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CN-121022791-B - Preparation method and application of natural nucleotide

CN121022791BCN 121022791 BCN121022791 BCN 121022791BCN-121022791-B

Abstract

The invention discloses a preparation method and application of natural nucleotide. The MrPPK mutant protein of the invention is A1) protein with polyphosphatase activity obtained by mutating 93 rd and 127 th amino acid residues on an amino acid sequence shown in SEQ ID NO.1, A2) protein with the same function and more than 80% of amino acid residues except the mutated amino acid residues in the protein shown in A1), and A3) protein obtained by connecting labels at the N end or/and the C end of the protein A1) or A2). According to the invention, sodium hexametaphosphate is used as a main phosphate group donor, mrPPK 2 mutants improve dGTP and dATP production rates, and under the combined use of SMC mutants and dTMP-K or dTMP-K, sodium hexametaphosphate is used as a main phosphate group donor, dTTP/dCTP is used as a secondary donor, so that dTTP and dCTP production rates are improved.

Inventors

  • CHENG SHUAI
  • WU WENQING
  • HE QIN
  • LIU WEI
  • YUAN KAIJING
  • LIU LIPEI
  • YANG CHENG
  • LU XIAOYUN
  • SUN JUAN

Assignees

  • 天津中合基因科技有限公司
  • 中合基因科技(常州)有限公司

Dates

Publication Date
20260505
Application Date
20251028

Claims (14)

  1. A mutant protein of MrPPK2 is characterized in that the amino acid sequence of the mutant protein of MrPPK is SEQ ID NO. 2.
  2. 2. A biological material related to the mutant protein according to claim 1, wherein the biological material is any one of the following B1) to B4): B1 A nucleic acid molecule encoding the mutant protein of claim 1; b2 An expression cassette comprising the nucleic acid molecule of B1); B3 A recombinant vector comprising the nucleic acid molecule of B1); b4 A recombinant microorganism comprising a nucleic acid molecule according to B1).
  3. 3. The biomaterial according to claim 2, characterized in that: b3 A recombinant vector comprising the expression cassette of B2); B4 A recombinant microorganism comprising the expression cassette of B2).
  4. 4. A biomaterial according to claim 2 or 3, characterized in that: b4 Is a recombinant microorganism comprising the recombinant vector of B3).
  5. 5. The method according to claim 1, wherein the mutant protein is C1-C4; c1 As a polyphosphate kinase; c2 Catalyzing substrate dATP to synthesize dATP; c3 Catalytic substrate dGMP synthesis dGTP; c4 The yield of deoxynucleoside triphosphates is improved, wherein the deoxynucleoside triphosphates are dATP or dGTP.
  6. 6. The method according to claim 2 to 4, wherein the biological material is C2-C5: c2 Catalyzing substrate dATP to synthesize dATP; c3 Catalytic substrate dGMP synthesis dGTP; c4 The output of the deoxynucleoside triphosphate is improved, wherein the deoxynucleoside triphosphate is dATP or dGTP; c5 Preparation of polyphosphate kinase.
  7. SMC mutant, characterized in that the amino acid sequence of the SMC mutant is SEQ ID NO. 5.
  8. 8. The use of the SMC mutant according to claim 7, wherein the use is any of the following: d1 Bio-enzymatic synthesis of deoxynucleoside triphosphates; d2 Catalyzing substrate dNMP to synthesize dNTP; d3 Increasing the yield of deoxynucleoside triphosphates; the deoxynucleoside triphosphate is dCTP or dTTP; The dNMP is dTMP or dTMP; The dNTP is dCTP or dTTP; The dNTP synthesized by the catalytic substrate dNMP comprises the following steps: dTMP is catalyzed by dTMP-K to synthesize dTMP, the SMC mutant catalyzes dTMP to synthesize dTMP, or dTMP is catalyzed by dTMP-K to synthesize dCTP, the SMC mutant catalyzes dTMP to synthesize dCTP; the amino acid sequence of dTMP-K is SEQ ID NO. 7; the amino acid sequence of dCMP-K is SEQ ID NO. 8.
  9. 9. A method for synthesizing dATP by a biological enzyme method is characterized by comprising the following steps of taking sodium hexametaphosphate as a phosphate group donor, and catalyzing substrate dATP by using the mutant protein according to claim 1 to realize the synthesis of dATP.
  10. 10. A method for synthesizing dGTP by a biological enzyme method is characterized by comprising the following steps of taking sodium hexametaphosphate as a phosphate group donor, catalyzing a substrate dGMP by using the mutant protein according to claim 1, and realizing synthesis of dGTP.
  11. 11. A method for improving the yield of dATP, which is characterized by comprising the step of catalyzing substrate dATP by using the mutant protein according to claim 1 by using sodium hexametaphosphate as a phosphate group donor to improve the yield of dATP.
  12. 12. A method for increasing dGTP production, characterized in that the method comprises the step of catalyzing substrate dGMP by using the mutant protein as claimed in claim 1 by using sodium hexametaphosphate as a phosphate group donor to realize the increase of dGTP production.
  13. 13. A method for synthesizing deoxynucleoside triphosphate by a biological enzyme method is characterized by comprising the following steps of dATP synthesis, dGTP synthesis, dTTP synthesis and dCTP synthesis; The method for synthesizing dATP comprises the steps of taking sodium hexametaphosphate as a phosphate group donor, catalyzing substrate dATP by using the mutant protein according to claim 1, and realizing synthesis of dATP; the step of synthesizing dGTP comprises the steps of taking sodium hexametaphosphate as a phosphate group donor, catalyzing a substrate dGMP by using the mutant protein of claim 1, and realizing synthesis of dGTP; The step of synthesizing dTTP comprises the steps of taking sodium hexametaphosphate as a phosphate group donor and dTTP as a secondary donor, and synthesizing dTTP by using the SMC mutant as claimed in claim 7 and dTMP-K catalytic substrate dTMP shown in SEQ ID NO. 7; The step of synthesizing dCTP comprises the step of synthesizing dCTP by using sodium hexametaphosphate as a phosphate group donor and dCTP as a secondary donor and using the SMC mutant as claimed in claim 7 and dCMP-K catalytic substrate dCMP as shown in SEQ ID NO. 8.
  14. 14. A method for improving the output of deoxynucleoside triphosphates is characterized by comprising the following steps of improving the output of dATP, improving the output of dGTP, improving the output of dTTP and improving the output of dCTP, and realizing the improvement of the output of deoxynucleoside triphosphates; The method for improving the dATP yield comprises the steps of taking sodium hexametaphosphate as a phosphate group donor, and catalyzing substrate dATP by using the mutant protein according to claim 1 to improve the dATP yield; The step of increasing dGTP yield comprises using sodium hexametaphosphate as a phosphate group donor, catalyzing substrate dGMP with the mutant protein of claim 1 to increase dGTP yield; The step of increasing dTTP yield comprises using sodium hexametaphosphate as a phosphate group donor and dTTP as a secondary donor, and using the SMC mutant as claimed in claim 7 and dTMP-K catalytic substrate dTMP shown in SEQ ID NO.7 to realize increasing dTTP yield; the step of increasing the productivity of dCTP comprises using sodium hexametaphosphate as a phosphate group donor and dCTP as a secondary donor, and using the SMC mutant as claimed in claim 7 and dCMP-K catalytic substrate dCMP as shown in SEQ ID NO.8 to achieve the improvement of dCTP productivity.

Description

Preparation method and application of natural nucleotide Technical Field The invention belongs to the technical field of bioengineering, and particularly relates to a preparation method and application of natural nucleotide. Background DNTPs represent deoxyribonucleotide triphosphates, and are molecules indispensable in DNA replication and repair processes. Each dNTP molecule consists of one phosphate group, one deoxyribose and one nitrogenous base. dNTPs can be classified into four types, dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate) and dCTP (deoxycytosine triphosphate), depending on the base. Among them, dATP and dGTP belong to purines, and dTTP and dCTP belong to pyrimidines. dNTPs play an important role in the field of bioscience, and are mainly used as raw materials for DNA replication and repair. In molecular biology research, dNTPs are the basis of experimental techniques such as PCR (polymerase chain reaction) for amplifying specific DNA fragments. dNTPs are key components in the biotechnology industry for synthesizing DNA fragments, gene editing and producing genetically engineered products. The traditional nucleotide synthesis method mainly comprises four methods of a nucleic acid degradation method, a chemical synthesis method, a microorganism direct fermentation method and an enzyme method. The method starts in the 60 th century of 20, and Japanese scientists degrade RNA by using 5' -phosphodiesterase to obtain nucleotide for the first time, thus opening the way of industrial production of nucleotide. The specific process comprises extracting RNA from yeast, degrading into a mixture of four nucleotides by nuclease P1, and separating and purifying to obtain nucleotide monomers. The method has the problems of longer production period, lower product purity, low fermentation level and low enzymolysis conversion rate. Chemical synthesis method this method uses phosphorus oxychloride (POCl 3) as phosphate donor, and uses nucleoside or deoxynucleoside dissolved in triethyl phosphate (TEP) or trimethyl phosphate (TMP), then adds POCl3 to make the phosphate group transferred to nucleoside or deoxynucleoside, and finally uses hydrolysis to obtain the nucleotide. The yield of the chemical synthesis method is higher, can reach more than 90 percent, and is more efficient than the nucleic acid degradation method. However, the method has the defects that a large amount of hydrochloric acid is accumulated in the production process, purine nucleotides (such as 5'-dGMP and 5' -dAMP) can be degraded, the used chemical reagent is expensive, the production cost is increased, and TEP, TMP and POCl3 used in the reaction are toxic reagents, so that the environmental pollution is caused easily due to improper treatment. Microorganism direct fermentation, a method for producing nucleotides directly by fermentation using a selected strain. The current more successful application is the production of inosinic acid (5' -IMP). For example, furuya et al have isolated a mutant of Brevibacterium ammoniagenes insensitive to Mn 2+ and have promoted accumulation of IMP by adjusting the concentration of Mn 2+ in fermentation media containing sufficient adenine. Nevertheless, the direct fermentation method still faces the problem that it is difficult to break the cell membrane barrier of the microorganism, and the natural enzyme system existing in the microorganism cell may degrade the nucleotide, making the direct fermentation method difficult to produce the nucleotide. The enzymatic synthesis method has mild reaction conditions, high speed, high conversion rate and less pollution, is the most suitable industrial production method for development at present, but is unfavorable for industrial production because the required energy donor ATP is expensive, the cost is higher, and more AMP and ADP byproducts are generated. Disclosure of Invention The technical problem solved by the invention is how to adopt a method for synthesizing deoxyribonucleoside triphosphates by biological enzymes. In order to solve the above technical problems, the first aspect of the present invention provides MrPPK a mutant protein, which is any one of the following: A1 The mutant protein shown in SEQ ID NO. 1 comprises the steps of mutating 93 rd and 127 th amino acid residues on the amino acid sequence shown in SEQ ID NO. 1, wherein the amino acid residues at other positions are unchanged, so as to obtain the protein with polyphosphatase activity; A2 A protein having more than 80% identity and the same function as the mutant amino acid residues in the protein shown in A1); A3 The mutant protein shown in the specification) comprises a protein shown in the sequence obtained by connecting a label at the N end or/and the C end of the protein shown in the specification A1) or A2). In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences c