Cytosine nucleoside production strain as well as construction method and application thereof

Abstract

The invention provides a cytidine production strain as well as a construction method and application thereof, the strain is constructed on an E.coliUR12 genome by utilizing a CRIPSR / Cas9 gene editing technology, and a cdd gene and a cmk gene are knocked out; a nucleoside triphosphate reductase gene nrdD is weakened; a nucleoside triphosphate pyrophosphate hydrolase gene nudG, a nucleoside diphosphate kinase gene ndk and a uridine monophosphate kinase gene pyrH (D93A) carrying a mutation point are overexpressed, and a cytidine triphosphate synthase gene pyrG (D160E, E162A, E168K, cgl) of corynebacterium glutamicum carrying a mutation point and a bifunctional nucleotidase gene PHM8 (sce) of saccharomyces cerevisiae are heterologously introduced and multi-copied; the obtained strain is good in hereditary stability and high in fermentation yield, and cytidine can be stably produced.

Description

Technical Field

[0001] The invention relates to the technical fields of metabolic engineering and genetic engineering, in particular to a cytosine nucleoside production strain and a construction method and application thereof. Background Art

[0002] Cytosine nucleoside, also known as cytosine ribonucleoside, or cytidine for short, is one of the pyrimidine nucleosides that constitute nucleic acids. It participates in various physiological and biochemical processes in organisms and plays an important role in metabolism, genetics and other regulatory processes in organisms. In addition, cytidine is also an important pharmaceutical synthesis intermediate. Because of its property of interfering with ribonucleic acid synthesis, it is often used as a raw material and intermediate for the synthesis of anti-tumor and antiviral drugs such as cytarabine, azacitidine, and zalcitabine. Nowadays, the application research and development of cytidine is still ongoing, and a more efficient synthesis method is urgently needed.

[0003] The production methods of cytidine nucleoside mainly include chemical synthesis and microbial fermentation. Traditional cytidine production processes mostly rely on chemical synthesis, but this method has many problems, including harsh reaction conditions, serious environmental pollution and high production costs. In order to overcome these problems, microbial fermentation has gradually become a research hotspot for cytidine preparation in recent years. Microbial fermentation can achieve efficient biosynthesis of cytidine by metabolic engineering of microorganisms. This method uses glucose as a raw material and produces cytidine through a fermentation process, which has the advantages of being green, environmentally friendly, low cost and mild production conditions. In recent years, the development of synthetic biology technology has further improved the efficiency of the fermentation method, making it the mainstream choice for industrial production. Therefore, constructing a cytidine nucleoside production strain with a clear genetic background, good stability and relatively considerable yield is a technical problem that needs to be solved urgently. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a cytosine nucleoside producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned cytosine nucleoside producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned cytosine nucleoside production strain.

[0007] In order to solve the above technical problems, the technical solution of the present invention is: A cytosine nucleoside production strain, which is a strain E.coli cyt-10, is a targeted transformation method in the starting strain E.coli It is obtained by further modification based on UR12, specifically: E.coliKnock out the cdd gene and cmk gene on the UR12 genome, weaken the ribonucleotide reductase gene nrdD, overexpress the nucleoside triphosphate pyrophosphohydrolase gene nudG, the nucleoside diphosphate kinase gene ndk, and the uridylate kinase gene pyrH with mutation sites (D93A) , and heterologously introduce and express Corynebacterium glutamicum Corynebacterium glutamicum The cytidine triphosphate synthase gene pyrG with mutation sites of ATCC 13032 (D160E、E162A、E168K，cgl) and the bifunctional nucleotide enzyme gene of Saccharomyces cerevisiae S288c PHM8 (sce) ; among them, the nucleoside triphosphate pyrophosphohydrolase gene nudG, the uridylate kinase gene pyrH with mutation sites (D93A) and the cytidine triphosphate synthase gene pyrG with mutation sites (D160E、E162A、E168K，cgl) are synchronously multi-copied through the artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk.

[0008] The above pyrH （D93A) is obtained by point mutation of the pyrH gene, that is, the 278th base is changed from A to C, resulting in the 93rd amino acid residue being changed from aspartic acid to alanine; pyrG (D160E、E162A、E168K，cgl) gene is obtained by point mutation of the pyrG gene of Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032, that is, the 480th base is changed from C to G, the 485th base is changed from A to C, and the 502nd base is changed from G to A, resulting in the 160th amino acid residue being changed from aspartic acid to glutamic acid, the 162nd amino acid residue being changed from glutamic acid to alanine, and the 168th amino acid residue being changed from glutamic acid to lysine.

[0009] Preferably, for the above cytosine nucleoside producing strain, the artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk includes the pyrH (D93A) gene, the ndk gene, and the pyrG (D160E、E162A、E168K，cgl) gene, specifically: (1) The gene order of the operon is the trc artificial promoter, pyrG (D160E、E162A、E168K，cgl) , pyrH (D93A) , ndk, and the rrnB T1 terminator; (2) The trc artificial promoter is directly connected end to end with the pyrG (D160E、E162A、E168K，cgl) gene; (3) The pyrG (D160E、E162A、E168K，cgl) gene, the pyrH (D93A)The genes are connected by linker-1, and the nucleotide sequence of linker-1 is shown in SEQ ID NO.12 of the sequence listing; (4)pyrH (D93A) The genes pyrG and ndk are connected by linker-2, and the nucleotide sequence of linker-2 is shown in SEQ ID NO.13 of the sequence listing; (5)ndk and the rrnB T1 terminator are directly connected head to tail.

[0010] The construction method of the above artificial operon is obtained by controlling the start of transcription of the operon by the same trc artificial promoter, controlling the termination of transcription of the operon by the same rrnB T1 terminator, and optimizing the gene connection mode and the position of the gene in the operon.

[0011] Preferably, in the above cytosine nucleoside-producing strain, the artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk has a nucleotide sequence shown in SEQ ID NO.14 of the sequence listing.

[0012] Preferably, in the above cytosine nucleoside-producing strain, the starting strain E.coli UR12 is the strain in Example 1.12 of CN202411087442.4 (a uridine-producing strain and its construction method and application) E.coli UR12.

[0013] Preferably, in the above cytosine nucleoside-producing strain, the directed modification method is to use the CRISPR / Cas9 gene editing technology to completely modify the starting strain E.coli UR12 on the chromosomal genome.

[0014] Preferably, in the above cytosine nucleoside-producing strain, the nucleotide sequence of the cdd gene knocked out on the genome is shown in SEQ ID NO.1 of the sequence listing; the nucleotide sequence of the cmk gene is shown in SEQ ID NO.2 of the sequence listing.

[0015] Preferably, in the above cytosine nucleoside-producing strain, the natural promoter of the nucleoside triphosphate reductase gene nrdD is replaced with the artificial promoter P BBa_j23114 , the nucleotide sequence of the nucleoside triphosphate reductase gene nrdD is shown in SEQ ID NO.3 of the sequence listing, the nucleotide sequence of the artificial promoter P BBa_j23114 is shown in SEQ ID NO.4 of the sequence listing, and the nucleotide sequence of the natural promoter of the nucleoside triphosphate reductase gene nrdD is shown in SEQ ID NO.5 of the sequence listing.

[0016] Preferably, in the above cytosine nucleoside-producing strain, the nucleoside triphosphate pyrophosphohydrolase gene nudG is successively integrated at three sites, namely fhiA, yjiV, and yeeL, and regulated by the artificial promoter trc; the nucleotide sequence of the artificial promoter trc is as shown in SEQ ID NO.6 in the sequence listing, and the nucleotide sequence of the nucleoside triphosphate pyrophosphohydrolase gene nudG is as shown in SEQ ID NO.7 in the sequence listing.

[0017] Preferably, in the above cytosine nucleoside-producing strain, the above artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH (D93A) -ndk is successively integrated at two sites, namely ycdN and yncK; the Corynebacterium glutamicum Corynebacterium glutamicum The cytidine triphosphate synthase gene pyrG with a mutation site carried by ATCC 13032 (D160E、E162A、E168K，cgl) The nucleotide sequence is as shown in SEQ ID NO.8 in the sequence listing, and the uridylate kinase gene pyrH with a mutation site carried (D93A) The nucleotide sequence is as shown in SEQ ID NO.9 in the sequence listing, and the nucleotide sequence of the nucleoside diphosphate kinase gene ndk is as shown in SEQ ID NO.10 in the sequence listing.

[0018] Preferably, in the above cytosine nucleoside-producing strain, the codon-optimized bifunctional nucleotide enzyme gene of Saccharomyces cerevisiae Saccharomyces cerevisiae S288c is successively integrated at two sites, namely ychG and yghE PHM8 (sce) , and regulated by the artificial promoter trc; the bifunctional nucleotide enzyme gene PHM8 (sce) The nucleotide sequence is as shown in SEQ ID NO.11 in the sequence listing.

[0019] The construction method of the above cytosine nucleoside-producing strain is as follows: (1) On the E. coli UR12 genome, knockout cdd The gene blocking product cytidine from flowing to uridine; knockout the cmk gene to prevent the precursor CMP from flowing back to CDP; (2) Replace the natural promoter of the nucleoside triphosphate reductase gene nrdD with the artificial promoter P BBa_j23114 Weaken the branch pathway of the precursor CTP flowing to the growth direction, and strengthen the accumulation of cytosine nucleoside while ensuring the growth of the bacteria; (3) Successively integrate the nucleoside triphosphate pyrophosphohydrolase gene nudG at three sites, namely fhiA, yjiV, and yeeL, and regulate it with the artificial promoter trc to overexpress the nucleoside triphosphate pyrophosphohydrolase gene nudG and strengthen the cytosine nucleoside synthesis pathway; (4) Integrate the above artificial operon pyrG successively at the ycdN and yncK loci (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk, overexpress the uridylate kinase gene pyrH carrying a mutation point (D93A) and the nucleoside diphosphate kinase gene ndk, heterologously introduce and multi-copy the cytidine triphosphate synthase gene Corynebacterium glutamicum pyrG carrying a mutation point of Corynebacterium glutamicum ATCC 13032 (D160E、E162A、E168K，cgl) , strengthen the cytosine nucleoside synthesis pathway; (5) Integrate the codon-optimized bifunctional nucleotide enzyme gene of Saccharomyces cerevisiae Saccharomyces cerevisiae S288c successively at the ychG and yghE loci PHM8 (sce) , regulate it with the artificial promoter trc, heterologously introduce and multi-copy the bifunctional nucleotide enzyme gene PHM8 (sce) , strengthen the cytosine nucleoside synthesis pathway.

[0020] Application of the above cytosine nucleoside-producing strain in the fermentation production of cytosine nucleoside.

[0021] Preferably, for the application of the above cytosine nucleoside-producing strain, shake flask fermentation is used to produce cytosine nucleoside, and the specific steps are as follows: (1) Seed activation and culture: Inoculate the bacterial liquid from the preservation tube and spread it evenly on the activation slant, culture at 37 °C for 12 h, transfer to the activation slant and continue to culture for 10 h, and then transfer to a shake tube containing the seed medium for seed culture; (2) Fermentation culture; Inoculate the seed liquid into a triangular flask containing the fermentation medium at an inoculation amount of 10-15%, seal it with nine layers of gauze, culture at 36 °C with shaking at 220 r / min, maintain the pH at 7.0-7.2 by adding ammonia water during the fermentation process; add 60% glucose solution to maintain the fermentation; the fermentation cycle is 30-32 h.

[0022] Preferably, for the application of the above cytosine nucleoside-producing strain, the slant medium used in seed activation is: glucose 2.0 g / L, peptone 10.0 g / L, yeast powder 5.0 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.

[0023] Preferably, for the application of the above cytosine nucleoside-producing strain, the seed medium used in seed culture is: yeast powder 8.0 g / L, peptone 3.0 g / L, (NH4)2SO4 3.0 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, 0.5 g / L of MgSO4·7H2O, and the rest is water.

[0024] Preferably, for the application of the above-mentioned cytosine nucleoside-producing strain, the fermentation medium used in the fermentation culture is: 10.0 g / L of yeast powder, 3.0 g / L of citric acid, 2.0 g / L of glutamic acid, 4.0 g / L of (NH4)2SO4, 8.0 g / L of KH2PO4, 2.0 g / L of MgSO4·7H2O, 40 mg / L of FeSO4·7H2O, V B1 , V B2 , V B3 , V B5 , V B12 1 mg / L each, V H 0.1 mg / L, 2% of phenol red, and the rest is water.

[0025] The above media can all be prepared by standard methods.

[0026] Beneficial effects: For the above-mentioned cytosine nucleoside-producing strain, the CRIPSR / Cas9 gene editing technology is first used to knockout cdd gene, blocking the flow of the product cytidine to uridine, knocking out the cmk gene to prevent the precursor CMP from flowing back to CDP; replacing the natural promoter of the ribonucleoside triphosphate reductase gene nrdD with the artificial promoter P BBa_j23114 , weakening the branch pathway of the precursor CTP flowing towards the growth direction, strengthening the accumulation of cytosine nucleoside while ensuring the growth of the bacteria; overexpressing the ribonucleoside triphosphate pyrophosphohydrolase gene nudG, the nucleoside diphosphate kinase gene ndk, and the uridylate kinase gene pyrH (D93A) with a mutation point, heterologously introducing and multi-copying the cytidine triphosphate synthase gene pyrG Corynebacterium glutamicum with a mutation point from Corynebacterium glutamicum ATCC 13032 (D160E、E162A、E168K，cgl) and the bifunctional nucleotide enzyme gene Saccharomyces cerevisiae from Saccharomyces cerevisiae PHM8 (sce) , synergistically strengthening the cytosine nucleoside synthesis pathway. The constructed strain does not contain plasmids, has no defects, does not require induction, has advantages such as good genetic stability and high fermentation yield, and is an excellent strain that can stably produce cytosine nucleoside. The strain efficiently synthesizes cytosine nucleoside de novo using glucose as a substrate. After 32 h of shake flask fermentation, the yield of cytosine nucleoside can reach as high as 11.8 g / L. Description of the drawings

[0027] Figure 1 Diagram of the method for directed modification of cytosine nucleoside-producing strains. Specific implementation manners

[0028] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described in detail below in conjunction with specific implementation manners.

[0029] In the examples, the percentage sign "%" refers to volume percentage if not otherwise specified; the percentage of the solution "% (m / v)" refers to the number of grams of solute contained in 100 ml of the solution.

[0030] The starting strain used in the examples is a uridine-producing strain E.coli UR12, which is the strain described in Example 1.12 of CN202411087442.4 (a uridine-producing strain and its construction method and application) E.coli UR12; the codon-optimized Saccharomyces cerevisiae Saccharomyces cerevisiae The bifunctional nuclease gene of S288c PHM8 (sce) Synthesized by Suzhou Genewiz Biotechnology Co., Ltd.; the corresponding promoter and gene are shown in the sequence listing.

[0031] The gene editing method used refers to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). The engineering plasmid pGRB involved in this method is based on pUC18 and includes the promoter J23100, the gRNA-Cas9 binding region sequence, the terminator sequence, and ampicillin resistance (working concentration: 100 mg / L). 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.

[0032] Table 1 Primers involved in the strain construction process Primer Sequence number Sequence (5'-end to 3'-end) cdd-QC-1 SEQ ID NO.15 TTATCAGGAAGGTGCATTTAGTTCGCT cdd-QC-2 SEQ ID NO.16 CCAGCGGAAGTAGTGCGAATG cdd-QC-3 SEQ ID NO.17 GCAGAAAAAGCCGATGCGC cdd-QC-4 SEQ ID NO.18 AGATATAAGGCGCGGTTTTCCAG pGRB-cdd-S SEQ ID NO.19 AGTCCTAGGTATAATACTAGTTGCCTTTGGGCCGAAAGATCGTTTTAGAGCTAGAA pGRB-cdd-A SEQ ID NO.20 TTCTAGCTCTAAAACGATCTTTCGGCCCAAAGGCAACTAGTATTATACCTAGGACT cmk-QC-1 SEQ ID NO.21 TTGTCATCTTTCGGGCTGGTC cmk-QC-2 SEQ ID NO.22 GCCGGAACCAGTGGCGCTACCGCTCGGTTACGATAGTCCAGCAGATGCCATTGC cmk-QC-3 SEQ ID NO.23 GAAGCGTTGCAATGGCATCTGCTGGACTATCGTAACCGAGCGGTAGC cmk-QC-4 SEQ ID NO.24 GCAACAACAACGCCACGAAC pGRB-cmk-S SEQ ID NO.25 GATTGCCGATGGCCGCGACAGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-cmk-A SEQ ID NO.26 TGTCGCGGCCATCGGCAATCACTAGTATTATACCTAGGACTGAGC DW-PnrdD-A SEQ ID NO.27 TCGCGCTGGGTTGGAATCA DW-PnrdD-js114-S SEQ ID NO.28 CCTAGGTACAATGCTAGCAGGAAACAGACCATGACACCGCATGTGATGAAACG UP-PnrdD-js114-A SEQ ID NO.29 TACCTAGGACTGAGCTAGCCATAAATATAGTGCTTTGCATCAAGGATGTTTGAG UP-PnrdD-S SEQ ID NO.30 AACTGGCAGCTTGTGATGCA Pjs114-JD-S SEQ ID NO.31 GCTCAGTCCTAGGTACAATGCTAGC pGRB-PnrdD-S SEQ ID NO.32 AGTCCTAGGTATAATACTAGTCGGCGTTGTGGATAAGCGGAGTTTTAGAGCTAGAA pGRB-PnrdD-A SEQ ID NO.33 TTCTAGCTCTAAAACTCCGCTTATCCACAACGCCGACTAGTATTATACCTAGGACT nudG-trc-S SEQ ID NO.34 TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAATGATTGAAGTTGTTGCCGCCA nudG-trc-A SEQ ID NO.35 AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTAATCCGCTGGTCTGGCG fhiA-1 SEQ ID NO.36 TTATTTAACGGCGACAGCCCAC fhiA-trc-2 SEQ ID NO.37 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATCGCCAGAATCATCATCCC fhiA-3 SEQ ID NO.38 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT CAAGCAGGAGCTGACGGTGT fhiA-4 SEQ ID NO.39 TGCACCAATGCTGGATACTTACA pGRB-S SEQ ID NO.40 AGTCCTAGGTATAATACTAGTTTTAAGCCTCGGTTACAAACGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.41 TTCTAGCTCTAAAACGTTTGTAACCGAGGCTTAAAACTAGTATTATACCTAGGACT yjiV-1 SEQ ID NO.42 TGTGACTGTGGAAGCCCTGTAT yjiV-trc-2 SEQ ID NO.43 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATTCGGGCTGTCCCTTGTC yjiV-3 SEQ ID NO.44 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTGGCACCTGAATGACGAACT yjiV-4 SEQ ID NO.45 TGGCGACATTCCCTTCCTT pGRB-S SEQ ID NO.46 AGTCCTAGGTATAATACTAGTGCGTAGTCGAAATTCTCAGCGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.47 TTCTAGCTCTAAAACGCTGAGAATTTCGACTACGCACTAGTATTATACCTAGGACT yeeL-1 SEQ ID NO.48 TTCATCGGGACGAGTGGAGA yeeL-trc-2 SEQ ID NO.49 TATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCACCATAGCATCGCCAATCTGA yeeL-3 SEQ ID NO.50 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATAAAGC yeeL-4 SEQ ID NO.51 CATTCCCTCTACAGAACTAGCCCT pGRB-S SEQ ID NO.52 AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.53 TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT pyrG-trc-S SEQ ID NO.54 TCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACCTCTAGTCGAAAAGTCCGT pyrG-mut-up-A SEQ ID NO.55 TTAAGGAATGGCTGGGATGCAATCTCACCGACGGTGCCACCG pyrG-mut-dw-S SEQ ID NO.56 GAGATTGCATCCCAGCCATTCCTTAAAGCAGCTCGCCAGGTACG pyrG-link1-A SEQ ID NO.57 CATGAATACTGTTTCCTCTAAGGGTGGACACGCAGC pyrH-link1-S SEQ ID NO.58 AGGAAACAGTATTCATGATGGCTACCAATGCAAAACCC pyrH-mut-up-A SEQ ID NO.59 GCAGCACGCATTGCCAGGCCGTTCATTACGGTCGC pyrH-mut-dw-S SEQ ID NO.60 CTGGCAATGCGTGCTGCACTGCACCGCGCCTATGT pyrH-link2-A SEQ ID NO.61 TTTCAATCTCCTATTATTTCCTTAGAAGCTTATTCCGTGATTAAAGTCCCTTCT ndk-link2-S SEQ ID NO.62 GCTTCTAAGGAAATAATAGGAGATTGAAAATGGCTATTGAACGTACTTTTTCCATCAT ndk-trc-A SEQ ID NO.63 ACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAACGGGTGCGCGGGCA ycdN-1 SEQ ID NO.64 GATTTTGACGCCACCAACACC ycdN-trc-2 SEQ ID NO.65 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAATCCACATCACACAATCCAT ycdN-3 SEQ ID NO.66 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAAGGGATTTTTGGCTATCAGGA ycdN-4 SEQ ID NO.67 CATATCGTATTCGCCAGGCTG pGRB-S SEQ ID NO.68 AGTCCTAGGTATAATACTAGTGCGGCATTTCAACAAGATGTGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.69 TTCTAGCTCTAAAACACATCTTGTTGAAATGCCGCACTAGTATTATACCTAGGACT yncK-1 SEQ ID NO.70 CATTTTTCACAACACCCGTGACCT yncK-trc-2 SEQ ID NO.71 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCATCAGGAAAACACAATGCCTTC yncK-3 SEQ ID NO.72 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCCCTGAAGGATGGGGTTTTAC yncK-4 SEQ ID NO.73 CGCAGCGTAAACCACTGGTTAC pGRB-S SEQ ID NO.74 AGTCCTAGGTATAATACTAGTATCGACCAGATATTGCATTCGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.75 TTCTAGCTCTAAAACGAATGCAATATCTGGTCGATACTAGTATTATACCTAGGACT PHM8-trc-S SEQ ID NO.76 TGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACAATTGCAAAAGATTATAGAACC PHM8-trc-A SEQ ID NO.77 CAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATGATGATTGTACGTTGATTTGTTG ychG-1 SEQ ID NO.78 GGGAGCCGTTTTCTTATGCCAC ychG-trc-2 SEQ ID NO.79 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGCGTTCTGCCGCTTAGTG ychG-3 SEQ ID NO.80 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATCAAAAACCCGTTATCCTGTGAAAC ychG-4 SEQ ID NO.81 CTGCGTCTTGATCAAGCAGG pGRB-S SEQ ID NO.82 AGTCCTAGGTATAATACTAGTAGGGACCATCCCGAAACGACGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.83 TTCTAGCTCTAAAACGTCGTTTCGGGATGGTCCCTACTAGTATTATACCTAGGACT yghE-1 SEQ ID NO.84 GTCAGGCACTGGCGAAAGAT yghE-trc-2 SEQ ID NO.85 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGCAAGCCATAAACCCACA yghE-3 SEQ ID NO.86 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTCCGACATCGAAATGCGT yghE-4 SEQ ID NO.87 AGGCGTTGTTGTGGCAGATT pGRB-S SEQ ID NO.88 AGTCCTAGGTATAATACTAGTGCTGAAAAAATATCGCCCACGTTTTAGAGCTAGAA pGRB-A SEQ ID NO.89 TTCTAGCTCTAAAACGTGGGCGATATTTTTTCAGCACTAGTATTATACCTAGGACT

[0033] Example 1 As Figure 1 shown, the specific process of constructing the genetic engineering strain is as follows: The starting strain E.coli UR12 was prepared according to the operations mentioned in CN 202411087442.4 E.coliElectrocompetent cells of UR12 / pRed-Cas9.

[0034] 1.1 Gene cdd knockout Using the Escherichia coli W3110 genome at available concentration as a template, PCR amplifications were performed with primers cdd-QC-1 and cdd-QC-2, cdd-QC-3 and cdd-QC-4 respectively to obtain the upstream homologous arm cdd-QC-UP and the downstream homologous arm cdd-QC-DW. Using the recovered upstream and downstream homologous arms as templates, overlap PCR was performed with primers cdd-QC-1 and cdd-QC-4 to obtain the complementation fragment Δcdd required for gene cdd knockout. Subsequently, the DNA fragment obtained by annealing primers pGRB-cdd-S and pGRB-cdd-A was ligated to the linearized plasmid pGRB to construct the pGRB-cdd plasmid. Finally, the pGRB-cdd plasmid and the overlapping fragment Δcdd were electrotransformed into E.coli the electrocompetent cells of UR12 / pRed-Cas9; then, using cdd-QC-1 and cdd-QC-4 as identification primers, positive transformants were screened to obtain strain E.coli cyt -1.

[0035] 1.2 Gene cmk knockout Using the Escherichia coli W3110 genome at available concentration as a template, PCR amplifications were performed with primers cmk-QC-1 and cmk-QC-2, cmk-QC-3 and cmk-QC-4 respectively to obtain the upstream homologous arm cmk-QC-UP and the downstream homologous arm cmk-QC-DW. Using the recovered upstream and downstream homologous arms as templates, overlap PCR was performed with primers cmk-QC-1 and cmk-QC-4 to obtain the complementation fragment Δcmk required for gene cmk knockout. Subsequently, the DNA fragment obtained by annealing primers pGRB-cmk-S and pGRB-cmk-A was ligated to the linearized plasmid pGRB to construct pGRB-cmk. Finally, the pGRB-cmk plasmid and the overlapping fragment Δcmk were electrotransformed into E.coli cyt the electrocompetent cells of -1 / pRed-Cas9; then, using cmk-QC-1 and cmk-QC-4 as identification primers, positive transformants were screened to obtain strain E.coli cyt -2.

[0036] 1.3 Gene nrdD promoter replacement (P nrdD ::P BBa_j23114 ) Using Escherichia coli W3110 as a template, PCR amplification was performed with primers UP-PnrdD-S and UP-PnrdD-js114-A, DW-PnrdD-A and DW-PnrdD-js114-S to obtain the upper homologous arm UP-PnrdD-js114 and the lower homologous arm DW-PnrdD. Using the recovered upper and lower homologous arms as templates, the target fragment PnrdD-js114 required for integration was obtained by overlap PCR with primers UP-PnrdD-S and DW-PnrdD-A. Subsequently, the DNA fragment obtained by annealing primers pGRB-PnrdD-S and pGRB-PnrdD-A was ligated to the linearized plasmid pGRB to construct the pGRB-PnrdD plasmid. Finally, the purified PnrdD-js114 integration fragment and the plasmid pGRB-PnrdD were simultaneously introduced into E.coli cyt the competent cells of -2 / pRed-Cas9 by electroporation, and then positive transformants were screened using primers Pjs114-JD-S and DW-PnrdD-A as identification primers, and finally the strain E.coli cyt-3 was obtained.

[0037] 1.4 Gene nudG Integration ( fhiA ::P trc - nudG integration) Using the Escherichia coli W3110 genome at available concentration as a template, PCR amplification was performed with primers fhiA-1 and fhiA-trc-2, fhiA-3 and fhiA-4, nudG-trc-S and nudG-trc-A to obtain the upper homologous arm fhiA-trc-UP, the lower homologous arm fhiA-DW and the middle target fragment P trc -nudG. Using the recovered upper and lower homologous arms and the middle target fragment as templates, the target fragment fhiA-P trc -nudG required for integration was obtained by overlap PCR with primers fhiA-1 and fhiA-4. Subsequently, the DNA fragment obtained by annealing primers pGRB-fhiA-S and pGRB-fhiA-A was ligated to the linearized plasmid pGRB to construct the pGRB-fhiA plasmid. Finally, the purified fhiA-P trc -nudG integration fragment and the plasmid pGRB-fhiA were simultaneously introduced into E.coli cyt the competent cells of -3 / pRed-Cas9 by electroporation, and then positive transformants were screened using primers fhiA-1 and fhiA-4 as identification primers, and finally the strain E.coli cyt -4 was obtained.

[0038] 1.5 Gene nudG Second copy (yjiV ::P trc - nudG Integration of Using the Escherichia coli W3110 genome at available concentration as a template, PCR amplification was performed with primers yjiV-1 and yjiV-trc-2, yjiV-3 and yjiV-4, nudG-trc-S and nudG-trc-A to obtain the upper homologous arm yjiV-trc-UP, the lower homologous arm yjiV-DW and the middle target fragment P trc -nudG. Using the recovered upper and lower homologous arms and the middle target fragment as templates, the target fragment yjiV-P required for integration was obtained by overlapping PCR with primers yjiV-1 and yjiV-4 trc -nudG. After that, the DNA fragment obtained by annealing primers pGRB-yjiV-S and pGRB-yjiV-A was ligated to the linearized plasmid pGRB to construct the plasmid pGRB-yjiV. Finally, the purified yjiV-P trc -nudG integration fragment and the plasmid pGRB-yjiV were simultaneously introduced into E.coli cyt the competent cells of -4 / pRed-Cas9 by electroporation, and then the positive transformants were screened using primers yjiV-1 and yjiV-4 as identification primers, and finally the strain E.coli cyt -5 was obtained.

[0039] 1.6 Gene nudG Triple copies of yeeL ::P trc - nudG Integration of Using the Escherichia coli W3110 genome at available concentration as a template, PCR amplification was performed with primers yeeL-1 and yeeL-trc-2, yeeL-3 and yeeL-4, nudG-trc-S and nudG-trc-A to obtain the upper homologous arm yeeL-trc-UP, the lower homologous arm yeeL-DW and the middle target fragment P trc -nudG. Using the recovered upper and lower homologous arms and the middle target fragment as templates, the target fragment yeeL-P required for integration was obtained by overlapping PCR with primers yeeL-1 and yeeL-4 trc -nudG. After that, the DNA fragment obtained by annealing primers pGRB-yeeL-S and pGRB-yeeL-A was ligated to the linearized plasmid pGRB to construct the plasmid pGRB-yeeL. Finally, the purified yeeL-P trc -nudG integration fragment and the plasmid pGRB-yeeL were simultaneously introduced into E.coli cytIn the competent cells of -5 / pRed-Cas9, the positive transformants were screened with primers yeeL-1 and yeeL-4 as identification primers, and finally the strain E.coli cyt -6 was obtained.

[0040] 1.7 Artificial operon gene pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -Integration of ndk ( ycdN ::P trc - pyrG (D160E、E162A、E168K，cgl) - pyrH （D93A) - ndk integration). Using the Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032 genome at available concentration as a template, with primers pyrG-trc-S and pyrG-mut-up-A, pyrG-mut-dw-S and pyrG-link1-A; using the Escherichia coli W3110 genome at available concentration as a template, with primers pyrH-link1-S and pyrH-mut-up-A, pyrH-mut-dw-S and pyrH-link2-A, ndk-link2-S and ndk-trc-A for PCR amplification to obtain fragment P trc -pyrG (D160E、E162A、E168K，cgl) -mut-UP, pyrG (D160E、E162A、E168K，cgl) -mut-DW, pyrH-mut-UP, pyrH-mut-DW and ndk. Using the recovered P trc -pyrG (D160E、E162A、E168K，cgl) -mut-UP and pyrG (D160E、E162A、E168K，cgl) -mut-DW fragments as templates, with primers pyrG-trc-S and pyrG-link1-A by overlap PCR to obtain fragment P trc -pyrG (D160E、E162A、E168K，cgl) . Using the recovered pyrH-mut-UP and pyrH-mut-DW fragments as templates, with primers pyrH-link1-S and pyrH-link2-A by overlap PCR to obtain fragment pyrH （D93A） . Using the recovered P trc -pyrG (D160E、E162A、E168K，cgl) , pyrH （D93A） and ndk fragments as templates, with primers pyrG-trc-S and ndk-trc-A by overlap PCR to obtain the artificial operon fragment P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A)-ndk. Using the Escherichia coli W3110 genome at an available concentration as a template, PCR amplification was performed with primers ycdN-1 and ycdN-trc-2, ycdN-3 and ycdN-4 to obtain the upper homologous arm ycdN-trc-UP and the lower homologous arm ycdN-DW. Using the recovered upper and lower homologous arms and the artificial operon fragment as templates, the target fragment ycdN-P required for integration was obtained by overlapping PCR with primers ycdN-1 and ycdN-4 trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk. Subsequently, the DNA fragment obtained by annealing primers pGRB-ycdN-S and pGRB-ycdN-A was ligated to the plasmid pGRB linear vector to construct the pGRB-ycdN plasmid. Finally, the purified ycdN-P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk integration fragment and the plasmid pGRB-ycdN were simultaneously introduced into E.coli cyt -6 / pRed-Cas9 competent cells by electroporation, and then positive transformants were screened using primers ycdN-1 and ycdN-4 as identification primers, and finally the strain E.coli cyt -7 was obtained

[0041] 1.8 Artificial operon gene pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk secondary copy ( yncK ::P trc - pyrG (D160E、E162A、E168K，cgl) - pyrH （D93A) - ndk integration) Using the Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032 genome at an available concentration as a template, PCR amplification was performed with primers pyrG-trc-S and pyrG-mut-up-A, pyrG-mut-dw-S and pyrG-link1-A; using the Escherichia coli W3110 genome at an available concentration as a template, with primers pyrH-link1-S and pyrH-mut-up-A, pyrH-mut-dw-S and pyrH-link2-A, ndk-link2-S and ndk-trc-A to obtain the fragment P trc -pyrG (D160E、E162A、E168K，cgl) -mut-UP, pyrG (D160E、E162A、E168K，cgl) -mut-DW, pyrH-mut-UP, pyrH-mut-DW and ndk. Using the recovered P trc -pyrG (D160E、E162A、E168K，cgl)-mut-UP and pyrG (D160E、E162A、E168K，cgl) Using the -mut-DW fragment as a template, fragment P was obtained by overlap PCR with primers pyrG-trc-S and pyrG-link1-A trc -pyrG (D160E、E162A、E168K，cgl) Using the recovered pyrH-mut-UP and pyrH-mut-DW fragments as templates, fragment pyrH was obtained by overlap PCR with primers pyrH-link1-S and pyrH-link2-A （D93A） Using the recovered P trc -pyrG (D160E、E162A、E168K，cgl) 、pyrH （D93A） and the ndk fragment as templates, the artificial operon fragment P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk was obtained by overlap PCR with primers pyrG-trc-S and ndk-trc-A. Using the available concentration of Escherichia coli W3110 genome as a template, PCR amplification was carried out with primers yncK-1 and yncK-trc-2, yncK-3 and yncK-4 to obtain the upper homologous arm yncK-trc-UP and the lower homologous arm yncK-DW. Using the recovered upstream and downstream homologous arms and the artificial operon fragment as templates, the target fragment yncK-P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk required for integration was obtained by overlap PCR. Subsequently, the DNA fragment obtained by annealing primers pGRB-yncK-S and pGRB-yncK-A was ligated to the linearized plasmid pGRB to construct the pGRB-yncK plasmid. Finally, the purified yncK-P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk integration fragment and the plasmid pGRB-yncK were simultaneously introduced into the competent cells of E.coli cyt -7 / pRed-Cas9 by electroporation, and then positive transformants were screened using primers yncK-1 and yncK-4 as identification primers, and finally strain E.coli cyt -8 was obtained.

[0042] 1.9 Gene PHM8 （sce) Integration ( ychG ::P trc - PHM8 (sce) Integration) Using the Escherichia coli W3110 genome at an available concentration as a template, with primers ychG-1 and ychG-trc-2, ychG-3 and ychG-4; using the codon-optimized bifunctional nuclease gene of Saccharomyces cerevisiae Saccharomyces cerevisiae S288c synthesized by GenScript PHM8 (sce) as a template, performing PCR amplification with primers PHM8-trc-S and PHM8-trc-A to obtain the upper homologous arm ychG-trc-UP, the lower homologous arm ychG-DW, and the middle target fragment P trc - PHM8 (sce) , using the recovered upstream and downstream homologous arms and the middle target fragment as templates, performing overlap PCR with primers ychG-1 and ychG-4 to obtain the target fragment ychG-P required for integration trc - PHM8 (sce) . Then, annealing the DNA fragments prepared from primers pGRB-ychG-S and pGRB-ychG-A and ligating them to the plasmid pGRB linear vector to construct the pGRB-ychG plasmid. Finally, simultaneously transforming the purified ychG-P trc - PHM8 (sce) integration fragment and the plasmid pGRB-ychG into the competent cells of E.coli cyt -8 / pRed-Cas9 by electroporation, and then screening for positive transformants using primers ychG-1 and ychG-4 as identification primers, finally obtaining the strain E.coli cyt -9.

[0043] 1.10 Gene PHM8 （sce) 's second copy ( yghE ::P trc - PHM8 (sce) integration) Using the Escherichia coli W3110 genome at an available concentration as a template, with primers yghE-1 and yghE-trc-2, yghE-3 and yghE-4; using the codon-optimized bifunctional nuclease gene of Saccharomyces cerevisiae Saccharomyces cerevisiae S288c synthesized by GenScript PHM8 (sce) as a template, performing PCR amplification with primers PHM8-trc-S and PHM8-trc-A to obtain the upper homologous arm yghE-trc-UP, the lower homologous arm yghE-DW, and the middle target fragment P trc - PHM8 (sce), using the recycled upstream and downstream homologous arms and the middle target fragment as templates, the target fragment yghE-P required for integration was obtained by overlapping PCR with primers yghE-1 and yghE-4 trc - PHM8 (sce) , then, the DNA fragment obtained by annealing primers pGRB-yghE-S and pGRB-yghE-A was ligated to the plasmid pGRB linear vector to construct the plasmid pGRB-yghE. Finally, the purified yghE-P trc - PHM8 (sce) integration fragment and the plasmid pGRB-yghE were simultaneously transferred into the competent cells of E.coli cyt -9 / pRed-Cas9 by electroporation, and then the positive transformants were screened using primers yghE-1 and yghE-4 as identification primers, and finally the strain E.coli cyt -10 was obtained.

[0044] The strains involved in the above construction process are shown in Table 2.

[0045] Strains involved in Table 2 Strain name Genotype -1 UR12,Δ -2 -1,Δ -3 <![CDATA E.coli cyt -2,P nrdD ::P BBa_j23114 > -4 <![CDATA E.coli cyt -3, fhiA ::P trc - nudG > -5 <![CDATA E.coli cyt -4, yjiV ::P trc - nudG > -6 <![CDATA E.coli cyt -5, yeeL ::P trc - nudG > -7 <![CDATA E.coli cyt -6, ycdN ::P trc - pyrG (D160E, E162A, E168K, cgl) - pyrH (D93A) - ndk > -8 <![CDATA E.coli cyt -7, yncK ::P trc - pyrG (D160E, E162A, E168K, cgl) - pyrH (D93A) - ndk > -9 <![CDATA E.coli cyt -8, ychG ::P trc - PHM8 (sce) > -10 <![CDATA E.coli cyt -9, yghE ::P trc - PHM8 (sce) >

[0046] Example 2 Using the cytosine nucleoside-producing strain described in Example 1 E.coli cyt -10 to produce cytosine nucleoside by shake flask fermentation.

[0047] 2.1 Medium 2.1.1 Slant medium Glucose 2.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, dissolved in water and adjusted to pH 7.0 - 7.2 with sodium hydroxide, made up to 500 ml, dispensed into test tubes (9 ml / tube) and eggplant-shaped flasks (45 ml / flask), and sterilized in a high-pressure steam autoclave at 121 °C for 20 min.

[0048] 2.1.2 Seed medium Yeast extract 8.0 g / L, peptone 3.0 g / L, (NH4)2SO4 3.0 g / L, KH2PO4 3.0 g / L, V B1 , V B2 , V B3 , V B5 , V B12 Each 2 mg / L, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, and the rest is water.

[0049] 2.1.3 Fermentation Medium Yeast powder 10.0 g / L, citric acid 3.0 g / L, glutamic acid 2.0 g / L, (NH4)2SO4 4.0 g / L, KH2PO4 8.0 g / L, MgSO4·7H2O 2.0 g / L, FeSO4·7H2O 40 mg / L, V B1 、V B2 、V B3 、V B5 、V B12 each 1 mg / L, V H 0.1 mg / L, phenol red 2%, and the rest is water.

[0050] 2.2.1 Seed Activation and Cultivation: Inoculate the bacterial liquid evenly on the activation slant from the preservation tube, culture at 37°C for 12 h, transfer to the activation slant for continued culture for 10 h, and then transfer to a shake flask containing 5 ml of seed medium for seed culture.

[0051] 2.2.2 Fermentation Culture: Inoculate the seed liquid into a 500 mL Erlenmeyer flask containing fermentation medium at an inoculation amount of 15% (the final volume is 30 mL), seal it with nine layers of gauze, culture at 36°C with shaking at 220 r / min, and maintain the pH at 7.0 - 7.2 by adding ammonia water during the fermentation process; add 60% (m / v) glucose solution to maintain the fermentation (using phenol red as an indicator, when the color of the fermentation broth no longer changes, it is considered sugar-deficient, and 1 - 2 mL of 60% (m / v) glucose solution is added when sugar-deficient). The fermentation cycle is 32 h, and no antibiotics or inducers are added during the fermentation process.

[0052] After 32 h of shake flask fermentation, the cytidine yield can reach as high as 11.8 g / L.

[0053] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made. The improvements and modifications such as strain transformation by those skilled in the art in this technical field using the method of the present invention or based on this method are all regarded as the protection scope of the present invention.

Claims

1. A cytosine nucleoside 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 The cdd gene and cmk gene were knocked out in the UR12 genome, the nucleoside triphosphate reductase gene nrdD was weakened, and the nucleoside triphosphate pyrophosphohydrolase gene nudG, the nucleoside diphosphate kinase gene ndk and the uridylate kinase gene pyrH carrying a mutation point were overexpressed. (D93A) , heterologous introduction into the expression of Corynebacterium glutamicum Corynebacterium glutamicum The cytidine triphosphate synthase gene pyrG carrying mutation point of ATCC 13032 (D160E、E162A、E168K，cgl) and the bifunctional nucleotidase gene of Saccharomyces cerevisiae S288c PHM8 (sce) ; Among them, the nucleoside triphosphate pyrophosphohydrolase gene nudG and the uridylate kinase gene pyrH carrying a mutation point (D93A) and the cytidine triphosphate synthase gene pyrG carrying a mutation point (D160E、E162A、E168K，cgl) Through the artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk to synchronize multiple copies.

2. The cytosine nucleoside producing strain according to claim 1, characterized in that: The artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) The nucleotide sequence of -ndk is shown in the sequence listing as SEQ ID NO.

14.

3. The cytosine nucleoside producing strain according to claim 1, characterized in that: The nucleotide sequence of the cdd gene knocked out on the genome is shown in the sequence list SEQ ID NO.1; the nucleotide sequence of the cmk gene is shown in the sequence list SEQ ID NO.

2.

4. The cytosine nucleoside producing strain according to claim 1, characterized in that: With artificial promoter P BBa_j23114 Replace the natural promoter of the nucleoside triphosphate reductase gene nrdD, the nucleotide sequence of the nucleoside triphosphate reductase gene nrdD is shown in the sequence table SEQ ID NO.3, the artificial promoter P BBa_j23114 The nucleotide sequence of is shown in the sequence listing SEQ ID NO.4, and the nucleotide sequence of the natural promoter of the nucleoside triphosphate reductase gene nrdD is shown in the sequence listing SEQ ID NO.

5.

5. The cytosine nucleoside producing strain according to claim 1, characterized in that: The nucleoside triphosphate pyrophosphohydrolase gene nudG is sequentially integrated at the three sites of fhiA, yjiV and yeeL and regulated by an artificial promoter trc; the nucleotide sequence of the artificial promoter trc is shown in the sequence table SEQ ID NO.6, and the nucleotide sequence of the nucleoside triphosphate pyrophosphohydrolase gene nudG is shown in the sequence table SEQ ID NO.

7.

6. The cytosine nucleoside producing strain according to claim 1, characterized in that: The artificial operon pyrG was sequentially integrated into the ycdN and yncK sites. (D160E、E162A、E168K，cgl) -pyrH (D93A) -ndk; Corynebacterium glutamicum Corynebacterium glutamicum The cytidine triphosphate synthase gene pyrG carrying mutation point of ATCC 13032 (D160E、E162A、E168K，cgl) The nucleotide sequence is shown in the sequence table SEQ ID NO.8, and the uridylate kinase gene pyrH carrying the mutation point (D93A) The nucleotide sequence is shown in SEQ ID NO.9 in the sequence list, and the nucleotide sequence of the nucleoside diphosphate kinase gene ndk is shown in SEQ ID NO.10 in the sequence list; the codon-optimized Saccharomyces cerevisiae was sequentially integrated into the ychG and yghE sites. Saccharomyces cerevisiae S288c bifunctional nucleotidase gene PHM8 (sce) and regulated by an artificial promoter trc; the bifunctional nucleotidase gene PHM8 (sce) The nucleotide sequence is shown in the sequence listing SEQ ID NO.

11.

7. The method for constructing the cytosine nucleoside producing strain according to any one of claims 1 to 6, characterized in that: The specific steps are as follows: (1) In the E. coli UR12 genome, knockout cd Gene blocks the flow of product cytidine to uridine; knocks out the cmk gene; (2) Using artificial promoter P BBa_j23114 Replace the natural promoter of the nucleoside triphosphate reductase gene nrdD; (3) The nucleoside triphosphate pyrophosphohydrolase gene nudG was sequentially integrated into the fhiA, yjiV, and yeeL sites, and the artificial promoter trc was used to regulate the overexpression of the nucleoside triphosphate pyrophosphohydrolase gene nudG; (4) Integrate the artificial operon pyrG at the ycdN and yncK sites in sequence (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk, overexpression of the pyrH gene carrying a mutation (D93A) and nucleoside diphosphate kinase gene ndk, heterologously introduced and multi-copy Corynebacterium glutamicum Corynebacterium glutamicum The cytidine triphosphate synthase gene pyrG carrying mutation point of ATCC 13032 (D160E、E162A、E168K，cgl) ; (5) Sequentially integrate the codon-optimized Saccharomyces cerevisiae at the ychG and yghE sites Saccharomyces cerevisiae S288c bifunctional nucleotidase gene PHM8 (sce) and regulated by the artificial promoter trc, heterologously introduced and multiple copies of the bifunctional nucleotidase gene PHM8 (sce) .

8. Use of the cytosine nucleoside producing strain according to any one of claims 1 to 6 in fermentation production of cytosine nucleoside.

9. The use according to claim 8, characterized in that: The specific steps for producing cytosine nucleoside using shake flask fermentation are as follows: (1) Seed activation and culture: Inoculate the bacterial solution from the bacterial preservation tube and evenly spread it on the activated slant, culture it at 37°C for 12 h, transfer it to the activated slant and continue to culture it for 10 h, then transfer it to the shake tube containing the seed culture medium for seed culture; (2) Fermentation culture: Inoculate the seed liquid into a triangular flask containing fermentation medium at a rate of 10-15%, seal the flask with nine layers of gauze, and culture at 36°C with shaking at 220 r / min. During the fermentation process, the pH value is maintained at 7.0-7.2 by adding ammonia water; 60% glucose solution is added to maintain the fermentation process; the fermentation period is 30-32 h.

10. The use according to claim 9, characterized in that: The slant medium used in seed activation was: glucose 2.0 g / L, peptone 10.0 g / L, yeast powder 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25%, the rest was water, pH 7.0-7.2; the seed medium used in seed culture was: yeast powder 8.0 g / L, peptone 3.0 g / L, (NH4)2SO4 3.0 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, and the rest is water; the fermentation medium used in the fermentation culture is: yeast powder 10.0 g / L, citric acid 3.0 g / L, glutamic acid 2.0 g / L, (NH4)2SO44.0 g / L, KH2PO48.0 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, phenol red 2%, and the rest is water.

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