Construction and use of pseudouridine production strain

Abstract

The invention discloses a genetically engineered bacterium for producing pseudouridine and a construction method of the genetically engineered bacterium, escherichia coli W3110 is used as a starting strain, the supply of precursor substances including aspartic acid, carbamyl phosphoric acid, glutamine and 5 '-phosphoribosyl pyrophosphate prpp is enhanced, pyrimidine nucleoside synthesis operon from bacillus subtilis is introduced, and the pseudouridine is produced. The invention discloses a pseudouridine-5 '-phosphosidase gene psuG and a preparation method thereof, and relates to the field of microorganisms, the field of genetic engineering and the field of fermentation engineering. The pseudouridine-5'-phosphosidase gene psuG is expressed by using high-copy plasmids, and the application of the pseudouridine-5 '-phosphosidase gene psuG to preparation of the pseudouridine-5'-phosphosidase gene psuG is provided. The strain is fermented in a 50L fermentation tank for 60h, the yield of pseudouridine reaches 50.8 g / L, the conversion rate is 18%, which is the highest value reported at present, and the strain has high industrial production value.

Description

Technical Field

[0001] The invention relates to the fields of microorganisms, genetic engineering and fermentation engineering, and in particular to a genetic engineering bacterium for producing pseudouridine and a construction method thereof. Background Art

[0002] Pseudouridine was the first modified nucleoside discovered in 1951. It is also the most abundant modified nucleoside on RNA and is known as the "fifth nucleoside" in RNA. Adding pseudouridine and its analogs to mRNA instead of uridine can solve the problem that mRNA drugs are easily recognized and cleared by the immune system and produce immune side effects, and has great application prospects in the field of biomedicine.

[0003] The production methods of pseudouridine mainly include chemical synthesis, chemical enzyme method and biological method, but they generally have the disadvantages of complicated steps, high production cost, low production efficiency and difficulty in product recovery and purification, which leads to high product prices and limits its large-scale application in the biological and pharmaceutical fields. Therefore, it is urgent to develop a cheap pseudouridine production process suitable for industrial scale-up.

[0004] The production of pseudouridine by fermentation of inexpensive glucose is an attractive option. In recent years, Escherichia coli has been widely used to construct cell factories for natural product biosynthesis due to its clear genetic background, efficient genetic manipulation methods, rapid growth, sufficient supply of metabolic precursors, ability to utilize inexpensive raw materials, and efficient fermentation process. In addition, Escherichia coli can efficiently produce pyrimidine nucleosides and has been used to produce uridine and cytidine from glucose by fermentation. Therefore, Escherichia coli was selected for genetic engineering to produce pseudouridine by fermentation.

[0005] To this end, the present invention provides a genetically engineered bacterium for producing pseudouridine and a construction method thereof, the yield of which is the highest value reported so far, which greatly reduces the production cost of pseudouridine and has good industrial production value. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a genetically engineered bacterium for producing pseudouridine and a construction method thereof, which solves the above problems.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: a genetically engineered bacterium for producing pseudouridine, which is Escherichia coli W3110 ( Escherichia coliW3110) was used as the starting strain, the supply of aspartic acid, carbamoyl phosphate, glutamine and 5'-phosphoribosyl pyrophosphate prpp required for the synthesis of pseudouridine was enhanced, the pyrimidine nucleoside synthesis operon from Bacillus subtilis was introduced, and the cytosine / isoguanine deaminase gene codA from Escherichia coli W3110 was overexpressed to enhance the synthesis of uracil, genes that were not conducive to uracil accumulation were knocked out, the accumulation of other types of nucleotides was reduced, and the pseudouridine-5'-phosphoglycosidase gene psuG was obtained. The pseudouridine-5'-phosphoglycosidase gene psuG was expressed using a high-copy plasmid, and a genetically engineered bacterium that can directly produce pseudouridine by glucose fermentation was constructed.

[0008] Preferably, the precursor substances are supplied by overexpressing the aspartate aminotransferase gene aspC, the carbamoyl phosphate synthase gene carAB, the phosphoenolpyruvate carboxylase gene ppc and the ribose-phosphate diphosphate kinase gene prs; The nucleotide sequence of the aspartate aminotransferase gene aspC is shown in SEQ ID No: 1; The nucleotide sequence of the carbamoyl phosphate synthase gene carAB is shown in SEQ ID No: 2; The nucleotide sequence of the phosphoenolpyruvate carboxylase gene ppc is shown in SEQ ID No: 3; The nucleotide sequence of the ribose-bisphosphate kinase gene prs is shown in SEQ ID No:4.

[0009] Preferably, the genes contained in the pyrimidine nucleoside synthesis operon from Bacillus subtilis include: pyrB, pyrC, pyrAA, pyrAB, pyrK, pyrD, pyrF and pyrE, and the cytosine / isoguanine deaminase gene codA from Escherichia coli W3110 is overexpressed by the strong promoter ptrc; The nucleotide sequence of the gene pyrB is shown in SEQ ID No: 5; The nucleotide sequence of the gene pyrC is shown in SEQ ID No: 6; The nucleotide sequence of the gene pyrAA is shown in SEQ ID No: 7; The nucleotide sequence of the gene pyrAB is shown in SEQ ID No: 8; The nucleotide sequence of the gene pyrK is shown in SEQ ID No: 9; The nucleotide sequence of the gene pyrD is shown in SEQ ID No: 10; The nucleotide sequence of the gene pyrF is shown in SEQ ID No: 11; The nucleotide sequence of the gene pyrE is shown in SEQ ID No: 12; The nucleotide sequence of the cytosine / isoguanine deaminase gene codA from Escherichia coli W3110 is shown in SEQ ID No: 13.

[0010] Preferably, the genes that are not conducive to uracil accumulation include DNA binding transcription repressor gene PurR, homoserine dehydrogenase gene thrA, ornithine carbamoyltransferase gene argF, uridine kinase udk, uracil phosphoribosyltransferase upp, pyrimidine specific ribonucleoside hydrolase rihA, non-specific ribonucleoside hydrolase rihC and cytidine / deoxycytidine deaminase cdd; The nucleotide sequence of the DNA binding transcription repressor protein gene PurR is shown in SEQ ID No: 14; The nucleotide sequence of the homoserine dehydrogenase gene thrA is shown in SEQ ID No: 15; The nucleotide sequence of the ornithine carbamoyltransferase gene argF is shown in SEQ ID No: 16; The nucleotide sequence of the uridine kinase udk is shown in SEQ ID No: 17; The nucleotide sequence of the uracil phosphoribosyltransferase upp is shown in SEQ ID No: 18; The nucleotide sequence of the pyrimidine-specific ribonucleoside hydrolase rihA is shown in SEQ ID No: 19; The nucleotide sequence of the non-specific ribonucleoside hydrolyzing rihC is shown in SEQ ID No: 20; The nucleotide sequence of the cytidine / deoxycytidine deaminase cdd is shown in SEQ ID No: 21.

[0011] Preferably, the pseudouridine-5'-phosphoglycosidase gene psuG is overexpressed by the pRB322 plasmid using the strong promoter ptac; The nucleotide sequence of the pseudouridine-5'-phosphatase gene psuG is shown in SEQ ID No: 22.

[0012] Preferably, the genetically engineered bacteria uses CRISPR / Cas9-mediated gene editing technology to perform gene knockout and integration.

[0013] Preferably, the genetically engineered bacteria is used to prepare pseudouridine.

[0014] Preferably, the pseudouridine preparation process comprises the following steps: Step a: Activate the genetically engineered bacteria for two generations on a slant; Step b: inoculate the genetically engineered bacteria after two generations of activation into 30 mL of liquid LB medium, and culture at 37° C. and 200 rpm / min for 5 h before inoculating into a seed tank; Step c: When the OD600 of the seed tank reaches 20-25, transplant the seeds at a 15%-20% inoculation rate; Step d: After transplanting, the culture is carried out in the fermentation medium for 58-63 hours. During the fermentation process, 60%-80% m / v glucose and 20%-25% ammonia water are added to maintain the residual sugar and pH stable.

[0015] Preferably, the LB medium contains: 5 g / L yeast powder, 10 g / L peptone, and 10 g / L NaCl; The seed tank components are 30 g / L glucose, 5 g / L yeast powder, 5 g / L peptone, 2 g / L KH2PO4, 2 g / L MgSO4, 3 g / L citric acid, 5 g / L corn steep liquor, 50 mg / L FeSO4, 20 mg / L MnSO4, 40 mg / L CoCl, 120 mg / L VB, 20 mg / L VH, and 40 mg / L ZnSO4. The fermentation medium components contain: 10 g / L glucose, 3 g / L yeast powder, 2 g / L peptone, 5 g / L KH2PO4, 2 g / L MgSO4, 1 g / L citric acid, 8 g / L corn steep liquor, 50 mg / L FeSO4, 20 mg / L MnSO4, 40 mg / L CoCl, 120 mg / L VB, 20 mg / L VH, 40 mg / L ZnSO4 and 40 mg / L CaCl2.

[0016] A method for constructing a genetically engineered bacterium for producing pseudouridine, the method comprising the following steps: Step 1: Starting from Escherichia coli W3110, knock out the lacI gene in the genome to obtain a chassis strain; Step 2: To enhance the supply of precursor substances, the aspartate aminotransferase gene aspC, the carbamoyl phosphate synthase gene carAB, the phosphoenolpyruvate carboxylase gene ppc and the ribose-phosphate diphosphate kinase gene prs were driven by the strong promoter ptrc and integrated into the genome of Escherichia coli W3110; Step 3: In order to enhance the synthesis ability of the product, the pyrimidine nucleoside synthesis operon from Bacillus subtilis is driven by the promoter ptrc and integrated into the genome of Escherichia coli W3110, wherein the genes include: pyrB, pyrC, pyrAA, pyrAB, pyrK, pyrD, pyrF and pyrE; the cytosine / isoguanine deaminase gene codA from Escherichia coli is driven by the promoter ptrc and integrated into the genome of Escherichia coli W3110; Step 4: knocking out genes that are not conducive to pseudouridine accumulation, including DNA binding transcription repressor gene PurR, homoserine dehydrogenase gene thrA, ornithine carbamoyltransferase gene argF, uridine kinase udk, uracil phosphoribosyltransferase upp, pyrimidine-specific ribonucleoside hydrolase rihA, non-specific ribonucleoside hydrolysis rihC and cytidine / deoxycytidine deaminase cdd, to obtain pseudouridine-5'-phosphoglycosidase gene psuG; Step 5: The pseudouridine-5'-phosphoglycosidase gene psuG is overexpressed from the pRB322 plasmid using the strong promoter ptac to obtain a genetically engineered bacterium that can directly produce pseudouridine by glucose fermentation.

[0017] Beneficial Effects The present invention provides a genetically engineered bacterium for producing pseudouridine and a construction method thereof. Compared with the prior art, it has the following beneficial effects: The present invention constructs a metabolic pathway for synthesizing pseudouridine from scratch in Escherichia coli, wherein the supply of precursors aspartic acid, carbamoyl phosphate, glutamine and 5'-phosphoribosyl pyrophosphate prpp required in the synthesis process of pseudouridine is enhanced, a pyrimidine nucleoside synthesis operon from Bacillus subtilis is introduced, and the cytosine / isoguanine deaminase gene codA from Escherichia coli is overexpressed to enhance the synthesis of uracil, genes that are not conducive to the accumulation of uracil are knocked out, and the accumulation of other types of nucleotides is reduced. Finally, a high-copy plasmid is used to express the pseudouridine-5'-phosphoglycosidase gene psuG, and a genetically engineered bacterium that can directly utilize glucose fermentation to produce pseudouridine is constructed. The strain is fermented in a 50L fermenter for 60 hours, and the pseudouridine yield reaches 50.8g / L, and the conversion rate is 18%, which is the highest value reported so far, and has a high industrial production value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the pseudouridine-5'-phosphatase gene psuG overexpression plasmid map in Example 1 of the present invention; Figure 2 It is a trend diagram of pseudouridine production and OD during the fermentation process of the pseudouridine production strain in Example 2 of the present invention. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1: See Figure 1 , a method for constructing a genetically engineered bacterium for producing pseudouridine, as follows: 1. The aspartate aminotransferase gene aspC was promoted by the strong promoter ptrc and integrated into the genome of Escherichia coli W3110. The specific steps are as follows: 1.1 Preparation of recombinant DNA fragments The aspC gene was integrated into the eaeH pseudogene site. The aspC gene was amplified using the E. coli W3110 genome as a template. The upstream and downstream homology arm fragments of eaeH were amplified using the E. coli W3110 genome as a template. The trc promoter was designed in the downstream primer of the upstream homology arm and the upstream primer of the aspC gene. Overlapping PCR was performed using the upstream and downstream homology arms and the target gene aspC as templates to obtain the fragment Ptrc-aspC.

[0021] 1.2 Construction of sgRNA plasmid The plasmid containing the target sequence was amplified by reverse PCR using the upstream and downstream primers of sgRNA, respectively. The template plasmid was removed by DPNⅠ digestion at 37℃ for 40min.

[0022] Take 10uL PCR reaction solution, add it to 100uL Jm109 competent cells, mix well, place on ice for 30min, heat shock at 42℃ for 60-100s, immediately place on ice for 3min, add 800uL LB medium, and resuscitate at 37℃ for 60-90min. Centrifuge at 5000rpm for 5min, discard the supernatant, keep 100uL to resuspend the bacteria, and then spread it on a plate containing 50mg / L tetracycline, turn the plate upside down, and culture it at 37℃ overnight. After the colonies grow on the plate, select the positive recombinants.

[0023] The selected positive recombinants were inoculated into a culture medium containing tetracycline resistance and cultured overnight to extract the plasmid for later use.

[0024] 1.3 Electroporation of sgRNA and recombinant DNA fragments The sgRNA and recombinant DNA fragments were simultaneously electro-transformed into the electro-transformation competent medium of the W3110 strain containing the pECas9 plasmid, and then revived for 60-90 minutes, plated on a plate containing kanamycin and streptomycin, and cultured overnight at 37°C. Colony PCR verification was performed using the upstream primer of the upper homology arm and the downstream primer of the lower homology arm to screen out positive recombinants.

[0025] 1.4 Elimination of tool plasmids 1.4.1 Elimination of pGRNA plasmid The positive recombinants were placed in LB medium containing rhamnose and kanamycin resistance and cultured at 37°C for three consecutive passages. After appropriate dilution, they were spread on plates containing kanamycin and cultured at 37°C. The single colonies that grew on the kanamycin plate but not on the tetracycline plate were selected for preservation on the LB plates containing tetracycline and kanamycin.

[0026] 1.4.2 Elimination of pECas9 plasmid The positive recombinants with pGRNA plasmid eliminated were inoculated into LB medium containing 50g / L sucrose, cultured at 37°C, subcultured twice, and spread onto LB plates containing 50g / L sucrose after appropriate dilution, and cultured at 37°C. The single colonies that grew on LB plates containing 50g / L sucrose but not on kanamycin plates were selected for preservation.

[0027] 2. The carbamoyl phosphate synthase gene carAB was promoted by the strong promoter ptrc and integrated into the genome of Escherichia coli W3110; the specific steps are as follows: 2.1 Preparation of recombinant DNA fragments The carA and carB genes were integrated into the pinH pseudogene site. The gene carAB was amplified using the E. coli W3110 genome as a template; the pinH upstream and downstream homology arm fragments were amplified using the E. coli W3110 genome as a template, and the trc promoter was designed in the downstream primer of the upstream homology arm and the upstream primer of the carAB gene. Overlapping PCR was performed using the upstream and downstream homology arms and the target gene carAB as templates to obtain the fragment Ptrc-carAB.

[0028] 2.2 Construction of sgRNA plasmid Same as step 1.2 2.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 2.4 Elimination of tool plasmids.

[0029] Same as step 1.4 3. The phosphoenolpyruvate carboxylase gene ppc is driven by the strong promoter ptrc and integrated into the genome of Escherichia coli W3110; the specific steps are as follows: 3.1 Preparation of recombinant DNA fragments The ppc gene was integrated into the yghX pseudogene site. The ppc gene was amplified using the E. coli W3110 genome as a template; the yghX upstream and downstream homology arm fragments were amplified using the E. coli W3110 genome as a template, and the trc promoter was designed in the downstream primer of the upstream homology arm and the upstream primer of the ppc gene. Overlapping PCR was performed using the upstream and downstream homology arms and the target gene ppc as templates to obtain the fragment Ptrc-ppc.

[0030] 3.2 Construction of sgRNA plasmid Same as step 1.2 3.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 3.4 Elimination of tool plasmids.

[0031] Same as step 1.4 4. The ribose-phosphate diphosphate kinase gene prs is driven by the strong promoter ptrc and integrated into the genome of Escherichia coli W3110; the specific steps are as follows: 4.1 Preparation of recombinant DNA fragments The prs gene was integrated into the yncI pseudogene site. The gene prs was amplified using the E. coli W3110 genome as a template; the yncI upstream and downstream homology arm fragments were amplified using the E. coli W3110 genome as a template, and the trc promoter was designed in the downstream primer of the upstream homology arm and the upstream primer of the prs gene. Overlap PCR was performed using the upstream and downstream homology arms and the target gene prs as templates to obtain the fragment Ptrc-prs.

[0032] 4.2 Construction of sgRNA plasmid Same as step 1.2 4.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 4.4 Elimination of tool plasmids.

[0033] Same as step 1.4 5. The pyrimidine nucleoside synthesis operon from Bacillus subtilis was initiated by the promoter ptrc and integrated into the genome of Escherichia coli W3110; the specific steps are as follows: 5.1 The Bacillus subtilis pyrimidine nucleoside synthesis operon was integrated into the rpnD pseudogene site in segments. The operon gene was amplified using the Bacillus subtilis genome as a template; the rpnD upstream and downstream homology arm fragments were amplified using the Escherichia coli W3110 genome as a template, and the trc promoter was designed in the downstream primer of the upstream homology arm and the upstream primer of the operon gene. Overlapping PCR was performed using the upstream and downstream homology arms and the target operon gene as templates to obtain fragments, and the operon gene was divided into four segments for integration.

[0034] 5.2 Construction of sgRNA plasmid Same as step 1.2 5.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 5.4 Elimination of tool plasmids.

[0035] Same as step 1.4 6. The cytosine / isoguanine deaminase gene codA from Escherichia coli is activated by the promoter ptrc and integrated into the genome of Escherichia coli W3110; specifically, the following steps are included: 6.1 Preparation of recombinant DNA fragments The codA gene was integrated into the ygaQ pseudogene site. The gene codA was amplified using the E. coli W3110 genome as a template; the upstream and downstream homology arm fragments of ygaQ were amplified using the E. coli W3110 genome as a template, and the trc promoter was designed in the downstream primer of the upstream homology arm and the upstream primer of the codA gene. Overlapping PCR was performed using the upstream and downstream homology arms and the target gene codA as templates to obtain the fragment Ptrc-codA.

[0036] 6.2 Construction of sgRNA plasmid Same as step 1.2 6.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 6.4 Elimination of tool plasmids.

[0037] Same as step 1.4 7. Knockout of DNA binding transcription repressor protein gene PurR; specifically divided into the following steps: 7.1 Preparation of recombinant DNA fragments Using the genome of Escherichia coli W3110 as a template, primers PurR-1, PurR-2, PurR -3, and PurR -4 were used to amplify the upstream and downstream homology arm fragments of mdh, and overlapping PCR was performed using the upstream and downstream homology arms as templates to obtain fragments.

[0038] 7.2 Construction of sgRNA plasmid Same as step 1.2 7.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 7.4 Elimination of tool plasmids.

[0039] Same as step 1.4 8. Knock out the homoserine dehydrogenase gene thrA; specifically divided into the following steps: 8.1 Preparation of recombinant DNA fragments Using the genome of Escherichia coli W3110 as a template, primers thrA -1, thrA -2, thrA -3, and thrA -4 were used to amplify the upstream and downstream homology arm fragments of thrA, and overlapping PCR was performed using the upstream and downstream homology arms as templates to obtain fragments.

[0040] 8.2 Construction of sgRNA plasmid Same as step 1.2 8.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 8.4 Elimination of tool plasmids.

[0041] Same as step 1.4 9. Knock out the ornithine carbamoyltransferase gene argF; specifically divided into the following steps: 9.1 Preparation of recombinant DNA fragments Using the Escherichia coli W3110 genome as a template, primers argF-1, argF-2, argF-3, and argF-4 were used to amplify the upstream and downstream homology arm fragments of rgF, and overlapping PCR was performed using the upstream and downstream homology arms as templates to obtain fragments.

[0042] 9.2 Construction of sgRNA plasmid Same as step 1.2 9.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 9.4 Elimination of tool plasmids.

[0043] Same as step 1.4 10. Knock out uridine kinase udk; specifically divided into the following steps: 10.1 Preparation of recombinant DNA fragments Using the Escherichia coli W3110 genome as a template, primers udk-1, udk-2, udk-3, and udk-4 were used to amplify the upstream and downstream homology arm fragments of udk, and overlapping PCR was performed using the upstream and downstream homology arms as templates to obtain fragments.

[0044] 10.2 Construction of sgRNA plasmid Same as step 1.2 10.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 10.4 Elimination of tool plasmids.

[0045] Same as step 1.4 11. Knockout of uracil phosphoribosyltransferase upp; specifically divided into the following steps: 11.1 Preparation of recombinant DNA fragments Using the Escherichia coli W3110 genome as a template, primers upp-1, upp-2, upp-3, and upp-4 were used to amplify the upp upstream and downstream homology arm fragments, and overlapping PCR was performed using the upstream and downstream homology arms as templates to obtain fragments.

[0046] 11.2 Construction of sgRNA plasmid Same as step 1.2 11.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 11.4 Elimination of tool plasmids.

[0047] Same as step 1.4 12. Knockout of pyrimidine-specific ribonucleoside hydrolase rihA; specifically divided into the following steps: 12.1 Preparation of recombinant DNA fragments Using the Escherichia coli W3110 genome as a template, primers rihA-1, rihA-2, rihA-3, and rihA-4 were used to amplify the upstream and downstream homology arm fragments of rihA, and overlapping PCR was performed using the upstream and downstream homology arms as templates to obtain fragments.

[0048] 12.2 Construction of sgRNA plasmid Same as step 1.2 12.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 12.4 Elimination of tool plasmids.

[0049] Same as step 1.4 13. Knockout of non-specific ribonucleoside hydrolysis rihC; specifically divided into the following steps: 13.1 Preparation of recombinant DNA fragments Using the Escherichia coli W3110 genome as a template, primers rihC-1, rihC-2, rihC-3, and rihC-4 were used to amplify the upstream and downstream homology arm fragments of rihC, and overlapping PCR was performed using the upstream and downstream homology arms as templates to obtain fragments.

[0050] 13.2 Construction of sgRNA Plasmid Same as step 1.2 13.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 13.4 Elimination of tool plasmids.

[0051] Same as step 1.4 14. Knockout of cytidine / deoxycytidine deaminase cdd; specifically divided into the following steps: 14.1 Preparation of recombinant DNA fragments Using the Escherichia coli W3110 genome as a template, primers cdd-1, cdd-2, cdd-3, and cdd-4 were used to amplify the cdd upstream and downstream homology arm fragments, and overlapping PCR was performed using the upstream and downstream homology arms as templates to obtain fragments.

[0052] 14.2 Construction of sgRNA Plasmid Same as step 1.2 14.3 Electroporation of sgRNA and recombinant DNA fragments Same as step 1.3 14.4 Elimination of tool plasmids.

[0053] Same as step 1.4 15. The pseudouridine-5'-phosphatase gene psuG was overexpressed from the pRB322 plasmid using the strong promoter ptac.

[0054] Using pRB322 plasmid as template, plasmid vector was obtained by PCR; using E. coli as template, target gene psuG was obtained by PCR. Using Novazon seamless cloning enzyme reagent C115, plasmid vector and target fragment were connected. Pseudouridine-5'-phosphatase gene psuG overexpression plasmid map is shown in Figure 1 shown.

[0055] The strain obtained in step 14 is used to prepare chemical transformation competent cells, and the obtained plasmid pRB322-psuG is introduced into the competent cells, and tetracycline resistance is used for screening. The genetically engineered bacteria that can grow normally on tetracycline plates are high-pseudouridine-producing bacteria.

[0056] Some of the primers involved in the above strain construction process are shown in Table 1: Table 1: Partial primer sequence list Embodiment 2: See also Figure 2 , using pseudouridine producing strains for fermentation production: Take the strain stored at -80℃, streak it on the slant, culture it at 37℃ for 12h, subculture it twice to restore the strain vitality. Use a sterile inoculation loop to take the strain and inoculate it into the seed culture medium, culture it at 37℃ for 7-10h. Then transfer it to 30mL liquid LB culture medium, culture it at 37℃, 200 rpm / min for 5h, and then inoculate it into the seed tank.

[0057] Take 30mL of cultured seeds and inoculate them into a sterilized seed tank, and the seed tank is set to 9L. The culture temperature is 36℃, the tank pressure is 0.05MPa, and the dissolved oxygen is controlled to be >30% by controlling the tank pressure, ventilation volume and stirring speed during the fermentation process. When OD600>20, the seeds are transplanted, and the transplant volume is 3L.

[0058] The fermentation tank was set to 18L, and the temperature was controlled at 36°C during the fermentation process. The pH was controlled at 7.2 when OD was <60 in the early stage, and adjusted to 7.0 after OD>60. During the fermentation process, the dissolved oxygen was controlled at 20-40% by controlling the tank pressure, ventilation volume and stirring speed. 60% glucose and 20% ammonia water were added to control the stability of residual sugar and pH. The fermentation cycle was 58-63h.

[0059] In 30mL liquid LB medium, culture at 37℃, 200 rpm / min for 5h, then inoculate into the seed tank. When the OD600 of the seed tank reaches 20-25, transplant according to 15%-20% inoculation amount. Fermentation culture for 58-63h, during the fermentation process, 60%-80% m / v glucose and 20%-25% ammonia water are fed to maintain residual sugar and pH stability.

[0060] The components of LB medium are: yeast powder 5g / L, peptone 10g / L, NaCl 10g / L; The seed tank components are 30 g / L glucose, 5 g / L yeast powder, 5 g / L peptone, 2 g / L KH2PO4, 2 g / L MgSO4, 3 g / L citric acid, 5 g / L corn steep liquor, 50 mg / L FeSO4, 20 mg / L MnSO4, 40 mg / L CoCl, 120 mg / L VB, 20 mg / L VH, and 40 mg / L ZnSO4. The fermentation medium components contain: 10 g / L glucose, 3 g / L yeast powder, 2 g / L peptone, 5 g / L KH2PO4, 2 g / L MgSO4, 1 g / L citric acid, 8 g / L corn steep liquor, 50 mg / L FeSO4, 20 mg / L MnSO4, 40 mg / L CoCl, 120 mg / L VB, 20 mg / L VH, 40 mg / L ZnSO4 and 40 mg / L CaCl2.

[0061] Among them, the pseudouridine production and OD trend during the fermentation of pseudouridine production strains are shown in the figure below. Figure 2 shown.

[0062] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0063] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0064] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A genetically engineered bacterium for producing pseudouridine, characterized in that: The Escherichia coli W3110 ( Escherichia coli W3110) was used as the starting strain, the supply of aspartic acid, carbamoyl phosphate, glutamine and 5'-phosphoribosyl pyrophosphate prpp required for the synthesis of pseudouridine was enhanced, the pyrimidine nucleoside synthesis operon from Bacillus subtilis was introduced, and the cytosine / isoguanine deaminase gene codA from Escherichia coli W3110 was overexpressed to enhance the synthesis of uracil, genes that were not conducive to uracil accumulation were knocked out, the accumulation of other types of nucleotides was reduced, and the pseudouridine-5'-phosphoglycosidase gene psuG was obtained. The pseudouridine-5'-phosphoglycosidase gene psuG was expressed using a high-copy plasmid, and a genetically engineered bacterium that can directly produce pseudouridine by glucose fermentation was constructed.

2. A genetically engineered bacterium for producing pseudouridine according to claim 1, characterized in that: The precursor substances are supplied by overexpressing aspartate aminotransferase gene aspC, carbamoyl phosphate synthase gene carAB, phosphoenolpyruvate carboxylase gene ppc and ribose-phosphate diphosphate kinase gene prs; The nucleotide sequence of the aspartate aminotransferase gene aspC is shown in SEQ ID No: 1; The nucleotide sequence of the carbamoyl phosphate synthase gene carAB is shown in SEQ ID No: 2; The nucleotide sequence of the phosphoenolpyruvate carboxylase gene ppc is shown in SEQ ID No: 3; The nucleotide sequence of the ribose-bisphosphate kinase gene prs is shown in SEQ ID No:

4.

3. A genetically engineered bacterium for producing pseudouridine according to claim 1, characterized in that: The genes contained in the pyrimidine nucleoside synthesis operon from Bacillus subtilis include: pyrB, pyrC, pyrAA, pyrAB, pyrK, pyrD, pyrF and pyrE, and the cytosine / isoguanine deaminase gene codA from Escherichia coli W3110 is overexpressed by the strong promoter ptrc; The nucleotide sequence of the gene pyrB is shown in SEQ ID No: 5; The nucleotide sequence of the gene pyrC is shown in SEQ ID No: 6; The nucleotide sequence of the gene pyrAA is shown in SEQ ID No: 7; The nucleotide sequence of the gene pyrAB is shown in SEQ ID No: 8; The nucleotide sequence of the gene pyrK is shown in SEQ ID No: 9; The nucleotide sequence of the gene pyrD is shown in SEQ ID No: 10; The nucleotide sequence of the gene pyrF is shown in SEQ ID No: 11; The nucleotide sequence of the gene pyrE is shown in SEQ ID No: 12; The nucleotide sequence of the cytosine / isoguanine deaminase gene codA from Escherichia coli W3110 is shown in SEQ ID No:

13.

4. A genetically engineered bacterium for producing pseudouridine according to claim 1, characterized in that: The genes that are not conducive to uracil accumulation include DNA binding transcription repressor gene PurR, homoserine dehydrogenase gene thrA, ornithine carbamoyltransferase gene argF, uridine kinase udk, uracil phosphoribosyltransferase upp, pyrimidine specific ribonucleoside hydrolase rihA, non-specific ribonucleoside hydrolysis rihC and cytidine / deoxycytidine deaminase cdd; The nucleotide sequence of the DNA binding transcription repressor protein gene PurR is shown in SEQ ID No: 14; The nucleotide sequence of the homoserine dehydrogenase gene thrA is shown in SEQ ID No: 15; The nucleotide sequence of the ornithine carbamoyltransferase gene argF is shown in SEQ ID No: 16; The nucleotide sequence of the uridine kinase udk is shown in SEQ ID No: 17; The nucleotide sequence of the uracil phosphoribosyltransferase upp is shown in SEQ ID No: 18; The nucleotide sequence of the pyrimidine-specific ribonucleoside hydrolase rihA is shown in SEQ ID No: 19; The nucleotide sequence of the non-specific ribonucleoside hydrolyzing rihC is shown in SEQ ID No: 20; The nucleotide sequence of the cytidine / deoxycytidine deaminase cdd is shown in SEQ ID No:

21.

5. The genetically engineered bacterium for producing pseudouridine according to claim 1, characterized in that: The pseudouridine-5'-phosphoglycosidase gene psuG was overexpressed by the pRB322 plasmid using the strong promoter ptac; The nucleotide sequence of the pseudouridine-5'-phosphatase gene psuG is shown in SEQ ID No:

22.

6. A genetically engineered bacterium for producing pseudouridine according to any of claims 1 to 5, characterized in that: The genetically engineered bacteria use CRISPR / Cas9-mediated gene editing technology to perform gene knockout and integration.

7. The genetically engineered bacteria according to claims 1-6 are used for preparing pseudouridine.

8. The pseudouridine according to claim 7, characterized in that The pseudouridine preparation process comprises the following steps: Step a: Activate the genetically engineered bacteria for two generations on a slant; Step b: The genetically engineered bacteria after two generations of activation were inoculated into 30 mL of liquid LB medium, and cultured at 37° C. and 200 rpm / min for 5 h before being inoculated into a seed tank; Step c: When the OD600 of the seed tank reaches 20-25, transplant the seeds at a 15%-20% inoculation rate; Step d: After transplanting, the culture is carried out in the fermentation medium for 58-63 hours. During the fermentation process, 60%-80% m / v glucose and 20%-25% ammonia water are added to maintain the residual sugar and pH stable.

9. The pseudouridine according to claim 8, characterized in that The LB medium contains 5 g / L yeast powder, 10 g / L peptone, and 10 g / L NaCl. The seed tank components include 30 g / L glucose, 5 g / L yeast powder, 5 g / L peptone, 2 g / L KH2PO4, 2 g / L MgSO4, 3 g / L citric acid, 5 g / L corn steep liquor, 50 mg / L FeSO4, 20 mg / L MnSO4, 40 mg / L CoCl, 20 mg / L VB1, 20 mg / L VH, and 40 mg / L ZnSO4. The fermentation medium components include: 10 g / L glucose, 3 g / L yeast powder, 2 g / L peptone, 5 g / L KH2PO4, 2 g / L MgSO4, 1 g / L citric acid, 8 g / L corn steep liquor, 50 mg / L FeSO4, 20 mg / L MnSO4, 40 mg / L CoCl, 20 mg / L VB1, 20 mg / L VH, 40 mg / L ZnSO4 and 40 mg / L CaCl2.

10. A method for constructing a genetically engineered bacterium for producing pseudouridine, characterized in that: The construction method comprises the following steps: Step 1: Starting from Escherichia coli W3110, knock out the lacI gene in the genome to obtain a chassis strain; Step 2: To enhance the supply of precursor substances, the aspartate aminotransferase gene aspC, the carbamoyl phosphate synthase gene carAB, the phosphoenolpyruvate carboxylase gene ppc and the ribose-phosphate diphosphate kinase gene prs were driven by the strong promoter ptrc and integrated into the genome of Escherichia coli W3110; Step 3: In order to enhance the synthesis ability of the product, the pyrimidine nucleoside synthesis operon from Bacillus subtilis is driven by the promoter ptrc and integrated into the genome of Escherichia coli W3110, wherein the genes include: pyrB, pyrC, pyrAA, pyrAB, pyrK, pyrD, pyrF and pyrE; the cytosine / isoguanine deaminase gene codA from Escherichia coli is driven by the promoter ptrc and integrated into the genome of Escherichia coli W3110; Step 4: knocking out genes that are not conducive to pseudouridine accumulation, including DNA binding transcription repressor gene PurR, homoserine dehydrogenase gene thrA, ornithine carbamoyltransferase gene argF, uridine kinase udk, uracil phosphoribosyltransferase upp, pyrimidine-specific ribonucleoside hydrolase rihA, non-specific ribonucleoside hydrolysis rihC and cytidine / deoxycytidine deaminase cdd, to obtain pseudouridine-5'-phosphoglycosidase gene psuG; Step 5: The pseudouridine-5'-phosphoglycosidase gene psuG is overexpressed from the pRB322 plasmid using the strong promoter ptac to obtain a genetically engineered bacterium that can directly produce pseudouridine by glucose fermentation.

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