A method for synthesizing pseudouridine by chemical enzymatic method

Abstract

The present invention discloses a method for synthesizing pseudouridine by chemical enzymatic method, wherein ribose-5-phosphate and pseudouridine-5-phosphoglycosidase mutant are used to synthesize pseudouridine monophosphate, and pseudouridine is obtained after dephosphorylation and separation; the amino acid sequence of the pseudouridine-5-phosphoglycosidase mutant is shown in SEQ ID NO: 1. The present invention synthesizes pseudouridine by chemical-enzymatic reaction, realizes mass production of pseudouridine at a relatively low cost through simple reaction, and promotes the application of industrial synthesis of pseudouridine.

Description

Technical Field

[0001] The invention relates to the technical field of pseudouridine synthesis, and in particular to a method for synthesizing pseudouridine using a chemical enzyme method. Background Art

[0002] In the middle of the last century, researchers discovered that there is an isomer similar to uridine in ribosomal RNA (rRNA) and transfer RNA (tRNA), in which the ribose is not connected to the N1 of uracil (U), but to C5, forming pseudouridine (ψ), which is abbreviated as pseudouridine. In the body, the uridine on RNA is cleaved by pseudouridine synthetase to form a CC bond between the ribose and the base, isomerizing into pseudouridine. The structural formula of pseudouridine is shown in formula (I).

[0003]

[0004] Later studies found that snRNA and other non-coding RNAs with high intracellular abundance are rich in pseudouridine modifications. With the development of sequencing technology in recent years, it has been found that mRNA is also rich in pseudouridine modifications. Studies have found that pseudouridine modification of mRNA can not only reduce the immune response caused by mRNA, but also improve the translation efficiency of coding genes. In recent years, there have been many successful cases in the research and development of nucleic acid drugs, especially mRNA vaccines and drugs. In all these mRNAs, pseudouridine is used instead of the original uridine. Therefore, the demand for pseudouridine is increasing, and its synthesis method has attracted widespread attention.

[0005] At present, the preparation of pseudouridine mainly relies on chemical synthesis, but the chemical method has problems such as long synthesis steps and low yield, and a large number of flammable and explosive reagents are used in the synthesis process. In terms of biosynthesis, some researchers use engineered Escherichia coli to express exogenous synthetic pathways to synthesize pseudouridine, but there are problems such as long culture cycle and complicated purification process. Therefore, there are certain defects in the rapid and convenient synthesis of pseudouridine in large quantities.

[0006] In view of the problems of long synthesis steps, low yield, and the use of more flammable and explosive reagents in chemical synthesis, and the long culture cycle and complex purification process in Escherichia coli engineering bacteria synthesis, it is necessary to seek a method for synthesizing pseudouridine through chemical-enzymatic reactions, so as to achieve mass production of pseudouridine at a lower cost through simple reactions. Summary of the invention

[0007] The purpose of the present invention is to provide a method for synthesizing pseudouridine by chemical enzymatic method, so as to at least solve one of the many defects existing in the prior art.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for synthesizing pseudouridine by chemical enzymatic method, comprising synthesizing pseudouridine monophosphate by ribose-5-phosphate and a pseudouridine-5-phosphatase mutant, and then dephosphorylating and separating the pseudouridine; the amino acid sequence of the pseudouridine-5-phosphatase mutant is shown in SEQ ID NO: 1.

[0010] According to an embodiment of the present invention, the nucleotide sequence of the pseudo-UR5-phosphatase mutant is shown in SEQ ID NO:3.

[0011] According to an embodiment of the present invention, the pseudouridine 5-phosphoglycosidase mutant is obtained by mutation of the pseudouridine 5-phosphoglycosidase with an amino acid sequence as shown in SEQ ID NO: 2. Preferably, the nucleotide sequence of the pseudouridine 5-phosphoglycosidase is as shown in SEQ ID NO: 4.

[0012] According to an embodiment of the present invention, the ribose-5-phosphate is obtained by hydrolysis of AMP.

[0013] According to an embodiment of the present invention, before dephosphorylation, the protein is denatured, centrifuged to remove protein precipitation, and the supernatant is filtered repeatedly until the uracil content is zero.

[0014] Preferably, the reaction solution is heated to denature the protein before dephosphorylation, and the supernatant is filtered after centrifugation, and then nanofiltration is performed until the conductivity of the filtrate drops to 10 mS / cm.

[0015] According to an embodiment of the present invention, the dephosphorylation step is to add ethanol to the filtrate, mix well, then add NaOH and react fully.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention utilizes a chemical method to synthesize ribose-5-phosphate and uses uracil as a substrate; a pseudouridine-5-phosphatase mutant is added to enzymatically synthesize pseudouridine monophosphate; the first step reaction solution is treated to remove impurities that affect subsequent separation and purification; and pseudouridine monophosphate is dephosphorylated to obtain pseudouridine.

[0018] 2. The pseudouridine synthesized by the chemical enzyme method of the present invention has a high conversion rate, a short reaction time, a simple purification process, and can be obtained with only simple reagents, and is suitable for industrial synthesis and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is the HPLC test result after dephosphorylation of the intermediate sample taken during the synthesis of pseudouridine monophosphate reaction of the present invention.

[0020] Figure 2 These are the HPLC results of the pseudouridine standard and uracil standard of the present invention.

[0021] Figure 3 The present invention adopts the mutant R146E / H156Q / K210A chemoenzymatic method to obtain pseudouridine HPLC results. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and beneficial technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0023] The invention provides a method for synthesizing pseudouridine by a chemoenzymatic method, comprising the steps of: performing molecular docking on a wild-type pseudouridine-5-phosphoglycosidase, ribose-5-phosphate and uracil, and then selecting amino acids near an active site for mutation to obtain a pseudouridine-5-phosphoglycosidase mutant R146E / H156Q / K210A; and chemically hydrolyzing and purifying AMP to obtain ribose-5-phosphate, and reacting the AMP with uracil in the pseudouridine-5-phosphoglycosidase mutant and then purifying the AMP to obtain pseudouridine.

[0024] In the present invention, all Escherichia coli strains and plasmids used were purchased from BioVector Plasmid Vector Bacteria Cell Protein Antibody Gene Collection Center; other reagents used in the experiment were commercially available.

[0025] Example 1 Chemical Synthesis of Ribose-5-Phosphate

[0026] Dissolve 50g AMP in 3L 1M HCl, heat to 100℃, and stir to react for 1h. Then cool to room temperature, adjust the pH of the system to 8.2 with saturated KOH solution, and add isopropanol to precipitate, which is crude ribose-5-phosphate. Add ion exchange resin to the crude ribose-5-phosphate solution, elute with 0.2, 0.4, 0.6, 0.8 and 1.0M chloroacetic acid solution, purify to obtain purified ribose-5-phosphate solution, and freeze-dry to obtain 30g ribose-5-phosphate. The purity of ribose-5-phosphate was 85% using HPLC equipped with an evaporative light scattering detector.

[0027] The detection method of ribose-5-phosphate is as follows:

[0028] Column: Waters XBridge BEH Amide, 250 × 4.6 mm, 5 μm;

[0029] Column temperature: 40°C;

[0030] Flow rate: 1 mL / min;

[0031] Mobile phase A: acetonitrile;

[0032] Mobile phase B: 2 g / L ammonium acetate;

[0033] Method: 70% A + 30% B, constant gradient;

[0034] Analysis time: 25min.

[0035] Example 2 Expression, purification and mutation of pseudouridine-5-phosphatase

[0036] 1) Expression and purification of pseudouridine 5-phosphatase

[0037] Pseudouridine-5-phosphoglycosidase was derived from Dorea formicigenerns ATCC 27755. Pseudouridine-5-phosphoglycosidase optimized for Escherichia coli was synthesized, and NdeI and XhoI restriction sites were used at both ends. The target gene and pET-28a plasmid were digested with restriction endonucleases NdeI and XhoI, and the target gene fragment and vector fragment were recovered by agarose gel electrophoresis. After the ligation reaction was carried out with T4 DNA ligase, the competent Escherichia coli DH5α was transformed and coated with a plate containing kanamycin. After picking a single clone for colony PCR verification, the successfully connected strain was selected to extract the plasmid for sequencing to verify the accuracy of the target fragment sequence.

[0038] After the correctly sequenced plasmid was transformed into the BL21 (DE3) strain, a single clone was picked and cultured overnight in an LB test tube. The next day, 200 mL of LB medium was inoculated at 1:100, cultured at 37 ° C for 6 hours, and inoculated into a 5L fermenter. When the OD was close to 30 at 37 ° C, the temperature was lowered to 20 ° C, 0.2 mM IPTG was added, and the fermentation was removed from the tank after induction for 12 hours. After the bacteria were collected by centrifugation at 7000g, they were resuspended in buffer, and the recombinant protein was released after high-pressure crushing. The target protein was purified by nickel column affinity chromatography.

[0039] 2) Selection of mutation sites of pseudouridine 5-phosphatase

[0040] According to the protein structure numbered 4GIM in PDB Bank, the protein structure of pseudouridine-5-phosphatase was established by homology modeling, and then molecular docking was performed with ribose-5-phosphate and uracil, and amino acids near the active site were selected for mutation to obtain the mutant R146E / H156Q / K210A (representing mutations of the 146th, 156th and 210th amino acids, respectively). The mutant gene synthesis, protein expression and purification were the same as the wild type.

[0041] The amino acid sequence of the pseudouridine 5-phosphatase mutant R146E / H156Q / K210A is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 3.

[0042] The amino acid sequence of pseudouridine-5-phosphatase is shown in SEQ ID NO:2, and the nucleotide sequence is shown in SEQ ID NO:4.

[0043] Example 3 Synthesis of pseudouridine monophosphate

[0044] 1) Synthesis

[0045] Weigh 220.8 g of ribose-5-phosphate and 4.8 g of MnCl2·4H2O, and dissolve them in 5 L of deionized water. Weigh 53.7 g of uracil, and dissolve it in 4.8 L of deionized water (heat to 80°C to help dissolution).

[0046] Pour the two prepared solutions into a 20L reactor, set the stirring speed to 150rpm, the temperature to 37℃, and the pH to 7.5. When the temperature rises to 37℃ and the pH is 7.5 (±0.05), add 0.5mg / ml pseudouridine-5-phosphatase and its mutants respectively, and use 2M NaOH solution to automatically adjust the pH. After 30 minutes of reaction, slowly add 4.8L of 100mM completely dissolved uracil solution. After 2 hours, take 500μL samples every 30 minutes and heat them in a boiling water bath for 10 minutes. Centrifuge and take the supernatant for dephosphorylation treatment and HPLC detection. Figure 1 As shown, the reaction is terminated after the detected pseudouridine production becomes stable.

[0047] 2) Intermediate processing

[0048] The reaction solution was heated to 80°C for 10 minutes to denature the protein, and then centrifuged to remove the protein precipitate after cooling. The supernatant was filtered and then nanofiltered using a 200D filter membrane. The filtrate was tested until the conductivity dropped to 10 mS / cm and uracil could not be detected.

[0049] 3) Dephosphorylation of pseudouridine monophosphate

[0050] Pour the nanofiltered solution into a 20L reactor, add an equal volume of ethanol, mix well and slowly add 10 times the equivalent of NaOH. Set the speed to 150rpm and the temperature to 60℃. After reacting for 1h, perform HPLC detection. The results are as follows Figure 3 As shown, after confirming that the reaction is complete, the reaction solution is collected to separate and purify pseudouridine.

[0051] The pseudouridine yield obtained by using the pseudouridine-5-phosphatase mutant in this example was 7.9 g / L, the conversion rate was 51%, and the purity was 98.3%.

[0052] Comparative Example

[0053] By using the same method as in Example 2 of the present invention, one or two active sites were mutated to obtain pseudouridine 5-phosphatase mutant R146E, pseudouridine 5-phosphatase mutant H156Q / K210A and pseudouridine 5-phosphatase mutant R146E / H156Q, respectively.

[0054] Then, the same method as in Example 3 of the present invention was used to perform enzymatic synthesis, intermediate treatment and dephosphorylation with the ribose-5-phosphate obtained in Example 1 to obtain pseudouridine. The yield and conversion rate were statistically compared with the results obtained with the mutant R146E / H156Q / K210A, and the results are shown in Table 1.

[0055] Table 1: Comparison of pseudouridine obtained by different methods

[0056]

[0057] It can be seen that there are great differences in the conversion rates of pseudouridine when different pseudouridine-5-phosphoglycosidases are used for the chemical enzymatic synthesis of pseudouridine. When only one or two amino acid mutations are present, the conversion rate is not significantly improved. When the R146E / H156Q / K210A mutations appear, the conversion rate is significantly improved. This proves that the amino acid mutations at three different sites have a synergistic effect on the improvement of enzyme activity.

[0058] Experimental example: Detection of pseudouridine and uracil

[0059] The commercially available pseudouridine standard and uracil standard were used as controls for HPLC detection. In addition, the pseudouridine obtained in Example 3 of the present invention was detected by HPLC according to the following method, and the conditions were as follows:

[0060] Column: Waters XBridge BEH Amide, 250 × 4.6 mm, 5 μm;

[0061] Wavelength: 262nm;

[0062] Column temperature: 30°C;

[0063] Flow rate: 1 mL / min;

[0064] Mobile phase A: 0.2% ammonium acetate aqueous solution (2 g ammonium acetate was weighed and dissolved in 1 L water);

[0065] Mobile phase B: ACN.

[0066] The elution gradient is shown in Table 2.

[0067] Table 2:

[0068]

[0069] like Figure 2 As shown, the HPLC results of pseudouridine standard and uracil standard obtained by using the detection conditions provided in this experimental example are as follows Figure 2 As shown in A and 2B, the peaks appear at 3.460min and 3.923min respectively; Figure 3 As shown, the pseudouridine obtained by the pseudouridine-5-phosphatase mutant R146E / H156Q / K210A enzymatic method elutes at 3.439 min, and the difference with the pseudouridine standard is within the normal range.

[0070] The present invention is not limited to what is described in the specification and implementation modes, and therefore additional advantages and modifications can be easily realized by those skilled in the art. Therefore, without departing from the spirit and scope of the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details, representative schemes, and examples described herein.

Claims

1. A method for synthesizing pseudouridine by chemical enzymatic method, characterized in that: The pseudouridine monophosphate is synthesized from ribose-5-phosphate and uracil under the catalysis of a pseudouridine-5-phosphatase mutant, and then dephosphorylated and separated to obtain pseudouridine; the amino acid sequence of the pseudouridine-5-phosphatase mutant is shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that The pseudouridine-5-phosphatase mutant is obtained by expressing the coding gene with the nucleotide sequence shown in SEQ ID NO:

3.

3. The method according to claim 1, characterized in that The pseudouridine 5-phosphatase mutant is obtained by mutating the pseudouridine 5-phosphatase whose amino acid sequence is shown in SEQ ID NO:

2.

4. The method according to claim 3, characterized in that The nucleotide sequence of the pseudouridine 5-phosphatase is shown in SEQ ID NO:

4.

5. The method according to claim 1, characterized in that The ribose-5-phosphate is obtained by hydrolysis of AMP.

6. The method according to claim 1, characterized in that Before the dephosphorylation, the protein is denatured, centrifuged to remove protein precipitation, and the supernatant is filtered repeatedly until the uracil content is zero.

7. The method according to claim 6, characterized in that Before the dephosphorylation, the reaction solution is heated to denature the protein, and after centrifugation, the supernatant is filtered and then nanofiltered until the conductivity of the filtrate drops to 10 mS / cm.

8. The method according to claim 1, characterized in that The dephosphorylation step is to add ethanol to the filtrate, mix well, then add NaOH and react fully.

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