A 5-methyluridine producing strain and its construction method and application

Abstract

The present invention provides a 5-methyluridine producing strain and a construction method and application thereof, wherein the strain utilizes CRIPSR / Cas9 gene editing technology to produce E.coli On the UR12 genome, the artificial promoter P BBa_j23114 Replacement of uridylate kinase gene pyrH The natural promoter of umpH , umpG and no Gene, to ensure bacterial growth while enhancing the accumulation of 5-methyluridine; heterologous introduction and multiple copies of flavin-dependent thymidylate synthase gene thyX , overexpression of alkaline phosphatase gene phoA, Synergistically enhance the 5-methyluridine synthesis pathway; finally, overexpression of the serine hydroxymethyltransferase gene glyA, The supply of CH2-THF, the coenzyme of the key enzyme ThyX, is maintained, and the obtained strain has good genetic stability, high fermentation yield, and can stably produce 5-methyluridine.

Description

Technical Field

[0001] The invention relates to the technical fields of metabolic engineering and genetic engineering, in particular to a 5-methyluridine producing strain and a construction method and application thereof. Background Art

[0002] At present, 5-Methyluridine is widely used in the pharmaceutical, cosmetic and health food industries, especially in the fields of antiviral drugs, nucleic acid drug precursors and functional additives. With the rapid growth of the pharmaceutical industry and health food market, the market demand for 5-Methyluridine is also continuously expanding.

[0003] At present, the main methods for industrial production of 5-methyluridine are chemical synthesis, enzyme catalysis and microbial fermentation. Among them, the chemical synthesis method is complicated to operate, usually requires multiple steps of reaction, and may introduce harmful by-products, causing great pollution to the environment; although the enzyme catalysis method has certain specificity and efficiency, the reaction process has high requirements for the purity of raw materials and the activity of enzymes, and the separation and recycling of enzymes are difficult. In contrast, the microbial fermentation method has the advantages of simple process, easy availability of raw materials, low cost and high safety, and is expected to become an important development direction for the production of 5-methyluridine.

[0004] However, the current 5-methyluridine production strains all carry plasmids, which have disadvantages such as poor genetic stability and heavy growth and metabolic burden, which will lead to industrial problems such as a significant decrease in product yield and difficulty in controlling the production process. Therefore, constructing a 5-methyluridine production strain with high acid production efficiency and good genetic stability is a technical problem that needs to be solved urgently. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a 5-methyluridine producing strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned 5-methyluridine producing strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned 5-methyluridine producing strain.

[0008] In order to solve the above technical problems, the technical solution of the present invention is:

[0009] A 5-methyluridine producing strain, which is a strain E.coli 5me-9 is a targeted modification method in the starting strain E.coli It is obtained by further modification based on UR12, specifically: E.coli Weakened uridylate kinase gene in UR12 genome pyrH(Gene ID:944989), knockout umpH Gene (Gene ID:945283), umpG Gene (Gene ID:947211) and tdk Gene (Gene ID: 945834), heterologously introduced into Streptomyces viridis Streptomyces viridochromogenes Tü57 ( STVIR ) in the flavin-dependent thymidylate synthase gene ThyX, overexpression of the alkaline phosphatase gene phoA (Gene ID:945041) and serine hydroxymethyltransferase (SHMT) gene glyA (Gene ID:947022).

[0010] Preferably, the above-mentioned 5-methyluridine producing strain, the starting strain E. coli UR12 is the strain obtained in Item 1.12 of Example 1 of CN202411087442.4 (A uridine producing strain and its construction method and application) E.coli UR12.

[0011] Preferably, in the above-mentioned 5-methyluridine producing strain, the directed transformation method is to completely modify the starting strain using CRISPR / Cas9 gene editing technology. E.coli The UR12 chromosome genome was transformed.

[0012] Preferably, the 5-methyluridine producing strain is expressed using an artificial promoter P BBa_j23114 Replacement of uridylate kinase gene pyrH The natural promoter of pyrH The nucleotide sequence of the promoter is shown in SEQ ID NO.1. BBa_j23114 The nucleotide sequence is shown in the sequence table SEQ ID NO.2; the uridylate kinase gene pyrH The natural promoter nucleotide sequence is shown in the sequence listing SEQ ID NO.3.

[0013] Preferably, the 5-methyluridine producing strain has a knockout gene on the genome umpH The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.4; umpG The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.5; tdk The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.6.

[0014] Preferably, the 5-methyluridine producing strain is yjiT , ychG , yncKThe three loci were integrated with codon-optimized Streptomyces viridis Streptomyces viridochromogenes Tü57 flavin-dependent thymidylate synthase gene ThyX (GenBank of ThyX gene before optimization: ELS54489.1), and regulated by artificial promoter trc; the amino acid sequence of the flavin-dependent thymidylate synthase gene ThyX is shown in the sequence listing SEQ ID NO.7, the nucleotide sequence is shown in the sequence listing SEQ ID NO.8, and the nucleotide sequence of the artificial promoter trc is shown in the sequence listing SEQ ID NO.9.

[0015] Preferably, the 5-methyluridine producing strain is mbhA Alkaline phosphatase gene phoA, The alkaline phosphatase gene is regulated by the promoter lac; phoA The nucleotide sequence of is shown in the sequence listing as SEQ ID NO.10, and the nucleotide sequence of the promoter lac is shown in the sequence listing as SEQ ID NO.11.

[0016] Preferably, the 5-methyluridine producing strain is yeeL Serine hydroxymethyltransferase gene glyA and regulated by the artificial promoter trc; serine hydroxymethyltransferase gene glyA The nucleotide sequence is shown in the sequence listing SEQ ID NO.12.

[0017] The method for constructing the above 5-methyluridine producing strain comprises the following specific steps:

[0018] (1) E.coli On the UR12 genome, the artificial promoter P BBa_j23114 Replacement of uridylate kinase gene pyrH (Gene ID: 944989) natural promoter, weakens the branching pathway of the precursor UMP flowing in the growth direction, and enhances the accumulation of 5-methyluridine while ensuring bacterial growth;

[0019] (2) Knockout umpH Gene (Gene ID:945283) and umpG Gene (Gene ID: 947211), blocking the branching pathway of the precursor UMP to uridine; knockout tdk gene (Gene ID: 945834), prevents the reflux of 5-methyluridine to 5-methyluridylic acid;

[0020] (3) yjiT , ychG , yncK The codon-optimized Streptomyces viridis was integrated into the three sitesStreptomyces viridochromogenes Tü57 flavin-dependent thymidylate synthase gene ThyX, which is regulated by the artificial promoter trc, heterologously introduced and multiple copies of flavin-dependent thymidylate synthase gene ThyX to enhance the 5-methyluridine synthesis pathway;

[0021] (4) mbhA Alkaline phosphatase gene phoA (Gene ID:945041) ， The alkaline phosphatase gene was overexpressed by the promoter lac phoA , strengthen the 5-methyluridine synthesis pathway;

[0022] (5) yeeL Serine hydroxymethyltransferase gene glyA (Gene ID:947022), and regulated by artificial promoter trc, overexpressing serine hydroxymethyltransferase gene glyA , strengthening the supply of coenzyme CH2-THF of the key enzyme ThyX.

[0023] Application of the 5-methyluridine producing strain in fermentation production of 5-methyluridine.

[0024] Preferably, the application of the above-mentioned 5-methyluridine production strain uses shake flask fermentation to produce 5-methyluridine, and the specific steps are as follows:

[0025] (1) Seed activation and culture: Spread the bacteria evenly on the activated slant, culture at 37°C for 12 h, transfer to the activated slant and continue to culture for 10 h, then transfer to a shake tube containing seed culture medium for seed culture;

[0026] (2) Fermentation culture: Inoculate the seed liquid into the fermentation medium at a rate of 10-15%, culture at 36°C and 220 r / min with shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; glucose solution is added to maintain the fermentation process; the fermentation cycle is 30-32 h.

[0027] Preferably, in the application of the above-mentioned 5-methyluridine producing strain, the slant culture medium used in the seed activation and culture is: glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, the rest is water, pH 7.0-7.2; the seed culture medium is: yeast powder 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 、V B2 、V B3 、V B5 、VB12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, and the rest is water; the fermentation medium comprises: yeast powder 10 g / L, folic acid 8 mg / L, serine 1.1 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 10.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 、V B2 、V B3 、V B5 、V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, phenol red 2%, and the rest is water.

[0028] Preferably, the application of the above-mentioned 5-methyluridine production strain uses a fermentation tank to produce 5-methyluridine, and the specific steps are as follows:

[0029] (1) Seed activation: Spread the strain evenly on the activation slope, culture at 37°C for 12 h, transfer to an eggplant-shaped bottle and continue culture for 12 h;

[0030] (2) Seed culture: Inoculate the bacterial suspension into the seed culture medium, pH 7.0, constant temperature at 36°C, dissolved oxygen at 30%-60%, and culture for 6.5-7 hours;

[0031] (3) Fermentation culture: Wait for the seed bacteria volume OD 600 To 20-25, add the fermentation medium according to the inoculation amount of 20%, start fermentation, control the pH value at 7.0, the temperature at 36℃, and the dissolved oxygen at 30%-60% during the fermentation process; when the glucose in the medium is consumed, add the glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1g / L, and add PLP together with the sugar solution.

[0032] Preferably, in the application of the above-mentioned 5-methyluridine producing strain, the slant culture medium used in the seed activation is: glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, and the rest is water, pH 7.0-7.2; the seed culture medium is: yeast powder 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, VB1 、V B2 、V B3 、V B5 、V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, and the rest is water; the fermentation medium is: glucose 10 g / L, yeast powder 10 g / L, folic acid 8 mg / L, serine 1.1 g / L, citric acid 2 g / L, (NH4)2SO42.5 g / L, KH2PO410.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 、V B2 、V B3 、V B5 、V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, PLP 5 mg / L (added with sugar), and the rest is water.

[0033] The above culture media can be prepared using standard methods.

[0034] Beneficial effects:

[0035] The above 5-methyluridine producing strain was used in E.coli On the UR12 genome, we first used the artificial promoter P BBa_j23114 Replacement of uridylate kinase gene pyrH The natural promoter of umpH and umpG The gene blocks the branching pathway of the precursor UMP to uridine, knocking out tdk Gene, prevent 5-methyluridine from flowing back to 5-methyluridylic acid, and enhance 5-methyluridine accumulation while ensuring bacterial growth; heterologous introduction and multi-copy flavin-dependent thymidylate synthase gene thyX , overexpression of alkaline phosphatase gene phoA, Synergistically enhance the 5-methyluridine synthesis pathway; finally, overexpress the serine hydroxymethyltransferase gene glyA, Maintains the supply of coenzyme CH2-THF (5,10-methylenetetrahydrofolate) for the key enzyme ThyX.

[0036] The obtained strain does not contain plasmids, has no defects, does not require induction, has the advantages of good genetic stability and high fermentation yield, and is an excellent strain that can stably produce 5-methyluridine. The strain can efficiently synthesize 5-methyluridine from scratch using glucose as a substrate. After 32 hours of shake flask fermentation, the 5-methyluridine production can be as high as 7.2 g / L. After 36 hours of 5L fermentation tank fermentation, the 5-methyluridine production reaches 28.5 g / L. The strain has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the directed transformation method of 5-methyluridine-producing strains.

[0038] Figure 2 For production strains E.coli Fermentation process curve of 5me-9 in a 5 L fermenter. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0040] The percentage sign "%" involved in the examples, unless otherwise specified, refers to mass percentage, and the percentage of a solution refers to the number of grams of a solute contained in 100 mL.

[0041] The starting strain used in the embodiment is a uridine high-yielding strain E.coli UR12, the strain is the strain obtained in Item 1.12 of Example 1 of CN202411087442.4 (A uridine-producing strain and its construction method and application) E.coli UR12; codon-optimized viridischromogenic Streptomyces Streptomyces viridochromogenes Tü57 flavin-dependent thymidylate synthase gene thyX Synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.; the corresponding promoter and gene are shown in the sequence listing.

[0042] The gene editing method used was referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coliUsing CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.), the engineering plasmid pGRB involved in this method is based on pUC18 as the skeleton, including the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence, and ampicillin resistance (working concentration: 100 mg / L). The professional terms such as gene integration and plasmid construction involved in the following examples can be explained in this article. The primers used in the strain construction process are shown in Table 1. The specific operation steps refer to the authorized patent "A genetically engineered bacterium for producing orotic acid and its construction method and application" (patent number: ZL202210417923.1). The professional terms such as gene integration and plasmid construction involved in the following examples can be explained in this article. The primers used in the strain construction process are shown in Table 1.

[0043] Table 1 Primers involved in strain construction

[0044] Primer Sequence number Sequence (5' to 3') DW-PpyrH-A SEQ ID NO.13 TAGCTTCTGCCCAGCTGTAGC DW-PpyrH-j114-S SEQ ID NO.14 CCTAGGTACAATGCTAGCAGGAAACAGACCATGGCTACCAATGCAAAACCC UP-PpyrH-j114-A SEQ ID NO.15 TACCTAGGACTGAGCTAGCCATAAAAATTAAGACTGCTTGGACATCGCAG UP-PpyrH-S SEQ ID NO.16 CTATGCACGTTGCTGCAAGC Pj114-JD-S SEQ ID NO.17 GCTCAGTCCTAGGTACAATGCTAGC pGRB-PpyrH-S SEQ ID NO.18 AGTCCTAGGTATAATACTAGTTAAATTCAGCTAACCCTTGTGTTTTAGAGCTAGAA pGRB-PpyrH-A SEQ ID NO.19 TTCTAGCTCTAAAACACAAGGGTTAGCTGAATTTAACTAGTATTATACCTAGGACT umpH-QC-1 SEQ ID NO.20 TGCCGGTGAAATCACCGAAG umpH-QC-2 SEQ ID NO.21 AGCGACTGACGGGTAAATCCGGCGACGTTATCGTGCATCAG umpH-QC-3 SEQ ID NO.22 CTGATGCACGATAACGTCGCCGGATTTACCCGTCAGTCGCT umpH-QC-4 SEQ ID NO.23 CGTAATGGTGATTGTCGATTGGTGAA pGRB-umpH-S SEQ ID NO.24 AGTCCTAGGTATAATACTAGTCGCAGCCATTAAACAAAATGCGTTTTAGAGCTAGAA pGRB-umpH-A SEQ ID NO.25 TTCTAGCTCTAAAACGCATTTTGTTTAATGCTGCGACTAGTATTATACCTAGGACT umpG-QC-1 SEQ ID NO.26 TCGAACAAGCAGCTGTCGC umpG-QC-2 SEQ ID NO.27 CAGCGGCGTGATGGAGACATAACGTCAGCAAACTCACGCAA umpG-QC-3 SEQ ID NO.28 TTGCGTGAGTTTGCTGACGTTATGTCTCCATCACGCCGCTG umpG-QC-4 SEQ ID NO.29 CAGGATTGCCGTTTGATATCCCGAA pGRB-umpG-S SEQ ID NO.30 AGTCCTAGGTATATACTAGTCGCCCGGACATTGTTGTGTCGTTTTAGAGCTAGAA pGRB-umpG-A SEQ ID NO.31 TTCTAGCTCTAAAACGACACAACAATGTCCGGGCGACTAGTATTATACCTAGGACT tdk-QC-1 SEQ ID NO.32 GTGAATAGTTACCAGCAGTCATTTACCC tdk-QC-2 SEQ ID NO.33 CGTTATAAGGTCTGCCTGCTTGATCACGAACTGACTTTCCCGGCAC tdk-QC-3 SEQ ID NO.34 GTGCCGGGAAAGTCAGTTCGTGATCAAGCAGGCAGACCTTATAACG tdk-QC-4 SEQ ID NO.35 CGACGATGTATAACGCCTAAACACAG pGRB-tdk-S SEQ ID NO.36 AGTCCTAGGTATAATACTTAGTGCAGCCAATACTTACTGGCAGTTTTAGAGCTAGA pGRB-tdk-A SEQ ID NO.37 TTCTAGCTCTAAAACTGCCAGTAAGTATTGGCTGCACTAGTATTATACCTAGGACT thyX-trc-S SEQ ID NO.38 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACCGTGACCCCGGTG thyX-trc-A SEQ ID NO.39 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCAAGCTTTTACGGGCTCACGC yjiT-1 SEQ ID NO.40 AATAGTTGTTGCCGCCTGAGT yjiT-trc-2 SEQ ID NO.41 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAACAGGCAGCAAAGTCCC yjiT-3 SEQ ID NO.42 ATCTGGAAAACAAAGGCCAGTAAAAGCACTACCTGTGAAGGGATGT yjiT-4 SEQ ID NO.43 CAGGGCTTCCACAGTCACAAT pGRB-S SEQ ID NO.44 AGTCCTAGGTATAATACTAGTAGGGATTATGAACGGCAATGGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.45 TTCTAGCTCTAAAACCATTGCCGTTCATAATCCCTACTAGTATTATACCTAGGACT yncK-1 SEQ ID NO.46 CGCAGCGTAAACCACTGGTTAC yncK-trc-2 SEQ ID NO.47 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCCCTGAAGGATGGGGTTTTAC yncK-3 SEQ ID NO.48 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCATCAGGAAAACACAATGCCTTC yncK-4 SEQ ID NO.49 CATTTTTCACAACACCCGTGACCT pGRB-S SEQ ID NO.50 AGTCCTAGGTATAATACTAGTATCGACCAGATATTGCATTCGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.51 TTCTAGCTCTAAAACGAATGCAATATCTGGTCGATACTAGTATTATACCTAGGACT ychG-1 SEQ ID NO.52 GGGAGCCGTTTTCTTATGCCAC ychG-trc-2 SEQ ID NO.53 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGCGTTCTGCCGCTTAGTG ychG-3 SEQ ID NO.54 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATCAAAAACCCGTTATCCTGTGAAAC ychG-4 SEQ ID NO.55 CTGCGTCTTGATCAAGCAGG pGRB-ychG-S SEQ ID NO.56 AGTCCTAGGTATAATACTAGTAGGGACCATCCCGAAACGACGTTTTAGAGCTAGAA pGRB-ychG-A SEQ ID NO.57 TTCTAGCTCTAAAACGTCGTTTCGGGATGGTCCCTACTAGTATTATACCTAGGACT phoA-lac-S SEQ ID NO.58 TCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGTGAAACAAAGCACTATTGCACTGG phoA-lac-A SEQ ID NO.59 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATTTCAGCCCCAGAGCGGC mbhA-1 SEQ ID NO.60 GCCAGCACGAACATAATCCC mbhA-lac-2 SEQ ID NO.61 ATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAACACGGTGGCAGGTTTTGG mbhA-3 SEQ ID NO.62 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-4 SEQ ID NO.63 CGGCGTAATCACAAACTGGC pGRB-mbhA-S SEQ ID NO.64 AGTCCTAGGTATAATACTAGTTACCGGGCATACCGATGCGAGTTTTAGAGCTAGAA pGRB-mbhA-A SEQ ID NO.65 TTCTAGCTCTAAAACTCGCATCGGTATGCCCGGTAACTAGTATTATACCTAGGACT glyA-trc-S SEQ ID NO.66 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTAAAGCGTGAAATGAACATTGCC glyA-trc-A SEQ ID NO.67 CCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATGCGTAAACCGGGTAACGTGC yeeL-1 SEQ ID NO.68 TTCATCGGGACGAGTGGAGA yeeL-trc-2 SEQ ID NO.69 TATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCACCATAGCATCGCCAATCTGA yeeL-3 SEQ ID NO.70 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATAAAGC yeeL-4 SEQ ID NO.71 CATTCCCTCTACAGAACTAGCCCT pGRB-yeeL-A SEQ ID NO.72 AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA pGRB-yeeL-S SEQ ID NO.73 TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT

[0045] Example 1

[0046] like Figure 1 As shown, the specific process of constructing genetically engineered strains is as follows:

[0047] The starting strain E.coli UR12 was prepared according to the operation mentioned in CN202411087442.4 E.coli UR12 / pRed-Cas9 electroporated competent cells.

[0048] 1.1 Gene pyrH promoter replacement (P pyrH ::P BBa_j23114 )

[0049] Using Escherichia coli W3110 as a template, primers UP-PpyrH-S and UP-PpyrH-j114-A, DW-PpyrH-A and DW-PpyrH-j114-S were used for PCR amplification to obtain the upper homology arm UP-PpyrH-js114 and the lower homology arm DW-PpyrH. Using the recovered upstream and downstream homology arms as templates, primers UP-PpyrH-S and DW-PpyrH-A were used for overlapping PCR to obtain the target fragment PpyrH-js114 required for integration. Afterwards, the DNA fragment obtained by annealing primers pGRB-PpyrH-S and pGRB-PpyrH-A was connected to the plasmid pGRB line to construct the pGRB-PpyrH plasmid. Finally, the purified PpyrH-js114 integration fragment and plasmid pGRB-PpyrH were simultaneously transferred intoE.coli UR12 / pRed-Cas9 competent cells were used, and primers Pj114-JD-S and DW-PpyrH-A were used as identification primers to screen positive transformants, and finally the strain was obtained. E.coli 5me-1.

[0050] 1.2 Genes umpH Knockout

[0051] Using the available concentration of Escherichia coli W3110 genome as a template, primers umpH-QC-1 and umpH-QC-2, umpH-QC-3 and umpH-QC-4 were used for PCR amplification to obtain the upstream homology arm umpH-QC-UP and the downstream homology arm umpH-QC-DW. Using the recovered upstream and downstream homology arms as templates, primers umpH-QC-1 and umpH-QC-4 were used for overlapping PCR to obtain the complementation fragment ΔumpH required for knocking out the gene umpH. Afterwards, the DNA fragment obtained by annealing primers pGRB-umpH-S and pGRB-umpH-A was connected to the plasmid pGRB line to construct the pGRB-umpH plasmid. Finally, the plasmid pGRB-umpH and the overlapping fragment ΔumpH were electroporated into E.coli 5me-1 / pRed-Cas9 electroporated competent cells; then umpH-QC-1 and umpH-QC-4 were used as identification primers to screen positive transformants and obtain strains E.coli 5me-2.

[0052] 1.3 Genes umpG Knockout

[0053] Using the available concentration of Escherichia coli W3110 genome as a template, PCR amplification was performed with primers umpG-QC-1 and umpG-QC-2, umpG-QC-3 and umpG-QC-4, respectively, to obtain the upstream homology arm umpG-QC-UP and the downstream homology arm umpG-QC-DW. Using the recovered upstream and downstream homology arms as templates, overlapping PCR was performed with primers umpG-QC-1 and umpG-QC-4 to obtain the complementation fragment ΔumpG required for knocking out the gene umpG. Afterwards, the DNA fragment obtained by annealing primers pGRB-umpG-S and pGRB-umpG-A was connected to the plasmid pGRB line to construct the pGRB-umpG plasmid. Finally, the plasmid pGRB-umpG and the overlapping fragment ΔumpG were electroporated into E.coli 5me-2 / pRed-Cas9 electroporated competent cells; then umpG-QC-1 and umpG-QC-4 were used as identification primers to screen positive transformants and obtain strains E.coli 5me-3.

[0054] 1.4 Genes tdk Knockout

[0055] Using the available concentration of Escherichia coli W3110 genome as a template, PCR amplification was performed with primers tdk-QC-1 and tdk-QC-2, tdk-QC-3 and tdk-QC-4, respectively, to obtain the upstream homology arm tdk-QC-UP and the downstream homology arm tdk-QC-DW. Using the recovered upstream and downstream homology arms as templates, overlapping PCR was performed with primers tdk-QC-1 and tdk-QC-4 to obtain the complementation fragment Δtdk required for knocking out the gene tdk. Afterwards, the DNA fragment obtained by annealing primers pGRB-tdk-S and pGRB-tdk-A was connected to the plasmid pGRB line to construct the pGRB-tdk plasmid. Finally, the pGRB-tdk plasmid and the overlapping fragment Δtdk were electroporated to E.coli 5me-3 / pRed-Cas9 electroporated competent cells; then tdk-QC-1 and tdk-QC-4 were used as identification primers to screen positive transformants and obtain strains E.coli 5me-4.

[0056] 1.5 Genes thyX (STVIR) Integration of yjiT ::P trc - thyX (STVIR) Integration of

[0057] The available concentration of Escherichia coli W3110 genome was used as a template, and primers yjiT-1 and yjiT-trc-2, yjiT-3 and yjiT-4 were used; the green chromogenic Streptomyces synthesized by Jinweizhi and codon-optimized Streptomyces viridochromogenes Tǖ57 flavin-dependent thymidylate synthase gene thyX As template, primer thyX -trc-S and thyX -trc-A was amplified by PCR to obtain the upper homology arm yjiT-trc-UP, the lower homology arm yjiT-DW and the middle target fragment P trc - thyX Using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, primers yjiT-1 and yjiT-4 were used to obtain the target fragment yjiT-P required for integration by overlapping PCR. trc - thyX Then, the DNA fragment obtained by annealing primers pGRB-yjiT-S and pGRB-yjiT-A was connected to the plasmid pGRB to construct the pGRB-yjiT plasmid. trc - thyXThe integration fragment and plasmid pGRB-yjiT were simultaneously transferred into E.coli 5me-4 / pRed-Cas9 competent cells were then used to screen positive transformants using primers yjiT-1 and yjiT-4 as identification primers to finally obtain the strain E.coli 5me-5.

[0058] 1.6 Genes thyX (STVIR) Second copy of ychG ::P trc - thyX (STVIR) Integration of

[0059] Using E. coli W3110 as a template, PCR amplification was performed with primers ychG-1 and ychG-trc-2, ychG-3 and ychG-4 to obtain the upper homology arm ychG-trc-UP and the lower homology arm ychG-DW. The recovered upper and downstream homology arms and the intermediate target fragment P trc - thyX As a template, primers ychG-1 and ychG-4 were used to obtain the target fragment ychG-P required for integration by overlapping PCR. trc - thyX Then, the DNA fragment obtained by annealing primers pGRB-ychG-S and pGRB-ychG-A was connected to the plasmid pGRB to construct the pGRB-ychG plasmid. trc - thyX The integration fragment and plasmid pGRB-ychG were simultaneously transferred into E.coli 5me-5 / pRed-Cas9 competent cells were used, and then primers ychG-1 and ychG-4 were used as identification primers to screen positive transformants, and finally the strain was obtained. E.coli 5me-6.

[0060] 1.7 Genes thyX (STVIR) Three copies of yncK ::P trc - thyX (STVIR) Integration of

[0061] Using E. coli W3110 as a template, PCR amplification was performed with primers yncK-1 and yncK-trc-2, yncK-3 and yncK-4 to obtain the upper homology arm yncK-trc-UP and the lower homology arm yncK-DW. The recovered upper and downstream homology arms and the intermediate target fragment P trc - thyXAs a template, primers yncK-1 and yncK-4 were used to obtain the target fragment yncK-P required for integration by overlapping PCR. trc - thyX Then, the DNA fragment obtained by annealing primers pGRB-yncK-S and pGRB-yncK-A was connected to the plasmid pGRB to construct the pGRB-yncK plasmid. trc - thyX The integration fragment and plasmid pGRB-yncK were simultaneously transferred into E.coli 5me-6 / pRed-Cas9 competent cells, and then use primers yncK-1 and yncK-4 as identification primers to screen positive transformants, and finally obtain the strain E.coli 5me-7.

[0062] 1.8 Genes phoA Integration mbhA ::P trc - phoA Integration of

[0063] Using Escherichia coli W3110 as template, primers mbhA-1 and mbhA-lac-2, mbhA-3 and mbhA-4, phoA-lac-S and phoA -lac-A was used for PCR amplification to obtain the upper homology arm mbhA-lac-UP, the lower homology arm mbhA-DW and the middle target fragment P lac - phoA Using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, primers mbhA-1 and mbhA-4 were used to obtain the target fragment mbhA-P required for integration by overlapping PCR. lac - phoA Then, the DNA fragment obtained by annealing primers pGRB-mbhA-S and pGRB-mbhA-A was connected to the plasmid pGRB to construct the pGRB-mbhA plasmid. lac - phoA The integration fragment and plasmid pGRB-mbhA were simultaneously transferred into E.coli 5me-7 / pRed-Cas9 competent cells were then used as identification primers to screen positive transformants, and finally the strain was obtained. E.coli 5me-8.

[0064] 1.9 Genes glyA Integration of yjiT ::P trc - glyA Integration of

[0065] Using the available concentration of Escherichia coli W3110 genome as a template, primers yeeL-1 and yeeL-trc-2, yeeL-3 and yeeL-4, glyA -trc-S and glyA -trc-A was amplified by PCR to obtain the upper homology arm yeeL-trc-UP, the lower homology arm yeeL-DW and the middle target fragment P trc - glyA Using the recovered upstream and downstream homology arms and the intermediate target fragment as templates, primers yeeL-1 and yeeL-4 were used to obtain the target fragment yeeL-P required for integration by overlapping PCR. trc - glyA Then, the DNA fragment obtained by annealing primers pGRB-yeeL-S and pGRB-yeeL-A was connected to the plasmid pGRB to construct the pGRB-yeeL plasmid. trc - glyA The integration fragment and plasmid pGRB-yeeL were simultaneously transferred into E.coli 5me-8 / pRed-Cas9 competent cells were then used to screen positive transformants using primers yeeL-1 and yeeL-4 as identification primers to finally obtain the strain E.coli 5me-9.

[0066] The strains involved in the above construction process are shown in Table 2.

[0067] Table 2 Strains involved

[0068]

[0069] Example 2

[0070] Utilize the 5-methyluridine production strain described in Example 1 E.coli Production of uridine by 5me-9 in shake flask fermentation.

[0071] 2.1 Culture medium

[0072] 2.1.1 Slant culture medium

[0073] Glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, dissolve in water, adjust pH to 7.0-7.2 with sodium hydroxide, make up to 500 ml, dispense into test tubes (9 ml / tube) and eggplant-shaped bottles (45 ml / bottle), and sterilize in a high-pressure steam cooker at 121°C for 20 min.

[0074] 2.1.2 Seed culture medium

[0075] Yeast powder 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 、V B2 、V B3 、V B5 、V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, and the rest is water.

[0076] 2.1.3 Fermentation medium

[0077] Yeast powder 10 g / L, folic acid 8 mg / L, serine 1.1 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 10.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 、V B2 、V B3 、V B5 、V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, phenol red 2%, and the rest is water.

[0078] 2.2 Cultivation methods

[0079] 2.2.1 Seed activation and cultivation:

[0080] The strain was evenly spread on the activated slant, cultured at 37°C for 12 h, transferred to the activated slant and continued to be cultured for 10 h, and then transferred to a shake tube containing 5 ml of seed culture medium for seed culture.

[0081] 2.2.2 Fermentation culture:

[0082] The seed liquid was inoculated at 15% inoculation into a 500 mL Erlenmeyer flask (final volume 30 mL) filled with fermentation medium, sealed with nine layers of gauze, and cultured at 36°C, 220 r / min shaking. During the fermentation process, ammonia water was added to maintain the pH at 7.0-7.2; 60% (m / v) glucose solution was added to maintain the fermentation process (phenol red was used as an indicator, and when the color of the fermentation liquid no longer changed, it was considered to be sugar-deficient. When sugar was deficient, 1-2 mL of 60% (m / v) glucose solution was added. The fermentation cycle was 32 h, and no antibiotics or inducers were added during the fermentation process.

[0083] After 32 h of shake flask fermentation, the uridine production could reach as high as 7.2 g / L.

[0084] Example 3

[0085] Utilize the 5-methyluridine production strain described in Example 1 E.coli 5-Methyluridine was produced by fermentation of 5me-9 in a 5 L fermenter.

[0086] 3.1 Culture medium

[0087] 3.1.1 Slant culture medium

[0088] Glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, dissolve in water, adjust pH to 7.0-7.2 with sodium hydroxide, make up to 500 ml, dispense into test tubes (9 ml / tube) and eggplant-shaped bottles (45 ml / bottle), and sterilize in a high-pressure steam cooker at 121°C for 20 min.

[0089] 3.1.2 Seed culture medium

[0090] Yeast powder 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 、V B2 、V B3 、V B5 、V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, and the rest is water.

[0091] 3.1.3 Fermentation medium

[0092] Glucose 10 g / L, yeast powder 10 g / L, folic acid 8 mg / L, serine 1.1 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 10.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 、V B2 、V B3 、V B5 、V B12 1 mg / L each, V H0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, PLP 5 mg / L (added with sugar), and the rest is water.

[0093] 3.2 Cultivation methods

[0094] 3.2.1 Seed activation: Spread the strain evenly on the activation slant, culture at 37℃ for 12 h, transfer to eggplant-shaped flask and continue culture for 12 h;

[0095] 3.2.2 Seed culture: Inoculate the bacterial suspension into the seed culture medium, pH 7.0, constant temperature at 36°C, dissolved oxygen at 30%-60%, and culture for 7 hours;

[0096] 3.2.3 Fermentation culture: wait for the seed bacteria to reach OD600 of 22, inoculate the fermentation medium at 20% inoculation rate, and start fermentation. During the fermentation process, control the pH at 7.0, the temperature at 36°C, and the dissolved oxygen at 30%-60%. When the glucose in the medium is consumed, feed 80% glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1 g / L, add PLP 5 mg / L following the sugar solution feed, and do not add antibiotics or inducers during the fermentation process.

[0097] like Figure 2 As shown in the figure, after 36 h of fermentation, the uridine production could reach as high as 28.5 g / L.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. A 5-methyluridine producing strain, characterized in that: The targeted transformation method is used to transform the starting strain E. coli It is obtained by further modification based on UR12, specifically: E. coli Weakened uridylate kinase gene in UR12 genome pyr , knockout umpH Gene, umpG Genes and tdk Streptomyces viridochromogenes Streptomyces viridochromogenes Flavin-dependent thymidylate synthase gene ThyX in Tü57, overexpression of alkaline phosphatase gene phoA and serine hydroxymethyltransferase gene glyA ;in, With artificial promoter P BBa_j23114 Replacement of uridylate kinase gene pyr The natural promoter of pyr The nucleotide sequence of the promoter is shown in SEQ ID NO.

1. BBa_j23114 The nucleotide sequence is shown in the sequence table SEQ ID NO.2; the uridylate kinase gene pyr The natural promoter nucleotide sequence is shown in the sequence listing SEQ ID NO.3; exist Yj , Yj , yncK The flavin-dependent thymidylate synthase gene ThyX is integrated into the three sites respectively and regulated by an artificial promoter trc; the nucleotide sequence of the flavin-dependent thymidylate synthase gene ThyX is shown in the sequence list SEQ ID NO.8, and the nucleotide sequence of the artificial promoter trc is shown in the sequence list SEQ ID NO.9; exist mbA Alkaline phosphatase gene phoA, The alkaline phosphatase gene is regulated by the promoter lac; phoA The nucleotide sequence of is shown in the sequence listing as SEQ ID NO.10, and the nucleotide sequence of the promoter lac is shown in the sequence listing as SEQ ID NO.11; exist yeeL Serine hydroxymethyltransferase gene glyA and regulated by the artificial promoter trc; serine hydroxymethyltransferase gene glyA The nucleotide sequence is shown in the sequence listing SEQ ID NO.

12.

2. The 5-methyluridine producing strain according to claim 1, characterized in that: Said umpH The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.4; umpG The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.5; tdk The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

6.

3. The method for constructing the 5-methyluridine producing strain according to claim 1 or 2, characterized in that: The specific steps are as follows: (1) E. coli On the UR12 genome, the artificial promoter P BBa_j23114 Replacement of uridylate kinase gene pyr The natural promoter of (2) Knockout umpH Gene, umpG Genes and tdk Gene; (3) Yj , Yj , yncK The codon-optimized Streptomyces viridis was integrated into the three sites Streptomyces viridochromogenes Tü57 flavin-dependent thymidylate synthase gene ThyX, and regulated by the artificial promoter trc; (4) mbA Alkaline phosphatase gene phoA, And regulated by the promoter lac; (5) yeeL Serine hydroxymethyltransferase gene glyA and regulated by the artificial promoter trc.

4. Use of the 5-methyluridine producing strain according to claim 1 or 2 in fermentation production of 5-methyluridine.

5. The use according to claim 4, characterized in that: The production of 5-methyluridine using shake flask fermentation is as follows: (1) Seed activation and culture: Spread the bacteria evenly on the activated slant, culture at 37°C for 12 h, transfer to the activated slant and continue to culture for 10 h, then transfer to a shake tube containing seed culture medium for seed culture; (2) Fermentation culture: Inoculate the seed liquid into the fermentation medium at a rate of 10-15%, culture at 36°C and 220 r / min with shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding ammonia water; glucose solution is added to maintain the fermentation process; the fermentation cycle is 30-32 hours.

6. The use according to claim 5, characterized in that: The slant medium used in the seed activation and cultivation is: glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, the rest is water, pH 7.0-7.2; the seed culture medium is: yeast powder 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 3.0 g / L, V B1 、V B2 、V B3 、V B5 、V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, and the rest is water; the fermentation medium comprises: yeast powder 10 g / L, folic acid 8 mg / L, serine 1.1 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 10.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 、V B2 、V B3 、V B5 、V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, phenol red 2%, and the rest is water.

7. The use according to claim 4, characterized in that: The specific steps of producing 5-methyluridine by fermentation in a fermenter are as follows: (1) Seed activation: Spread the strain evenly on the activation slope, culture at 37°C for 12 hours, transfer to an eggplant-shaped bottle and continue to culture for 12 hours; (2) Seed culture: Inoculate the bacterial suspension into the seed culture medium, pH 7.0, constant temperature at 36°C, dissolved oxygen at 30%-60%, and culture for 6.5-7 hours; (3) Fermentation culture: Wait for the seed bacteria volume OD 600 To 20-25, add the fermentation medium according to the inoculation amount of 20%, start fermentation, control the pH value at 7.0, the temperature at 36℃, and the dissolved oxygen at 30%-60% during the fermentation process; when the glucose in the medium is consumed, add the glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1g / L, and add PLP together with the sugar solution.

8. The use according to claim 7, characterized in that: The slant culture medium used in the seed activation is: glucose 2 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, the rest is water, pH 7.0-7.2; the seed culture medium is: yeast powder 8.0 g / L, peptone 2.0 g / L, (NH4)2SO4 2.0 g / L, KH2PO43.0 g / L, V B1 、V B2 、V B3 、V B5 、V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O0.5 g / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, and the rest is water; the fermentation medium is: glucose 10 g / L, yeast powder 10 g / L, folic acid 8 mg / L, serine 1.1 g / L, citric acid 2 g / L, (NH4)2SO4 2.5 g / L, KH2PO4 10.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 、V B2 、V B3 、V B5 、V B12 1 mg / L each, V H 0.1 mg / L, ammonium molybdate 0.32 mg / L, boric acid 4.5 mg / L, CoCl2·6H2O 1.6 mg / L, PLP 5 mg / L, and the rest is water; the PLP is added along with the sugar.

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