Methods and applications for enhancing cytidine production

Abstract

The invention discloses a method and application for enhancing cytidine production, and belongs to the field of genetic engineering and microbial engineering. The present invention knocks out cytidine deaminase gene cdd, uridine kinase gene udk, pyrimidine-specific ribonucleoside hydrolase gene rihB, nucleotide 5'-monophosphate nucleosidase gene ppnN, pyruvate oxidase gene poxB and integrates and expresses 5'-CTP diphosphohydrolase, orotate phosphoribosyltransferase, uridylate kinase mutant gene PyrH (R92G / D93G), as well as phosphoribosyl pyrophosphate synthetase gene prs, glucose-6-phosphate dehydrogenase gene zwf and 6-phosphogluconate dehydrogenase gene gnd, and constructs a recombinant Escherichia coli with enhanced cytidine synthesis ability, and the cytidine yield reaches 61.9g / L in a 30L fermenter without adding antibiotics and inducers, which can be used to realize the industrial production of cytidine and realize green biomanufacturing.

Description

Technical Field

[0001] The invention relates to a method for enhancing cytidine production and application, and belongs to the field of genetic engineering and microbial engineering. Background Art

[0002] 1-β-D-furanosylcytosine (1-beta-ribofuranosylcytosine) is also known as cytosine nucleoside and cytidine. Cytidine is a compound formed by connecting the N-1 of cytosine and the C-1 of D-ribose through a β-glycosidic bond. Cytidine exists in all organisms and is a component of RNA. The most important industrial use of cytidine is as a raw material for chemical synthesis of antiviral and antitumor drugs. Many cytidine structural analogs have the effect of interfering with cellular RNA synthesis. Based on this principle, a variety of cytidine analogs have been developed for the treatment of viral infections and tumors, many of which are widely used as clinical specific drugs and first-choice drugs. For example: Zalcitabine (2', 3'-dideoxycytidine nucleoside) can competitively inhibit reverse transcriptase activity and is widely used to treat HIV infection and other retroviral infections. Capecitabine (5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]cytidine) is also a cytidine analogue, which is clinically used to treat various tumors such as colorectal cancer, gastric cancer, liver cancer and breast cancer. These cytidine structural analogues are important clinical drugs, all of which are produced by chemical synthesis using cytidine as the raw material. Therefore, cytidine is an important pharmaceutical intermediate.

[0003] So far, the industrial production methods of cytidine include RNA hydrolysis, chemical synthesis and microbial fermentation. Although the RNA hydrolysis method for producing cytidine has the advantages of simple technology, easy operation and control, it has high requirements for raw materials, complex process, low product yield and high production cost. It has been basically replaced by other methods. Dozens of chemical synthesis methods have been developed for the production of cytidine. The latest cytidine synthesis process uses di-p-nitrophenol phosphate as a catalyst to catalyze the reaction of N4-acetylcytosine and tetra-O-acetyl-β-D-furanose, and removes the acetyl group to generate cytidine. The development of the technology for producing cytidine by chemical synthesis has significantly reduced the price of cytidine. However, due to the inherent characteristics of the chemical synthesis method, such as long reaction route, harsh reaction conditions, expensive catalysts, many by-products and potential environmental pollution, it is almost impossible to further reduce the production cost. Due to the problem of cytidine production cost, its application in the field of medicine and other uses is limited.

[0004] The production of cytidine by microbial fermentation includes the fermentation method with added precursors and the direct fermentation method. The production of cytidine by the fermentation method with added precursors mainly utilizes the salvage pathway of cytidine synthesis. The salvage pathway of cytidine synthesis refers to the direct generation of UMP by the catalysis of uracil ribose phosphotransferase (pyrR / upp) from uracil added to the culture medium and 5-phosphoribosyl pyrophosphate (PRPP) in the microbial cells, and then generates cytidine through UDP, UTP, and CTP. The direct fermentation method of cytidine mainly utilizes the "from scratch" generation of UMP by microorganisms, and then generates cytidine through UDP, UTP, and CTP. The fermentation production of cytidine using the salvage pathway of microorganisms requires uracil as a precursor for fermentation, resulting in high raw material costs and low production efficiency, which is not the direction of technological development. The direct fermentation method can select high-yield cytidine fermentation strains through microbial genetic breeding technology, use glucose as the main raw material, and produce high-quality cytidine under mild conditions through relatively simple processes. The direct fermentation method can further reduce the production cost of cytidine, the production process is relatively environmentally friendly, and there is a large space for technological improvement.

[0005] CN106754602A discloses a recombinant microorganism for producing cytidine and a method for producing cytidine, knocks out cytidine deaminase, ribonucleoside hydrolase, cytidine / uridine kinase, nucleoside transporter, and overexpresses cytidine triphosphate pyrophosphorylase and cytidine monophosphate phosphorylase, removes the feedback inhibition of pyrimidine nucleoside pathway, and constructs a recombinant Escherichia coli, which can reach 20g / L cytidine production level in 5L fermentor. However, the strain constructed by the method needs to add antibiotics during fermentation, and when fermenting cytidine, it is necessary to add inducer IPTG, and the yield is not high. The inducer and antibiotic are expensive and toxic to cells, and if the accumulation concentration of cytidine is further improved, its industrial production will be further promoted. Summary of the invention

[0006] The technical problem to be solved by the present invention is that the prior art lacks a recombinant strain capable of producing high cytidine, in particular, lacks a recombinant strain capable of producing high cytidine without adding an inducer and an antibiotic.

[0007] The present invention provides a recombinant Escherichia coli, in which a cytidine deaminase gene cdd, a uridine kinase gene udk, a pyrimidine-specific ribonucleoside hydrolase gene rihB, a nucleotide 5'-monophosphate nucleosidase gene ppnN, a pyruvate oxidase gene poxB and a ribonucleoside triphosphate reductase gene nrdD on the genome are knocked out, and 5'-CTP diphosphohydrolase and / or orotate phosphoribosyltransferase are integrated and expressed.

[0008] In one embodiment, the nucleotide sequence of the cytidine deaminase gene cdd is shown as SEQ ID NO.1, the nucleotide sequence of the uridine kinase gene udk is shown as SEQ ID NO.2, the nucleotide sequence of the pyrimidine-specific ribonucleoside hydrolase gene rihB is shown as SEQ ID NO.3, and the nucleotide sequence of the nucleotide 5'-monophosphonucleotide enzyme gene ppnN is shown as SEQ ID NO.4.

[0009] In one embodiment, the 5'-CTP diphosphohydrolase has an amino acid sequence shown in SEQ ID NO.6.

[0010] In one embodiment, the amino acid sequence of the orotate phosphoribosyltransferase is shown as SEQ ID NO.8 or SEQ ID NO.10.

[0011] In one embodiment, a 5'-CTP diphosphohydrolase gene is integrated into the umpH site; the gene sequence of the 5'-CTP diphosphohydrolase is shown in SEQ ID NO.5.

[0012] In one embodiment, the orotate phosphoribosyltransferase gene pyrE and / or pyrE / K26H is integrated at the umpH site; the gene sequence of the orotate phosphoribosyltransferase gene pyrE is shown in SEQ ID NO.7; the gene sequence of the pyrE / K26H is shown in SEQ ID NO.9.

[0013] In one embodiment, the nucleotide sequence of the pyruvate oxidase gene poxB is shown as SEQ ID NO.13, and the ribonucleoside triphosphate reductase gene nrdD is shown as SEQ ID NO.18.

[0014] In one embodiment, the recombinant Escherichia coli also integrates and expresses the uridylate kinase mutant gene PyrH (R92G / D93G) at the location of the pyruvate oxidase gene poxB.

[0015] In one embodiment, the uridylate kinase mutant PyrH (R92G / D93G) has an amino acid sequence as shown in SEQ ID NO.14, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.15.

[0016] In one embodiment, the recombinant Escherichia coli integrates and expresses the phosphoribosyl pyrophosphate synthetase gene prs, the glucose-6-phosphate dehydrogenase gene zwf and the 6-phosphogluconate dehydrogenase gene gnd at the location of the ribonucleoside triphosphate reductase gene nrdD.

[0017] In one embodiment, the phosphoribosyl pyrophosphate synthetase gene prs, glucose-6-phosphate dehydrogenase gene zwf and 6-phosphogluconate dehydrogenase gene gnd are expressed in tandem; the RBS sequence AGGA is contained between prs and zwf; the RBS sequence AGGA is contained between zwf and gnd, and the tandem order is zwf-RBS-gnd-RBS-prs.

[0018] In one embodiment, the nucleotide sequence of the phosphoribosyl pyrophosphate synthetase gene prs is shown as SEQ ID NO.19, the nucleotide sequence of the glucose-6-phosphate dehydrogenase gene zwf is shown as SEQ ID NO.20, and the nucleotide sequence of the 6-phosphogluconate dehydrogenase gene gnd is shown as SEQ ID NO.21.

[0019] The present invention also provides a method and application for enhancing cytidine production, which comprises knocking out a pyruvate oxidase gene poxB and a ribonucleoside triphosphate reductase gene nrdD on the Escherichia coli genome, integrating and expressing a uridylate kinase mutant gene PyrH (R92G / D93G), integrating and expressing a phosphoribosyl pyrophosphate synthetase gene prs, a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd, and promoting efficient accumulation of cytidine.

[0020] In one embodiment, the Escherichia coli is selected from: DH5α, BL21 (DE3), JM109, HB101 or MG1655.

[0021] The present invention also provides a method for producing cytidine by fermentation, which comprises fermenting the recombinant Escherichia coli in a medium containing glucose.

[0022] In one embodiment, the fermentation process is performed without adding any antibiotics and inducer (IPTG).

[0023] In one embodiment, the fermentation is to inoculate the strain into LB medium, culture at 37°C and 200rpm for 5-15h; for shake flask fermentation, inoculate into a 500mL Erlenmeyer flask containing 50-150mL LB medium at a 0.1-10% inoculation rate, culture at 37°C and 200rpm for 40-75h.

[0024] In one embodiment, the fermentation is to transfer the seed liquid in the LB medium to the fermentation medium according to the inoculation amount of 0.5-15%, and carry out fermentation culture at 35°C and 200rpm. The glucose concentration in the fermentation process is controlled to be 15±2g / L, and the dissolved oxygen is controlled to be 25%; when the dissolved oxygen is lower than 25%, the stirring speed, ventilation volume and tank pressure are increased; and ammonia water is used to control the pH to 6-8 during the fermentation process.

[0025] In one embodiment, the culture medium for fermentation contains: glucose 12-85 g / L, MgSO4 0.5-5 g / L, sodium citrate 1.6-19 mg / L, calcium chloride 7-560 mg / L, disodium hydrogen phosphate 0.1-7.9 g / L, sodium dihydrogen phosphate 0.6-10.3 g / L, zinc chloride 0.7-72 mg / L, yeast powder 0.3-11 g / L, peptone 0.5-21 g / L, copper chloride 5-145 mg / L, zinc sulfate 1-9.2 mg / L, and sodium molybdate 1.3-165 mg / L.

[0026] In one embodiment, the fermentation process is further fed; the feed medium contains: 350-820 g / L glucose, 0.5-20 g / L peptone, and 0.5-20 g / L yeast powder.

[0027] The present invention also provides application of the recombinant Escherichia coli in fermentation production of cytidine.

[0028] Beneficial effects:

[0029] (1) The present invention adopts gene knockout technology to integrate and express the uridylate kinase mutant gene PyrH (R92G / D93G) to weaken the rate-limiting step in the synthesis pathway of the cytidine precursor 5'-CMP and knock out the pyruvate oxidase gene poxB to enhance the accumulation of the precursor substance pyruvate, integrate and express the phosphoribosyl pyrophosphate synthetase gene prs, the glucose-6-phosphate dehydrogenase gene zwf and the 6-phosphogluconate dehydrogenase gene gnd to increase the carbon flux of the PPP pathway to enhance the supply of the precursor substance PRPP and knock out the ribonucleoside triphosphate reductase gene nrdD to block the flux from CTP to dCTP, so that cytidine can be efficiently produced. The yield in a 30L fermenter reaches 61.9g / L, and there is basically no accumulation of intermediate metabolites, which is conducive to the separation and purification of cytidine.

[0030] (2) The recombinant Escherichia coli provided by the present invention can use glucose as a substrate and obtain cytidine by one-step fermentation without adding an inducer IPTG and antibiotics. The cytidine concentration in the fermentation supernatant is high, which is convenient for separation and purification, and the process is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of cytidine metabolic pathway;

[0032] Figure 2 is the cytidine production level of different recombinant strains (shake flask);

[0033] Figure 3 This is a schematic diagram of the PPP metabolic pathway;

[0034] Figure 4The curve of cytidine production by the recombinant strain E. coli Cr09 (30L fermenter);

[0035] Figure 5 This is the HPLC chart of cytidine production by the recombinant strain E. coli Cr09. DETAILED DESCRIPTION

[0036] Table 1: Strains involved in the specific implementation mode:

[0037]

[0038]

[0039] HPLC detection conditions for cytidine: liquid chromatograph Shimadzu 10A, chromatographic column: INERTSIL ODS-SP 5μm 4.6*250mm, mobile phase: phosphate buffer: acetonitrile = 97:3, phosphate buffer: disodium hydrogen phosphate 1.884g, sodium dihydrogen phosphate 0.726g plus water 1L, plus 30.9mL acetonitrile. Column temperature: 35 degrees, wavelength: 260nm, flow rate: 1.0mL / min.

[0040] Primer sequences required for constructing recombinant strains:

[0041] Table 2 Primer sequences used

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] Construction of recombinant plasmids: The plasmids used for knockout or integration gene construction are as follows: 1) Primers sg-ppnN-FW and pTargetF-RS were used to construct plasmid pTargetF-ppnN by whole plasmid PCR with plasmid pTargetF as template. 2) Primers pEtac-pyrE-FW and pEtac-pyrE-RS were used to construct plasmid pEtac-pyrE. 3) Primers pEtac-nudG-FW and pEtac-nudG-RS were used to construct plasmid pEtac-nudG. 4) Gene udk, sg-udk-FW and pTargetF-RS were used to construct plasmid pTargetF-udk by whole plasmid PCR with plasmid pTargetF as template. 5) For gene umpH, primers sg-umpH-FW and pTargetF-RS were used to construct plasmid pTargetF-umpH by whole plasmid PCR with plasmid pTargetF as template. 6) For gene umpG, primers sg-umpG-FW and pTargetF-RS were used to construct plasmid pTargetF-umpG by whole plasmid PCR with plasmid pTargetF as template. 7) For gene cdd, primers sg-cdd-FW and pTargetF-RS were used to construct plasmid pTargetF-cdd by whole plasmid PCR with plasmid pTargetF as template. 8) For rihB, primers sg-rihB-FW and pTargetF-RS were used to construct plasmid pTargetF-rihB by whole plasmid PCR with plasmid pTargetF as template. 9) For plasmid pEtac-pyrH, primers pEtac-pyrH-FW and pEtac-pyrH-RS were used to construct plasmid pEtac-pyrH. 10) Primers pEtac-PPP-FW and pEtac-PPP-RS were used to construct plasmid pEtac-PPP. 11) For gene poxB, primers sg-poxB-FW and pTargetF-RS were used to construct plasmid pTargetF-poxB by whole plasmid PCR with plasmid pTargetF as template. 12) For gene nrdD, primers sg-nrdD-FW and pTargetF-RS were used to construct plasmid pTargetF-nrdD by whole plasmid PCR with plasmid pTargetF as template.

[0048] Gene knockout: CRISPR Cas9 gene editing technology was used to knock out genes in the 5'-CMP metabolic pathway on the E. coli MG1655 genome, taking the ppnN gene knockout as an example. Primers were designed based on the ppnN gene sequence of E. coli MG1655 in NCBI (Table 1), and PCR amplified the knockout frame upstream homologous arm gene fragment ppnN-up and downstream homologous arm gene fragment ppnN-down. Among them, ppnN-up and ppnN-down have a 40bp overlap region. At the same time, fusion PCR was performed using ppnN-up and ppnN-down as templates to obtain the knockout frame ppnN-G1 for knocking out the ppnN gene.

[0049] Using pTargetF plasmid as template, primers sg-ppnN-L and pTarget-R were used to perform full plasmid PCR and purify the fragment. After digesting the plasmid template with restriction endonuclease DpnⅠ, it was transferred into E.coli JM109, spread on LB plates containing spectinomycin, and cultured at 37℃ for 12h. Single colonies on the plate were picked and cultured in 10mL LB medium for 8h, and plasmids were extracted and sequenced for verification.

[0050] The constructed knockout frame ppnN-G1 and plasmid pTargetF-ppnN (sgRNA plasmid) were transferred into E. coli MG1655 pCas containing plasmid and cultured at 30℃ for 2h. Spread on LB plates containing spectinomycin resistance and kanamycin resistance and cultured at 30℃. Colony PCR was performed on single colonies using primers ΔppnN-PCR-FW and ΔppnN-PCR-RS. The PCR product size of the positive clone was 1279bp. The size of the original fragment PCR product on the genome was 1579bp, indicating that the gene knockout was successful, and sequencing verification was performed at the same time.

[0051] Positive transformants were selected and cultured in LB medium containing IPTG (final concentration was 1 mM) for 2 h. 100 μL of the culture solution was spread on an LB plate containing kanamycin and cultured at 30°C to eliminate the pTargetF-ppnN plasmid. After a single colony grew out, it was transferred to an LB plate containing kanamycin and an LB plate containing spectinomycin and kanamycin at the same time. The colonies that grew on the kanamycin resistance medium but not on the spectinomycin resistance and kanamycin resistance medium were selected, which were clones that eliminated the pTargetF-ppnN plasmid.

[0052] Pick the clones that have eliminated the pTargetF-ppnN plasmid and streak them on the LB plate containing kanamycin resistance, and culture them at 30℃ for 12h. Pick a single colony and streak it on the LB plate, and culture it at 42℃ for 12h. After the single colony grows, transfer the single colony to the kanamycin LB plate and the LB plate without resistance at the same time. Select the clones that do not grow on the kanamycin resistance plate but grow on the LB plate without resistance, and name them E.coli Cr04 (pCas plasmid has been eliminated). Use the same method to obtain recombinant strains with other gene knockouts.

[0053] Olotate phosphoribosyltransferase pyrE enzyme activity assay: The reaction mixture (150 μL) contained 20 mM Tris-HCl, pH 7.6, 2 mM magnesium chloride, 100 μM PRPP, 20 μL enzyme and 50 μM orotate. The enzyme was incubated with all components except PRPP for 1 min. The reaction was started by adding 100 μM PRPP, reacted for 2 min, and the consumption of orotate was measured. Enzyme activity definition unit: The amount of orotate consumed per minute under the above reaction conditions is one enzyme activity unit (U).

[0054] Shake flask fermentation method: the strain was inoculated into LB medium, cultured at 37°C and 200 rpm for 8 h; for shake flask fermentation, 1% inoculation was inoculated into a 500 mL Erlenmeyer flask containing 100 mL LB medium, cultured at 37°C and 200 rpm for 60 h.

[0055] The fermentation medium of 30L fermenter is: glucose 12-85g / L, MgSO4 0.5-5g / L, sodium citrate 1.6-19mg / L, calcium chloride 7-560mg / L, disodium hydrogen phosphate 0.1-7.9g / L, sodium dihydrogen phosphate 0.6-10.3g / L, zinc chloride 0.7-72mg / L, yeast powder 0.3-11g / L, peptone 0.5-21g / L, copper chloride 5-145mg / L, zinc sulfate 1-9.2mg / L, sodium molybdate 1.3-165mg / L). Feed medium: glucose 350-820g / L, peptone 0.5-20g / L, yeast powder 0.5-20g / L.

[0056] Fermentation in a 30L fermenter: The seed liquid in the LB medium is transferred to the fermentation medium (30L fermenter, the initial liquid volume is 12L) according to the inoculation amount of 0.5-15%, and the fermentation culture is carried out at 35°C and 200rpm. The glucose concentration is controlled to be 15±2g / L by measuring the glucose in the fermentation liquid; the dissolved oxygen is controlled to be 25%, and when it is lower than 25%, the stirring speed, ventilation volume and tank pressure are increased. Ammonia water is used to control the pH to 6-8. After the fermentation is completed, the bacteria are centrifuged and the supernatant is taken, and the cytidine content is determined by HPLC.

[0057] Example 1: Blocking the degradation pathway of cytidine and its precursor CMP to increase cytidine accumulation level

[0058] In order to achieve the accumulation of cytidine and block the degradation of cytidine, the knockout plasmid pTargetF-cdd was constructed, and the cytidine deaminase gene cdd was knocked out in the strain E. coli MG1655 to obtain the recombinant strain E. coli Cr01. The strain was inoculated into LB medium and cultured at 37°C and 200rpm for 8h; for shake flask fermentation, 1% inoculation was inoculated into a 500mL Erlenmeyer flask containing 100mL LB medium and cultured at 37°C and 200rpm for 60h. Figure 2 As shown, the cytidine production of E. coli Cr01 can reach 60.2 mg / L (shake flask level) at 60 h of fermentation, which is significantly higher than that of the control strain E. coli MG1655. This indicates that knocking out the cytidine deaminase gene cdd blocks the pyrimidine metabolic flux from cytidine to uridine and uracil, which has a positive effect on improving cytidine accumulation.

[0059] In order to achieve the accumulation of cytidine and block the conversion pathway of cytidine to CMP, the knockout plasmid pTargetF-udk was constructed, and the uridine / cytosine nucleoside kinase gene udk of the strain E. coli Cr01 was knocked out to obtain the recombinant strain E. coli Cr02. The strain was inoculated into LB medium and cultured at 37°C and 200rpm for 8h; for shake flask fermentation, it was inoculated into a 500mL Erlenmeyer flask containing 100mL LB medium at a 1% inoculum and cultured at 37°C and 200rpm for 60h. Figure 2 As shown, the cytidine production of E. coli Cr02 can reach 110.1 mg / L (shake flask level) at 60 h of fermentation, which is significantly higher than that of the control strain E. coli Cr01. This indicates that knocking out the uridine / cytosine nucleoside kinase gene udk blocks the pyrimidine metabolic flux of cytidine to CMP, which has a positive effect on improving cytidine accumulation.

[0060] In order to achieve the accumulation of cytidine and block the conversion pathway of cytidine to CMP, the knockout plasmid pTargetF-rihB was constructed, and the pyrimidine-specific ribonucleoside hydrolase gene rihB was knocked out in the strain E. coli Cr02 to obtain the recombinant strain E. coli Cr03. Figure 2 As shown, the cytidine production of E. coli Cr03 can reach 310.6 mg / L (shake flask level) at 60 h of fermentation, which is significantly higher than that of the control strain E. coli Cr02. This indicates that knocking out the pyrimidine-specific ribonucleoside hydrolase gene rihB blocks the pyrimidine metabolic flux of cytidine to CMP, which has a positive effect on improving cytidine accumulation.

[0061] The nucleotide 5'-monophosphate nucleosidase encoded by the gene ppnN is of great significance for the efficient accumulation of cytidine in E. coli. In order to achieve the accumulation of cytidine and block the conversion pathway of cytidine to CMP, a knockout plasmid pTargetF-ppnN was constructed, and the pyrimidine-5'-nucleotide nucleosidase gene ppnN was knocked out in the strain E. coli Cr03 to obtain the recombinant strain E. coli Cr04. Figure 2 As shown, the cytidine production of E. coli Cr04 can reach 362.4 mg / L (shake flask level) at 60 h of fermentation, which is significantly higher than that of the control strain E. coli Cr03. This indicates that knocking out the pyrimidine-5'-nucleotide nucleosidase gene ppnN blocks the metabolic flux of CMP to cytosine, which has a positive effect on improving cytidine accumulation.

[0062] Example 2: Enhancing the metabolic flux of the cytidine synthesis pathway to increase its accumulation level

[0063] The recombinant strain E. coli Cr04 constructed in Example 1 was used as the starting strain, and the 5'-CTP diphosphohydrolase gene nudG was integrated and expressed at the gene site umpH on the genome to increase the metabolic flux from CTP to cytidine, thereby constructing the recombinant strain E. coli Cr05. Figure 2 As shown, the cytidine production of the recombinant strain E. coli Cr05 increased from 362.4 mg / L of the strain E. coli Cr04 to 378.2 mg / L after 60 h of shake flask fermentation. The results show that the integrated expression of the nudG gene has a positive effect on improving the accumulation of cytidine in E. coli.

[0064] In order to further improve the metabolic flux of the cytidine synthesis pathway, the production level of cytidine was increased by reducing the accumulation of orotic acid. The orotic acid phosphoribosyltransferase gene pyrE was integrated and expressed at the gene umpG site on the genome of the recombinant strain E. coli Cr05 to construct the recombinant strain E. coli Cr06. The plasmid pTargetF-umpG was constructed by full plasmid PCR using primers sg-umpG-FW and pTargetF-RS and plasmid pTargetF as a template. Primers ΔumpG::pyrE / pyrE / K26H-up-FW, ΔumpG::pyrE / pyrE / K26H-up-RS, ΔumpG::pyrE / pyrE / K26H-down-FW, and ΔumpG::pyrE / pyrE / K26H-down-RS were used to obtain the upstream and downstream homology arms pyrE-G1 and pyrE-G2 of the integration frame using the E. coli MG1655 genome as a template. Primers ΔumpG::pyrE / pyrE / K26H-mid-FW and ΔumpG::pyrE / pyrE / K26H-mid-RS were used to obtain the expression cassette pyrE-Z using plasmid pEtac-pyrE as a template. Primers ΔumpG::pyrE / pyrE / K26H-up-FW and ΔumpG::pyrE / pyrE / K26H-down-RS were used to perform fusion PCR with pyrE-G1, pyrE-G2, and pyrE-Z as templates to obtain the Donor DNA fragment. pTargetF-umpG and Donor DNA were introduced into the electroporation competent state of the recombinant strain E.coli Cr05 containing the pCAS plasmid, and the recombinant strain with the integrated pyrE gene was screened. The plasmids pCAS plasmid and pTarget plasmid were eliminated to obtain recombinant E.coli Cr06. After 60 hours of horizontal fermentation in shake flasks, the cytidine production of E.coli Cr06 reached 396.5 mg / L, which was significantly higher than that of the control strain E.coli Cr05 ( Figure 2 ). The results showed that the integrated expression of the orotate phosphoribosyltransferase gene umpG on the genome increased the metabolic flux of the cytidine synthesis pathway and further increased the production level of cytidine.

[0065] In order to further improve the metabolic flux of the cytidine synthesis pathway, the production level of cytidine was increased by reducing the accumulation of orotic acid. The software SWISS MODEL was used for homology modeling to construct the tertiary structure of pyrE. According to the bioinformatics analysis of the tertiary structure of pyrE, it was found that amino acid residues K26 and H105 formed a "basic bridge". In the study, the enzyme mutant pyrE / K26H with high enzyme activity was constructed by replacing the 26th basic amino acid Lys with the basic amino acid His with specificity and a shorter side chain. The plasmid pET28a was used to express the enzyme mutant pyrE / K26H (the enzyme cutting sites for connection were EcoRⅠ and Hind III), and the strain was inoculated into LB medium at 37℃ and 200rpm for 8h; for shake flask fermentation, it was inoculated into a 500mL triangular flask containing 100mL LB medium at a 1% inoculation rate, and cultured for 60h at 37℃ and 200rpm. The cells were collected, subjected to ultrasonic treatment, and then centrifuged to obtain the supernatant. The specific enzyme activity (0.13U / mg) of the enzyme mutant pyrE / K26H purified by nickel column was increased by 82% relative to the control enzyme pyrE (0.07U / mg). In addition, when the basic amino acid Lys at position 26 was replaced by the amino acid Gly, the specific enzyme activity (0.026U / mg) of the mutant pyrE / K26G (amino acid sequence as shown in SEQ ID NO.12; nucleotide sequence as shown in SEQ ID NO.11) was significantly reduced to 37% of the specific enzyme activity of the control enzyme pyrE. The gene umpG site on the genome of the recombinant strain E.coli Cr05 was integrated and expressed to obtain the recombinant strain E.coli Cr07. After 60h of shake flask fermentation, the cytidine production of E. coli Cr07 reached 469.3mg / L, which was significantly higher than that of the control strain E. coli Cr06 ( Figure 2 The results showed that the integrated expression of the orotate phosphoribosyltransferase gene pyrE on the genome increased the metabolic flux of the cytidine synthesis pathway and further increased the production level of cytidine.

[0066] Example 3: Enhancing the metabolic flux of the cytidine synthesis pathway to increase its accumulation level

[0067] To further improve the metabolic flux of the cytidine synthesis pathway, primers pyrH-FW and pyrH-RS were used to perform PCR with the E. coli MG1655 genome as a template to obtain the gene fragment, which was then connected to the T vector plasmid pMD-18T to construct the recombinant plasmid pMD-18T-pyrH. Plasmid pMD-18T-pyrH was used as a template and primers pyrH-R92G / D93G-FW and pyrH-R92G / D93G-RS were used to perform whole plasmid PCR to obtain the mutant gene pyrH-R92G / D93G containing the mutation sites R92G and D93G. The gene PyrH (R92G / D93G) was integrated and expressed at the gene poxB site on the E. coli Cr07 genome, and the pyruvate oxidase gene poxB was knocked out (or silenced) to enhance the accumulation of the precursor substance pyruvate, and the recombinant E. coli Cr08 was constructed. Compared with E. coli Cr07, the pyruvate oxidase gene poxB was knocked out in the genome of the recombinant strain E. coli Cr08, and the uridylate kinase mutant gene PyrH (R92G / D93G) was integrated and expressed in the genome. At the same time, the pyruvate oxidase gene poxB was knocked out (inactivated) to enhance the accumulation of the precursor substance pyruvate. The specific experimental steps are: using primers sg-poxB-FW and pTargetF-RS, plasmid pTargetF as a template, full plasmid PCR was performed to construct plasmid pTargetF-poxB. Primers ΔpoxB::PyrH(R92G / D93G)-up-FW, ΔpoxB::PyrH(R92G / D93G)-up-RS, ΔpoxB::PyrH(R92G / D93G)-down-FW, ΔpoxB::PyrH(R92G / D93G)-down-RS were used to E.coli The MG1655 genome was used as a template to obtain the upstream and downstream homology arms of the integration frame PyrH(R92G / D93G)-G1 and PyrH(R92G / D93G)-G2. Primers ΔpoxB::PyrH(R92G / D93G)-mid-FW and ΔpoxB::PyrH(R92G / D93G)-mid-RS were used to obtain the expression cassette PyrH(R92G / D93G)-Z using plasmid pEtac-PyrH(R92G / D93G) as a template. Fusion PCR was performed using primers ΔpoxB::PyrH(R92G / D93G)-up-FW and ΔpoxB::PyrH(R92G / D93G)-down-RS, and PyrH(R92G / D93G)-G1, PyrH(R92G / D93G)-G2, and PyrH(R92G / D93G)-Z as templates to obtain the Donor DNA fragment.pTargetF-poxB and Donor DNA were introduced into the electroporation competent cell of the recombinant strain E. coli Cr07 containing the pCAS plasmid, and the recombinant strain E. coli Cr08 integrated with the PyrH (R92G / D93G) gene was obtained by screening.

[0068] like Figure 2 As shown, the cytidine production of E. coli Cr08 can reach 613.1 mg / L when the shake flask is fermented for 60 hours. It can be inferred from the results that the knockout of the pyruvate oxidase gene poxB down-regulated the gene expression of enzymes related to glycolysis, reduced the carbon flux of glycolysis, inhibited the consumption of pyruvate, and promoted the synthesis of cytidine precursors such as glutamate and aspartate. The integrated expression of the uridylate kinase mutant gene PyrH (R92G / D93G) in the genome significantly weakened the feedback inhibition caused by UTP and GTP, further increasing the accumulation of cytidine in E. coli. At the same time, the accumulation of the precursor substance pyruvate was enhanced, further increasing the accumulation of cytidine in the cell.

[0069] In the same way, primers pyrH-D93A-FW and pyrH-D93A-RS were used to perform whole plasmid PCR to obtain the mutant gene pyrH-D93A (shown in SEQ ID NO.17) containing the mutation site D93A. The same method as that used to construct the recombinant strain E. coli Cr08 was used to integrate the expression gene PyrH (D93A) at the gene poxB site on the E. coli Cr07 genome, and to knock out (or silence) the pyruvate oxidase gene poxB to enhance the accumulation of the precursor substance pyruvate, thereby constructing the recombinant E. coliCr08A. Figure 2 As shown, the cytidine production of E. coli Cr08A can only reach 507.5 mg / L after 60 h of shake flask fermentation, which further indicates that the uridylate kinase mutant gene PyrH (R92G / D93G) has a greater advantage in cytidine accumulation than PyrH (D93A).

[0070] Example 4: Improving the metabolic flux of the PPP pathway and enhancing the accumulation of the precursor substance pyruvate to increase the accumulation of cytidine

[0071] The prs, zwf and gnd genes were integrated and expressed at the nrdD gene locus on the genome of strain E. coli Cr08 (the three genes were expressed in series, with an RBS sequence (AGGA) in the middle; the schematic diagram of the series is zwf-RBS-gnd-RBS-prs), which increased the carbon flux of the PPP pathway and enhanced the supply of the precursor substance PRPP. The ribonucleoside triphosphate reductase gene nrdD was knocked out to block the flux of CTP to dCTP ( Figure 3) to construct the recombinant strain E. coli Cr09. The zwf-RBS-gnd-RBS-prs gene fusion fragment construction method: using primers pEtac-Z-zwf-RBS-FW, pEtac-Z-zwf-RBS-RS, pEtac-zwf-RBS-gnd-RBS-FW, pEtac-zwf-RBS-gnd-RBS-RS, pEtac-zwf-RBS-gnd-RBS-prs-RBS-FW, pEtac-zwf-RBS-gnd-RBS-prs-RBS-RS, a one-step cloning kit was used to construct the fusion gene fragment zwf-RBS-gnd-RBS-prs. The recombinant strain construction method is: using primers sg-nrdD-FW and pTargetF-RS, using plasmid pTargetF as a template, whole plasmid PCR was performed to construct plasmid pTargetF-nrdD. Primers ΔnrdD::PPP-up-FW, ΔnrdD::PPP-up-RS, ΔnrdD::PPP-down-FW, ΔnrdD::PPP-down-RS were used to obtain the upstream and downstream homology arms PPP-G1 and PPP-G2 of the integration frame using the E. coli MG1655 genome as a template, and primers ΔnrdD::PPP-mid-FW and ΔnrdD::PPP-mid-RS were used to obtain the expression frame PPP-Z using the plasmid pEtac-zwf-RBS-gnd-RBS-prs as a template. Primers ΔnrdD::PPP-up-FW and ΔnrdD::PPP-down-RS were used to perform fusion PCR using PPP-G1, PPP-G2, and PPP-Z as templates to obtain the Donor DNA fragment. pTargetF-nrdD and Donor DNA were introduced into the electroporation competent cell of the recombinant strain E.coli Cr08 containing the pCAS plasmid, and the recombinant strains with integrated prs, zwf and gnd genes were screened. The pCAS plasmid and the pTarget plasmid were eliminated to obtain the recombinant E.coli Cr09. When the shake flask horizontal fermentation lasted for 60 hours, the cytidine production of E.coli Cr09 reached 675.0 mg / L, which was significantly higher than that of the control strain E.coli Cr08 ( Figure 2 ). The above results prove that the integration and expression of prs, zwf and gnd genes at the nrdD site on the genome increases the carbon flux of the PPP pathway, enhances the supply of the precursor substance PRPP, and blocks the flux from CTP to dCTP, further increasing the accumulation of cytidine in cells.

[0072] Similarly, when the order of tandem expression of the three genes zwf, gnd, and prs was adjusted to gnd-RBS-prs-RBS-zwf (the primers for constructing the fusion expression gene fragment included: pEtac-Z-gnd-RBS-FW, pEtac-Z-gnd-RBS-RS, pEtac-gnd-RBS-prs-FW, pEtac-gnd-RBS-prs-RS, pEtac-gnd-RBS-prs-RBS-zwf-FW, pEtac-gnd-RBS-prs-RBS-zwf-RS, and pTarget-RBS-RS), the same method as that for constructing the recombinant strain E. coli Cr09 was used to construct the recombinant strain E. coli Cr09B. Figure 2 As shown, at the shake flask level, the cytidine production of E. coli Cr09B can only reach 630.3 mg / L when fermented to 60h. This shows that the order of gene concatenation has an impact on the expression level of the gene, and this concatenation mode zwf-RBS-gnd-RBS-prs is more conducive to the accumulation of cytidine in the recombinant strain E. coli Cr09.

[0073] Example 5: Production of cytidine by fermentation of recombinant bacteria

[0074] The fermentation medium of 30L fermenter is: glucose 60g / L, MgSO4 2g / L, sodium citrate 6mg / L, calcium chloride 230mg / L, disodium hydrogen phosphate 6g / L, sodium dihydrogen phosphate 5g / L, zinc chloride 29mg / L, yeast powder 7g / L, peptone 13g / L, copper chloride 76mg / L, zinc sulfate 2mg / L, sodium molybdate 5mg / L. Feed medium: glucose 730g / L, peptone 11g / L, yeast powder 15g / L. The fermentation conditions of the 30L fermenter are as follows: the strain is inoculated into LB medium, cultured at 37°C and 200rpm for 10h, the seed liquid in the LB medium is transferred to the fermentation medium (30L fermenter, initial liquid volume is 12L) at an inoculation rate of 8%, and fermentation is carried out at 35°C and 200rpm. The glucose concentration is controlled to be 15±2g / L by measuring the glucose in the fermentation broth; the dissolved oxygen is controlled to be 25%, and when it is lower than 25%, the stirring speed, ventilation volume and tank pressure are increased. Ammonia water is used to control the pH to 6.7. After the fermentation is completed, the bacteria are centrifuged to remove the supernatant, and the cytidine content therein is determined by HPLC. When a 30L fermenter is used for high-density fermentation, E. coli Cr09 can efficiently produce cytidine after 48h of fermentation, with a yield of 61.9g / L, and the content of impurities such as uracil is very low ( Figure 4 , Figure 5 ).

[0075] Comparative Example 1:

[0076] The specific implementation method is the same as Example 2, except that when the prs, zwf and gnd genes are integrated and expressed at the nrdD gene site on the genome of strain E. coli Cr08, the RBS sequence (AGGA) is not added between the genes prs and zwf; when the RBS sequence (AGGA) is not added between the genes zwf and gnd, the tandem schematic diagram is zwf-gnd-prs (the method for constructing the fusion expression gene fragment includes: pEtac-Z-gnd-FW, pEtac-Z-gnd-RS, pEtac-gnd-prs-FW, pEtac-gnd-prs-RS, pEtac-gnd-prs-zwf-FW, pEtac-gnd-prs-zwf-RS, pTarget-RS). The cytidine production of the recombinant strain E. coli Cr09A at the shake flask level obtained by the construction is only 619.1 mg / L.

[0077] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli, wherein the cytidine deaminase gene cdd, the uridine kinase gene udk, the pyrimidine-specific ribonucleoside hydrolase gene rihB, the nucleotide 5'-monophosphate nucleosidase gene ppnN, the pyruvate oxidase gene poxB and the ribonucleoside triphosphate reductase gene nrdD on the genome are knocked out, and 5'-CTP diphosphohydrolase and / or orotate phosphoribosyltransferase are integrated and expressed.

2. The recombinant Escherichia coli according to claim 1, characterized in that The amino acid sequence of the 5'-CTP diphosphohydrolase is shown in SEQ ID NO.6; the amino acid sequence of the orotate phosphoribosyltransferase is shown in SEQ ID NO.8 or SEQ ID NO.

10.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that The genes encoding 5'-CTP diphosphohydrolase and / or orotate phosphoribosyltransferase are integrated into the umpH site.

4. The recombinant Escherichia coli according to claim 3, characterized in that The gene sequence of the 5'-CTP diphosphohydrolase is shown in SEQ ID NO.5; the gene sequence of the orotate phosphoribosyltransferase is shown in SEQ ID NO.7 or SEQ ID NO.

9.

5. The recombinant Escherichia coli according to claim 4, characterized in that A uridylate kinase mutant is integrated and expressed at the location of the pyruvate oxidase gene poxB; the uridylate kinase mutant has an amino acid sequence shown in SEQ ID NO.

14.

6. The recombinant Escherichia coli according to any one of claims 1 to 2, 4 to 5, characterized in that: The recombinant Escherichia coli integrates and expresses the phosphoribosyl pyrophosphate synthetase gene prs, the glucose-6-phosphate dehydrogenase gene zwf and the 6-phosphogluconate dehydrogenase gene gnd at the position of the ribonucleoside triphosphate reductase gene nrdD.

7. The recombinant Escherichia coli according to any one of claims 1 to 2, 4 to 5, characterized in that: The Escherichia coli is selected from: DH5α, BL21 (DE3), JM109, HB101 or MG1655.

8. The recombinant Escherichia coli according to claim 6, characterized in that The Escherichia coli is selected from: DH5α, BL21 (DE3), JM109, HB101 or MG1655.

9. A method for producing cytidine by fermentation, characterized in that: The recombinant Escherichia coli according to any one of claims 1 to 8 is fermented in a medium containing glucose.

10. The method according to claim 9, characterized in that The culture medium used for fermentation contains: glucose 12-85g / L, MgSO4 0.5-5g / L, sodium citrate 1.6-19mg / L, calcium chloride 7-560mg / L, disodium hydrogen phosphate 0.1-7.9g / L, sodium dihydrogen phosphate 0.6-10.3g / L, zinc chloride 0.7-72mg / L, yeast powder 0.3-11g / L, peptone 0.5-21g / L, copper chloride 5-145mg / L, zinc sulfate 1-9.2mg / L, sodium molybdate 1.3-165mg / L.

11. Use of the recombinant Escherichia coli according to any one of claims 1 to 8 in the fermentation production of cytidine.

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