A whole-cell catalyst for efficiently catalyzing the preparation of pseudouridine and its preparation and application

Abstract

The invention discloses a whole-cell catalyst for efficiently catalyzing the preparation of pseudouridine, a preparation method and an application thereof. The whole-cell catalyst is a genetically engineered Escherichia coli bacterium expressing a yeiN or yeiN-102 mutant, the amino acid sequence of the yeiN is shown in SEQ ID NO.1, and the yeiN-102 mutant is a mutant in which the amino acid residue at position 102 of yeiN is substituted. Based on the whole-cell catalyst, the invention develops a method for preparing pseudouridine by a whole-cell method. In the method of the invention, pseudouridine can be directly obtained by increasing the concentration of bacterial cells without the need for a separate dephosphorylation treatment, and the synthesis efficiency of pseudouridine is increased to 92.96% by optimizing the reaction conditions. The invention has important significance for the green synthesis of pseudouridine.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a whole-cell catalyst for efficiently catalyzing the preparation of pseudouridine, and a preparation method and application thereof. Background Art

[0002] Pseudouridine is mainly found in the coding regions of tRNA, rRNA, and even mRNA. Studies have shown that the replacement of uridine in the coding region of mRNA with pseudouridine can change the selection of aminoacyl-tRNA and the synthesis rate of ribosomal proteins, thereby affecting the translation effect of mRNA. Studies have confirmed that the replacement of uridine with pseudouridine can reduce the ability of RNA delivered into cells to be recognized by the innate immune system and significantly improve the translation ability of mRNA. In addition, studies on surface pseudouridine-modified Cas9 or Cpf1 mRNA can improve the efficiency of gene cutting in response to CRISPR gene editing systems. It can be seen that pseudouridine modification of mRNA is not only related to the success or failure of mRNA vaccines, but also an important part of the practical application of CRISPR gene editing technology in the future. Therefore, strengthening the research on its synthesis process has important application value.

[0003] At present, chemical synthesis is still the main means of commercial preparation of pseudouridine in China and even internationally, so the development of pseudouridine synthesis processes based on enzymes and whole cells is in line with current development requirements. In recent years, bioenzymatic synthesis has been proven to be applicable to the biosynthesis of pseudouridine, and the whole cell biosynthesis method has a more significant cost advantage and will be the next stop for enzymatic synthesis, with important industrial application value. Summary of the invention

[0004] In view of this, the present invention aims to develop a whole-cell method for synthesizing pseudouridine.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] The first aspect of the present invention provides a whole-cell catalyst for efficiently catalyzing the preparation of pseudouridine, specifically a genetically engineered Escherichia coli expressing yeiN or a yeiN mutant, wherein the amino acid sequence of yeiN is as shown in SEQ ID NO.1, and the amino acid sequence of the yeiN mutant is substituted at the 102nd amino acid residue relative to the sequence shown in SEQ ID NO.1.

[0007] Preferably, in the above-mentioned whole-cell catalyst, the yeiN mutant is obtained by mutating the valine (Val, V) at position 102 of yeiN to aspartic acid (Asp, D), or leucine (Leu, L), or glutamic acid (Glu, E), or lysine (Lys, K), or isoleucine (Ile, I), or alanine (Ala, A), that is, specifically one of yeiN-V102D, yeiN-V102L, yeiN-V102E, yeiN-V102K, yeiN-V102I, and yeiN-V102A.

[0008] The experimental results of the present invention show that, compared with the wild-type yeiN, the catalytic efficiency of the bacteria expressing the yeiN mutant is significantly improved.

[0009] The second aspect of the present invention provides a method for preparing the whole-cell catalyst of the present invention, specifically: constructing a gene sequence encoding yeiN or a yeiN mutant into a pET32a vector, transforming the obtained expression vector into an E. coli BL21 (DE3) strain to obtain genetically engineered Escherichia coli; after culturing the genetically engineered Escherichia coli, a whole-cell suspension is obtained, which can be used as a whole-cell catalyst for synthesizing pseudouridine.

[0010] Preferably, in the above method, the gene sequence encoding yeiN is as shown in SEQ ID NO.2, and the gene sequence encoding the yeiN mutant is specifically:

[0011] yeiN-V102D, the GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with GAT encoding aspartic acid;

[0012] yeiN-V102L, the GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with CTG encoding leucine;

[0013] yeiN-V102E, replacing GTG encoding valine at position 102 in SEQ ID NO.2 with GAA encoding glutamic acid;

[0014] yeiN-V102K, replacing GTG encoding valine at position 102 in SEQ ID NO.2 with AAA encoding lysine;

[0015] yeiN-V102I, the GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with ATT encoding isoleucine;

[0016] yeiN-V102A, the GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with GCC encoding alanine;

[0017] More preferably, in the above method, the gene sequence encoding the yeiN mutant is obtained by constructing the nucleotide sequence encoding yeiN into a pET32a vector and obtaining it by overlap extension PCR using site-directed mutagenesis primers.

[0018] The third aspect of the present invention provides the use of the whole-cell catalyst of the present invention in producing pseudouridine.

[0019] The fourth aspect of the present invention provides a method for preparing pseudouridine by a whole-cell method, which specifically comprises the following steps:

[0020] S1, preparing a whole-cell catalytic reaction solution with a whole-cell catalyst, uracil and ribose pentaphosphate to carry out a reaction;

[0021] S2. After the reaction is completed, the supernatant is concentrated and purified to obtain pseudouridine.

[0022] Preferably, in the whole-cell catalytic reaction solution, the final concentration of the whole-cell catalyst is greater than or equal to 20.0 mg / ml.

[0023] The data of the examples of the present invention show that when the bacterial concentration in the reaction solution is high enough, the pseudouridine monophosphate generated by the reaction will be directly dephosphorylated to generate pseudouridine, and there is no need to perform a separate dephosphorylation reaction, so the preparation process is more convenient.

[0024] Preferably, in the whole-cell catalytic reaction solution, the molar concentration ratio of uracil to ribose pentaphosphate is 1:2.

[0025] Preferably, in the whole-cell catalytic reaction solution, step S1 specifically comprises:

[0026] S11, resuspend the genetically engineered E. coli in the reaction buffer and incubate at 30°C for 15-30 minutes;

[0027] S12. Add uracil and ribose pentaphosphate to the reaction solution of S11 for reaction.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] (1) The present invention first obtains a soluble yeiN recombinant protein through prokaryotic expression and purification, and confirms that it has the activity of catalyzing the synthesis of pseudouridine monophosphate from uracil and ribose pentaphosphate; on this basis, in order to further increase the output rate of pseudouridine, the inventors mutated several amino acid sites in the yeiN enzyme activity center and found that except for the 102nd amino acid site mutation, the other sites had no enzyme activity after mutation, thereby obtaining a series of yeiN-102 mutants that can significantly improve the efficiency of pseudouridine synthesis.

[0030] (2) Based on the yeiN protein and the yeiN-102 mutant, the present invention obtains BL21 (DE3) bacteria that highly express yeiN or its mutants through induction culture. The experimental data of the present invention show that when the bacteria obtained by the present invention are used as whole-cell catalysts to produce pseudouridine, at low bacterial concentrations, the pseudouridine monophosphate produced by whole-cell catalysis is much higher than the output by the enzymatic method, while under high-concentration bacterial reactions, the generated pseudouridine monophosphate is further dephosphorylated to generate pseudouridine.

[0031] (3) The present invention further optimizes the whole-cell method for preparing pseudouridine, such as by optimizing the ratio of substrate uracil to ribose pentaphosphate and whole-cell catalyst pretreatment, and successfully increases the synthesis efficiency of pseudouridine to 92.96%.

[0032] (4) The method of the present invention not only simplifies the preparation process but also saves production and purification costs, and lays a solid research foundation for the subsequent establishment of a whole-cell biosynthesis method for pseudouridine triphosphate and a subsequent bioenzymatic synthesis of N1-methylpseudouridine. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram showing the agarose gel electrophoresis detection results of the vector and yeiN gene amplified in Example 1 of the present invention.

[0034] Figure 2 This is a diagram showing the results of SDS-PAGE detection of the expression and purification of the recombinant vector pET23a-yeiN in Example 1 of the present invention.

[0035] Figure 3 This is an identification and analysis diagram of the activity of the recombinant protein His-yeiN of the present invention in enzymatically catalyzing CC bonds to synthesize pseudouridine monophosphate.

[0036] Figure 4 This is a comparative analysis of the products of ribose phosphate and uracil-free catalysis by the whole-cell method at different yeiN bacterial concentrations.

[0037] Figure 5 This is a liquid chromatogram of pseudouridine monophosphate after CIP treatment in Example 2 of the present invention.

[0038] Figure 6 This is a graph showing the detection results of whole-cell catalytic reaction products of different yeiN-102 mutants of the present invention.

[0039] Figure 7 This is a diagram showing the results of reaction product detection after the conditions for pseudouridine synthesis catalyzed by the cell method of the present invention are optimized. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0041] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0042] Example 1 Expression of yeiN and yeiN-102 mutants and identification of catalytic activity

[0043] In this case, the recombinant protein His-yeiN and its mutants were first prepared by prokaryotic expression, and the activity of catalyzing the synthesis of pseudouridine monophosphate by CC bonding was verified. The specific experimental process is as follows:

[0044] (1) Construction of expression vector.

[0045] The yeiN gene sequence (NC_004337.2) was found through NCBI and constructed into the pET32a vector after codon optimization.

[0046] Site-directed mutagenesis primers were designed, and the yeiN mutant gene sequence was obtained by overlap extension PCR and constructed into an expression vector. The site-directed mutagenesis primer sequences are shown in Table 1.

[0047] Table 1 Primer information

[0048] Primer name Primer sequence (5'-3') yeiN V102E-F taccttttGAAgttgccgccggaaaaaatggcg(SEQ ID NO.4) yeiN-V102E-R gcggcaacTTCaaaaggtaaatcgcgac(SEQ ID NO.5) yeiN-V102K-F taccttttAAAgttgccgccggaaaaaatggcg(SEQ ID NO.6) yeiN-V102K-R gcggcaacTTTaaaaggtaaatcgcgac(SEQ ID NO.7) yeiN-V102L-F taccttttCTGgttgccgccggaaaaaatggcg(SEQ ID NO.8) yeiN-V102L-R gcggcaacCAGaaaaggtaaatcgcgac(SEQ ID NO.9) yeiN-V102A-F taccttttGCCgttgccgccggaaaaaatggcg(SEQ ID NO.10) yeiN-V103A-R gcggcaacGGCaaaaggtaaatcgcgac(SEQ ID NO.11) yeiN-V102I-F taccttttATTgttgccgccggaaaaaatggcg(SEQ ID NO.12) yeiN-V103I-R gcggcaacAATaaaaggtaaatcgcgac(SEQ ID NO.13) yeiN-V102D-F atttaccttttGATgttgccgccggaaaaaatggcg(SEQ ID NO.14) yeiN-V102D-R attttttccggcggcaacATCaaaaggtaaatcgcgac(SEQ ID NO.15)

[0049] The amplified vector and yeiN gene were detected by 0.8% agarose gel electrophoresis, and the detection results were as follows: Figure 1 As shown, a is the PCR amplification detection of the recombinant vector pET23a (M is 1Kb marker, lane 1 is the reverse amplification of the pET23a vector), and b is the yeiN gene amplification detection (M is DL2000 marker, 1-8 are the PCR amplification products of yeiN and its mutants respectively). Figure 1 It can be seen that the size of the PCR amplification product is consistent with the expected result. The cloned plasmid was sent to Shanghai Bioengineering for sequencing verification to confirm that the mutation site is consistent with the expected result.

[0050] (2) Induced expression.

[0051] The constructed expression vector was transformed into E. coli BL21-DE3 strain, and the cells were collected after low-temperature induction of expression with 0.5 mM IPTG for 18 h. After high-pressure bacterial lysis and nickel column purification, 10% SDS-PAGE gel electrophoresis and Western blot based on anti-His primary antibody were performed to verify the expression of the recombinant protein.

[0052] Figure 2 The figure is the result of SDS-PAGE detection of expression and purification of the recombinant vector pET23a-yeiN, wherein M is a protein marker, and lanes 1-3 are the purified and concentrated His-yeiN recombinant protein (with a His tag, and the amino acid sequence is shown in SEQ ID NO.3). Figure 2 The results showed that the His-yeiN recombinant protein was consistent with the expected size of 34.3KDa.

[0053] (3) Enzymatic preparation of pseudouridine monophosphate (PsiMP) and its detection.

[0054] Prepare 25mM HEPES (pH 7.1) buffer, add 5mM uracil (uracil is insoluble in water, can be dissolved in the reaction buffer at 80°C first, and will not precipitate after cooling) and 5mM ribose pentaphosphate and 0.5mM MnCl2 to a final concentration. Place the reaction mixture in a water bath with a magnetic stirrer at 30°C for 15 minutes in the dark, then add the recombinant protein His-yeiN or its mutant (200U) prepared in Example 1; after 5 hours of constant temperature reaction at 30°C, place the sample in a water bath at 80°C for 15 minutes to terminate the reaction, centrifuge at 12000r / min for 10 minutes, take the supernatant, filter with a 0.22μm microporous filter membrane, and perform HPLC detection. Wherein, chromatographic column: Ultimate LP-C18 (4.6mm×250mm, 5μm); mobile phase: 20mM ammonium dihydrogen phosphate (pH 5.4): methanol = 90:10; flow rate: 0.8mL / min; ultraviolet detector, detection wavelength 254nm.

[0055] Figure 3 The results of the identification and analysis of the activity of yeiN in catalyzing the CC bond synthesis of pseudouridine monophosphate, where a is a schematic diagram of the synthesis of ribose pentaphosphate, uracil and synthetic pseudouridine monophosphate, b is a HPLC detection diagram of uracil standard, and c is a HPLC identification result diagram of the reaction product. Figure 3 From b and 3c, we can see that the peak time of PsiMP is 2.96 min, and the peak time of uracil is 3.56 min.

[0056] The experimental results of the present invention show that the recombinant protein His-yeiN and the yeiN-102 mutant both have the activity of catalyzing CC bonding to synthesize pseudouridine monophosphate.

[0057] Example 2 Synthesis of pseudouridine catalyzed by wild-type yeiN whole cells

[0058] In order to shorten the time and cost of pseudouridine synthesis, based on Example 1, this example takes wild-type yeiN as an example to preliminarily establish a whole-cell method for preparing pseudouridine. The specific steps are as follows:

[0059] (1) Cultivation of enzyme-producing strains and preparation of whole-cell suspension.

[0060] According to the inoculation amount of 1%, the BL21 (DE3) / Pet32a-yeiN bacteria prepared in Example 1 were inoculated into a 500mL Erlenmeyer flask containing 100mL sterilized LB medium (containing 0.5mg / mL ampicillin), and cultured at 37°C and 220r / min until OD600 reached 0.6-0.8, and IPTG with a final concentration of 0.5mM was added, and induced at 18°C ​​for 18h. After the induction, the fermentation broth was centrifuged at 4°C and 8000r / min for 10min, the supernatant was discarded to obtain a bacterial precipitate, the wet weight of the bacterial body was weighed, and the bacterial body was resuspended with 25mM HEPES buffer (pH7.1) to a bacterial concentration of 200mg / mL, and the yeiN whole cell suspension was obtained.

[0061] (2) Whole-cell catalytic preparation of pseudouridine and its detection.

[0062] The reaction conditions are the same as those in step (4) of Example 1. After balancing in a water bath at 30°C for 15 minutes, the whole cell suspension prepared in step (1) (final concentration 20 mg / ml) is added and the mixture is kept in a water bath at 30°C for 5 hours. 1 mL of the whole cell catalytic reaction solution is taken and centrifuged at 12000 r / min for 5 minutes. The whole cell precipitate is discarded and the supernatant is taken and filtered through a 0.22 μm microporous filter membrane before HPLC detection. The chromatographic column is Ultimate LP-C18 (4.6 mm×250 mm, 5 μm); the mobile phase is 20 mM diammonium phosphate (pH 5.4): methanol = 85:15; the flow rate is 1.0 mL / min; the UV detector has a detection wavelength of 254 nm.

[0063] Referring to the above experimental process, it was found that after increasing the concentration of yeiN enzyme-producing bacteria, the pseudouridine monophosphate generated by the reaction would be dephosphorylated to generate pseudouridine. Figure 4 As shown, Figure 4 a is the product of the catalytic reaction of yeiN bacteria at a final concentration of 2.0 mg / ml. The product is mainly pseudouridine monophosphate and uracil residue, containing a small amount of pseudouridine; Figure 4 b is the final concentration of 20.0 mg / ml. The product of the catalytic reaction of eiN bacteria is mainly pseudouridine, containing very little pseudouridine monophosphate. Figure 4 The peak time of pseudouridine monophosphate was 2.96 min, the peak time of pseudouridine was 3.28 min, and the peak time of uracil was 3.56 min.

[0064] To further confirm that the product of the whole-cell method is pseudouridine, the yeiN enzymatic method in step (4) of Example 1 was used to prepare pseudouridine monophosphate and incubate it with CIP alkaline phosphatase. The specific operation was as follows: 89 μL of the enzymatic reaction product was taken, 10 μL of 10×cutsmart buffer and 1 μL of alkaline phosphatase (CIP) were added, and the mixture was incubated in a 37°C metal bath for 1 hour, inactivated at 80°C for 15 minutes, and then centrifuged at 12000 r / min for 5 minutes. The supernatant was taken, filtered through a 0.22 μm microporous membrane, and then tested by HPLC. The test results are as follows: Figure 5 As shown, the peak time of pseudouridine is 3.605 min. It can be seen that after increasing the bacterial concentration of the whole-cell catalyst of the present invention, the catalytic product of the whole-cell method is indeed pseudouridine.

[0065] Example 3 Synthesis of pseudouridine catalyzed by whole cells of different yeiN-102 mutants

[0066] Referring to step (1) of Example 2, this example first prepared whole cell suspensions of different yeiN-102 mutants, and then referring to step (2) of Example 2, different whole cell suspensions were used to carry out catalytic reactions. Figure 6 As shown, a is the HPLC detection of the whole cell catalytic product of wild-type yeiN, b is the HPLC detection of the whole cell catalytic product of yeiN-V102D mutant, c is the HPLC detection of the whole cell catalytic product of yeiN-V102L mutant, d is the HPLC detection of the whole cell catalytic product of yeiN-V102E mutant, e is the HPLC detection of the whole cell catalytic product of yeiN-V102K mutant, f is the HPLC detection of the whole cell catalytic product of yeiN-V102I mutant, and g is the HPLC detection of the whole cell catalytic product of yeiN-V102A alanine mutant.

[0067] At the same time, in order to facilitate the detection of enzymatic reaction efficiency, in this example, uracil was dissolved and diluted into different concentrations (0.1mM, 0.2mM, 0.5mM, 2.0mM, 5.0mM, 10.0mM), and the HPLC sample volume was unified to 5μL. The uracil standard curve was generated with the peak area as the ordinate and the uracil concentration as the abscissa.

[0068] According to the remaining uracil, the catalytic efficiency of pseudouridine reaction of wild-type and different mutants was calculated according to the obtained uracil concentration standard curve. The results showed that the catalytic efficiency of wild-type yeiN was 23.89%, the catalytic efficiency of yeiN-V102D mutant was 30.4%, the catalytic efficiency of yeiN-V102L mutant was 29.43%, the catalytic efficiency of yeiN-V102E mutant was 31.81%, the catalytic efficiency of yeiN-V102K mutant was 27.57%, the catalytic efficiency of yeiN-V102I mutant was 29.16%, and the catalytic efficiency of yeiN-V102A mutant was 28.11%; among them, the catalytic efficiency of yeiN-V102E mutant was the highest, with a catalytic efficiency increase of 33.15%.

[0069] Example 4 Process Optimization of Whole Cell Catalytic Synthesis of Pseudouridine

[0070] Although Examples 2 and 3 preliminarily established the process of synthesizing pseudouridine by the whole cell method, the reaction efficiency was low and could not meet the production requirements. Therefore, this example optimized the reaction conditions of the whole cell method. After a lot of attempts, it was found that:

[0071] (1) The concentrations of the substrates uracil and ribose pentaphosphate have a significant effect on the reaction efficiency, and the optimal concentration ratio of uracil to ribose pentaphosphate in the whole-cell reaction solution is 5mM:10mM;

[0072] (2) Since yeiN and its mutants are expressed intracellularly in E. coli, when the freeze-thaw method is used, the enzyme and substrate are fully contacted by adding surfactants such as Tween 20, Triton X-100, etc. and ultrasonic disruption. It was found that after ultrasonic disruption, the enzyme activity system of E. coli was destroyed, resulting in the inability to proceed with the reaction (e.g. Figure 7 a, uracil peak time is 3.889min);

[0073] (3) Unlike enzyme-catalyzed reactions, whole-cell catalysis can effectively improve the reaction efficiency by first resuspending the collected bacteria in the reaction buffer and incubating them in a 30°C water bath for 15-30 min before adding the substrate for reaction (e.g. Figure 7 b, the peak time of pseudouridine is 3.28min, and the peak time of uracil is 3.56min).

[0074] After optimization, the optimal reaction conditions for preparing pseudouridine by the whole-cell method established by the present invention are: first, 25mM HEPES buffer (pH 7.1), bacterial concentration 20mg / ml and 0.5mM MnCl2 are incubated at 30°C for 30min, and then 5mM uracil and 10mM ribose pentaphosphate are added to react, and no inactivation is required after the reaction, and the bacterial cells are collected at 4000rm / min. In addition, the reaction buffer and substrate can be added again as described above to further improve the yield of pseudouridine.

[0075] Using the above optimized conditions, taking the yeiN-V102E mutant as an example, the uracil residue and reaction efficiency were calculated according to the method described in Example 3, and the uracil reaction efficiency was as high as 62.4%; and the bacteria after the reaction were recycled and reused again, and they also had very good catalytic activity (such as Figure 7 c) The uracil reaction efficiency is as high as 92.96%, of which pseudouridine accounts for 82.9% of the total products after the reaction.

[0076] In summary, yeiN and yeiN-102 mutant proteins are firstly obtained by prokaryotic expression and purification, and it is confirmed that they have the activity of catalyzing the synthesis of pseudouridine monophosphate from uracil and ribose pentaphosphate, and then the genetically engineered Escherichia coli cells with high expression of yeiN or yeiN-102 mutants are obtained by induction culture; experimental data show that at low concentrations of cells, the pseudouridine monophosphate produced by whole cell catalysis is much higher than the output of the enzymatic method, and at high concentrations of cells, the generated pseudouridine monophosphate is further dephosphorylated to generate pseudouridine; in addition, the present invention improves the synthesis efficiency of pseudouridine to 92.96% by optimizing the reaction conditions. The present invention not only simplifies the preparation process of pseudouridine, but also saves the production and purification costs, and is of great significance to the production and research of pseudouridine.

[0077] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A whole-cell catalyst for efficiently catalyzing the preparation of pseudouridine, characterized in that: The whole-cell catalyst is a genetically engineered Escherichia coli expressing yeiN or a yeiN mutant, the amino acid sequence of the yeiN is shown in SEQ ID NO.1, the amino acid sequence of the yeiN mutant is substituted relative to the 102nd amino acid residue of the sequence shown in SEQ ID NO.1, the yeiN mutant is one of yeiN-V102D, yeiN-V102L, yeiN-V102E, yeiN-V102K, yeiN-V102I, and yeiN-V102A, and the genetically engineered Escherichia coli is based on E. coli BL21-DE3 as the basic strain.

2. A method for preparing the whole cell catalyst as claimed in claim 1, characterized in that: The gene sequence encoding yeiN or yeiN mutant is cloned into pET32a vector, and the obtained expression vector is transformed into E. coli BL21-DE3 strain to obtain genetically engineered E. coli; wherein, The gene sequence encoding yeiN is shown in SEQ ID NO.2; The gene sequence encoding yeiN-V102D is as follows: GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with GAT encoding aspartic acid; The gene sequence encoding yeiN-V102L is as follows: GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with CTG encoding leucine; The gene sequence encoding yeiN-V102E is as follows: GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with GAA encoding glutamic acid; The gene sequence encoding yeiN-V102K is as follows: GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with AAA encoding lysine; The gene sequence encoding yeiN-V102I is as follows: GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with ATT encoding isoleucine; The gene sequence encoding yeiN-V102A is as follows: GTG encoding valine at position 102 in SEQ ID NO.2 is replaced with GCC encoding alanine.

3. The method according to claim 2, characterized in that The gene sequence encoding the yeiN mutant is obtained by constructing the gene sequence encoding the yeiN mutant into a pET32a vector and obtaining the gene sequence by overlapping extension PCR using site-directed mutagenesis primers.

4. Use of the whole-cell catalyst as claimed in claim 1 in the production of pseudouridine.

5. The use according to claim 4, characterized in that: The following steps are involved: S1, preparing a whole-cell catalytic reaction solution with a whole-cell catalyst, uracil and ribose pentaphosphate to carry out a reaction; S2. After the reaction is completed, the supernatant is concentrated and purified to obtain pseudouridine.

6. The use according to claim 5, characterized in that: In the whole-cell catalytic reaction solution, the final bacterial concentration of the whole-cell catalyst is greater than or equal to 20.0 mg / ml.

7. The use according to claim 5, characterized in that: In the whole-cell catalytic reaction solution, the molar concentration ratio of uracil to ribose pentaphosphate is 1:

2.

8. The use according to claim 5, characterized in that: Step S1 is specifically as follows: S11, resuspend the genetically engineered E. coli in the reaction buffer and incubate at 30°C for 15-30 minutes; S12. Add uracil and ribose pentaphosphate to the reaction solution of S11 for reaction.

Images