Methods for enhancing 5'-cytidylic acid accumulation and uses thereof

Abstract

The application discloses a method for enhancing 5'-cytidylic acid accumulation and application, and belongs to the field of genetic engineering and microbial engineering. The application knocks out ppnN, ushA, yrfG, yjjG, umpH and umpG genes, overexpresses 5'-CTP diphosphohydrolase gene nudG and orotidine-5'-phosphate ribosyltransferase gene pyrE, integrates expression of uridine phosphorylase mutant gene PyrH(R92G / D93G), further knocks out cytidine deaminase gene cdd and ribonucleoside-triphosphate reductase gene nrdD, and integrates expression of uridine-cytidine kinase gene udk, phosphoribosyl pyrophosphate synthetase gene prs, glucose-6-phosphate dehydrogenase gene zwf and 6-phosphogluconate dehydrogenase gene, so that the 5'-cytidylic acid accumulation capacity of Escherichia coli is improved. Under the condition that no antibiotic and inducer are added, the 5'-cytidylic acid yield of the recombinant bacteria reaches 39.6 g / L in a 60L fermenter.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for enhancing 5'-cytidylic acid accumulation and application, belonging to the field of genetic engineering and microbial engineering. BACKGROUND

[0002] Nucleotides are mainly involved in the composition of nucleic acids and have important biological functions. Nucleotides and their derivatives can be widely used in agricultural production, food, medicine and other fields. Chemical synthesis of nucleotides usually uses phosphorus oxychloride as a phosphate donor to phosphorylate nucleosides to produce corresponding nucleotides. However, when using this chemical method to synthesize 5'-cytidylic acid, a protecting group needs to be added to the 2', 3'-hydroxyl group of ribose, and then phosphorylation reaction is carried out. The chemical synthesis of nucleotides has many operation steps, long route, poor stereoselectivity, expensive and toxic reagents, poor operability, and high production cost, which is not suitable for large-scale green production.

[0003] Ribonucleic acid (RNA) degradation can also be used to produce 5'-cytidylic acid (5'-CMP). This method can obtain four products at a time, in addition to 5'-cytidylic acid, the other three are by-products of the reaction, which need to be further separated and purified to obtain pure 5'-cytidylic acid. At the same time, as the raw material for degradation-RNA, there are many limitations, such as the source of RNA is not extensive, and the separation operation in the process of degrading RNA to produce 5'-cytidylic acid is complex, and the yield of the target product is reduced.

[0004] CN 111269870 A discloses a recombinant Escherichia coli with high yield of cytidylic acid and its application. The cytidylyl kinase gene cloned from the cytidylic acid production strain has good catalytic activity and stability after the recombinant bacteria are broken. Cytidine, adenosine triphosphate (ATP) and Mg 2+ are added, and cytidylic acid is obtained by reaction. However, this method needs to break the recombinant bacteria, then separate the enzyme, and the yield is not high. CN 202111280840.4 discloses a method for producing 5'-cytidylic acid. The ushA gene of cytidine monophosphate pyrophosphorylase and the ppnN gene of nucleotide 5'-monophosphate nucleosidase of Escherichia coli are knocked out by gene knockout technology, and the udk gene of uridine kinase is overexpressed to obtain an Escherichia coli mutant that can accumulate 5'-cytidylic acid (5'-CMP). However, the above-mentioned recombinant strain needs to add inducer IPTG and antibiotics to accumulate 5'-CMP, which is expensive and toxic to cells. At the same time, if the accumulation concentration of 5'-cytidylic acid (5'-CMP) is further improved, it will further promote its industrial production. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a recombinant strain that can produce 5'-cytidylic acid at high yield without adding inducers and antibiotics.

[0006] The present application provides a recombinant Escherichia coli, which is knocked out of nucleotide 5'-monophosphonucleoside enzyme gene ppnN, cytidine monophosphate pyrophosphorylase gene ushA, purine nucleotide enzyme gene yrfG, pyrimidine 5'-nucleotide enzyme gene yjjG, UMP phosphatase gene umpH and 5'-nucleotide enzyme gene umpG on the genome.

[0007] In an embodiment, the nucleotide sequence of the 5'-monophosphonucleoside enzyme gene ppnN is shown in SEQ ID NO. 1; the nucleotide sequence of the cytidine monophosphate pyrophosphorylase gene ushA is shown in SEQ ID NO. 2; the nucleotide sequence of the purine nucleotide enzyme gene yrfG is shown in SEQ ID NO. 3; the nucleotide sequence of the pyrimidine 5'-nucleotide enzyme gene yjjG is shown in SEQ ID NO. 4; the nucleotide sequence of the UMP phosphatase gene umpH is shown in SEQ ID NO. 5; and the nucleotide sequence of the 5'-nucleotide enzyme gene umpG is shown in SEQ ID NO. 6.

[0008] In an embodiment, the recombinant Escherichia coli further expresses 5'-CTP diphosphohydrolase; the amino acid sequence of the 5'-CTP diphosphohydrolase is shown in SEQ ID NO. 8.

[0009] In an embodiment, the recombinant Escherichia coli further expresses orotidine-5'-phosphate ribosyltransferase.

[0010] In an embodiment, the amino acid sequence of the orotidine-5'-phosphate ribosyltransferase is shown in SEQ ID NO. 10 or SEQ ID NO. 12.

[0011] In an embodiment, the recombinant Escherichia coli integrates 5'-CTP diphosphohydrolase gene nudG at the umpH site; the nucleotide sequence of the 5'-CTP diphosphohydrolase gene is shown in SEQ ID NO. 7.

[0012] In an embodiment, the recombinant Escherichia coli integrates orotidine-5'-phosphate ribosyltransferase gene pyrE at the umpG site.

[0013] In an embodiment, the recombinant Escherichia coli integrates orotidine-5'-phosphate ribosyltransferase gene pyrE at the umpG site and integrates gene PyrH(R92G / D93G) at the poxB site.

[0014] In an embodiment, the nucleotide sequence of the orotidine-5'-phosphate ribosyltransferase gene pyrE is as shown in SEQ ID NO. 9, and the nucleotide sequence of the gene PyrH(R92G / D93G) is as shown in SEQ ID NO. 11.

[0015] In an embodiment, the recombinant E. coli further has a cytosine deaminase gene cdd knocked out, and the nucleotide sequence of the gene cdd is as shown in SEQ ID NO. 15.

[0016] In an embodiment, the recombinant E. coli further integrates expression of a uridine-cytidine kinase gene udk, a ribose phosphate pyrophosphokinase gene prs, a glucose-6-phosphate dehydrogenase gene zwf, and a 6-phosphogluconate dehydrogenase gene gnd.

[0017] In an embodiment, the recombinant E. coli integrates the uridine-cytidine kinase gene udk at the rihC site.

[0018] In an embodiment, the recombinant E. coli integrates the ribose phosphate pyrophosphokinase gene prs, the glucose-6-phosphate dehydrogenase gene zwf, and the 6-phosphogluconate dehydrogenase gene gnd at the nrdD site.

[0019] In an embodiment, the nucleotide sequence of the uridine-cytidine kinase gene udk is as shown in SEQ ID NO. 18; the nucleotide sequence of the ribose phosphate pyrophosphokinase gene prs is as shown in SEQ ID NO. 20; the nucleotide sequence of the glucose-6-phosphate dehydrogenase gene zwf is as shown in SEQ ID NO. 22; and the nucleotide sequence of the 6-phosphogluconate dehydrogenase gene gnd is as shown in SEQ ID NO. 24.

[0020] In an embodiment, the nucleotide sequence of the rihC is as shown in SEQ ID NO. 16; and the nucleotide sequence of the nrdD is as shown in SEQ ID NO. 17.

[0021] In an embodiment, the E. coli is taken as a starting strain of DH5a, BL21(DE3), JM109, HB101, or MG1655.

[0022] The present application also provides a method for fermentatively producing 5'-cytidylic acid, which comprises fermenting the recombinant E. coli in a culture medium containing glucose.

[0023] In an embodiment, the fermentation process does not add any antibiotic and inducer (TPTG)

[0024] In an embodiment, the fermentation is inoculating the strain into LB medium, 37℃ and 200rpm, culturing for 6-16h; for shake flask fermentation, inoculating into 250ml flask containing 30-100ml LB medium at 0.2-10% inoculation amount, 37℃ and 200rpm, culturing for 50-80h.

[0025] In an embodiment, the fermentation is inoculating the seed liquid in LB medium at 1-12% inoculation amount into fermentation medium (60L fermenter, initial liquid volume 28L), carrying out fermentation culture at 37℃ and 200rpm, controlling the glucose concentration to be 10±2g / L by measuring the glucose in the fermentation broth; controlling the dissolved oxygen to be 30%, when the dissolved oxygen is lower than 30%, increasing the stirring speed, aeration amount and tank pressure. Ammonia is used to control the pH to be 6-8.

[0026] In an embodiment, the fermentation medium for fermentation contains: glucose 20-70g / L, ferric chloride 2-15mg / L, MgSO40.1-3g / L, sodium citrate 1-20mg / L, calcium chloride 10-500mg / L, disodium hydrogen phosphate 1-6g / L, sodium dihydrogen phosphate 1-9g / L, zinc chloride 1-60mg / L, yeast powder 0.5-12g / L, proteose peptone 0.2-15g / L, trace elements 1-30ml (containing copper chloride 2-51g / L, zinc sulfate 3-28g / L, sodium molybdate 3-50g / L).

[0027] In an embodiment, the fermentation process also carries out feeding; the feeding medium contains: glucose 400-800g / L, proteose peptone 1-16g / L, yeast powder 1-15g / L.

[0028] The application also provides the use of the recombinant E. coli in the production of 5'-cytidylic acid-containing products in the medical field.

[0029] Beneficial effects:

[0030] The present application adopts gene knockout technology to knockout gene ppnN to block the degradation pathway of 5'-CMP to cytosine, knockout genes ushA, yrfG, yjjG, umpH and umpG respectively to block the degradation pathway of 5'-CMP to cytosine, overexpress 5'-CTP diphosphatase gene nudG, express orfE to reduce the accumulation of intermediate metabolite orotidine-5'-phosphate, integrate expression of uridine kinase mutant gene PyrH(R92G / D93G) or PyrH(D93A) to weaken the rate-limiting step in the synthesis pathway of 5'-CMP, knockout cytosine deaminase gene cdd to block the metabolism of cytosine and increase the metabolic flux of cytosine to 5'-CMP, knockout ribonucleoside triphosphate reductase gene nrdD to block the flux of CTP to dCTP, integrate expression of uridine-cytidine kinase gene udk to promote the catalysis of cytosine to 5'-CMP, integrate expression of phosphoribosyl pyrophosphate synthetase gene prs, glucose-6-phosphate dehydrogenase gene zwf and 6-phosphogluconate dehydrogenase gene gnd to improve the carbon flux of the PPP pathway and enhance the supply of precursor PRPP, so that 5'-cytidine monophosphate (5'-CMP) can be produced efficiently. The yield reaches 39.6 g / L after 60 h of fermentation in a 60 L fermenter, and the intermediate metabolite is not accumulated, which is beneficial to the separation and purification of 5'-cytidine monophosphate.

[0031] The recombinant E. coli provided by the present application can ferment 5'-cytidine monophosphate in one step under the condition that glucose is used as the substrate and no inducer IPTG and antibiotic are supplemented, the concentration of 5'-cytidine monophosphate in the fermentation supernatant is high, the separation and purification are convenient, and the process is simple. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a schematic diagram of the metabolic pathway of 5'-CMP;

[0033] Figure 2 It is the production level of 5'-CMP of different recombinant strains (shake flask);

[0034] Figure 3 It is a schematic diagram of the PPP metabolic pathway;

[0035] Figure 4 It is the HPLC diagram of the production of 5'-cytidine monophosphate by the recombinant strain. DETAILED DESCRIPTION

[0036] The HPLC detection conditions of cytidine monophosphate are as follows: liquid chromatograph Shimadzu 10A, chromatographic column: INERTSIL ODS-SP 5μm 4.6*250mm, mobile phase: buffer preparation: 0.1 mol / L potassium dihydrogen phosphate aqueous solution: 0.01 mol / L tetrabutylammonium hydroxide: methanol = 95: 95: 10, then adjust the pH to 4.5 with phosphoric acid, wavelength: 276 nm, flow rate: 1.0 mL / min.

[0037] Primer sequences required to construct recombinant strains:

[0038] Primer sequences used in Table 1

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] Strains involved in the specific embodiments of Table 2:

[0046] Strain name Genotype E. coli MG1655 Wild type E. coli str. K12 substr. MG1655, starting strain E. coli MG1655 pCas E. coli MG1655, containing plasmid pCas E. coli CMP1 E. coli MG1655, ΔppnN E. coli CMP2 E. coli CMP1, ΔushA E. coli CMP3 E. coli CMP2, ΔyrfG E. coli CMP4 E. coli CMP3, ΔyjjG E. coli CMP5 E. coli CMP4, ΔumpH E. coli CMP6 E. coli CMP5, ΔumpG E. coli CMP7 E. coli CMP4, ΔumpH::nudG E. coli CMP8 E. coli CMP7, ΔumpG::pyrE E. coli CMP9 E. coli CMP8, ΔpoxB::pyrH(R92G / D93G) E. coli CMP0 E. coli CMP8, ΔpoxB::pyrH(D93A) E. coli CMP10 E. coli CMP9, Δcdd E. coli CMP11 E. coli CMP10, ΔrihC::udk E. coli CMP12 E. coli CMP11, ΔnrdD::PPP

[0047] Construction of recombinant plasmids: The plasmids used for construction of knock-out or integration of genes were constructed as follows: 1) gene ppnN, using primers sg-ppnN-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-ppnN by whole plasmid PCR. 2). Using primers pEtac-pyrE-FW and pEtac-pyrE-RS, to construct plasmid pEtac-pyrE. 3) Using primers pEtac-pyrH-FW and pEtac-pyrH-RS, to construct plasmid pEtac-pyrH. 4) Using primers pEtac-nudG-FW and pEtac-nudG-RS, to construct plasmid pEtac-nudG. 5) Using primers pEtac-udk-FW and pEtac-udk-RS, to construct plasmid pEtac-udk. 6) gene ushA, using primers sg-ushA-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-ushA by whole plasmid PCR. 7) gene yrfG, using primers sg-yrfG-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-yrfG by whole plasmid PCR. 8) gene yjjG, using primers sg-yjjG-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-yjjG by whole plasmid PCR. 9) gene umpH, using primers sg-umpH-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-umpH by whole plasmid PCR. 10) gene umpG, using primers sg-umpG-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-umpG by whole plasmid PCR. 11) gene poxB, using primers sg-poxB-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-poxB by whole plasmid PCR. 12) gene cdd, using primers sg-cdd-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-cdd by whole plasmid PCR. 13) gene rihC, using primers sg-rihC-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-rihC by whole plasmid PCR. 14) gene PPP, using primers sg-PPP-FW and pTargetF-RS, with plasmid pTargetF as template, to construct plasmid pTargetF-PPP by whole plasmid PCR.

[0048] Gene knockout: using CRISPR Cas9 gene editing technology, knockout the gene on E. coli MG1655 genome that decomposes 5'-CMP metabolic pathway, taking ppnN gene knockout as an example. According to the ppnN gene sequence of E. coli MG1655 in NCBI, primers are designed (Table 1), and the upstream homologous arm gene fragment ppnN-up and the downstream homologous arm gene fragment ppnN-down of the knockout frame are amplified by PCR. Among them, ppnN-up and ppnN-down have an overlapping region of 40 bp, and fusion PCR is carried out with ppnN-up and ppnN-down as templates to obtain the knockout frame ppnN-G1 for knocking out ppnN gene.

[0049] Using the pTargetF plasmid as a template, whole plasmid PCR is performed with primers sg-ppnN-L and pTarget-R, and the fragment is purified. After digestion of the plasmid template with restriction endonuclease Dpn I, it is transferred into E. coli JM109, and coated on LB plates containing spectinomycin and cultured at 37°C for 12 h. Single colonies on the plate are picked into 10 mL of LB medium and cultured for 8 h, and the plasmid is extracted and sequenced.

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

[0051] The positive transformant is picked and cultured in LB medium containing IPTG (final concentration is 1 mM) for 2 h, and 100 μL of culture solution is coated on LB plates containing kanamycin and cultured at 30°C to eliminate the pTargetF-ppnN plasmid. After the single colony grows, the single colony is transferred to LB plates containing kanamycin, LB plates containing spectinomycin and kanamycin, and colonies that grow on kanamycin-resistant medium but do not grow on spectinomycin-resistant and kanamycin-resistant medium are selected, which are clones that have eliminated the pTargetF-ppnN plasmid.

[0052] The clones which have eliminated pTargetF-ppnN plasmid were streaked on LB plates containing kanamycin and incubated at 30°C for 12 hours. Single colonies were streaked on LB plates and incubated at 42°C for 12 hours. After the single colonies grew, the single colonies were inoculated on kanamycin LB plates and LB plates without antibiotic at the same time. The clones which did not grow on the kanamycin LB plates but grew on the LB plates without antibiotic were selected and named as E. coli CMP1 (pCas plasmid eliminated). Other recombinant strains were obtained in the same way.

[0053] The fermentation medium in 60L fermenter was as follows: glucose 20-70 g / L, ferric chloride 2-15 mg / L, MgSO4 0.1-3 g / L, sodium citrate 1-20 mg / L, calcium chloride 10-500 mg / L, disodium hydrogen phosphate 1-6 g / L, sodium dihydrogen phosphate 1-9 g / L, zinc chloride 1-60 mg / L, yeast extract 0.5-12 g / L, proteose peptone 0.2-15 g / L, trace elements 1-30 mL.

[0054] The trace element solution was as follows: copper chloride 2-51 g / L, zinc sulfate 3-28 g / L, sodium molybdate 3-50 g / L.

[0055] The feed medium was as follows: glucose 400-800 g / L, proteose peptone 1-16 g / L, yeast extract 1-15 g / L.

[0056] Shaking flask fermentation: the strain was inoculated into LB medium and incubated at 37°C and 200 rpm for 6-16 hours; for shaking flask fermentation, the strain was inoculated into 250 mL flask containing 50 mL LB medium at an inoculation amount of 1% and incubated at 37°C and 200 rpm for 72 hours.

[0057] 60L fermenter fermentation: the seed liquid in LB medium was inoculated into the fermentation medium at an inoculation amount of 6% (the initial liquid volume in the 60L fermenter was 28L), and the fermentation culture was carried out at 37°C and 200 rpm. The glucose concentration was controlled at 10±2 g / L by measuring the glucose in the fermentation broth. The dissolved oxygen was controlled at 30%, and when it was lower than 30%, the stirring speed, aeration and tank pressure were increased. Ammonia was used to control the pH at 6.8. After the fermentation was completed, the bacteria were removed by centrifugation, and the supernatant was taken to determine the content of 5'-CMP by HPLC.

[0058] Example 1: Blocking the degradation pathway of 5'-CMP to cytosine to improve its accumulation level

[0059] The nucleotide 5'-monophosphatase encoded by the gene ppnN is important for the efficient accumulation of 5'-CMP in E. coli. After shake flask fermentation of wild type E. coli MG1655, the accumulated 5'-CMP concentration is too low to be detected by HPLC. By knocking out the gene ppnN on the genome of E. coli MG1655 through CRISPR technology, the recombinant strain E. coli CMP1 was constructed. After 72 h shake flask fermentation of the recombinant strain E. coli CMP1, the 5'-CMP yield reached 20.58 mg / L (shake flask level), which was 34.3 times higher than that of the original strain (0.6 mg / L) Figure 2

[0060] Example 2: Blocking the degradation pathway of 5'-CMP to cytidine improves its accumulation level

[0061] Using CRISPR-Cas9 gene editing technology, five key genes for 5'-CMP catabolism, ushA, yrfG, yjjG, umpH, and umpG, were knocked out to block the degradation pathway of 5'-CMP to cytidine and systematically improve its accumulation level in E. coli. Using E. coli CMP1 as the starting strain, recombinant strains E. coli CMP2, E. coli CMP3, E. coli CMP4, E. coli CMP5, and E. coli CMP6 were constructed by knocking out the above genes (Table 2). After knocking out the gene ushA in the recombinant strain E. coli CMP1, the 5'-CMP yield of E. coli CMP2 strain after 72 h shake flask fermentation increased to 106.97 mg / L, which was 80.5% higher than that of the control strain E. coli CMP1. The results showed that knocking out the gene ushA, which has 5'-nucleotidase and uridine diphosphate-sugar hydrolase activities, is important for the efficient accumulation of 5'-CMP.

[0062] Using E. coli CMP2 strain as the starting strain, the strain E. coli CMP4 obtained by knocking out the genes yrfG and yjjG had a 5'-CMP yield of 117.89 mg / L after 72 h shake flask fermentation, which was 9.3% higher than that of the control strain E. coli CMP2 Figure 2 ​). The strain E. coli CMP6 was constructed after the double knockout of genes umpH and umpG, and the yield of 5'-CMP was 133.4 mg / L, which was 24.7% higher than that of the control strain E. coli CMP2, indicating that the double knockout of genes umpH and umpG was beneficial to the accumulation of CMP. With the accumulation of gene knockout, the accumulation of 5'-CMP in the fermentation broth gradually increased, and finally the yield of CMP of the recombinant strain E. coli CMP6 was 133.4 mg / L at 72 h, which was 6.48 times higher than that of E. coli CMP1, indicating that blocking the 5'-CMP degradation pathway could significantly improve the accumulation of 5'-CMP.

[0063] Example 3: Enhancing the metabolic flux of 5'-CMP synthesis pathway to improve its accumulation level

[0064] The recombinant strain E. coli CMP6 constructed in Example 2 was used as the starting strain, and the 5'-CTP diphosphohydrolase gene nudG was integrated at the umpH site on its genome to improve the metabolic flux of CTP to 5'-CMP, and the recombinant strain E. coli CMP7 was constructed. As shown in Figure 2 , the yield of 5'-CMP of the recombinant strain E. coli CMP7 was increased to 216 mg / L after 72 h of shake flask fermentation, which was 17.1% higher than that of the strain E. coli CMP6 (133.4 mg / L), and was 17.1% higher than that of the control strain. The results showed that the integration of the nudG gene had a positive effect on the accumulation of 5'-CMP in E. coli.

[0065] To further improve the metabolic flux of 5'-CMP synthesis pathway, the production level of 5'-CMP was improved by reducing the accumulation of orotic acid. The orotate phosphoribosyltransferase gene pyrE was integrated at the umpG site on the genome of the recombinant strain E. coli CMP7, and the recombinant strain E. coli CMP8 was constructed. The yield of 5'-CMP of E. coli CMP8 was 223 mg / L after 72 h of fermentation, which was 4.7% Figure 2 ) higher than that of the control strain E. coli CMP7 (216 mg / L). Compared with the control strain E. coli CMP7, the accumulation of orotic acid in the recombinant E. coli CMP8 after integration of pyrE was reduced from 18.52 mg / L to 15.24 mg / L. The results proved that the overexpression of the orotate phosphoribosyltransferase gene pyrE on the genome reduced the accumulation of orotic acid, improved the metabolic flux of the 5'-CMP synthesis pathway, and further improved the production level of 5'-CMP.

[0066] The recombinant E. coli CMP9 was constructed by integrating and expressing the gene PyrH (R92G / D93G) at the poxB site on the genome of E. coli CMP8. The plasmid pTargetF-poxB was constructed by performing whole plasmid PCR using the plasmid pTargetF as a template and using the primers sg-poxB-FW and pTargetF-RS. The plasmid pEtac-pyrH (R92G / D93G) was constructed by performing whole plasmid PCR using the plasmid pEtac-pyrH as a template and using the primers pyrH-R92G / D93G-FW and pyrH-R92G / D93G-RS. The homologous arms G1 and G2 were obtained by performing PCR using the genome of E. coli MG1655 as a template and using the primers ΔpoxB::pyrH-up-L, ΔpoxB::pyrH-up-R, ΔpoxB::pyrH-down-L, and ΔpoxB::pyrH-down-R. The expression frame Z was obtained by performing PCR using the plasmid pEtac-pyrH (R92G / D93G) as a template and using the primers ΔpoxB::pyrH-mid-L and ΔpoxB::pyrH-mid-R. The Donor DNA fragment was obtained by performing fusion PCR using G1, G2, and Z as templates and using the primers ΔpoxB::pyrH-up-L and ΔpoxB::pyrH-down-R. The plasmid pTargetF-poxB and the Donor DNA were introduced into electrocompetent cells of the recombinant strain E. coli CMP8 containing the plasmid pCAS, and the recombinant strain in which the gene PyrH (R92G / D93G) was integrated was screened. The plasmid pCAS and the plasmid pTarget were eliminated, and the recombinant E. coli CMP9 was obtained.

[0067] Compared with E. coli CMP8, the pyruvate oxidase gene poxB on the genome of the recombinant strain E. coli CMP9 was knocked out, and after the uridine phosphorylase mutant gene PyrH (R92G / D93G) was integrated and expressed at the position, the 5’-CMP yield of E. coli CMP9 after 72 h of fermentation reached 285.1 mg / L. 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 precursor substances such as glutamic acid and aspartic acid for 5’-CMP. The integration and expression of the uridine phosphorylase mutant gene PyrH (R92G / D93G) on the genome significantly weakened the feedback inhibition caused by UTP and GTP, further improving the accumulation of 5’-CMP in E. coli.

[0068] Example 4: Blocking cytidine degradation to enhance the metabolic flux of cytidine to 5’-CMP to improve the accumulation of 5’-CMP

[0069] To improve the yield of 5'-CMP, block the degradation of cytidine, and increase the metabolic flux of cytidine to 5'-CMP, the gene cdd was knocked out in the recombinant strain E. coli CMP9 to obtain the recombinant strain E. coli CMP10. The process of knocking out the cdd gene mainly includes: using primers sg-cdd-FW and pTargetF-RS, and plasmid pTargetF as a template, to perform whole plasmid PCR to construct plasmid pTargetF-cdd. Using primers △cdd-up-FW, △cdd-up-RS, △cdd-down-FW and △cdd-down-RS, and E. coli MG1655 genome as a template to obtain the homologous arms G3' and G4' of the upper and lower flanking regions of the knockout frame. Using primers △cdd-up-FW and △cdd-down-RS, and G3' and G4' as a template to obtain Donor DNA. Introducing pTargetF-cdd and Donor DNA into the electrotransformation competent cells of the recombinant strain E. coli CMP9 containing plasmid pCAS, and screening to obtain the recombinant strain with the cdd gene knocked out. Eliminating plasmid pCAS and pTarget to obtain the recombinant E. coli CMP10. As shown in Table 1, the yield of 5'-CMP of E. coli CMP10 can reach 290 mg / L at 72 h of fermentation, which is significantly improved compared with the yield of 5'-CMP of the control strain E. coli CMP9. Figure 2

[0070] ​On the basis of the recombinant strain E. coli CMP10, the udk gene was integrated and expressed at the rihC site on the genome to obtain the recombinant strain E. coli CMP11. The process of integrating and expressing the udk gene mainly includes: using primers sg-rihC-FW and pTargetF-RS, and taking plasmid pTargetF as a template, performing whole plasmid PCR to construct plasmid pTargetF-rihC. Using primers △rihC::udk-up-FW, △rihC::udk-up-RS, △rihC::udk-down-FW, and △rihC::udk-down-RS, and taking the E. coli MG1655 genome as a template, udk-G5 and udk-G6, which are homologous arms on the integration frame, are obtained. Using primers △rihC::udk-mid-FW and △rihC::udk-mid-RS, and taking plasmid pEtac-udk as a template, udk-D, which is an expression frame, is obtained. Using primers △rihC::udk-up-FW and △rihC::udk-down-RS, and taking udk-G5, udk-G6, and udk-D as templates, fusion PCR is performed to obtain a Donor DNA fragment. pTargetF-rihC and the Donor DNA are introduced into electrotransformation competent cells of the recombinant strain E. coli CMP10 containing the pCAS plasmid, and a recombinant strain in which the udk gene is integrated and expressed at the rihC site on the genome is screened and obtained. The pCAS plasmid and the pTarget plasmid are eliminated, and the recombinant E. coli CMP11 is obtained. The 5'-CMP yield of the recombinant strain E. coli CMP11 after 72 h of fermentation can reach 356 mg / L Figure 2 ). Compared with the 5'-CMP yield of the control strain E. coli CMP10, the 5'-CMP yield is significantly improved. The results show that overexpression of uridine cytidine kinase has a positive effect on improving the metabolic flux of cytidine to 5'-CMP and improving the accumulation amount of 5'-CMP.

[0071] Example 5: Improving the metabolic flux of the PPP pathway to improve the accumulation of 5'-CMP

[0072] The prs, zwf, and gnd genes are integrated and expressed at the nrdD gene site on the genome of the strain E. coli CMP11 to improve the carbon flux of the PPP pathway and enhance the supply of the precursor PRPP, and the recombinant strain E. coli CMP12 is constructed. The 5'-CMP yield of E. coli CMP12 after 72 h of fermentation can reach 456 mg / L, which is significantly improved compared with the 5'-CMP yield of the control strain E. coli CMP11 Figure 2). The above research results prove that the integration of the expression of the prs, zwf and gnd genes at the nrdD site on the genome improves the carbon flux of the PPP pathway, enhances the supply of the precursor PRPP, and further improves the accumulation of 5'-CMP in the cell. High-density fermentation was carried out in a 60 L fermenter system under the following fermentation conditions: the seed liquid in the LB medium was inoculated into the fermentation medium at an inoculation amount of 6%, and the initial liquid volume in the 60 L fermenter was 28 L. The fermentation culture was carried out at 37°C and 200 rpm, and the glucose concentration in the fermentation broth was controlled to be 10±2 g / L by measuring the glucose. The dissolved oxygen was controlled to be 30%, and when it was lower than 30%, the stirring speed, the aeration amount and the tank pressure were increased. Ammonia was used to control the pH to be 6.8. After the fermentation was completed, the bacteria were removed by centrifugation, and the content of 5'-CMP in the supernatant was determined. The 5'-CMP can be produced efficiently at 60 h, and the yield reaches 39.6 g / L, and the contents of impurities such as uracil and guanosine are very low. Figure 4

[0073] Comparative Example 1

[0074] The specific implementation is the same as that in Example 3, except that the uridine acid kinase mutant gene PyrH(D93A) is expressed at the poxB site on the genome of E. coli CMP8. The yield of 5'-CMP of the obtained recombinant strain E. coli CMP0 is only 253 mg / L.

[0075] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.​

Claims

1. A recombinant Escherichia coli, characterized in that, Knocking out the nucleotide 5'-monophosphatase gene on the wild-type Escherichia coli MG1655 genome ppnN , the cytidine monophosphate pyrophosphatase gene ushA, , the purine nucleotidase gene yrfG, , the pyrimidine 5'-nucleotidase gene yjjG, , the UMP phosphatase gene umpH , and the 5'-nucleotidase gene umpG ; and in umpG site-integrated orfelin gene pyrE and in poxB site-integrated gene PyrH(R92G / D93G) ; the nucleotide sequence of the orfelin gene pyrE is shown in SEQ ID NO. 9; the nucleotide sequence of the gene PyrH(R92G / D93G) is shown in SEQ ID NO.

11.

2. The recombinant E. coli of claim 1, wherein, The recombinant E. coli further expresses a 5'-CTP diphosphohydrolase; the amino acid sequence of the 5'-CTP diphosphohydrolase is shown as SEQ ID NO. 8 。 3. The recombinant E. coli according to claim 1 or 2, characterized in that, The recombinant Escherichia coli further expresses orotidine-5'-phosphate ribosyltransferase; the amino acid sequence of the orotidine-5'-phosphate ribosyltransferase is shown as SEQ ID NO. 10 or SEQ ID NO.

12.

4. The recombinant E. coli of claim 2, wherein, The recombinant E. coli in umpH Site-integrated 5'-CTP diphosphatase gene nudG The nucleotide sequence of the 5'-CTP diphosphatase gene is shown as SEQ ID NO. 7 。 5. The recombinant E. coli of claim 3, wherein, The recombinant E. coli also has a cytosine deaminase gene knocked out cdd ; the nucleotide sequence of the gene cdd is shown as SEQ ID NO. 15 。 6. The recombinant E. coli of any one of claims 1-2, 4-5, wherein, The recombinant E. coli also knocks out the cytosine deaminase gene cdd ; the nucleotide sequence of the gene cdd is shown as SEQ ID NO. 15 。 7. The recombinant E. coli of any one of claims 1-2, 4-5, wherein, The recombinant E. coli also integrates expression of a uridine-cytidine kinase gene udk, a phosphoribosyl pyrophosphate synthetase gene prs a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd .

8. The recombinant E. coli of claim 3, wherein, The recombinant E. coli also integrates expression of a uridine-cytidine kinase gene udk, a phosphoribosyl pyrophosphate synthetase gene prs a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd .

9. The recombinant E. coli of claim 6, wherein, The recombinant E. coli also integrates expression of a uridine-cytidine kinase gene udk, a phosphoribosyl pyrophosphate synthetase gene prs a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd .

10. A method for fermentatively producing 5'-cytidylic acid, characterized in that the recombinant Escherichia coli according to any one of claims 1 to 9 is fermented in a culture medium containing glucose.

11. Use of the recombinant Escherichia coli according to any one of claims 1 to 9 in the medical field for producing products containing 5'-cytidylic acid.

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