10 kDa, Millipore) (4 ℃, 3400 r, 30 min) to a final volume of 1 mL. 10 mL of protein buffer was added, and the concentration was increased to 1 mL. This process was repeated once to obtain MrPPK2 solution, MrPPK2 mutant D127P solution, and MrPPK2 mutant V93Y solution.

[0169] (4) The concentration of the concentrated protein solution in (3) was detected by BCA kit and diluted to 10 mg / mL to obtain the purified protein MrPPK2 solution and the purified MrPPK2 mutant D127P / V93Y solution.

[0170] The same method was used to prepare purified SMC and SMC mutant T119R solutions.

[0171] Example 4: Functional identification of the MrPPK2 mutant

[0172] The substrate used in the embodiments of the present invention is:

[0173] (1) Deoxyribonucleic acid monophosphate (dNMP), including adenine deoxyribonucleic acid monophosphate, guanine deoxyribonucleic acid monophosphate, cytosine deoxyribonucleic acid monophosphate, and thymine deoxyribonucleic acid monophosphate;

[0174] (2) Deoxyribonucleic acid diphosphate (dNDP), including adenine deoxyribonucleic acid diphosphate, guanine deoxyribonucleic acid diphosphate, cytosine deoxyribonucleic acid diphosphate, and thymine deoxyribonucleic acid diphosphate;

[0175] The product of this invention is a deoxyribonucleic acid triphosphate (dNTP), including adenine deoxyribonucleic acid triphosphate, guanine deoxyribonucleic acid triphosphate, cytosine deoxyribonucleic acid triphosphate, and thymine deoxyribonucleic acid triphosphate.

[0176] In vitro complex enzyme reaction

[0177] Reaction system with different substrates (200 μL): 50 mM potassium dihydrogen phosphate, 10 mM MgCl2, 1 mM dNMP, 2 mM sodium hexametaphosphate, 200 μg / mL of each enzyme, balance water, pH 7.5.

[0178] In the above system, dNMP is either dAMP or dGMP.

[0179] The enzymes in the above system are MrPPK2 mutant D127P / V93Y (denoted as D127P-V93Y in the figure) and wild-type MrPPK2.

[0180] Reaction conditions: The reaction system containing each dNMP was directly added to a shallow well plate (200 μL system) and shaken at 37°C and 120 rpm for 3 h. The reaction product was then terminated at 100°C for 10 min and filtered into a liquid chromatography vial using a 0.22 μm filter for UPLC detection.

[0181] Liquid phase detection parameters

[0182] Liquid chromatography instrument model: Fuli Instruments (LC5190)

[0183] Chromatographic column (silica gel based): C18 5um, 4.6mm × 250mm

[0184] Setting conditions:

[0185] Column temperature: 30℃; Injection volume: 10ul

[0186] Mobile phase ratio (isocratic): A / B 65% / 35%

[0187] Mobile phase A: H2O + 10mM TBAH + 10mM KH2PO4

[0188] Mobile phase B: CH3OH + 10mM TBAH

[0189] UV: 254nm

[0190] The yield of dNTPs is obtained by substituting the detected signal values ​​into the standard curve function of Example 2.

[0191] The detection results after the reaction are as follows: Figure 3As shown in Figures A and 3B, the MRPPK2 and MrPPK2 mutants D127P / V93Y catalyze the production of dATP (deoxyribonucleic acid triphosphate) from dAMP (deoxyribonucleic acid monophosphate) substrates at yields of 0.56 mM and 0.64 mM, respectively. Similarly, the MRPPK2 and MrPPK2 mutants D127P / V93Y catalyze the production of dGTP (deoxyribonucleic acid triphosphate) from dGMP (deoxyribonucleic acid monophosphate) substrates at yields of 0.04 mM and 0.62 mM, respectively. This indicates that the MRPPK2 mutant promotes the production of dA / GTP (deoxyribonucleic acid triphosphate) from sodium hexametaphosphate as a phosphate donor, with the effect of dGMP on dGTP formation being particularly pronounced.

[0192] Example 5: Functional Verification of SMC Mutants

[0193] The substrate used in the embodiments of the present invention is:

[0194] (1) Deoxyribonucleic acid monophosphate (dNMP), including adenine deoxyribonucleic acid monophosphate, guanine deoxyribonucleic acid monophosphate, cytosine deoxyribonucleic acid monophosphate, and thymine deoxyribonucleic acid monophosphate;

[0195] (2) Deoxyribonucleic acid diphosphate (dNDP), including adenine deoxyribonucleic acid diphosphate, guanine deoxyribonucleic acid diphosphate, cytosine deoxyribonucleic acid diphosphate, and thymine deoxyribonucleic acid diphosphate;

[0196] The product of this invention is a deoxyribonucleic acid triphosphate (dNTP), including adenine deoxyribonucleic acid triphosphate, guanine deoxyribonucleic acid triphosphate, cytosine deoxyribonucleic acid triphosphate, and thymine deoxyribonucleic acid triphosphate.

[0197] In vitro complex enzyme reaction

[0198] Reaction system with different substrates (200 μL): 50 mM potassium dihydrogen phosphate, 10 mM MgCl2, 1 mM dNDP, 2 mM sodium hexametaphosphate, 50 μg / mL of each enzyme, balance water, pH 7.5.

[0199] In the above system, dNDP is either dTDP or dCDP.

[0200] The enzymes used in the above reactions are either the SMC mutant T119R or the wild-type SMC.

[0201] Reaction conditions: The above reaction system was directly added to a shallow well plate (200 μL system) and shaken at 37°C and 120 rpm for 3 h. The reaction product was then taken and the reaction was terminated at 100°C for 10 min. The product was filtered through a 0.22 μm filter membrane into a liquid chromatography vial for UPLC detection.

[0202] Liquid phase detection parameters

[0203] Liquid chromatography instrument model: Fuli Instruments (LC5190)

[0204] Chromatographic column (silica gel based): C18 5um, 4.6mm × 250mm

[0205] Setting conditions:

[0206] Column temperature: 30℃; Injection volume: 10ul

[0207] Mobile phase ratio (isocratic): A / B 65% / 35%

[0208] Mobile phase A: H2O + 10mM TBAH + 10mM KH2PO4

[0209] Mobile phase B: CH3OH + 10mM TBAH

[0210] UV: 254nm

[0211] The yield of dNTPs is obtained by substituting the detected signal values ​​into the standard curve function of Example 2.

[0212] The detection results after the reaction are as follows: Figure 3 As shown in Figure C, the yields of dTTP catalyzed by SMC and the SMC mutant T119R in the formation of dTTP from dTDP substrates were 0.01 mM and 0.38 mM, respectively. The yields of dCTP catalyzed by SMC and the SMC mutant T119R in the formation of dCTP from dCDP substrates were 0.007 mM and 0.55 mM, respectively. This indicates that the SMC mutant T119R promotes the use of sodium hexametaphosphate as a phosphate donor, thereby increasing the yield of dTTP / dCTP (deoxyribonucleic acid triphosphate) from dTDP / dCDP (deoxyribonucleic acid monophosphate) substrates.

[0213] Example 6: Transformation of four types of dNMP (5mM) and their functional verification in PCR reactions

[0214] The substrate used in the embodiments of the present invention is:

[0215] (1) Deoxyribonucleic acid monophosphate (dNMP), including adenine deoxyribonucleic acid monophosphate, guanine deoxyribonucleic acid monophosphate, cytosine deoxyribonucleic acid monophosphate, and thymine deoxyribonucleic acid monophosphate;

[0216] (2) Deoxyribonucleic acid diphosphate (dNDP), including adenine deoxyribonucleic acid diphosphate, guanine deoxyribonucleic acid diphosphate, cytosine deoxyribonucleic acid diphosphate, and thymine deoxyribonucleic acid diphosphate;

[0217] The product of this invention is a deoxyribonucleic acid triphosphate (dNTP), including adenine deoxyribonucleic acid triphosphate, guanine deoxyribonucleic acid triphosphate, cytosine deoxyribonucleic acid triphosphate, and thymine deoxyribonucleic acid triphosphate.

[0218] I. Conversion of 4 types of dNMP (5mM)

[0219] In vitro complex enzyme reaction:

[0220] The reaction system for the dAMP substrate (200 μL) consisted of 50 mM potassium dihydrogen phosphate, 10 mM MgCl2, 5 mM dAMP, 3 mM sodium hexametaphosphate, 200 μg / ml MrPPK2 mutant D127P / V93Y, with the remainder being water, and the pH was 7.5.

[0221] The reaction system for the dGMP substrate (200 μL) consisted of 50 mM potassium dihydrogen phosphate, 10 mM MgCl2, 5 mM dGMP, 3 mM sodium hexametaphosphate, 200 μg / ml MrPPK2 mutant D127P / V93Y, with the remainder being water, and a pH of 7.5.

[0222] The reaction system for the dTMP substrate (200 μL) consisted of 50 mM potassium dihydrogen phosphate, 10 mM MgCl2, 5 mM dTMP, 3 mM sodium hexametaphosphate, 1.5 mM dNTP, 100 μg / ml SMC mutant T119R, 300 μg / ml dTMP-K (SEQ ID NO: 7), with the remainder being water, and a pH of 7.5.

[0223] The reaction system for the dCMP substrate (200 μL) consisted of 50 mM potassium dihydrogen phosphate, 10 mM MgCl2, 5 mM dCMP, 3 mM sodium hexametaphosphate, 1.5 mM dNTP, 100 μg / ml SMC mutant T119R, 300 μg / ml dCMP-K (SEQ ID NO: 8), with the remainder being water, and a pH of 7.5.

[0224] Reaction conditions: The above reaction systems were directly added to a shallow well plate (200 μL system) and shaken at 37°C and 120 rpm for 3 h. The reaction products were then taken and the reaction was terminated at 100°C for 10 min. The mixture was filtered through a 0.22 μm filter membrane into a liquid chromatography vial for UPLC detection.

[0225] Liquid phase detection parameters

[0226] Liquid chromatography instrument model: Fuli Instruments (LC5190)

[0227] Chromatographic column (silica gel based): C18 5um, 4.6mm × 250mm

[0228] Setting conditions:

[0229] Column temperature: 30℃; Injection volume: 10ul

[0230] Mobile phase ratio (isocratic): A / B 65% / 35%

[0231] Mobile phase A: H2O + 10mM TBAH + 10mM KH2PO4

[0232] Mobile phase B: CH3OH + 10mM TBAH

[0233] UV: 254nm

[0234] The yield of dNTPs is obtained by substituting the detected signal values ​​into the standard curve function of Example 2.

[0235] The detection results after the reaction are as follows: Figure 4 As shown, the target dNTP is obtained.

[0236] II. Functional Verification for PCR Reactions

[0237] The dNTPs prepared above were used to perform PCR reactions on different fragments (gene13, gene14, gene15, gene16, gene17, gene18, gene19, gene20, and gene21) of the substrate DNA using different primer pairs. PCR reactions were performed using Phanta Max Super-Fidelity DNA Polymerase P505 from Novizan, with the dNTPs from this product serving as controls.

[0238] The 25ul commercial control PCR reaction system consisted of: 12.5ul buffer, 0.5ul Phanta Max Super-Fidelity DNA Polymerase, 1ul substrate DNA (SEQ ID NO: 9), 0.5ul each of upstream and downstream primers, 0.2mMP505dNTP, and water to a final volume of 25ul.

[0239] The only difference between the 25 μL PCR reaction system of this invention and the above-mentioned commercial control PCR reaction system is that the P505 dNTP is replaced with the 0.1 mM and 0.2 mM of the dNTPs prepared above.

[0240] The upstream and downstream primer sequences for amplifying gene 13 are SEQ ID NO: 10 and SEQ ID NO: 11, respectively, as listed in the sequence listing; the upstream and downstream primer sequences for amplifying gene 14 are SEQ ID NO: 12 and SEQ ID NO: 13, respectively, as listed in the sequence listing; the upstream and downstream primer sequences for amplifying gene 15 are SEQ ID NO: 14 and SEQ ID NO: 15, respectively, as listed in the sequence listing; the upstream and downstream primer sequences for amplifying gene 16 are SEQ ID NO: 16 and SEQ ID NO: 17, respectively, as listed in the sequence listing; the upstream and downstream primer sequences for amplifying gene 17 are SEQ ID NO: 18 and SEQ ID NO: 19, respectively, as listed in the sequence listing; the upstream and downstream primer sequences for amplifying gene 18 are SEQ ID NO: 20 and SEQ ID NO: 21, respectively, as listed in the sequence listing; the upstream and downstream primer sequences for amplifying gene 19 are SEQ ID NO: 22 and SEQ ID NO: 23, respectively, as listed in the sequence listing; the upstream and downstream primer sequences for amplifying gene 20 are SEQ ID NO: 24 and SEQ ID NO: 15, respectively, as listed in the sequence listing; NO: 25; The upstream and downstream primer sequences used to amplify gene21 are SEQ ID NO: 26 and SEQ ID NO: 27, respectively, as listed in the sequence listing.

[0241] The PCR reaction conditions are as follows: Step 1: 95°C denaturation for 3 minutes; Step 2: 95°C denaturation for 15 seconds; Step 3: 58°C annealing for 15 seconds; Step 4: 72°C extension for 30 seconds; Step 5: Repeat steps 2 to 4 29 times; Step 6: 72°C extension for 5 minutes. The PCR amplification product is obtained through the above steps.

[0242] The PCR amplification products were detected, and the results are as follows: Figure 5 As shown in Figure A, it can be seen that the PCR reaction of the 0.1mM / 0.2mM dNTP prepared above yields the same PCR results as the 0.1mM dNTP in P505.

[0243] PCR results were sent to Genewiz for sequencing to check accuracy, and the results were as follows: Figure 5B, genes 19, 20, and 21 are commercial dNTPs at 0.2 mM, genes 13, 14, and 15 are dNTPs invented in this invention at 0.1 mM, and genes 16, 17, and 18 are dNTPs invented in this invention at 0.2 mM. It can be seen that the sequencing results of the self-made samples of this invention are consistent with those of commercial samples.

[0244] 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 MrPPK2, characterized by: The amino acid sequence of the mutant protein of MrPPK2 is SEQ ID NO:

2.

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 molecules described in B1); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1).

3. The biomaterial according to claim 2, characterized in that: B3) A recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the expression cassette described in B2).

4. The biomaterial according to claim 2 or 3, characterized in that: B4) refers to recombinant microorganisms containing the recombinant vector described in B3).

5. The application of the mutant protein according to claim 1, characterized in that: The application is any one of the following C1-C4; C1) acts as a polyphosphokinase; C2) Catalyzes the synthesis of dATP from the substrate dAMP; C3) Catalytic synthesis of dGTP from dGMP substrate; C4) Increase the yield of deoxynucleoside triphosphates; the deoxynucleoside triphosphates are dATP or dGTP.

6. The application of the biomaterial according to any one of claims 2-4, characterized in that: The application is any one of the following C2-C5: C2) Catalyzes the synthesis of dATP from the substrate dAMP; C3) Catalytic synthesis of dGTP from dGMP substrate; C4) Increase the yield of deoxynucleoside triphosphates; wherein the deoxynucleoside triphosphates are dATP or dGTP; C5) to prepare polyphosphokinase.

7. An SMC mutant, characterized by: The amino acid sequence of the SMC mutant is SEQ ID NO:

5.

8. The application of the SMC mutant according to claim 7, characterized in that: The application is any one of the following: D1) Bioenzymatic synthesis of deoxyribonucleoside triphosphates; D2) Catalyzes the synthesis of dNTPs from dNMP substrates; D3) Increases the yield of deoxyribonucleoside triphosphates; The deoxynucleoside triphosphate is dCTP or dTTP; The dNMP is either dTMP or dCMP; The dNTP is either dCTP or dTTP; The synthesis of dNTPs from the catalytic substrate dNMP includes the following steps: The SMC mutant catalyzes the synthesis of dTDP from dTMP using dTMP-K, and then catalyzes the synthesis of dTTP from dTDP; or the SMC mutant catalyzes the synthesis of dCDP from dCMP using dCMP-K, and then catalyzes the synthesis of dCTP from dCDP. The amino acid sequence of the dTMP-K is SEQ ID NO: 7; The amino acid sequence of the dCMP-K is SEQ ID NO:

8.

9. A method for synthesizing dATP using a biological enzymatic method, characterized in that: The method includes the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein of claim 1 to catalyze the substrate dAMP to achieve the synthesis of dATP.

10. A method for synthesizing dGTP using a bioenzymatic method, characterized in that: The method includes the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein of claim 1 to catalyze the substrate dGMP to achieve the synthesis of dGTP.

11. A method for increasing dATP production, characterized in that: The method includes the following steps: using sodium hexametaphosphate as a phosphate donor, the mutant protein of claim 1 catalyzes the substrate dAMP to increase dATP production.

12. A method for increasing dGTP production, characterized in that: The method includes the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein of claim 1 to catalyze the substrate dGMP to increase dGTP production.

13. A method for synthesizing deoxynucleoside triphosphates via a bioenzymatic process, characterized in that: The method includes the following steps: synthesizing dATP, synthesizing dGTP, synthesizing dTTP, and synthesizing dCTP; The method for synthesizing dATP includes the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein of claim 1 to catalyze the substrate dAMP to achieve the synthesis of dATP; The steps for synthesizing dGTP include: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein of claim 1 to catalyze the substrate dGMP to achieve the synthesis of dGTP; The steps for synthesizing dTTP include: using sodium hexametaphosphate as a phosphate donor and dTTP as a secondary donor, and using the SMC mutant described in claim 7 and the dTMP-K catalytic substrate dTMP shown in SEQ ID NO: 7 to synthesize dTTP; The steps for synthesizing dCTP include: using sodium hexametaphosphate as a phosphate donor and dCTP as a secondary donor, and using the SMC mutant described in claim 7 and the dCMP-K catalytic substrate dCMP shown in SEQ ID NO: 8 to synthesize dCTP.

14. A method for increasing the yield of deoxynucleoside triphosphates, characterized in that: The method includes the following: increasing dATP production, increasing dGTP production, increasing dTTP production, and increasing dCTP production to achieve increased deoxynucleoside triphosphate production; The method for increasing dATP production includes the following steps: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein of claim 1 to catalyze the substrate dAMP to increase dATP production; The steps for increasing dGTP production include: using sodium hexametaphosphate as a phosphate donor, and using the mutant protein of claim 1 to catalyze the substrate dGMP to increase dGTP production; The steps for increasing dTTP yield include: using sodium hexametaphosphate as a phosphate donor and dTTP as a secondary donor, and using the SMC mutant described in claim 7 and the dTMP-K catalytic substrate dTMP shown in SEQ ID NO: 7 to increase dTTP yield; The steps for increasing dCTP yield include: using sodium hexametaphosphate as a phosphate donor and dCTP as a secondary donor, and using the SMC mutant described in claim 7 and the dCMP-K catalytic substrate dCMP shown in SEQ ID NO: 8 to increase dCTP yield.

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