Cytidine producing strain as well as construction method and application thereof

Abstract

The invention belongs to the technical field of metabolic engineering and genetic engineering, and particularly relates to a cytidine production strain as well as a construction method and application thereof. The cytidine production strain provided by the invention is constructed by taking wild bacillus subtilis as a starting strain and utilizing a modular metabolic engineering strategy. The strain has good genetic stability, does not contain plasmids, has no nutritional deficiency, can stably and efficiently produce cytidine without induction, is easy to control in the production process, and can be used for industrial production of cytidine. The invention also provides a construction method of the strain, and provides a method for producing cytidine by fermentation of the strain. The yield of 31.41 g / L can be achieved within 48 hours when the cytidine production strain is used for fermentation production of cytidine.

Description

Technical Field

[0001] The present invention belongs to the technical fields of metabolic engineering and genetic engineering, and particularly relates to a cytidine-producing strain and a construction method and application thereof. Background Art

[0002] In recent years, cytidine has been widely used in the pharmaceutical, food, and health supplement industries, particularly as a precursor for antiviral and anti-tumor drugs and a dietary supplement. With the rapid development of the pharmaceutical industry and the health food market, market demand for cytidine continues to expand.

[0003] Currently, the main methods for industrial production of cytidine include chemical synthesis, enzymatic methods, and microbial fermentation. Among them, the chemical synthesis method, which uses pyrimidine bases and ribose as the main raw materials, has problems such as multiple reaction steps, harsh reaction conditions, cumbersome operation, the need for multiple protection and deprotection steps, severe environmental pollution, and high production costs. Although the enzymatic method has certain specificity and efficiency, it is difficult to maintain the catalytic activity of the enzyme and the enzyme preparation cannot be recycled after use, resulting in high production costs and difficulty in scaling up production. In contrast, the microbial fermentation method has become the preferred method for producing cytidine due to its advantages such as low cost, simple conditions, easy control, few by-products, high yield, and environmental friendliness.

[0004] However, in industrial production, engineered bacteria carrying plasmids have disadvantages such as poor genetic stability and a high growth and metabolic burden. This can lead to industrial problems such as significantly reduced cytidine production and difficulty in controlling the production process. Therefore, constructing a genetically stable industrial production strain that can stably and efficiently synthesize cytidine is a technical challenge that needs to be solved. Summary of the Invention

[0005] To address the above issues, the present invention provides a cytidine-producing strain, a method for its construction, and its application. The cytidine-producing strain provided by the present invention is an engineered bacterium derived from a wild-type strain of Bacillus subtilis through genetic modification. It exhibits excellent genetic stability, lacks plasmids, and is free of auxotrophic defects. It can stably and efficiently produce cytidine without the need for induction, and the production process is easily controlled, making it suitable for the industrial production of cytidine.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a cytidine-producing strain: the strain is constructed by genetically modifying Bacillus subtilis as a starting strain; the genetic modification method comprises: not expressing cytidine deaminase, cytidine kinase, pyrimidine operon regulatory protein, spermine succinate synthetase, ornithine carbamoyltransferase, homoserine dehydrogenase, glutamine phosphoribosylpyrophosphate aminotransferase, and ATP phosphoribosyltransferase; overexpressing the pyr operon gene (Gene ID: 937734); and overexpressing glutamate aminotransferase, glutamate synthase, PRPP synthase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase.

[0007] The present invention utilizes a modular metabolic engineering strategy to perform the aforementioned genetic modification on Bacillus subtilis. The resulting cytidine-producing strain is capable of stable and efficient cytidine production, and exhibits excellent genetic stability. Furthermore, the strain is plasmid-free and auxotrophic, and can stably and efficiently produce cytidine without the need for induction. This makes the production process easy to control, making it more suitable for industrialized cytidine production.

[0008] Preferably, the cytidine deaminase is cytidine deaminase CDD.

[0009] Preferably, the method of not expressing the cytidine deaminase is not expressing cdd gene (Gene ID: 937885).

[0010] Preferably, the cytidine kinase is cytidine kinase UDK.

[0011] Preferably, the method of not expressing the cytidine kinase is not expressing udk gene (Gene ID: 937560).

[0012] Preferably, the pyrimidine operon regulatory protein is PyrR protein.

[0013] Preferably, the method of not expressing the pyrimidine operon regulatory protein is not expressing pyR gene (Gene ID:938030).

[0014] Preferably, the spermine succinate synthetase is spermine succinate synthetase argG.

[0015] Preferably, the method of not expressing the spermine succinate synthetase is not expressing argG gene (Gene ID:937348).

[0016] Preferably, the ornithine carbamoyltransferase is ornithine carbamoyltransferase argF.

[0017] Preferably, the method of not expressing the ornithine transcarbamylase encoding the enzyme is not expressing argF gene (GeneID: 936386).

[0018] Preferably, the homoserine dehydrogenase is homoserine dehydrogenase hom.

[0019] Preferably, the method of not expressing the homoserine dehydrogenase is not expressing man gene (Gene ID:936654).

[0020] Preferably, the glutamine phosphoribosyl pyrophosphate aminotransferase is glutamine phosphoribosyl pyrophosphate aminotransferase purF.

[0021] Preferably, the method of not expressing the glutamine phosphoribosylpyrophosphate aminotransferase is not expressing pure gene (Gene ID: 936046).

[0022] Preferably, the ATP phosphoribosyltransferase is ATP phosphoribosyltransferase hisG.

[0023] Preferably, the method of not expressing the ATP phosphoribosyltransferase is not expressing this gene (Gene ID:936568).

[0024] Preferably, the glutamate aminotransferase is glutamate aminotransferase aspB.

[0025] Preferably, the method for overexpressing the glutamate aminotransferase is overexpressing aspB gene (Gene ID:939037).

[0026] Preferably, the glutamate synthase is glutamate synthase gltA.

[0027] Preferably, the method for overexpressing the glutamate synthase is overexpressing gltA Gene (Gene ID: 940024) and / or gltB gene (Gene ID: 940053).

[0028] Preferably, the PRPP synthase is PRPP synthase prs.

[0029] Preferably, the method for overexpressing the PRPP synthase is to overexpress for gene (Gene ID: 936985).

[0030] Preferably, the 6-phosphogluconate dehydrogenase is 6-phosphogluconate dehydrogenase gndA.

[0031] Preferably, the method for overexpressing the 6-phosphogluconate dehydrogenase is to overexpress gndA gene (Gene ID:938695).

[0032] Preferably, the glucose-6-phosphate dehydrogenase is glucose-6-phosphate dehydrogenase zwf.

[0033] Preferably, the method for overexpressing the glucose-6-phosphate dehydrogenase is to overexpress zwf gene (Gene ID:938690).

[0034] Preferably, the method for overexpressing the pyr operon gene is: using promoter P 43 Replace the promoter of the pyr operon, the P 43 The nucleotide sequence of the promoter is shown in SEQ ID No. 1 in the sequence listing.

[0035] Preferably, the method of overexpressing the glutamate aminotransferase, glutamate synthase, PRPP synthase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase is to increase the copy number of the encoding gene of each enzyme, or to use promoter P 43 Replace the natural promoter of each enzyme encoding gene, the P 43 The nucleotide sequence of the promoter is shown in the sequence table SEQ ID No. 1. gltA Genes and gltB Genes share the same promoter, and this promoter is replaced by promoter P 43 back, gltA Genes and gltB All genes can be overexpressed.

[0036] Preferably, the method for not expressing the cytidine deaminase, cytidine kinase, pyrimidine operon regulatory protein, spermine succinate synthetase, ornithine carbamoyltransferase, homoserine dehydrogenase, glutamine phosphoribosyl pyrophosphate aminotransferase and ATP phosphoribosyltransferase is: knocking out the corresponding coding genes, or using CRISPRi silencing technology to make the corresponding coding genes not expressed.

[0037] Preferably, the starting strain is Bacillus subtilis B. subtilis 168, ATCC23857.

[0038] The second aspect of the present invention provides a method for constructing the above-mentioned cytidine-producing strain, which specifically comprises the following steps: S1. Using Bacillus subtilis as the starting strain, the genes encoding cytidine deaminase, cytidine kinase, and pyrimidine operon regulatory protein are not expressed, and the pyr operon gene is overexpressed; S2, suppressing the expression of genes encoding spermine succinate synthetase, ornithine transcarbamoylase, and homoserine dehydrogenase; and overexpressing genes encoding glutamate aminotransferase and glutamate synthase; S3. The genes encoding glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase are not expressed, and the genes encoding PRPP synthase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase are overexpressed.

[0039] In this construction method, S1 blocks the cytidine degradation pathway by inactivating the genes encoding cytidine deaminase and cytidine kinase, relieves transcriptional repression by inactivating the genes encoding pyrimidine operon regulatory proteins, and enhances the synthesis flux of UMP by overexpressing the pyr operon genes. Thus, S1 enhances the de novo synthesis of pyrimidine nucleosides and promotes cytidine accumulation. S2 blocks the consumption of carbamyl phosphate and L-aspartate by inactivating the genes encoding spermine succinate synthetase, ornithine carbamoyltransferase, and homoserine dehydrogenase, while overexpressing the genes encoding glutamate aminotransferase and glutamate synthase, thereby increasing the production of L-aspartate and glutamine, the precursors for cytidine synthesis. By de-expressing the genes encoding glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase and overexpressing the genes encoding PRPP synthase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase, S3 modifies the PP pathway, increasing PP pathway flux, reducing PRPP loss, and increasing PRPP, another precursor for cytidine synthesis, thereby promoting cytidine accumulation. Therefore, this construction method can produce a cytidine-producing strain with stable genetic properties and high cytidine production.

[0040] Preferably, the method for preventing the expression of the genes encoding cytidine deaminase, cytidine kinase and pyrimidine operon regulatory protein in S1 is to knock out the genes encoding them.

[0041] More preferably, the method for not expressing the coding genes of cytidine deaminase, cytidine kinase and pyrimidine operon regulatory protein in S1 is to knock out cdd Gene, udk Genes and pyR The present invention knocks out S1 cdd Gene, udk Genes and pyR The order of genes is not limited, and knocking out the genes in any order can achieve the purpose of the present invention and is within the scope of protection of the present invention.

[0042] Preferably, the method for overexpressing the pyr operon gene in S1 is to use the promoter P 43 The present invention knocks out the coding genes of cytidine deaminase, cytidine kinase and pyrimidine operon regulatory protein in S1 and replaces the promoter of pyr operon with promoter P. 43The order of replacing the promoter of the pyr operon is not limited. Knocking out each gene first, replacing the promoter first, or alternating gene knockout and promoter replacement can all achieve the purpose of the present invention and are within the scope of protection of the present invention.

[0043] Preferably, the method for preventing the expression of genes encoding sperminosuccinate synthetase, ornithine transcarbamylase and homoserine dehydrogenase in S2 is to knock out the genes encoding them.

[0044] More preferably, the method for not expressing the genes encoding spermine succinate synthetase, ornithine transcarbamylase and homoserine dehydrogenase in S2 is to knock out argG Gene ,argF Genes and man The present invention knocks out the gene in S2 argG Gene ,argF Genes and man The order of genes is not limited, and knocking out the genes in any order can achieve the purpose of the present invention and is within the scope of protection of the present invention.

[0045] Preferably, the method for overexpressing the coding genes of glutamate aminotransferase and glutamate synthase in S2 is: aspB Genes and gltA / B The natural promoter of the gene is replaced by promoter P 43 The present invention replaces S2 aspB Genes and gltA / B The order of the natural promoter of the gene is not limited, replace it first aspB The natural promoter of the gene may be replaced first gltA / B Any natural promoter of any gene can achieve the objectives of the present invention and is within the scope of protection of the present invention. Furthermore, the present invention does not limit the order in which the genes encoding spermine succinate synthetase, ornithine transcarbamylase, and homoserine dehydrogenase are knocked out in S2, or the promoters are replaced. The objectives of the present invention can be achieved by first knocking out the gene, then replacing the promoter, or alternating between knocking out the gene and replacing the promoter, and is within the scope of protection of the present invention.

[0046] Preferably, the method for preventing the expression of the genes encoding glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase in S3 is to knock out the genes encoding them.

[0047] More preferably, the method for not expressing the coding genes of glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase in S3 is to knock out pure Genes and this The present invention does not limit the order of knocking out genes in S3. pure Gene or knockout first thisAll genes can achieve the purpose of the invention and are within the scope of protection of the invention.

[0048] Preferably, the method for overexpressing PRPP synthase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in S3 is: for Gene 、gndA Genes and zwf The natural promoter of the gene is replaced by promoter P 43 . The present invention does not limit the order of replacing the natural promoters of each gene in S3. The replacement of the promoters in any order can achieve the purpose of the invention and is within the scope of protection of the present invention. In addition, the present invention does not limit the order of knocking out the coding genes of glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase in S3 and replacing each promoter. The purpose of the invention can be achieved by knocking out the genes first, replacing the promoter first, or alternating between gene knockout and promoter replacement, and are within the scope of protection of the present invention.

[0049] Further preferably, each coding gene is knocked out using CRISPR / Cas9 gene editing technology.

[0050] For example, CRISPR / Cas9 gene editing technology is used to knock out cdd Gene, udk Gene, pyR Gene, argG Gene, argF Gene, man Gene, pure Gene, this The genetic method can be performed as follows: Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the sgRNA sequence of the gene to be knocked out was introduced to obtain the recombinant plasmid pJOE8999-cdd / udk / pyrR / argG / argF / hom / purF / hisG - sgRNA; using the genome of the starting strain as a template, SfiI The primer pair at the site amplifies the upstream and downstream homology arms of the gene to be knocked out, and then connects the upstream and downstream homology arms into a sequence to introduce SfiI Cut the linearized recombinant plasmid pJOE8999-cdd / udk / pyrR / argG / argF / hom / purF / hisG-sgRNA into the competent strain. Then, electroporate the resulting recombinant plasmid into the competent strain. After identification, select the positive transformants to complete the gene knockout. When performing the first gene knockout, the competent strain is the starting strain. When performing gene knockout in other steps, the competent strain is the strain that underwent the previous gene editing step.

[0051] For example, the promoter P 43 replace pyR The following operations can be used for gene promoters: Using the genome of the starting strain as a template, amplify pyR The upstream and downstream homology arm sequences of the gene were amplified using the pWB980 plasmid as a template and primers. 43 The promoter sequence is then pyR Gene upstream and downstream homology arm fragments, P 43 Promoter fragment and salt The single enzyme-cut pJOE8999-pyrB-sgRNA plasmid was seamlessly connected, and the resulting recombinant plasmid was transformed into the competent strain by electroporation. After identification, positive transformants were screened to complete the promoter replacement.

[0052] For example, the promoter P 43 replace aspB Gene 、gltA / B Gene, for Gene, gndA Gene, zwf The following operations can be used to identify the promoter of a gene: Using the genome of the starting strain as a template, the upstream and downstream homology arm sequences of the gene to be replaced were amplified, and the amplified fragment was seamlessly cloned and connected with the pUC19 linearized vector; using the pWB980 plasmid as a template and primers to amplify the P 43 Promoter sequence; the aspB / gltA / prs / gndA / zwf-pUC19 plasmid with correct sequencing was used as a template to reverse-amplify the aspB / gltA / prs / gndA / zwf-pUC19 vector, and then P 43 The gene fragment was seamlessly cloned and connected with the aspB / gltA / prs / gndA / zwf-pUC19 reverse amplification vector to obtain aspB / gltA / prs / gndA / zwf-P 43 -pUC19 plasmid; with sequenced aspB / gltA / prs / gndA / zwf-P 43 -pUC19 plasmid as a template, amplify and repair homology arms, and then align the repaired homology arms with salt The single-enzyme-cut pJOE8999-aspB / gltA / prs / gndA / zwf-sgRNA plasmid was ligated to obtain the recombinant plasmid pJOE8999_aspB / gltA / prs / gndA / zwf; finally, the purified pJOE8999_aspB / gltA / prs / gndA / zwf plasmid was electroporated into the competent cells of the strain that had undergone gene editing in the previous step. After identification, positive transformants were screened to complete the promoter.

[0053] The third aspect of the present invention provides the use of the above-mentioned cytidine-producing strain in the fermentation production of cytidine.

[0054] A fourth aspect of the present invention provides a method for producing cytidine by fermentation, wherein the cytidine-producing strain is used to produce cytidine by fermentation.

[0055] Preferably, after the cytidine-producing strain is activated and cultured, it is inoculated into a fermentation medium for fermentation, and during the fermentation process, the pH of the fermentation medium is maintained at 7.0-7.2, and glucose or culture components are added to maintain the glucose concentration in the fermentation medium at 3-10 g / L to ensure normal fermentation.

[0056] Further preferably, the method specifically includes the following steps: first, inoculating the cytidine-producing bacteria into a slant culture medium, culturing at 30-40°C until a uniform bacterial lawn forms on the surface of the slant culture medium, transferring the culture medium to a liquid culture medium and culturing the culture medium until the logarithmic growth phase, then inoculating the culture medium into a seed culture medium and culturing the culture medium at 30-40°C until the strain reaches the logarithmic growth phase again, thereby obtaining a seed solution of the cytidine-producing bacteria; transferring the seed solution into a fermentation medium at an inoculum size of 10%-25%, and fermenting the culture medium at 30-40°C. During the fermentation process, the dissolved oxygen is maintained at 30%-60%, ammonia is added to maintain the pH at 7.0-7.2, and glucose or culture components are added to maintain the glucose concentration in the fermentation medium at 3-10 g / L. Preferably, the activation culture and fermentation are performed at 37±1°C.

[0057] Further preferably, the composition of the slant culture medium is: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 15 g / L agar powder, and the pH is 7.0-7.2.

[0058] Further preferably, the components of the liquid culture medium are: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, and pH 7.0-7.2.

[0059] Further preferably, the components of the seed culture medium are: 20 g / L glucose, 10 g / L yeast powder, 5 g / L peptone, 2.5 g / L NaCl, 1 g / L MgSO4•7H2O, 1 g / L KH2PO4, 1.5 g / L sodium glutamate, and the pH is 7.0~7.2.

[0060] Further preferably, the components of the fermentation medium are: 80 g / L glucose, 10 g / L peptone, 15 g / L yeast powder, 2.5 g / L NaCl, 3 g / L MgSO4.7H2O, 2.5 g / L KH2PO4, 6 g / L (NH4)2SO4, 5 g / L sodium glutamate, 5 mg / L biotin, phenol red 2%, and pH 7.0~7.2.

[0061] Further preferably, the components of the fermentation medium are: 80 g / L glucose, 20 g / L yeast powder, 5 g / L (NH4)2SO4, 1 g / L KH2PO4, 5 g / L K2HPO4, 10 g / L sodium citrate, 20 g / L sodium glutamate, 1.5 g / L MgSO4•7H2O, 0.02 g / L MnSO4, 0.02 g / L ZnSO4, 5 mg / L biotin, pH = 7.0; the culture component is a feed medium, whose components are: 500 g / L glucose, 10 g / L yeast extract, 5 g / L (NH4)2SO4, 5 g / L K2HPO4 and 0.5 g / L MgSO4•7H2O.

[0062] The beneficial effects of the present invention are as follows: the cytidine production strain provided by the present invention blocks the cytidine degradation pathway by not expressing cytidine deaminase and cytidine kinase, relieves transcriptional repression by pyrimidine operon regulatory proteins, and enhances the synthesis flux of UMP by overexpressing the pyr operon; increases the supply of precursor substances L-aspartate and glutamine by not expressing spermine succinate synthetase, ornithine transcarbamoylase, and homoserine dehydrogenase and overexpressing glutamate aminotransferase and glutamate synthase; reduces the flow of PRPP to histidine and purine nucleosides by not expressing glutamine phosphoribosyl pyrophosphate aminotransferase and ATP phosphoribosyltransferase; and increases the PP pathway flux and the supply of another cytidine precursor, PRPP, by overexpressing the encoding genes of PRPP synthase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase, thereby achieving rapid accumulation of cytidine. The cytidine production strain does not contain plasmids, has no auxotrophic defects, does not require induction, has good genetic stability, and has a high fermentation yield, making it an excellent strain capable of stably and highly producing cytidine. The cytidine-producing strain constructed by the present invention can efficiently synthesize cytidine from scratch using glucose as a substrate. The cytidine yield can reach 31.41 g / L after 48 hours of fermentation, which is much higher than the level of cytidine production by existing Bacillus subtilis. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Schematic diagram of the modular metabolic engineering strategy for constructing a cytidine-producing strain in Example 1 of the present invention; Figure 2This is a fermentation process curve diagram of cytidine production using the cytidine-producing strain BSNX14 in Example 3 of the present invention. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0065] Currently, the engineered bacteria carrying plasmids used to produce cytidine by microbial fermentation have disadvantages such as poor genetic stability and a heavy growth and metabolic burden, leading to industrial problems such as significantly reduced cytidine production and difficulty in controlling the production process.

[0066] To address this problem, the present invention uses a wild-type Bacillus subtilis as the starting strain and utilizes a modular metabolic engineering strategy to construct a cytidine-producing strain. The genetic modification method is as follows: cytidine deaminase, cytidine kinase, pyrimidine operon regulatory protein, spermine succinate synthetase, ornithine transcarbamoylase, homoserine dehydrogenase, glutamine phosphoribosyl pyrophosphate aminotransferase, and ATP phosphoribosyltransferase are de-expressed; pyr operon genes are over-expressed; and glutamate aminotransferase, glutamate synthase, PRPP synthase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase are over-expressed. This strain has good genetic stability, is plasmid-free, and has no auxotrophic deficiencies. It can stably and efficiently produce cytidine without induction, and the production process is easy to control, making it suitable for the industrial production of cytidine.

[0067] The embodiment of the present invention also provides a method for constructing the cytidine-producing strain.

[0068] The embodiment of the present invention also provides the use of the cytidine-producing strain in the fermentation production of cytidine.

[0069] The embodiments of the present invention also provide a method for producing cytidine by fermentation.

[0070] The solutions of the present invention are described below through specific embodiments.

[0071] In the following examples, the percentage sign "%" refers to mass percentage unless otherwise specified, and the percentage of a solution refers to the number of grams of solute contained in 100 mL.

[0072] The starting strain used in the following examples is the wild type B. subtilis 168, for B. subtilis ATCC23857 (commercially available).

[0073] The CRIPSR / Cas9 gene editing technology and operation steps used in the following examples are referenced to the literature (Altenbuchner J. Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System. Appl Environ Microbiol. 2016; 82(17):5421-5427.). The technical terms such as gene integration and plasmid construction involved in the following examples are explained in this article.

[0074] The primers used in the strain construction are shown in Table 1.

[0075] Table 1 Primers involved in strain construction

[0076] Unless otherwise specified, the raw materials, reagents, drugs, or instruments used in the following examples are all commercially available products. The methods used in the following examples are all conventional methods in the art unless otherwise specified.

[0077] Example 1 This embodiment provides a cytidine-producing strain and a construction method thereof.

[0078] by B. subtilis 168 strains were used as the starting strain and were genetically modified using a modular metabolic engineering strategy (e.g. Figure 1 During the genetic modification process, the starting strain B. subtilis The preparation and transformation of 168 competent cells and the preparation and transformation of recombinant competent cells obtained in each step were all referred to the literature (Jin L, Li L, Zhou L, ZhangR, Xu Y, Li J. Improving expression of bovine lactoferrin n-lobe by promoter optimization and codon engineering in Bacillus subtilis and its antibacterial activity. J Agric Food Chem. 2019; 67(35):9749-9756.). The specific process of genetic modification is as follows: 1.1 cdd gene knockout Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the cdd-sgRNA sequence was introduced to obtain the recombinant plasmid pJOE8999-cdd-sgRNA. B. subtilis 168 genome as template, with SfiI Locus primer pairs cdd-UF / cdd-UR and cdd-DF / cdd-DR amplification cdd The upstream and downstream homology arms of the gene are then connected into a sequence and introduced SfiI Cut the linearized pJOE8999-cdd-sgRNA plasmid to obtain the recombinant plasmid pJOE8999_cdd. Use electroporation to transform the recombinant plasmid into B. subtilis In 168 competent cells, cdd-JD-F and cdd-JD-R were used as identification primers to screen positive transformants and obtain strain BSNX1.

[0079] 1.2 udk gene knockout Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the udk-sgRNA sequence was introduced to obtain the recombinant plasmid pJOE8999-udk-sgRNA. B. subtilis 168 genome as template, with SfiI Locus primer pairs udk-UF / udk-UR and udk-DF / udk-DR amplify udk The upstream and downstream homology arms of the gene are then connected into a sequence and introduced SfiI The linearized pJOE8999-cdd-sgRNA plasmid was cut to obtain the recombinant plasmid pJOE8999_udk. The recombinant plasmid was transformed into BSNX1 competent cells by electroporation. Positive transformants were screened using primers udk-JD-F and udk-JD-R to obtain strain BSNX2.

[0080] 1.3 pyR gene knockout Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the pyrR-sgRNA sequence was introduced to obtain the recombinant plasmid pJOE8999-pyrR-sgRNA. B. subtilis 168 genome as template, with SfiI The locus primer pairs pyrR-UF / pyrR-UR and pyrR-DF / pyrR-DR were used for amplification. pyR The upstream and downstream homology arms of the gene are then connected into a sequence and introduced SfiIThe linearized pJOE8999-pyrR-sgRNA plasmid was cut to obtain the recombinant plasmid pJOE8999_pyrR. The recombinant plasmid was transformed into BSNX2 competent cells by electroporation. Positive transformants were screened using pyrR-JD-F and pyrR-JD-R primers to obtain strain BSNX3.

[0081] 1.4 pyR Gene promoter replacement (P pyrB :P 43 ) To extract B. subtilis 168 DNA was used as template to amplify the PCR product using primers pyrB-UF / pyrB-UR and pyrB-DF / pyrB-DR. pyR The upstream and downstream homology arm sequences of the gene promoter were extracted using the pWB980 plasmid as a template and pyrB-P 43 -F / pyrB-P 43 -R primer amplifies P 43 The promoter sequence is then pyR Gene upstream and downstream homology arm fragments, P 43 Promoter fragment and salt The single-enzyme-digested pJOE8999-pyrB-sgRNA plasmid was seamlessly ligated to obtain the recombinant plasmid pJOE8999_pyrB. Finally, the purified pJOE8999_pyrB plasmid was electroporated into BSNX3 competent cells, and positive transformants were screened using pyrB-JD-F and pyrB-JD-R primers to obtain strain BSNX4.

[0082] 1.5 argG gene knockout Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the argG-sgRNA sequence was introduced to obtain the recombinant plasmid pJOE8999-argG-sgRNA. B. subtilis 168 genome as template, with SfiI The locus primer pairs argG-UF / argG-UR and argG-DF / argG-DR were used for amplification. argG The upstream and downstream homology arms of the gene are then connected into a sequence and introduced SfiI The linearized pJOE8999-argG-sgRNA plasmid was cut to obtain the recombinant plasmid pJOE8999_argG. The recombinant plasmid was transformed into BSNX4 competent cells by electroporation. Positive transformants were screened using primers argG-JD-F and argG-JD-R to obtain strain BSNX5.

[0083] 1.6 argF gene knockout Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the argF-sgRNA sequence was introduced to obtain the recombinant plasmid pJOE8999-argF-sgRNA. B. subtilis 168 genome as template, with SfiI Locus primer pairs argF-UF / argF-UR and argF-DF / argF-DR amplify argF The upstream and downstream homology arms of the gene are then connected into a sequence and introduced SfiI The linearized pJOE8999-argF-sgRNA plasmid was cut to obtain the recombinant plasmid pJOE8999_argF. The recombinant plasmid was transformed into BSNX4 competent cells by electroporation. Positive transformants were screened using argF-JD-F and argF-JD-R primers to obtain strain BSNX6.

[0084] 1.7 man gene knockout Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the hom-sgRNA sequence was introduced to obtain the recombinant plasmid pJOE8999-hom-sgRNA. B. subtilis 168 genome as template, with SfiI Locus primer pairs hom-UF / hom-UR and hom-DF / hom-DR amplification man The upstream and downstream homology arms of the gene are then connected into a sequence and introduced SfiI The linearized pJOE8999-hom-sgRNA plasmid was cut to obtain the recombinant plasmid pJOE8999_hom. The recombinant plasmid was transformed into BSNX4 competent cells by electroporation. Positive transformants were screened using hom-JD-F and hom-JD-R primers to obtain strain BSNX7.

[0085] 1.8 aspB Gene promoter replacement (P aspB :P 43 ) To extract B. subtilis 168 DNA was used as a template to amplify the PCR product using primers aspB-19-F and aspB-19-R. aspB The upstream and downstream homology arm sequences of the gene promoter were detected, and the amplified fragment was seamlessly cloned and connected with the pUC19 linearized vector. 43 -aspB-F / R amplification P43 Gene fragment. Using the correctly sequenced aspB-pUC19 plasmid as template, primers aspB-P 43 -F / R reverse amplification of aspB-pUC19 vector. 43 The gene fragment was seamlessly cloned and connected with the aspB-pUC19 reverse amplification vector to obtain aspB-P 43 -pUC19 plasmid. 43 -pUC19 plasmid was used as a template, and the repair homology arms were amplified with primers aspB-salI-F / R, and the repaired homology arms were combined with salt The single-enzyme-digested pJOE8999-aspB-sgRNA plasmid was ligated to obtain the recombinant plasmid pJOE8999_aspB. Finally, the purified pJOE8999_aspB plasmid was electroporated into BSNX7 competent cells, and positive transformants were screened using aspB-JD-F and aspB-JD-R primers to obtain strain BSNX8.

[0086] 1.9 gltA Gene promoter replacement (P gltA :P 43 ) To extract B. subtilis 168 DNA was used as template to amplify the gene using primers gltA-19-F and gltA-19-R. gltA The upstream and downstream homology arm sequences of the gene promoter were detected, and the amplified fragment was seamlessly cloned and connected with the pUC19 linearized vector. 43 -gltA-F / R amplification P 43 Gene fragment. Using the correctly sequenced gltA-pUC19 plasmid as a template, primers gltA-P 43 -F / R reverse amplification of gltA-pUC19 vector. 43 The gene fragment was seamlessly cloned and connected with the gltA-pUC19 reverse amplification vector to obtain gltA-P 43 -pUC19 plasmid. At the same time, the correct gltA-P 43 -pUC19 plasmid was used as a template, and the repair homology arms were amplified with primers gltA-salI-F / R, and the repaired homology arms were aligned with salt The single-enzyme-digested pJOE8999-gltA-sgRNA plasmid was ligated to obtain the recombinant plasmid pJOE8999_gltA. Finally, the purified pJOE8999_gltA plasmid was electroporated into BSNX8 competent cells, and positive transformants were screened using gltA-JD-F / R primers to obtain strain BSNX9.

[0087] 1.10 pure gene knockout Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the purF-sgRNA sequence was introduced to obtain the recombinant plasmid pJOE8999-purF-sgRNA. B. subtilis 168 genome as template, with SfiI Locus primer pairs purF-UF / purF-UR and purF-DF / purF-DR amplify pure The upstream and downstream homology arms of the gene are then connected into a sequence and introduced SfiI The linearized pJOE8999-purF-sgRNA plasmid was cut to obtain the recombinant plasmid pJOE8999_purF. The recombinant plasmid was transformed into BSNX9 competent cells by electroporation, and positive transformants were screened using purF-JD-F and purF-JD-R primers to obtain strain BSNX10.

[0088] 1.11 this gene knockout Use restriction enzymes BsaI The plasmid pJOE8999 was linearized and the hisG-sgRNA sequence was introduced to obtain the recombinant plasmid pJOE8999-hisG-sgRNA. B. subtilis 168 genome as template, with SfiI The primer pairs hisG-UF / hisG-UR and hisG-DF / hisG-DR were used to amplify the locus this The upstream and downstream homology arms of the gene are then connected into a sequence and introduced SfiI The linearized pJOE8999-hisG-sgRNA plasmid was cut to obtain the recombinant plasmid pJOE8999_hisG. The recombinant plasmid was transformed into BSNX10 competent cells by electroporation. Positive transformants were screened using hisG-JD-F and hisG-JD-R primers to obtain strain BSNX11.

[0089] 1.12 for Gene promoter replacement (P prs :P 43 ) To extract B. subtilis 168 DNA was used as template to amplify the gene using primers prs-19-F and prs-19-R. for The upstream and downstream homology arm sequences of the gene promoter were detected, and the amplified fragment was seamlessly cloned and connected with the pUC19 linearized vector. 43 -prs-F / R amplification P43 Gene fragment. Using the correctly sequenced prs-pUC19 plasmid as template, primers prs-P 43 -F / R reverse amplification of prs-pUC19 vector. 43 The gene fragment was seamlessly cloned and connected with the prs-pUC19 reverse amplification vector to obtain prs-P 43 -pUC19 plasmid. 43 -pUC19 plasmid was used as a template, and the repair homology arms were amplified with primers prs-salI-F / R, and the repaired homology arms were aligned with salt The pJOE8999-prs-sgRNA plasmids were digested with a single enzyme and ligated to obtain the recombinant plasmid pJOE8999_prs. Finally, the purified pJOE8999_prs plasmid was electroporated into BSNX11 competent cells, and positive transformants were screened using prs-JD-F / R primers to obtain strain BSNX12.

[0090] 1.13 gndA Gene promoter replacement (P gndA :P 43 ) To extract B. subtilis 168 DNA was used as template to amplify the gene using primers gndA-19-F / R. gndA The upstream and downstream homology arm sequences of the gene promoter were detected, and the amplified fragment was seamlessly cloned and connected with the pUC19 linearized vector. 43 -gndA-F / R amplification P 43 Gene fragment. Using the correctly sequenced gndA-pUC19 plasmid as template, primers gndA-P 43 -F / R reverse amplification of gndA-pUC19 vector. 43 The gene fragment was seamlessly cloned and connected with the gndA-pUC19 reverse amplification vector to obtain gndA-P 43 -pUC19 plasmid. At the same time, the correct sequenced gndA-P 43 -pUC19 plasmid was used as a template, and the repair homology arms were amplified with primers gndA-salI-F / R, and the repaired homology arms were aligned with salt The single-enzyme-digested pJOE8999-gndA-sgRNA plasmid was ligated to obtain the recombinant plasmid pJOE8999_gndA. Finally, the purified pJOE8999_gndA plasmid was electroporated into BSNX12 competent cells, and positive transformants were screened using gndA-JD-F / R primers to obtain strain BSNX13.

[0091] 1.14 zwfGene promoter replacement (P zwf :P 43 ) To extract B. subtilis 168 DNA was used as template to amplify the DNA using primers zwf-19-F / R zwf The upstream and downstream homology arm sequences of the gene promoter were detected, and the amplified fragment was seamlessly cloned and connected with the pUC19 linearized vector. 43 -zwf-F / R amplification P 43 Gene fragment. Using zwf-pUC19 plasmid as template, primers zwf-P 43 -F / R reverse amplification zwf-pUC19 vector. 43 The gene fragment was seamlessly cloned and connected with the zwf-pUC19 reverse amplification vector to obtain zwf-P 43 -pUC19 plasmid. 43 -pUC19 plasmid was used as a template, and the repair homology arms were amplified with primers zwf-salI-F / R, and the repaired homology arms were aligned with salt The single-enzyme-digested pJOE8999-zwf-sgRNA plasmid was ligated to obtain the recombinant plasmid pJOE8999_zwf. Finally, the purified pJOE8999_zwf plasmid was electroporated into BSNX13 competent cells, and positive transformants were screened using the zwf-JD-F / R primers to obtain strain BSNX14.

[0092] Example 2 In this example, the cytidine-producing strain BSNX14 constructed in Example 1 was used to produce cytidine by shake flask fermentation.

[0093] 1. Culture medium 1.1 Slant culture medium Prepare an aqueous solution containing 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, and 15 g / L agar powder, adjust the pH to 7.0, dispense into test tubes (8 mL / tube), and sterilize in a high-pressure steam cooker at 121°C for 15 min.

[0094] 1.2 Liquid culture medium Prepare an aqueous solution containing 10 g / L peptone, 5 g / L yeast powder, and 10 g / L NaCl, adjust the pH to 7.0, dispense into test tubes (8 mL / tube), and sterilize in a high-pressure steam cooker at 121°C for 15 min.

[0095] 1.3 Seed culture medium Prepare an aqueous solution containing 20 g / L glucose, 10 g / L yeast powder, 5 g / L peptone, 2.5 g / L NaCl, 1 g / LMgSO4•7H2O, 1 g / L KH2PO4, and 1.5 g / L sodium glutamate, adjust the pH to 7.0, and dispense into 100 mL Erlenmeyer flasks (30 mL / flask). Sterilize in a high-pressure steam cooker at 121°C for 15 min.

[0096] 1.4 Fermentation medium Prepare an aqueous solution containing 80 g / L glucose, 10 g / L peptone, 15 g / L yeast powder, 2.5 g / L NaCl, 3 g / LMgSO4•7H2O, 2.5 g / L KH2PO4, 6 g / L (NH4)2SO4, 5 g / L sodium glutamate, 5 mg / L biotin, and 2% phenol red, adjust the pH to 7.0, and dispense into 500 mL Erlenmeyer flasks (50 mL / flask), and sterilize in a high-pressure steam cooker at 121°C for 15 min.

[0097] 2. Cultivation methods 2.1 Seed activation and cultivation: The strain stored at -80°C was inoculated into a slant medium and cultured at 37°C for 12 h until a uniform bacterial lawn formed on the surface of the slant medium. The strain was then transferred to a liquid medium and cultured for 12 h until the strain reached the logarithmic growth phase. The strain was then inoculated into a seed medium and cultured at 37°C for 8 h until the strain reached the logarithmic growth phase again, thereby obtaining the seed solution of the cytidine-producing strain BSNX14. 2.2 Fermentation culture: The seed solution was transferred to a 500 mL Erlenmeyer flask containing fermentation medium at a 10% inoculum volume and cultured at 37°C with shaking at 200 rpm for 48 hours. During the fermentation process, ammonia was added to maintain the pH value at 7.0-7.2. 50% (m / v) glucose solution was added to maintain the glucose concentration in the fermentation medium at 3-8 g / L to ensure normal fermentation (using phenol red as an indicator, glucose supplementation was considered necessary when the color of the fermentation liquid no longer changed).

[0098] After 48 h of shake flask fermentation, the cytidine production reached 7.03 g / L.

[0099] Example 3 In this example, the cytidine-producing strain BSNX14 constructed in Example 1 was used to ferment cytidine in a 5 L fermenter.

[0100] 1. Culture medium 1.1 Slant culture medium Prepare an aqueous solution containing 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, and 15 g / L agar powder, adjust the pH to 7.0, dispense into test tubes (8 mL / tube), and sterilize in a high-pressure steam cooker at 121°C for 15 min.

[0101] 1.2 Liquid culture medium Prepare an aqueous solution containing 10 g / L peptone, 5 g / L yeast powder, and 10 g / L NaCl, adjust the pH to 7.0, dispense into test tubes (8 mL / tube), and sterilize in a high-pressure steam cooker at 121°C for 15 min.

[0102] 1.3 Seed culture medium Prepare an aqueous solution containing 20 g / L glucose, 10 g / L yeast powder, 5 g / L peptone, 2.5 g / L NaCl, 1 g / LMgSO4•7H2O, 1 g / L KH2PO4, and 1.5 g / L sodium glutamate, adjust the pH to 7.0, and dispense into 500 mL Erlenmeyer flasks (100 mL / flask). Sterilize in a high-pressure steam cooker at 121°C for 15 min.

[0103] 1.4 Fermentation medium Prepare an aqueous solution containing 80 g / L glucose, 20 g / L yeast powder, 5 g / L (NH4)2SO4, 1 g / L KH2PO4, 5 g / LK2HPO4, 10 g / L sodium citrate, 20 g / L sodium glutamate, 1.5 g / L MgSO4•7H2O, 0.02 g / L MnSO4, 0.02 g / LZnSO4, and 5 mg / L biotin, adjust the pH to 7.0, place the solution in a sterilized fermentation tank, and sterilize the tank body at 121°C for 15 min.

[0104] 1.5 Feed medium Contains 500 g / L glucose, 10 g / L yeast extract, 5 g / L (NH4)2SO4, 5 g / L K2HPO4 and 0.5 g / L MgSO4•7H2O, adjusted to pH 7.0, and sterilized in an autoclave at 121℃ for 15 min.

[0105] 2. Cultivation methods 2.1 Seed activation: The strain stored at -80℃ was inoculated into a slant culture medium and cultured at 37℃ for 12 h. A uniform bacterial lawn formed on the surface of the slant culture medium. The strain was then transferred to a triangular flask containing liquid culture medium and cultured at 37℃ for another 12 h until the strain reached the logarithmic growth phase. 2.2 Seed cultivation: The bacterial suspension was inoculated into the seed culture medium and cultured at 37°C with shaking for 8 h until the strain reached the logarithmic growth phase again, thus obtaining the seed solution of the cytidine-producing strain BSNX14. 2.3 Fermentation culture: Seed liquid in the logarithmic growth phase was inoculated into the fermentation medium at a 20% inoculum rate. During the fermentation process, 50% ammonia was automatically added to maintain the pH between 7.0 and 7.2, the fermentation temperature at 37°C, and the dissolved oxygen level between 30% and 60%. The glucose concentration in the fermentation medium was maintained at 5 to 10 g / L by controlling the feed medium flow rate. No antibiotics or inducers were added during the fermentation process.

[0106] Cytidine content, glucose content and OD value of fermentation medium 600nm (When measuring, dilute first and then measure the absorbance to make the absorbance between 0.2-0.8, and then convert the measured absorbance according to the dilution multiple to get the value of Figure 2 The numerical value corresponding to the coordinates) Figure 2 As shown in the figure, the cytidine production reached 31.41 g / L after 48 h of fermentation in the fermenter.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cytidine-producing strain, characterized in that The cytidine production strain is constructed by genetically modifying Bacillus subtilis as a starting strain; the genetic modification method comprises the following steps: not expressing cytidine deaminase, cytidine kinase, pyrimidine operon regulatory protein, spermine succinate synthetase, ornithine carbamoyltransferase, homoserine dehydrogenase, glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase; overexpressing the pyr operon gene; and overexpressing glutamate aminotransferase, glutamate synthase, PRPP synthase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase.

2. The cytidine-producing strain according to claim 1, characterized in that The cytidine deaminase is cytidine deaminase CDD; and / or The cytidine kinase is cytidine kinase UDK; and / or The pyrimidine operon regulatory protein is a PyrR protein; and / or The spermine succinate synthetase is spermine succinate synthetase argG; and / or The ornithine transcarbamylase is ornithine transcarbamylase argF; and / or The homoserine dehydrogenase is homoserine dehydrogenase hom; and / or The glutamine phosphoribosyl pyrophosphate aminotransferase is glutamine phosphoribosyl pyrophosphate aminotransferase purF; and / or The ATP phosphoribosyltransferase is ATP phosphoribosyltransferase hisG; and / or The glutamate aminotransferase is glutamate aminotransferase aspB; and / or The glutamate synthase is glutamate synthase gltA; and / or The PRPP synthase is PRPP synthase prs; and / or The 6-phosphogluconate dehydrogenase is 6-phosphogluconate dehydrogenase gndA; and / or The glucose-6-phosphate dehydrogenase is glucose-6-phosphate dehydrogenase zwf; and / or The method for overexpressing the pyr operon gene is: using promoter P 43 Replace the promoter of the pyr operon, the P 43 The nucleotide sequence of the promoter is shown in SEQ ID No. 1; and / or The method for overexpressing the glutamate aminotransferase, glutamate synthase, PRPP synthase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase is to increase the copy number of the coding gene of each enzyme, or to use the promoter P 43 Replace the natural promoter of the gene encoding each enzyme; and / or The method for not expressing the cytidine deaminase, cytidine kinase, pyrimidine operon regulatory protein, spermine succinate synthetase, ornithine transcarbamoylase, homoserine dehydrogenase, glutamine phosphoribosyl pyrophosphate aminotransferase and ATP phosphoribosyltransferase is: knocking out the corresponding coding genes, or using CRISPRi silencing technology to make the corresponding coding genes not expressed; and / or The starting strain is Bacillus subtilis B. subtilis 168, ATCC23857.

3. The cytidine-producing strain according to claim 1 or 2, characterized in that The method of not expressing the cytidine deaminase is not expressing cdd Genes; and / or The method of not expressing the cytidine kinase is not expressing udk Genes; and / or The method of not expressing the pyrimidine operon regulatory protein is not expressing pyrR Genes; and / or The method of not expressing the spermine succinate synthetase is not expressing argG Genes; and / or The method of not expressing the ornithine transcarbamylase encoding the invention is not expressing argF Genes; and / or The method of not expressing the homoserine dehydrogenase is not expressing hom Genes; and / or The method of not expressing the glutamine phosphoribosyl pyrophosphate aminotransferase is not expressing purF Genes; and / or The method of not expressing the ATP phosphoribosyltransferase is not expressing hisG Genes; and / or The method for overexpressing the glutamate aminotransferase is to overexpress aspB Genes; and / or The method for overexpressing the glutamate synthase is to overexpress gltA Genes and / or gltB Genes; and / or The method for overexpressing the PRPP synthase is to overexpress prs Genes; and / or The method for overexpressing the 6-phosphogluconate dehydrogenase is to overexpress gndA Genes; and / or The method for overexpressing the glucose-6-phosphate dehydrogenase is to overexpress zwf Gene.

4. The method for constructing the cytidine-producing strain according to any one of claims 1 to 3, characterized in that: The specific steps include: S1. Using Bacillus subtilis as the starting strain, the genes encoding cytidine deaminase, cytidine kinase, and pyrimidine operon regulatory protein are not expressed, and the pyr operon gene is overexpressed; S2, suppressing the expression of genes encoding spermine succinate synthetase, ornithine transcarbamoylase, and homoserine dehydrogenase; and overexpressing genes encoding glutamate aminotransferase and glutamate synthase; S3. The genes encoding glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase are not expressed, and the genes encoding PRPP synthase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase are overexpressed.

5. The construction method according to claim 4, characterized in that: The method for preventing the expression of the genes encoding cytidine deaminase, cytidine kinase and pyrimidine operon regulatory protein in S1 is to knock out the genes encoding them; and / or The method for overexpressing pyr operon genes in S1 is to use promoter P 43 replacing the promoter of the pyr operon; and / or The method for preventing the expression of genes encoding spermine succinate synthetase, ornithine transcarbamylase and homoserine dehydrogenase in S2 is to knock out the genes encoding them; and / or The method for overexpressing the genes encoding glutamate aminotransferase and glutamate synthase in S2 is as follows: aspB Genes and gltA / B The natural promoter of the gene is replaced by promoter P 43 and / or The method for preventing the expression of the genes encoding glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase in S3 is to knock out the genes encoding them; and / or The method for overexpressing PRPP synthase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in S3 is as follows: prs Gene 、gndA Genes and zwf The natural promoter of the gene is replaced by promoter P 43 .

6. The construction method according to claim 4 or 5, characterized in that: The method of not expressing the genes encoding cytidine deaminase, cytidine kinase and pyrimidine operon regulatory protein in S1 is to knock out cdd Gene, udk Genes and pyrR Genes; and / or The method for not expressing the genes encoding spermine succinate synthetase, ornithine transcarbamylase and homoserine dehydrogenase in S2 is to knock out argG Gene argF Genes and hom Genes; and / or The method for not expressing the genes encoding glutamine phosphoribosylpyrophosphate aminotransferase and ATP phosphoribosyltransferase in S3 is to knock out purF Genes and hisG Genes; and / or CRISPR / Cas9 gene editing technology was used to knock out each coding gene.

7. Use of the cytidine-producing strain according to any one of claims 1 to 3 in the fermentation production of cytidine.

8. A method for producing cytidine by fermentation, characterized in that: Cytidine is produced by fermentation using the cytidine-producing strain according to any one of claims 1 to 3.

9. The method according to claim 8, characterized in that After the cytidine production strain is activated and cultured, it is inoculated into a fermentation medium for fermentation. During the fermentation process, the pH of the fermentation medium is maintained at 7.0-7.2, and glucose or culture components are added to maintain the glucose concentration in the fermentation medium at 3-10 g / L.

10. The method according to claim 9, characterized in that Specifically, the method includes the following operations: first, inoculating the cytidine-producing bacteria into a slant culture medium, culturing at 30-40° C. until a uniform bacterial lawn is formed on the surface of the slant culture medium, transferring the bacteria into a liquid culture medium and culturing the bacteria into a logarithmic growth phase, then inoculating the bacteria into a seed culture medium and culturing the bacteria at 30-40° C. until the bacteria reaches a logarithmic growth phase again, thereby obtaining a seed solution of the cytidine-producing bacteria; transferring the seed solution into a fermentation culture medium at an inoculum rate of 10%-25%, and fermenting the solution at 30-40° C., maintaining the dissolved oxygen at 30%-60% during the fermentation process, adding ammonia water to maintain the pH value at 7.0-7.2, and adding glucose or culture components to maintain the glucose concentration in the fermentation culture medium at 3-10 g / L.

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