2'-deoxycytidine-5'-monophosphate and enzymatic synthesis method therefor

Abstract

2'-deoxycytidine-5'-monophosphate and an enzymatic synthesis method therefor, relating to the technical field of biosynthesis. The synthesis method comprises: constructing a first engineered strain and a second engineered strain; respectively carrying out fermentation culture and protein separation and purification on the first engineered strain and the second engineered strain to obtain high-purity deoxycytidine kinase and high-purity polyphosphate kinase; using 2'-deoxycytidine as a deoxycytidine substrate and adenosine triphosphate as a phosphate donor, cyclically feeding polyphosphate, and keeping the ratio of adenosine triphosphate to polyphosphate to a preset ratio to form an enzymatic reaction system; and performing an enzymatic reaction to obtain 2'-deoxycytidine-5'-monophosphate. High-concentration deoxycytidine monophosphate can be quickly and effectively produced, so that pollution hazards caused by a chemical synthesis method can be solved, and the input amount of adenosine triphosphate as a phosphate donor in a biosynthesis system can also be reduced, thereby reducing production costs.

Description

2'-deoxycytidine monophosphate and enzymatic synthesis method thereof Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and in particular relates to 2'-deoxycytidine monophosphate and an enzymatic synthesis method thereof. Background Art

[0002] Nucleotides are small molecule compounds composed of three substances: purine or pyrimidine bases, ribose or deoxyribose, and phosphate. They form the material basis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), the genetic material of organisms. The close connection between nucleotides and DNA and RNA suggests the significant role of nucleotide compounds in biological genetic metabolism. In the field of scientific research, polymerase chain reaction (PCR) technology has made unprecedented contributions to all aspects of life sciences since its inception, rapidly driving the development of biotechnology. Deoxynucleoside triphosphates are the material foundation of PCR technology. Deoxynucleoside monophosphates are one of the essential raw materials for the synthesis of deoxynucleoside triphosphates. Currently, chemical synthesis is generally used to synthesize deoxynucleoside monophosphates, but this method not only poses certain hazards to humans and the environment, but also sometimes results in low nucleotide yields. To alleviate these issues, biosynthesis can be used. This method not only effectively reduces raw material costs and increases nucleotide production in the manufacturing industry, but also helps alleviate the environmental pollution, high raw material costs, and low product yields associated with chemical synthesis, thus having important implications for sustainable social development. Therefore, it is more suitable for industrial-scale production processes and has promising application prospects.

[0003] The presence of various deoxynucleoside kinases in organisms is a key factor influencing the enzymatic synthesis of deoxynucleotides using deoxynucleosides as substrates. Related research has shown that deoxynucleoside kinases from Drosophila melanogaster play an important role in the synthesis of deoxynucleotides and their analogs, exhibiting excellent catalytic activity towards deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine, converting them into their corresponding deoxynucleoside monophosphates. However, the synthesis of deoxynucleosides from deoxynucleoside monophosphates catalyzed by deoxynucleoside kinases primarily utilizes adenosine triphosphate (ATP) as the phosphate donor, adenosine triphosphate being expensive, significantly increasing production costs.

[0004] Studies have shown that the use of a phosphate donor regeneration system can effectively reduce the amount of phosphate donors in the supply system, such as acetyl phosphate, phosphoenolpyruvate, adenosine triphosphate, and guanosine triphosphate. This allows for the enzymatic synthesis of products such as glucose hexaphosphate, L-alanyl-L-glutamine (Ala-Gln), and N1-methyl-pseudouridine monophosphate, significantly reducing production costs. Among the various phosphate donors, polyphosphates are relatively simple to prepare, offer excellent stability, and are relatively inexpensive, thus attracting widespread attention from researchers.

[0005] Existing methods utilize a polyphosphate-based phosphate donor regeneration system, employ unpurified crude enzyme solution, and achieve enzymatic synthesis of N1-methyl-pseudouridine monophosphate via a single-feed method. However, this method suffers from low substrate conversion rates and product purity, long reaction times, and a high concentration of impurities.

[0006] Summary of the Invention

[0007] To solve the above problems, the present invention provides an enzymatic synthesis method of 2'-deoxycytidine monophosphate, comprising:

[0008] The first engineered strain was constructed based on the deoxycytidine kinase from Drosophila melanogaster; and the second engineered strain was constructed based on the polyphosphate kinase from Sphingobacterium siyangensis;

[0009] Fermenting and culturing the first engineered strain and the second engineered strain and separating and purifying the protein, respectively, to obtain high-purity deoxycytidine kinase corresponding to the first engineered strain and high-purity polyphosphate kinase corresponding to the second engineered strain;

[0010] An enzymatic reaction system is formed by using 2'-deoxycytidine as a deoxycytidine substrate, adenosine triphosphate as a phosphate donor, and cyclically feeding polyphosphate as a phosphate donor substrate while maintaining a ratio of adenosine triphosphate to polyphosphate at a preset ratio;

[0011] In the enzymatic reaction system, the deoxycytidine substrate is contacted with the high-purity deoxycytidine kinase and the high-purity polyphosphate kinase to perform an enzymatic reaction to obtain the 2'-deoxycytidine monophosphate;

[0012] Preferably, the preset ratio is (1-200):100.

[0013] Preferably, the first engineered strain is constructed by expressing codon-optimized deoxycytidine kinase derived from Drosophila melanogaster in Escherichia coli;

[0014] The second engineered strain is constructed by expressing the codon-optimized polyphosphate kinase derived from Sphingobacterium siyangensis in Escherichia coli.

[0015] Preferably, the Escherichia coli includes any one or more of E. coli BL21 (DE3) strain, E. coli Rosetta strain and E. coli OrigamiB (DE3) strain.

[0016] Preferably, the amino acid sequence of the deoxycytidine kinase is as shown in SEQ ID NO: 1;

[0017] The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO: 3;

[0018] Preferably, the nucleotide sequence of the deoxycytidine kinase is shown in SEQ ID NO: 2;

[0019] The nucleotide sequence of the polyphosphate kinase is shown in SEQ ID NO: 4.

[0020] Preferably, the construction method of the first engineered bacteria and the second engineered bacteria includes:

[0021] The deoxycytidine kinase encoding gene based on Drosophila melanogaster and the polyphosphate kinase encoding gene based on Sphingobacterium siyangensis are cloned into plasmid vectors respectively, and then the plasmids are transferred into host cells by any one of heat shock method, chemical transformation method and electroporation method.

[0022] Preferably, the plasmid vector is a vector suitable for expression in Escherichia coli;

[0023] Preferably, 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(+).

[0024] Preferably, in the enzymatic reaction system, the concentration of 2'-deoxycytidine is 1 mM-500 mM;

[0025] The concentration of the adenosine triphosphate is 1mM-20mM;

[0026] 0.1 g-10 g of the high-purity deoxycytidine kinase and 0.1 g-10 g of the high-purity polyphosphate kinase are added to each 1 L of the enzymatic reaction system.

[0027] Preferably, in the enzymatic reaction system, the initial concentration of polyphosphate is 1 mM-50 mM;

[0028] During the enzymatic reaction, polyphosphate is added once at a preset time interval so as to maintain a preset ratio of adenosine triphosphate to polyphosphate;

[0029] Preferably, the preset ratio is (1-200):100;

[0030] Preferably, the polyphosphate is added 1 to 10 times;

[0031] Preferably, during the enzymatic reaction, after the 20th minute of reaction time, the preset time is started to be added, and the preset time interval for the first four times is 10 minutes; thereafter, the preset time interval for each time is 30 minutes; 1 mL-50 mL of polyphosphate is added each time;

[0032] Preferably, the added concentration of the polyphosphate is 1 mM-15 mM.

[0033] Preferably, the enzymatic reaction system further comprises an additive;

[0034] Preferably, the additive is one of magnesium chloride or magnesium sulfate;

[0035] Preferably, the additive is added at a concentration of 1 mM-100 mM.

[0036] Preferably, the reaction conditions of the enzymatic reaction include:

[0037] Reaction temperature: 35°C-37°C;

[0038] pH: 7.0-7.4;

[0039] Reaction time: 2 hours to 4 hours.

[0040] In addition, to solve the above problems, the present invention also provides a 2'-deoxycytidine monophosphate, which is prepared by the enzymatic synthesis method of 2'-deoxycytidine monophosphate as described above.

[0041] The present invention provides 2'-deoxycytidine monophosphate and an enzymatic synthesis method thereof. The enzymatic synthesis method comprises: constructing a first engineered strain based on deoxycytidine kinase from Drosophila melanogaster; and constructing a second engineered strain based on polyphosphate kinase from Sphingobacterium siyangensis; fermenting and purifying the first and second engineered strains, respectively. High-purity deoxycytidine kinase corresponding to the first engineered strain and high-purity polyphosphate kinase corresponding to the second engineered strain are obtained; 2'-deoxycytidine is used as a deoxycytidine substrate, adenosine triphosphate is used as a phosphate donor, and polyphosphate as a phosphate donor substrate is cyclically fed while maintaining a preset ratio of adenosine triphosphate to polyphosphate to form an enzymatic reaction system; in the enzymatic reaction system, the deoxycytidine substrate is contacted with the high-purity deoxycytidine kinase and the high-purity polyphosphate kinase to carry out an enzymatic reaction to obtain the 2'-deoxycytidine monophosphate; the present invention is the first to synthesize high-concentration 2'-deoxycytidine monophosphate through a one-pot method using deoxycytidine kinase and polyphosphate kinase, thereby enabling rapid and efficient production of high-concentration deoxycytidine monophosphate, thereby eliminating the pollution hazards caused by chemical synthesis methods and reducing the input amount of adenosine triphosphate as a phosphate donor in the biosynthesis system, thereby lowering production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG1 is a schematic flow diagram of the enzymatic synthesis method of 2′-deoxycytidine monophosphate of the present invention;

[0043] FIG2 is a schematic diagram of a pathway for biosynthesizing high-concentration deoxynucleoside monophosphate in the enzymatic synthesis method of 2'-deoxycytidine monophosphate of the present invention (wherein Poly(P)n and Poly(P)n-1 are polyphosphates);

[0044] FIG3 is a schematic diagram of protein expression of DNK and PPK2 in Example 2 of the present invention (lane 2: expression of DNK protein; lane 3: expression of PPK2 protein);

[0045] FIG4 is a schematic diagram showing the results of thin-layer chromatography detection of substrate consumption and product generation during the synthesis of 2'-deoxycytidine monophosphate in Example 3 of the present invention (dC is 2'-deoxycytidine, representing the substrate; dCMP is 2'-deoxycytidine monophosphate, representing the product);

[0046] FIG5 is a diagram of the reaction process detected by high performance liquid chromatography in Example 3 of the present invention (comparison of UV signals of substrate and product, wherein dC is 2'-deoxycytidine, representing the substrate; dCMP is 2'-deoxycytidine monophosphate, representing the product);

[0047] FIG6 is a schematic diagram showing the specific results of the HPLC analysis of substrate consumption and product formation in Example 3 of the present invention (the results shown in the figure are three biological replicates).

[0048] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] The following are some of the names, numbers and their meanings used in this invention:

[0051] dNK, DNK, DmdNK, dnk, deoxynucleoside kinase, representative kinases - nucleoside kinases from Drosophila melanogaster.

[0052] PPK2, ppk2, polyphosphate kinase representative kinase - polyphosphate kinase from Sphingobacterium siyangensis.

[0053] dCMP (2'-Deoxycytidine-5'-monophosphate) is a nucleotide, which means 2'-deoxycytidine monophosphate in Chinese. It is a monophosphate formed by deoxycytidine (deoxycytidine) combining with phosphate.

[0054] Referring to FIG1 , the present invention provides an enzymatic synthesis method of 2′-deoxycytidine monophosphate, comprising:

[0055] Step S1, constructing a first engineered strain based on deoxycytidine kinase from Drosophila melanogaster; and constructing a second engineered strain based on polyphosphate kinase from Sphingobacterium siyangensis;

[0056] Step S2, fermenting and culturing the first engineered strain and the second engineered strain and separating and purifying the protein, respectively, to obtain high-purity deoxycytidine kinase corresponding to the first engineered strain and high-purity polyphosphate kinase corresponding to the second engineered strain;

[0057] Step S3, using 2'-deoxycytidine as a deoxycytidine substrate, adenosine triphosphate as a phosphate donor, and cyclically feeding polyphosphate as a phosphate donor substrate to maintain a preset ratio of adenosine triphosphate to polyphosphate, thereby forming an enzymatic reaction system;

[0058] Step S4: contacting the deoxycytidine substrate with the high-purity deoxycytidine kinase and the high-purity polyphosphate kinase in the enzymatic reaction system to perform an enzymatic reaction to obtain the 2'-deoxycytidine monophosphate.

[0059] Wherein, the preset ratio is (1-200):100. For example, it can be 1:100, 10:100, 20:100, 30:100, 50:100, 70:100, 80:100, 100:100, 120:100, 150:100, 160:100, 180:100, 200:100 or the like. It should be noted that the multiple deoxynucleoside kinases present in organisms are one of the key influencing factors for the enzymatic synthesis of deoxynucleotides using deoxynucleosides as substrates. In recent years, research on deoxynucleoside kinases has emerged in an endless stream, and most of them are mainly studied around enzymatic properties, metabolism and regulatory mechanisms. However, research and reports on deoxynucleotide synthesis, especially large-scale synthesis, are relatively few. Related studies have shown that deoxynucleoside kinases from insects play an important role in the synthesis of deoxynucleotides and analogs. They have good catalytic activity for deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine, and can convert them into the corresponding deoxynucleoside monophosphates.

[0060] The aforementioned studies were all conducted at the laboratory level; currently, there are no reports of industrial-scale biosynthesis of deoxynucleoside monophosphates. Based on these research findings and the potential for industrial application, improving the efficiency and product purity of biosynthesis of high-concentration deoxynucleoside monophosphates has significant economic and environmental implications.

[0061] In the present invention, a first engineered strain is constructed using deoxycytidine kinase derived from Drosophila melanogaster; and a second engineered strain is constructed based on polyphosphate kinase derived from Sphingobacterium siyangensis. The expression products of the two strains after cultivation are reduced by a dual-bacteria-dual-enzyme method in the reaction system, thereby reducing the input amount of phosphate donors such as adenosine triphosphate in the reaction system, thereby reducing the cost of synthesizing high-concentration 2'-deoxycytidine monophosphate.

[0062] The present invention provides a one-pot enzymatic method for the biosynthesis of high-concentration 2'-deoxycytidine monophosphate. The strategy comprises constructing an enzyme-expressing recombinant bacterium, fermenting and purifying the recombinant strain, and then cyclically feeding polyphosphate to maintain the enzymatic reaction system for efficient synthesis of high-purity deoxycytidine kinase and polyphosphate kinase and 2'-deoxycytidine. The enzymatic reaction time is less than 3 hours, the substrate conversion rate is close to 100%, and the reaction system is relatively clean, with no detected impurities.

[0063] The synthesis method provided by the present invention, and its reaction principle, refer to Figure 2 (Pathway for the Biosynthesis of High-Concentration Deoxynucleoside Monophosphates). The present invention utilizes a dual-bacterium-dual-enzyme combination to rapidly and efficiently produce high-concentration deoxycytidine monophosphate. This method can be used to efficiently catalyze the production of high-concentration 2'-deoxycytidine monophosphate from 2'-deoxycytidine and polyphosphate. This method not only addresses the pollution hazards associated with chemical synthesis but also significantly reduces the input of the expensive phosphate donor, adenosine triphosphate, in the reaction system, significantly lowering production costs. Furthermore, the method achieves high product recovery and purity, demonstrating significant potential for application.

[0064] Furthermore, the first engineered strain is constructed by expressing a codon-optimized deoxycytidine kinase derived from Drosophila melanogaster in Escherichia coli;

[0065] The second engineered strain is constructed by expressing the codon-optimized polyphosphate kinase derived from Sphingobacterium siyangensis in Escherichia coli.

[0066] As mentioned above, in the present invention, deoxycytidine kinase (dNK) from Drosophila melanogaster and polyphosphate kinase (PPK2) from Sphingobacterium siyangensis are expressed in Escherichia coli, respectively. To this end, the genes encoding these two enzymes, dNK and PPK2, are integrated into the Escherichia coli genome by plasmid transformation technology to obtain recombinant bacteria.

[0067] As mentioned above, the terms "recombinant bacteria" and "(genetically) engineered bacteria (strains)" herein have the same meaning, both referring to the deoxycytidine kinase and polyphosphate kinase or expression strains that have been genetically modified.

[0068] Furthermore, the Escherichia coli includes any one or more of E. coli BL21 (DE3) strain, E. coli Rosetta strain and E. coli OrigamiB (DE3) strain.

[0069] Furthermore, the amino acid sequence of the deoxycytidine kinase is shown in SEQ ID NO: 1;

[0070] The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO: 3.

[0071] In the above-mentioned one-pot enzymatic high-concentration 2'-deoxycytidine monophosphate synthesis pathway, an adenosine triphosphate cycle pathway is introduced to reduce the amount of adenosine triphosphate added, thereby simultaneously improving the synthesis efficiency of deoxycytidine monophosphate and reducing production costs. The deoxynucleoside kinase can be dNK, NCBI accession number Dmel_CG5452, and the polyphosphate kinase can be PPK2, NCBI accession number IQ31_RS01295.

[0072] It should be noted that the deoxycytidine kinase and polyphosphate kinase that can be expressed in the E. coli engineered bacteria of the present invention are not limited to Dmel_CG5452 and IQ31_RS01295, but can also be any enzyme type that is used for deoxycytidine monophosphate synthesis and performs the function of deoxycytidine kinase and any enzyme type that is used for the adenosine triphosphate cycle pathway and performs the function of polyphosphate kinase, and there is no limitation on the source.

[0073] The deoxycytidine kinase and polyphosphate kinase are both exogenous and can be derived from Drosophila melanogaster and Sphingobacterium siyangensis, or from other microbial species.

[0074] In step S1, when exogenous deoxynucleoside kinases and / or polyphosphate kinases derived from other microbial species are expressed in engineered Escherichia coli, the expression genes of these enzymes may be codon-optimized in order to optimally express these exogenous enzymes in the strain.

[0075] Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of the gene of interest. Different organisms often show a particular preference for one of several codons encoding the same amino acid due to mutational propensity and natural selection.

[0076] For example, in fast-growing microorganisms, the optimized codons reflect the composition of their respective genomic tRNA pools. Thus, in fast-growing microorganisms, a low-frequency codon for an amino acid can be replaced with a high-frequency codon for the same amino acid.

[0077] Thus, expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0078] For ease of description, the terms "dnk" and "deoxycytidine kinase" are sometimes used interchangeably in this document, such as for example, deoxycytidine kinase (DNK) and its encoding gene (DNA). These terms are understood by those skilled in the art to represent different substances in different contexts. These terms are readily understood by those skilled in the art based on the context and language. For example, when describing the functions or classes of deoxycytidine kinase and polyphosphate kinase, the terms "Dmel_CG5452" and "IQ31_RS01295" refer to proteins; when describing a gene, the terms "dnk" and "deoxycytidine kinase" refer to the genes encoding the enzymes.

[0079] This study, published in the journal Nature Communications, for the first time, discovered that introducing the ATP cycle pathway—the ppk2 gene—can both reduce the amount of ATP added and accelerate the reaction process. Furthermore, through system optimization, the substrate is completely converted into the desired product without the generation of byproducts. Furthermore, the amount of ATP added in the 2'-deoxycytidine monophosphate synthesis system is significantly reduced, thereby reducing production costs. This research finding provides new insights and strategies for the modification of biosynthetic deoxynucleoside monophosphates, potentially leading to more efficient product synthesis.

[0080] Furthermore, the nucleotide sequence of the deoxycytidine kinase is shown in SEQ ID NO: 2;

[0081] The nucleotide sequence of the polyphosphate kinase is shown in SEQ ID NO: 4.

[0082] Furthermore, the construction method of the first engineered bacteria and the second engineered bacteria includes:

[0083] The deoxycytidine kinase encoding gene based on Drosophila melanogaster and the polyphosphate kinase encoding gene based on Sphingobacterium siyangensis are cloned into plasmid vectors respectively, and then the plasmids are transferred into host cells by any one of heat shock method, chemical transformation method and electroporation method.

[0084] It should be noted that, in the present invention, the method for transferring the plasmid into the host cell adopts heat shock method, chemical transformation method and electroporation method, which may specifically include the following:

[0085] (1) The heat shock method is a plasmid transformation method that is primarily applicable to bacteria. This method utilizes a brief high temperature stimulation to create temporary pores in the bacterial cell membrane, thereby increasing the chances of plasmid entry into the cell. The general steps include mixing the plasmid and cells, briefly heat shocking them at high temperatures, and then allowing the bacteria to resume growth in a culture medium.

[0086] (2) Chemical transformation is another plasmid transformation method. This method uses chemical methods to change the properties of the bacterial cell wall, enabling it to effectively absorb exogenous plasmids. The general steps include treating the plasmid and bacterial cells together, and changing the permeability of the cell wall by adding chemicals (such as calcium ions) or electric pulses to promote the entry of the plasmid into the cell.

[0087] (3) Electroporation is a transformation method that uses electric field pulses to guide plasmids into cells. This method applies high-intensity electric field pulses to create temporary pores in the bacterial cell membrane, allowing the plasmid to enter the cell. The general steps include mixing the plasmid and bacteria, applying an electric pulse, and then allowing the bacteria to resume growth in a culture medium.

[0088] Furthermore, the plasmid vector is a vector suitable for expression in Escherichia coli;

[0089] Furthermore, 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(+).

[0090] Furthermore, in the enzymatic reaction system, the concentration of 2'-deoxycytidine is 1 mM-500 mM, for example, 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, 80 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM, etc.

[0091] The concentration of the adenosine triphosphate is 1 mM-20 mM; for example, it can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 9 mM, 10 mM, 11 mM, 13 mM, 15 mM, 17 mM, 19 mM, 20 mM, etc.

[0092] 0.1 g to 10 g of the highly purified deoxycytidine kinase and 0.1 g to 10 g of the highly purified polyphosphate kinase are added to each 1 L of the enzymatic reaction system. For example, the amounts of highly purified deoxycytidine kinase and highly purified polyphosphate kinase added can be 0.1 g, 0.3 g, 0.5 g, 0.8 g, 1 g, 2 g, 3 g, 5 g, 8 g, 10 g, etc., respectively.

[0093] Furthermore, in the enzymatic reaction system, the initial concentration of polyphosphate is 1 mM-50 mM; for example, it can be 1 mM, 3 mM, 5 mM, 7 mM, 9 mM, 10 mM, 13 mM, 15 mM, 17 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, etc.

[0094] During the enzymatic reaction, polyphosphate is added once at a preset time interval so as to keep the ratio of ATP to polyphosphate constant;

[0095] Furthermore, the polyphosphate is added 1 to 10 times; for example, it can be 1 time, 2 times, 4 times, 6 times, 8 times, 9 times, 10 times, etc.

[0096] Furthermore, during the enzymatic reaction, after the 20th minute of reaction time, the preset time is added, and the preset time interval for the first four times is 10 minutes; thereafter, the preset time interval for each time is 30 minutes; 1-50 mL of polyphosphate is added each time;

[0097] Furthermore, the polyphosphate is added at a concentration of 1 mM to 15 mM, for example, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, and the like.

[0098] Furthermore, the enzymatic reaction system further includes additives;

[0099] Furthermore, the additive is one of magnesium chloride or magnesium sulfate;

[0100] Furthermore, the additive is added at a concentration of 1 mM to 100 mM, for example, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 80 mM, 100 mM, and the like.

[0101] Furthermore, the reaction conditions of the enzymatic reaction include:

[0102] Reaction temperature: 35°C-37°C; for example, 35°C, 36°C, 37°C, etc.

[0103] pH: 7.0-7.4; for example, it can be 7.0, 7.1, 7.2, 7.3, 7.4, etc.

[0104] Reaction time: 2 hours to 4 hours. For example, it can be 2 hours, 3 hours, 4 hours, etc.

[0105] In addition, the present invention also provides 2'-deoxycytidine monophosphate, which is prepared by the above-mentioned enzymatic synthesis method of 2'-deoxycytidine monophosphate.

[0106] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0107] The culture medium and reagents used in the examples are:

[0108] 1. LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride (LB solid medium plus 20 g / L agar powder);

[0109] 2. Protein purification buffer: weigh 2.4228 g of tris (hydroxymethyl)aminomethane, 37.224 g of potassium chloride, 10% glycerol, dilute to 1 L with double-distilled water, and adjust the pH to 7.9;

[0110] 3. 4-Hydroxyethylpiperazineethanesulfonic acid buffer: 200 mM 4-Hydroxyethylpiperazineethanesulfonic acid. Weigh 47.6462 g of 4-Hydroxyethylpiperazineethanesulfonic acid powder, dissolve it in a small amount of double-distilled water, and then dilute to 1 L. Adjust the pH to 7.4.

[0111] 4, 2'-deoxycytidine;

[0112] 5. Adenosine triphosphate;

[0113] 6. Magnesium chloride hexahydrate;

[0114] 7. 0.6 M polyphosphate stock solution: Weigh 367.056 g of polyphosphate powder, dissolve it in 200 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer, and dilute to 1 L.

[0115] 8. Escherichia coli BL21 (DE3) was purchased from the German Culture Collection (DSMZ).

[0116] 9. The plasmids expressing the genes DNK and / or PPK2 and the plasmids used for gene editing operations in the examples are constructed and preserved by Sangon Biotech (Shanghai) Co., Ltd. Any organization or individual may obtain the plasmids for verification of the present invention, but may not use them for other purposes, including development, utilization, scientific research, and teaching, without the permission of Sangon Biotech (Shanghai) Co., Ltd.

[0117] The plasmids expressing the genes DNK and / or PPK2 and the plasmids used for gene editing operations in the examples are constructed and preserved by Sangon Biotech (Shanghai) Co., Ltd. Any unit or individual may obtain the plasmids for verification of the present invention, but may not be used for other purposes, including development, utilization, scientific research, and teaching, without the permission of Sangon Biotech (Shanghai) Co., Ltd.

[0118] The enzymatic reaction system for the one-pot enzymatic synthesis of 2'-deoxycytidine monophosphate used in the examples is as follows:

[0119] Table 1. Reaction system

[0120] a: Z = (1000 mL - 50 mL - XY), first add an appropriate volume of 200 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer to dissolve each material, and finally fill the system to 1 L.

[0121] b: After the reaction has proceeded for 20 min, polyphosphate was added. 20 mL was added every 10 min for the first 4 times. Starting from the 5th time, 20 mL was added every 30 min for a total of 8 times. After the last addition of polyphosphate, the reaction was continued for 30-40 min to terminate the reaction.

[0122] c: Calculate based on the protein concentration of the enzyme. Assuming the protein concentration of DNK is 10 mg / mL and the final concentration of DNK added to the system is 1 mg / mL, the volume of DNK added is:

[0123] 1 (mg / mL) / 10 (mg / mL) × total reaction volume. The calculation of the amount of PPK2 added is similar.

[0124] Method for detecting the remaining 2'-deoxycytidine content and the generated 2'-deoxycytidine monophosphate content in the reaction system:

[0125] (1) Thin layer chromatography detection

[0126] Prepare a detection solution with a ratio of dichloromethane to methanol of 10:3. Take 20 μL of the initial reaction solution from the experimental group and the control group, and take 20 μL of the reaction solution from the experimental group and the control group after each addition of polyphosphate. Heat at 85°C for 5-10 minutes, centrifuge at 12,000 rpm for 1 minute, take 0.5 μL of the supernatant and spot it on a thin layer chromatography plate, place it in the detection solution, stop when the liquid level reaches 1 / 10 of the distance from the edge of the plate, take it out and dry it, and observe the results at 254 nm.

[0127] (2) High performance liquid chromatography

[0128] Take 20 μL of the initial reaction solution of the experimental group and the control group, and take 20 μL of the reaction solution of the experimental group and the control group after each addition of polyphosphate, heat at 85°C for 5-10 minutes, centrifuge at 12000 rpm for 1 minute, dilute the experimental group and the control group with ddH2O 300 times, and prepare 1 mM of the corresponding 2'-deoxycytidine and 2'-deoxycytidine monophosphate standards, and detect by high performance liquid chromatography.

[0129] Example 1:

[0130] In this example, the construction and preservation of plasmids and engineered strains were carried out.

[0131] Experimental methods and steps:

[0132] According to the DNK and PPK2 coding genes reported in the literature, the relevant gene sequences were obtained using the NCBI database, the target genes were synthesized and the recombinant strains were constructed.

[0133] (1) Codon optimization and gene synthesis were performed on the deoxycytidine kinase encoding gene dnk (SEQ ID NO: 1; SEQ ID NO: 2) from Drosophila melanogaster and the polyphosphate kinase encoding gene ppk2 (SEQ ID NO: 3; SEQ ID NO: 4) from Sphingobacterium siyangensis according to the codon preference of Escherichia coli;

[0134] (2) They were constructed into pET-21a vector respectively, and the above enzymes were expressed in Escherichia coli strain BL21 (DE3).

[0135] Example 2:

[0136] In this example, fermentation of the recombinant strain, protein expression and purification were performed.

[0137] Experimental methods:

[0138] (1) Take out the pET-21a / DNK / BL21(DE3) and pET-21a / PPK2 / BL21(DE3) strains stored at -80°C and inoculate them into 5 mL LB medium containing 100 μg / mL of LB medium at a 1% inoculum size. Incubate at 37°C for 12-16 h.

[0139] (2) After that, take 2 μL of bacterial solution and streak it on LB plate (containing 1.5% agar) containing ampicillin sodium antibiotics and culture it at 37℃ for 12-24h. When single colonies visible to the naked eye grow on the plate, pick three single colonies and inoculate them into 5mL LB medium tubes containing ampicillin sodium and culture them in a constant temperature shaking incubator at 37℃ until OD 600 = around 1.0.

[0140] (3) The above culture was inoculated into a 500 mL Erlenmeyer flask containing 200 mL of LB medium (containing 1‰ 100 mg / mL Ampicillin) at a 1% inoculum size and cultured in a constant temperature shaking incubator at 37°C for 2-3 h to an OD600 of 0.6-0.8. IPTG was added to a final concentration of 0.5 mM and induced at 16°C, 160 rpm for 16-20 h.

[0141] (4) After induction, the cells were collected by centrifugation at 8000 rpm for 10 min at 4°C, and an appropriate amount of protein purification buffer was added to resuspend the cells. The cells were then disrupted using a cell homogenizer at 800 psi for 3 to 4 times until the solution became clear.

[0142] (5) After disruption, centrifuge at 6000 g for 50 min and collect the supernatant for the next step of purification. Use 1 mL of Ni-NTA for every 1.5 g of wet weight of bacterial cells. Wash with 5 column volumes of protein purification buffer to remove the ethanol that preserves the filler. Incubate the filler and sample on ice with shaking for at least 30 min before loading onto the column and collecting the flow-through.

[0143] (6) Wash the column with 5 column volumes of 100% protein purification buffer at a flow rate to filler volume ratio of 1:1. Wash the column with 5-7.5% 1 M imidazole mixed with protein purification buffer. Elute with 25% 1 M imidazole mixed with protein purification buffer and collect the eluate.

[0144] Experimental results:

[0145] The collected fractions were identified by SDS-PAGE, and the results are shown in Figure 3.

[0146] The analysis results showed that the purity of the purified DNK could reach 99% (lane 2), and the purity of PPK2 was 94.4% (lane 3).

[0147] Example 3:

[0148] In this example, based on the above examples, a one-pot enzymatic synthesis of high-concentration 2'-deoxycytidine monophosphate was performed.

[0149] Experimental methods:

[0150] (1) Biosynthesis of high concentrations of 2'-deoxycytidine monophosphate using recombinant DNK and PPK2.

[0151] Reaction system: 1 mg / mL purified deoxycytidine kinase and polyphosphate kinase, 300 mM 2'-deoxycytidine, 20 mM adenosine triphosphate, 70 mM magnesium chloride, and 30 mM polyphosphate were added to 200 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer (pH 7.4). The reaction was carried out at 37°C. 12 mM polyphosphate was added every 10 minutes during the initial reaction and every 20 minutes thereafter until the reaction was complete. Samples were collected every 20 minutes to monitor the reaction progress.

[0152] (2) After the reaction, the reaction solution was placed in an 85°C water bath for 5 minutes and centrifuged at 12,000 rpm for 1 minute. The supernatant was used for thin-layer chromatography and high-performance liquid chromatography.

[0153] Experimental results:

[0154] Thin layer chromatography showed that after the eighth addition of polyphosphate and a further 30 min of reaction (a total of 2.5 h), 2'-deoxycytidine was completely converted into 2'-deoxycytidine monophosphate ( FIG4 ).

[0155] Furthermore, HPLC analysis revealed that after eight additional additions of polyphosphate and a further 30 minutes of reaction (a total of 2.5 hours), the presence of the substrate 2'-deoxycytidine could no longer be detected in the reaction system, while the concentration of the product 2'-deoxycytidine monophosphate increased significantly, with a substrate conversion rate of >99.5% ( Figures 5 and 6 ).

[0156] The above results indicate that by optimizing the concentrations of various substances in the initial reaction system and the method of feeding the phosphate donor, the catalytic efficiency of the dual-enzyme reaction can be effectively improved, thereby achieving a rapid and efficient one-pot synthesis of 2'-deoxycytidine monophosphate.

[0157] In summary, the synthesis method provided by the present invention, which utilizes a dual-bacterium-dual-enzyme combination, can rapidly and efficiently produce high-concentration deoxycytidine monophosphate. It can be used to efficiently catalyze the generation of high-concentration 2'-deoxycytidine monophosphate from 2'-deoxycytidine and polyphosphate. This method not only addresses the hazards associated with chemical synthesis but also significantly reduces the input of the expensive phosphate donor, adenosine triphosphate, in the reaction system, greatly reducing production costs. Furthermore, the product recovery rate and purity are both high, and the method has enormous application potential.

[0158] The above description is the preferred embodiment and corresponding examples of the present invention. It should be noted that, for those skilled in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to adjustments in proportions, processes, dosages and reaction vessels, such as the use of a continuous flow reactor, all of which fall within the scope of protection of the present invention. The above description is the preferred embodiment and corresponding examples of the present invention. It should be noted that, for those skilled in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to adjustments in proportions, processes, dosages and reaction vessels, all of which fall within the scope of protection of the present invention.

Claims

1. A method for enzymatically synthesizing 2'-deoxycytidine monophosphate, characterized in that: include: The first engineered strain was constructed based on deoxycytidine kinase from Drosophila melanogaster; and constructing a second engineered strain based on the polyphosphate kinase from Sphingobacterium siyangensis; The first engineering strain and the second engineering strain are subjected to fermentation culture and protein separation and purification respectively to obtain high-purity deoxycytidine kinase corresponding to the first engineering strain and high-purity polyphosphate kinase corresponding to the second engineering strain; An enzymatic reaction system is formed by using 2'-deoxycytidine as a deoxycytidine substrate, adenosine triphosphate as a phosphate donor, and cyclically feeding polyphosphate as a phosphate donor substrate and maintaining the ratio of adenosine triphosphate to polyphosphate at a preset ratio; In the enzymatic reaction system, the deoxycytidine substrate is contacted with the high-purity deoxycytidine kinase and the high-purity polyphosphate kinase to perform an enzymatic reaction to obtain the 2'-deoxycytidine monophosphate; Preferably, the preset ratio is (1-200):

100.

2. The method for enzymatic synthesis of 2'-deoxycytidine monophosphate according to claim 1, characterized in that: The first engineered strain is constructed by expressing codon-optimized deoxycytidine kinase derived from Drosophila melanogaster in Escherichia coli; The second engineered strain is constructed by expressing the codon-optimized polyphosphate kinase derived from Sphingobacterium siyangensis in Escherichia coli; Preferably, the Escherichia coli includes any one or more of E. coli BL21 (DE3) strain, E. coli Rosetta strain and E. coli OrigamiB (DE3) strain.

3. The method for enzymatic synthesis of 2'-deoxycytidine monophosphate according to claim 1, characterized in that: The amino acid sequence of the deoxycytidine kinase is shown in SEQ ID NO: 1; The amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO: 3; Preferably, the nucleotide sequence of the deoxycytidine kinase is as shown in SEQ ID NO: 2; The nucleotide sequence of the polyphosphate kinase is shown in SEQ ID NO:

4.

4. The method for enzymatic synthesis of 2'-deoxycytidine monophosphate according to claim 1, characterized in that: The construction method of the first engineering bacteria and the second engineering bacteria comprises: The deoxycytidine kinase encoding gene based on Drosophila melanogaster and the polyphosphate kinase encoding gene based on Sphingobacterium siyangensis are cloned into plasmid vectors respectively, and then the plasmids are transferred into host cells by any one of heat shock method, chemical transformation method and electrotransformation method.

5. The method for enzymatic synthesis of 2'-deoxycytidine monophosphate according to claim 4, characterized in that: The plasmid vector is a vector suitable for expression in Escherichia coli; Preferably, 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(+).

6. The method for enzymatic synthesis of 2'-deoxycytidine monophosphate according to claim 1, characterized in that: In the enzymatic reaction system, the concentration of 2'-deoxycytidine is 1 mM-500 mM; The concentration of adenosine triphosphate is 1mM-20mM; 0.1 g-10 g of the high-purity deoxycytidine kinase and 0.1 g-10 g of the high-purity polyphosphate kinase are added to each 1 L of the enzymatic reaction system.

7. The method for enzymatic synthesis of 2'-deoxycytidine monophosphate according to claim 1, characterized in that: In the enzymatic reaction system, the initial concentration of polyphosphate is 1 mM-50 mM; During the enzymatic reaction, polyphosphate is added once at a preset time interval so as to maintain a preset ratio of adenosine triphosphate to polyphosphate; Preferably, the preset ratio is (1-200): 100; Preferably, the polyphosphate is added 1 to 10 times; Preferably, during the enzymatic reaction, after the 20th minute of reaction time, the preset time starts to be added, and the preset time interval for the first 4 times is 10 minutes; thereafter, the preset time interval for each time is 30 minutes; 1 mL-50 mL of polyphosphate is added each time; Preferably, the polyphosphate is added at a concentration of 1 mM-15 mM.

8. The method for enzymatic synthesis of 2'-deoxycytidine monophosphate according to claim 1, characterized in that: The enzymatic reaction system further includes additives; Preferably, the additive is one of magnesium chloride or magnesium sulfate; Preferably, the additive is added at a concentration of 1 mM-100 mM.

9. The method for enzymatic synthesis of 2'-deoxycytidine monophosphate according to claim 1, characterized in that: The reaction conditions of the enzymatic reaction include: Reaction temperature: 35℃-37℃; pH: 7.0-7.4; Reaction time: 2 hours to 4 hours.

10. A 2'-deoxycytidine monophosphate, characterized in that The 2'-deoxycytidine monophosphate is prepared by the enzymatic synthesis method of any one of claims 1 to 9.

Images