Cytosine nucleoside production strain and construction method and application thereof

Abstract

The application provides a cytosine nucleoside producing strain and a construction method and application thereof, and the strain is constructed by using CRIPSR / Cas9 gene editing technology E. coli The cdd gene and cmk gene are knocked out on the UR12 genome, the nucleoside triphosphate reductase gene nrdD is weakened, and the nucleoside triphosphate pyrophosphatase gene nudG, the nucleoside diphosphate kinase gene ndk and the uridine acid kinase gene pyrH carrying a mutation point are overexpressed (D93A) The cytosine triphosphate synthase gene pyrG carrying a mutation point is introduced into the Corynebacterium glutamicum heterologously and in multiple copies (D160E、E162A、E168K，cgl) The bifunctional nucleotidase gene of Saccharomyces cerevisiae PHM8 (sce) The obtained strain has good genetic stability, high fermentation yield and can stably produce cytosine nucleoside.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of metabolic engineering and genetic engineering, and particularly relates to a cytosine nucleoside production strain and a construction method and application thereof. BACKGROUND

[0002] Cytosine nucleoside, also known as cytosine ribonucleoside, is one of pyrimidine nucleosides constituting nucleic acid, participates in various physiological and biochemical processes in organisms, and plays an important role in the regulation of metabolism and heredity in organisms. In addition, cytosine is also an important intermediate for the synthesis of medicine. Due to its property of interfering with the synthesis of ribonucleic acid, it is often used as a raw material and intermediate for the synthesis of antitumor and antiviral drugs such as cytarabine, azacitidine and zalcitabine. Nowadays, research and development on the application of cytosine are still ongoing, and a more efficient synthesis method is urgently needed.

[0003] The production method of cytosine nucleoside mainly includes chemical synthesis and microbial fermentation. Traditional cytosine production process mainly relies on chemical synthesis, but this method has many problems, including harsh reaction conditions, serious environmental pollution and high production cost. In order to overcome these problems, in recent years, microbial fermentation method has gradually become a research hotspot for the preparation of cytosine. Through metabolic engineering of microorganisms, microbial fermentation method can realize the efficient biosynthesis of cytosine. This method uses glucose as raw material to produce cytosine through fermentation process, which has the advantages of green environmental protection, low cost and mild production conditions. In recent years, the development of synthetic biology technology has further improved the efficiency of fermentation method, making it the mainstream choice for industrial production. Therefore, it is a technical problem to be solved at present to construct a cytosine nucleoside production strain with clear genetic background, good stability and considerable yield. SUMMARY

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

[0005] Another technical problem to be solved by the present application is to provide a construction method of the cytosine nucleoside production strain.

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

[0007] To solve the above technical problems, the technical scheme of the present application is as follows:

[0008] A cytosine nucleoside production strain, which is a strain E. coli cyt-10, is obtained by further modification of the starting strain E. coli UR12 by using a directed modification method, and specifically, the modification is as follows: in the cytosine nucleoside production strain E. coliThe UR12 genome is knocked out of cdd gene, cmk gene, weakened nucleoside triphosphate reductase gene nrdD, overexpresses nucleoside triphosphate pyrophosphohydrolase gene nudG, nucleoside diphosphate kinase gene ndk and carries a mutation point of uridine acid kinase gene pyrH (D93A) , heterologous introduction of Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032 carrying a mutation point of cytidine triphosphate synthase gene pyrG (D160E、E162A、E168K，cgl) and the bifunctional nucleotide enzyme gene of Saccharomyces cerevisiae S288c PHM8 (sce) ; wherein the nucleoside triphosphate pyrophosphohydrolase gene nudG, the uridine acid kinase gene pyrH (D93A) carrying a mutation point and the cytidine triphosphate synthase gene pyrG (D160E、E162A、E168K，cgl) are synchronously multi-copied by the artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk.

[0009] The above-mentioned 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; the 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.

[0010] Preferably, the above-mentioned cytosine nucleoside production strain, the artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk includes the pyrH (D93A) gene, the ndk gene, the pyrG (D160E、E162A、E168K，cgl) gene, specifically:

[0011] (1) The operon gene order is trc artificial promoter, pyrG (D160E、E162A、E168K，cgl) , pyrH (D93A) , ndk, rrnB T1 terminator;

[0012] (2) The trc artificial promoter is directly connected at the head and tail of the pyrG (D160E、E162A、E168K，cgl) gene;

[0013] (3) The pyrG (D160E、E162A、E168K，cgl)pyrH (D93A) The genes are connected using linker-1, the nucleotide sequence of which is shown in SEQ ID NO. 12 of the sequence listing;

[0014] (4) pyrH (D93A) The genes are connected using linker-2, the nucleotide sequence of which is shown in SEQ ID NO. 13 of the sequence listing;

[0015] (5) ndk, rrnB T1 terminator are directly connected at the head and tail.

[0016] The construction method of the artificial operon described above is controlled by the same trc artificial promoter to start transcription of the operon, and by the same rrnB T1 terminator to control termination of transcription of the operon, and is obtained by optimizing the connection mode of the genes and the position of the genes in the operon.

[0017] Preferably, in the cytosine nucleoside production strain described above, the nucleotide sequence of the artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) The nucleotide sequence of ndk is shown in SEQ ID NO. 14 of the sequence listing.

[0018] Preferably, in the cytosine nucleoside production strain described above, the starting strain E. coli UR12 is the strain in Example 1.12 of CN202411087442.4 (a uridine production strain and a construction method and application thereof). E. coli UR12.

[0019] Preferably, in the cytosine nucleoside production strain described above, the directed modification method is to use the CRISPR / Cas9 gene editing technology to completely modify the starting strain E. coli UR12 on the chromosome genome.

[0020] Preferably, in the cytosine nucleoside production strain described above, the nucleotide sequence of the cdd gene knocked out on the genome is shown in SEQ ID NO. 1 of the sequence listing; and the nucleotide sequence of the cmk gene is shown in SEQ ID NO. 2 of the sequence listing.

[0021] Preferably, in the cytosine nucleoside production strain described above, the natural promoter of the nucleoside triphosphate reductase gene nrdD is replaced with an 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, and the artificial promoter P BBa_j23114The nucleotide sequence of is shown in the sequence listing as 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 as SEQ ID NO.5.

[0022] Preferably, the above-mentioned cytosine nucleoside production strain integrates the nucleoside triphosphate pyrophosphohydrolase gene nudG at the three sites fhiA, yjiV, and yeeL in sequence, and is regulated by the artificial promoter trc; the nucleotide sequence of the artificial promoter trc is shown in the sequence listing SEQ ID NO.6, and the nucleotide sequence of the nucleoside triphosphate pyrophosphohydrolase gene nudG is shown in the sequence listing SEQID NO.7.

[0023] Preferably, the above-mentioned cytidine nucleoside production strain integrates the above-mentioned artificial operon pyrG at the two sites ycdN and yncK in sequence. (D160E、E162A、E168K，cgl) -pyrH (D93A) -ndk; the Corynebacterium glutamicum Corynebacterium glutamicum The cytidine triphosphate synthase gene pyrG carrying a mutation point in ATCC 13032 (D160E、E162A、E168K，cgl) The nucleotide sequence is shown in the sequence table SEQ ID NO.8, the uridylate kinase gene pyrH carrying the mutation point (D93A) The nucleotide sequence is shown in the sequence listing as SEQ ID NO.9, and the nucleotide sequence of the nucleoside diphosphate kinase gene ndk is shown in the sequence listing as SEQ ID NO.10.

[0024] Preferably, the above-mentioned cytosine nucleoside production strain is sequentially integrated with codon-optimized Saccharomyces cerevisiae at 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.

[0025] The method for constructing the above-mentioned cytosine nucleoside production strain comprises the following specific steps:

[0026] (1) In the E. coli UR12 genome, knockout cdd Genetic blockade of the product cytidine to uridine; knockout of the cmk gene prevents the precursor CMP from flowing back to CDP;

[0027] (2) Using artificial promoter P BBa_j23114 Replace the natural promoter of the nucleoside triphosphate reductase gene nrdD, weaken the branching pathway of the precursor CTP flowing in the direction of growth, and enhance the accumulation of cytosine nucleoside while ensuring bacterial growth;

[0028] (3) Integrate the nucleoside triphosphate pyrophosphohydrolase gene nudG into the three sites of fhiA, yjiV and yeeL in sequence, and regulate by the artificial promoter trc, overexpress the nucleoside triphosphate pyrophosphohydrolase gene nudG, and strengthen the cytosine nucleoside synthesis pathway;

[0029] (4) Integrate the above artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk into the two sites of ycdN and yncK in sequence, overexpress the uridine kinase gene pyrH (D93A) carrying a mutation point and the nucleoside diphosphate kinase gene ndk, heterologously introduce and multiply the cytosine triphosphate synthetase gene pyrG Corynebacterium glutamicum carrying a mutation point of Corynebacterium glutamicum (D160E、E162A、E168K，cgl) ATCC 13032, and strengthen the cytosine nucleoside synthesis pathway;

[0030] (5) Integrate the codon-optimized bifunctional nucleotide hydrolase gene Saccharomyces cerevisiae of Saccharomyces cerevisiae PHM8 (sce) S288c into the two sites of ychG and yghE in sequence, and regulate by the artificial promoter trc, heterologously introduce and multiply the bifunctional nucleotide hydrolase gene PHM8 (sce) , and strengthen the cytosine nucleoside synthesis pathway.

[0031] Application of the above cytosine nucleoside production strain in fermentation production of cytosine nucleoside.

[0032] Preferably, the application of the above cytosine nucleoside production strain uses shake flask fermentation to produce cytosine nucleoside, and the specific steps are as follows:

[0033] (1) Seed activation and culture: inoculate the bacteria evenly on the activation slope from the bacteria preservation tube, cultivate at 37°C for 12 h, continue to cultivate the activation slope for 10 h, and then transfer to the seed culture medium in the shake flask for seed culture;

[0034] (2) Fermentation culture: inoculate the seed liquid into the triangular flask containing the fermentation medium at an inoculation amount of 10-15%, seal with nine layers of gauze, cultivate at 36°C with 220 r / min shaking, maintain the pH at 7.0-7.2 by supplementing ammonia water during the fermentation process, add 60% glucose solution to maintain the fermentation, and the fermentation period is 30-32 h.

[0035] Preferably, in the application of the above cytosine nucleoside production strain, the slant medium used in seed activation is: glucose 2.0 g / L, proteose 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.

[0036] Preferably, in the application of the above cytosine nucleoside production strain, the seed culture medium used in seed culture is: yeast powder 8.0 g / L, proteose 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, the rest is water.

[0037] Preferably, in the application of the above cytosine nucleoside production strain, the fermentation medium used in fermentation culture is: 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%, the rest is water.

[0038] The above culture media can be prepared by standard methods.

[0039] Beneficial effects:

[0040] The above cytosine nucleoside production strain uses CRIPSR / Cas9 gene editing technology to first knock out cdd the gene, block the product cytidine to uridine, knock out the cmk gene to prevent the precursor CMP from flowing back to CDP; replace the natural promoter of the nucleoside triphosphate reductase gene nrdD with an artificial promoter P BBa_j23114 , weaken the branch pathway of the precursor CTP flowing to the growth direction, strengthen the accumulation of cytosine nucleoside under the condition of ensuring the growth of the bacterial body; overexpress the nucleoside triphosphate pyrophosphohydrolase gene nudG, the nucleoside diphosphate kinase gene ndk, and the uridine acid kinase gene pyrH (D93A)heterologously introduced and multicopy Corynebacterium glutamicum Corynebacterium glutamicum Cytidine triphosphate synthase gene pyrG of ATCC 13032 carrying a mutation point (D160E、E162A、E168K，cgl) and Saccharomyces cerevisiae Saccharomyces cerevisiae Bifunctional nucleotidase gene of S288c PHM8 (sce) The constructed strain does not contain a plasmid, is not defective, does not need to be induced, has the advantages of good genetic stability, high fermentation yield, etc., is an excellent strain for stably producing cytosine nucleosides, and efficiently synthesizes cytosine nucleosides from scratch using glucose as a substrate. After 32 h of shake flask fermentation, the cytosine nucleoside yield can be as high as 11.8 g / L. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Diagram of the method for targeted modification of a cytosine nucleoside production strain. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solutions of the present application, the technical solutions described in the present application will be further described in detail below in combination with specific embodiments.

[0043] The percentage sign “%” involved in the embodiments refers to volume percentage, unless otherwise specified; the percentage “% (m / v)” of a solution refers to the number of grams of solute contained in 100 ml of solution.

[0044] The starting strain used in the embodiments is a uridine production strain E. coli UR12, which is the strain described in CN202411087442.4 (a uridine production strain and its construction method and application) embodiment 1.12 E. coli UR12; Saccharomyces cerevisiae Saccharomyces cerevisiae Bifunctional nucleotidase gene of S288c PHM8 (sce) Synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.; see the sequence listing for the corresponding promoters and genes, etc.

[0045] The gene editing method used refers to 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, including promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence and ampicillin resistance (working concentration: 100 mg / L). The professional terms involved in the following examples, such as gene integration and plasmid construction, can be explained in this article. The primers used in the process of strain construction are shown in Table 1.

[0046] Table 1 Primers involved in the process of strain construction

[0047] Primer Sequence No. 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 TATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCACCATAGCATCGCCAATCTGA TCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACCTCTAGTCGAAAAGTCCGT pyrG-mut-up-A SEQ ID NO. 55 TTAAGGAATGGCTGGGATGCAATCTCACCGACGGTGCCACCG pyrG-mut-dw-S SEQ ID NO. 56 GAGATTGCATCCCAGCCATTCCTTAAAGCAGCTCGCCAGGTACG pyrG-linkl-A SEQ ID NO. 57 CATGAATACTGTTTCCTCTAAGGGTGGACACGCAGC pyrH-linkl-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

[0048] Example 1

[0049] As shown in the following: Figure 1 The specific process of constructing a genetically engineered strain is as follows:

[0050] The starting strain E. coli UR12 was prepared according to the operation mentioned in CN 202411087442.4. E. coli UR12 / pRed-Cas9 was electrotransformed into the competent cells.

[0051] 1.1 Knockout of gene cdd

[0052] The E. coli W3110 genome at a usable concentration was used as a template, and primers cdd-QC-1 and cdd-QC-2, cdd-QC-3 and cdd-QC-4 were used for PCR amplification to obtain the upstream homologous arm cdd-QC-UP and the downstream homologous arm cdd-QC-DW. The recovered upstream and downstream homologous arms were used as templates, and primers cdd-QC-1 and cdd-QC-4 were used for overlap PCR to obtain the complementation fragment Δcdd required for knocking out the gene cdd. Then, the DNA fragment obtained by annealing primers pGRB-cdd-S and pGRB-cdd-A was ligated with plasmid pGRB to construct plasmid pGRB-cdd. Finally, the plasmid pGRB-cdd was electrotransformed into the electrocompetent cells of E. coli UR12 / pRed-Cas9; and cdd-QC-1 and cdd-QC-4 were used as identification primers to screen positive transformants to obtain strain E. coli cyt -1.

[0053] 1.2 Knockout of gene cmk

[0054] ​​The E. coli W3110 genome with available concentration as template, primer cmk-QC-1 and cmk-QC-2, cmk-QC-3 and cmk-QC-4 were used for PCR amplification, and the upstream homologous arm cmk-QC-UP and the downstream homologous arm cmk-QC-DW were obtained. The recovered upstream and downstream homologous arms were used as templates, and the primers cmk-QC-1 and cmk-QC-4 were used for overlap PCR to obtain the complementation fragment Δcmk required for knocking out the gene cmk. Then, the DNA fragment obtained by annealing the primers pGRB-cmk-S and pGRB-cmk-A was ligated with the plasmid pGRB to construct pGRB-cmk. Finally, the pGRB-cmk plasmid and the overlap fragment Δcmk were electroporated into the electrocompetent cells of E. coli cyt -1 / pRed-Cas9; and the primers cmk-QC-1 and cmk-QC-4 were used as identification primers to screen positive transformants, and the strain E. coli cyt -2 was obtained.

[0055] 1.3 Gene nrdD Promoter (P nrdD ::P BBa_j23114 )

[0056] The E. coli W3110 as template, primer UP-PnrdD-S and UP-PnrdD-js114-A, DW-PnrdD-A and DW-PnrdD-js114-S were used for PCR amplification to obtain the upper homologous arm UP-PnrdD-js114 and the lower homologous arm DW-PnrdD. The recovered upstream and downstream homologous arms were used as templates, and the primers UP-PnrdD-S and DW-PnrdD-A were used for overlap PCR to obtain the target fragment PnrdD-js114 required for integration. Then, the DNA fragment obtained by annealing the primers pGRB-PnrdD-S and pGRB-PnrdD-A was ligated with the plasmid pGRB to construct the pGRB-PnrdD plasmid. Finally, the purified PnrdD-js114 integration fragment and the pGRB-PnrdD plasmid were simultaneously electroporated into the electrocompetent cells of E. coli cyt -2 / pRed-Cas9, and the primers Pjs114-JD-S and DW-PnrdD-A were used as identification primers to screen positive transformants, and finally the strain E. coli cyt-3 was obtained.

[0057] 1.4 Gene nudG Integration (P fhiA ::P trc - nudG Integration of P

[0058] PCR amplification was performed with the primers fhiA-1 and fhiA-trc-2, fhiA-3 and fhiA-4, nudG-trc-S and nudG-trc-A using the available concentration of E. coli W3110 genome as the template to obtain the upper homologous arm fhiA-trc-UP, the lower homologous arm fhiA-DW and the intermediate target fragment P trc PCR amplification was performed with the primers fhiA-1 and fhiA-4 using the recovered upper and lower homologous arms and the intermediate target fragment as the template to obtain the target fragment fhiA-P required for integration trc PCR amplification was performed with the primers fhiA-1 and fhiA-4 using the recovered upper and lower homologous arms and the intermediate target fragment as the template to obtain the target fragment fhiA-P required for integration trc The purified fhiA-P E. coli cyt The primers fhiA-1 and fhiA-4 were used as the identification primers to screen the positive transformants in the competent cells of E. coli cyt -4.

[0059] 1.5 genes nudG of the second copy yjiV ::P trc - nudG of the integration

[0060] PCR amplification was performed with the primers yjiV-1 and yjiV-trc-2, yjiV-3 and yjiV-4, nudG-trc-S and nudG-trc-A using the available concentration of E. coli W3110 genome as the template to obtain the upper homologous arm yjiV-trc-UP, the lower homologous arm yjiV-DW and the intermediate target fragment P trc PCR amplification was performed with the primers yjiV-1 and yjiV-4 using the recovered upper and lower homologous arms and the intermediate target fragment as the template to obtain the target fragment yjiV-P required for integration trc PCR amplification was performed with the primers yjiV-1 and yjiV-4 using the recovered upper and lower homologous arms and the intermediate target fragment as the template to obtain the target fragment yjiV-P required for integration trc The purified yjiV-P E. coli cyt The primers yjiV-1 and yjiV-4 were used as the identification primers to screen the positive transformants in the competent cells ofE. coli cyt -5.

[0061] 1.6 Genes nudG Three copies of yeeL ::P trc - nudG Integration of

[0062] Using the available concentration of Escherichia coli W3110 genome 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 homology arm yeeL-trc-UP, the lower homology arm yeeL-DW and the middle target fragment P trc -nudG, 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 -nudG, then the DNA fragment prepared by annealing primers pGRB-yeeL-S and pGRB-yeeL-A was ligated with the plasmid pGRB to construct the pGRB-yeeL plasmid. trc -nudG integration fragment and plasmid pGRB-yeeL were simultaneously transformed into E. coli cyt -5 / pRed-Cas9 competent cells, and then the primers yeeL-1 and yeeL-4 were used as identification primers to screen positive transformants, and finally the strain was obtained. E. coli cyt -6.

[0063] 1.7 Artificial operon gene pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -NDK integration ( ycdN ::P trc - pyrG (D160E、E162A、E168K，cgl) - pyrH （D93A) - ndk Integration of

[0064] Corynebacterium glutamicum at available concentrations Corynebacterium glutamicum ATCC 13032 genome was used as template, primers pyrG-trc-S and pyrG-mut-up-A, pyrG-mut-dw-S and pyrG-link1-A were used; the available concentration of Escherichia coli W3110 genome was used as template, primers pyrH-link1-S and pyrH-mut-up-A, pyrH-mut-dw-S and pyrH-link2-A, ndk-link2-S and ndk-trc-A were used for PCR amplification to obtain fragment Ptrc -pyrG (D160E、E162A、E168K，cgl) -mut-UP, pyrG (D160E、E162A、E168K，cgl) -mut-DW, pyrH-mut-UP, pyrH-mut-DW and ndk. The P trc -pyrG (D160E、E162A、E168K，cgl) -mut-UP and pyrG (D160E、E162A、E168K，cgl) -mut-DW fragments as templates, the P trc -pyrG (D160E、E162A、E168K，cgl) The pyrH-mut-UP and pyrH-mut-DW fragments were recycled as templates, and the P （D93A） The P trc -pyrG (D160E、E162A、E168K，cgl) , pyrH （D93A） and ndk fragments as templates, the artificial operon fragment P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk. The E. coli W3110 genome was used as template for PCR amplification with primers ycdN-1 and ycdN-trc-2, ycdN-3 and ycdN-4 to obtain the upper homology arm ycdN-trc-UP and the lower homology arm ycdN-DW. The upper and lower homology arms and the artificial operon fragment were used as templates for overlap PCR with primers ycdN-1 and ycdN-4 to obtain the target fragment ycdN-P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk. Then, the DNA fragment obtained by annealing primers pGRB-ycdN-S and pGRB-ycdN-A was ligated with plasmid pGRB to construct plasmid pGRB-ycdN. Finally, the purified ycdN-P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk was transformed into E. coli cyt -6 / pRed-Cas9 competent cells, and primers ycdN-1 and ycdN-4 were used as identification primers to screen positive transformants, and finally the strain E. coli cyt -7 was obtained.

[0065] 1.8 Artificial operon gene pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A)- Second copy of ndk ( yncK ::P trc - pyrG (D160E、E162A、E168K，cgl) - pyrH （D93A) - ndk Integration of

[0066] Corynebacterium glutamicum at available concentrations Corynebacterium glutamicum ATCC 13032 genome was used as template, primers pyrG-trc-S and pyrG-mut-up-A, pyrG-mut-dw-S and pyrG-link1-A were used; the available concentration of Escherichia coli W3110 genome was used as template, primers pyrH-link1-S and pyrH-mut-up-A, pyrH-mut-dw-S and pyrH-link2-A, ndk-link2-S and ndk-trc-A were used 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. trc -pyrG (D160E、E162A、E168K，cgl) -mut-UP and pyrG (D160E、E162A、E168K，cgl) -mut-DW fragment was used as template and primers pyrG-trc-S and pyrG-link1-A were used to obtain fragment P by overlapping PCR. trc -pyrG (D160E、E162A、E168K，cgl) The recovered pyrH-mut-UP and pyrH-mut-DW fragments were used as templates and primers pyrH-link1-S and pyrH-link2-A were used to obtain the fragment pyrH. （D93A） . With recycled P trc -pyrG (D160E、E162A、E168K，cgl) 、pyrH （D93A） The artificial operon fragment P was obtained by overlapping PCR using primers pyrG-trc-S and ndk-trc-A. trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) Using the available concentration of Escherichia coli W3110 genome as a template, primers yncK-1 and yncK-trc-2, yncK-3 and yncK-4 were used for PCR amplification to obtain the upper homology arm yncK-trc-UP and the lower homology arm yncK-DW. Using the recovered upstream and downstream homology arms and the artificial operon fragment as templates, primers yncK-1 and yncK-4 were used for overlapping PCR to obtain the target fragment yncK-P required for integration. trc-pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk, then the DNA fragment prepared by annealing primers pGRB-yncK-S and pGRB-yncK-A was ligated with plasmid pGRB to construct pGRB-yncK plasmid. Finally, the purified yncK-P trc -pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) -ndk integration fragment and plasmid pGRB-yncK were simultaneously transformed into E. coli cyt -7 / pRed-Cas9 competent cells, and primers yncK-1 and yncK-4 were used as identification primers to screen positive transformants, and finally strain E. coli cyt -8.

[0067] 1.9 Gene PHM8 （sce) integration ( ychG ::P trc - PHM8 (sce) integration)

[0068] The E. coli W3110 genome at a usable concentration was used as a template, and primers ychG-1 and ychG-trc-2, ychG-3 and ychG-4 were used for PCR amplification. Saccharomyces cerevisiae S288c bifunctional nucleotidase gene PHM8 (sce) The upper homology arm ychG-trc-UP, the lower homology arm ychG-DW, and the intermediate target fragment P trc - PHM8 (sce) The recovered upper and lower homology arms and the intermediate target fragment were used as templates, and primers ychG-1 and ychG-4 were used for overlap PCR to obtain the target fragment ychG-P trc - PHM8 (sce) , then the DNA fragment prepared by annealing primers pGRB-ychG-S and pGRB-ychG-A was ligated with plasmid pGRB to construct pGRB-ychG plasmid. Finally, the purified ychG-P trc - PHM8 (sce) The integration fragment and plasmid pGRB-ychG were simultaneously transformed into E. coli cyt -8 / pRed-Cas9 competent cells, and primers ychG-1 and ychG-4 were used as identification primers to screen positive transformants, and finally strainE. coli cyt -9.

[0069] 1.10 Gene PHM8 （sce) yghE ::P trc - PHM8 (sce)

[0070] The E. coli W3110 genome with available concentration as template, primers yghE-1 and yghE-trc-2, yghE-3 and yghE-4; the codon-optimized S. cerevisiae Saccharomyces cerevisiae bifunctional nucleotidase gene of S288c synthesized by Genewiz as template, primers PHM8-trc-S and PHM8-trc-A were used for PCR amplification to obtain the upper homology arm yghE-trc-UP, the lower homology arm yghE-DW and the middle target fragment P PHM8 (sce) trc - PHM8 (sce) The recovered upper and lower homology arms and the middle target fragment were used as templates, and primers yghE-1 and yghE-4 were used for overlap PCR to obtain the target fragment yghE-P required for integration. trc - PHM8 (sce) After that, the DNA fragment prepared by annealing primers pGRB-yghE-S and pGRB-yghE-A was ligated with plasmid pGRB to construct plasmid pGRB-yghE. Finally, the purified yghE-P trc - PHM8 (sce) integration fragment and plasmid pGRB-yghE were simultaneously transformed into the competent cells of E. coli cyt -9 / pRed-Cas9 by electroporation, and primers yghE-1 and yghE-4 were used as identification primers to screen positive transformants, and finally the strain E. coli cyt -10.

[0071] The strains involved in the above construction process are shown in Table 2.

[0072] Strains involved in Table 2

[0073] 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) ]]>

[0074] Example 2

[0075] The cytosine nucleoside production strain E. coli cyt -10 described in Example 1 was used for shake flask fermentation to produce cytosine nucleoside.

[0076] 2.1 Medium ​​​

[0077] 2.1.1 Inclined surface culture medium

[0078] Glucose 2.0 g / L, Proteose 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%, dissolved with water, pH adjusted to 7.0-7.2 with sodium hydroxide, constant volume to 500 ml, divided into test tubes (9 ml / tube) and tomato-shaped bottles (45 ml / bottle), sterilized in a high-pressure steam kettle at 121°C for 20 min.

[0079] 2.1.2 Seed culture medium

[0080] Yeast powder 8.0 g / L, Proteose peptone 3.0 g / L, (NH4)2SO4 3.0 g / L, KH2PO4 3.0 g / L, Vitamin B1 2 mg / L, Vitamin B6 2 mg / L, Vitamin B12 1 mg / L, MgSO4·7H2O 0.5 g / L, and the rest is water. B1 , Vitamin B6 2 mg / L, B2 , Vitamin B12 1 mg / L, B3 , Vitamin B12 1 mg / L, B5 , Vitamin B12 1 mg / L, B12 , Vitamin B12 1 mg / L, H 1 mg / L, MgSO4·7H2O 0.5 g / L, and the rest is water.

[0081] 2.1.3 Fermentation culture medium

[0082] 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, Vitamin B1 2 mg / L, Vitamin B6 2 mg / L, Vitamin B12 1 mg / L, MgSO4·7H2O 0.5 g / L, and the rest is water. B1 , Vitamin B6 2 mg / L, B2 , Vitamin B12 1 mg / L, B3 , Vitamin B12 1 mg / L, B5 , Vitamin B12 1 mg / L, B12 , Vitamin B12 1 mg / L, H 0.1 mg / L, Phenol red 2%, and the rest is water.

[0083] 2.2.1 Seed activation and culture:

[0084] Inoculate the bacteria evenly on the activated slant from the bacteria preservation tube, cultivate at 37°C for 12 h, continue to cultivate on the activated slant for 10 h, and then transfer to a shaking tube containing 5 ml of seed culture medium for seed culture.

[0085] 2.2.2 Fermentation culture:

[0086] Seed liquid was inoculated into 500 mL triangular flask containing fermentation medium at 15% inoculation amount (final volume 30 mL), and the flask was sealed with nine layers of gauze and incubated at 36℃ with shaking at 220 r / min. The pH was maintained at 7.0-7.2 by adding ammonia water during the fermentation process. The fermentation was maintained by adding 60% (m / v) glucose solution (phenol red was used as indicator, and the fermentation was considered to be sugar-deficient when the color of the fermentation broth no longer changed, and 1-2 mL of 60% (m / v) glucose solution was added when the fermentation was sugar-deficient). The fermentation period was 32 h, and no antibiotics or inducers were added during the fermentation process.

[0087] After 32 h of shake flask fermentation, the cytidine yield was as high as 11.8 g / L.

[0088] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and the improvements and refinements made by those skilled in the art based on the method of the present application or based on the method are considered to be within the protection scope of the present application.

Claims

1. A cytidine-producing strain, characterized in that: The directed transformation method is used to transform the starting strain E. coli It is obtained by further transformation based on UR12, specifically: E. coli The cdd and cmk genes 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) , heterologously introduced into Corynebacterium glutamicum Corynebacterium glutamicum The cytidine triphosphate synthase gene pyrG carrying a mutation point in ATCC 13032 (D160E、E162A、E168K，cgl) and the bifunctional nucleotidase gene of Saccharomyces cerevisiae S288c PHM8 (sce) Among them, the nucleoside diphosphate kinase gene ndk and the uridylate kinase gene pyrH carrying mutation points (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 of the artificial operon pyrG (D160E、E162A、E168K，cgl) -pyrH （D93A) The nucleotide sequence of -ndk is shown in the sequence table SEQ ID NO.14; 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 the nucleoside triphosphate reductase gene nrdD 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; the nucleoside triphosphate pyrophosphohydrolase gene nudG is sequentially integrated into the fhiA, yjiV, and yeeL sites and regulated by the artificial promoter trc, the nucleotide sequence of the artificial promoter trc is shown in SEQ ID NO.6 of the sequence listing, and the nucleotide sequence of the nucleoside triphosphate pyrophosphohydrolase gene nudG is shown in SEQ ID NO.7 of the sequence listing; the artificial operon pyrG is sequentially integrated into the ycdN and yncK sites. (D160E、E162A、E168K，cgl) -pyrH (D93A) -ndk; the Corynebacterium glutamicum Corynebacterium glutamicum The cytidine triphosphate synthase gene pyrG carrying a mutation point in ATCC 13032 (D160E、E162A、E168K，cgl) The nucleotide sequence is shown in the sequence table SEQ ID NO.8, the uridylate kinase gene pyrH carrying the mutation point (D93A) The nucleotide sequence is shown in SEQ ID NO.9 of the sequence list, and the nucleotide sequence of the nucleoside diphosphate kinase gene ndk is shown in SEQ ID NO.10 of 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.

2. The cytidine-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 listing SEQ ID NO.1; the nucleotide sequence of the cmk gene is shown in the sequence listing SEQ ID NO.

2.

3. The method for constructing the cytidine-producing strain according to claim 1 or 2, characterized in that: The specific steps are as follows: (1) In the E. coli UR12 genome, knockout cdd Gene blocks the flow of cytidine product 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 regulated by the artificial promoter trc to overexpress the nucleoside triphosphate pyrophosphohydrolase gene nudG; (4) Integrate the artificial operon pyrG at the ycdN and yncK sites sequentially (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 a mutation point in 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 multi-copy bifunctional nucleotidase gene PHM8 (sce) .

4. Use of the cytidine nucleoside producing strain according to claim 1 or 2 in fermentative production of cytidine nucleoside.

5. The use according to claim 4, characterized in that: Cytosine nucleoside is produced by shake flask fermentation. The specific steps are as follows: (1) Seed activation and culture: Inoculate the bacterial solution from the bacterium preservation tube and evenly spread it on the activated slant, culture it at 37℃ for 12 hours, transfer it to the activated slant and continue to culture it for 10 hours, and then transfer it to the shake tube containing 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 and 220 r / min with shaking. During the fermentation process, add ammonia water to maintain the pH at 7.0-7.2; add 60% glucose solution 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 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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