Method for improving cytidine production level and application

Abstract

The invention belongs to the technical field of genetic engineering and microbial engineering, and particularly relates to a method for improving the production level of cytidine and application. According to the recombinant escherichia coli, a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd are subjected to overexpression. Specifically, the fusion expression of a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd is carried out, and the fusion expression is carried out by adopting Linkers with different properties. During application, the recombinant escherichia coli can be used for producing the cytidine by fermentation, so that the production level of the cytidine is improved. According to the invention, recombinant Escherichia coli which is over-expressed and fused with a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd is constructed, and the recombinant Escherichia coli can be used for efficiently fermenting and producing cytidine.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial engineering technology, specifically relating to a method and application for improving cytidine production levels. Background Technology

[0002] 1-β-D-furanoside cytosine, also known as cytosine nucleoside or cytidine, is a compound formed by the N-1 of cytosine and the C-1 of D-ribose linked by a β-glycosidic bond. Cytidine is present in all living organisms and is a component of RNA. Its most important industrial use is as a raw material for the chemical synthesis of antiviral and antitumor drugs. Many cytidine structural analogs interfere with cellular RNA synthesis. Based on this principle, various cytidine analogs have been developed for the treatment of viral infections and tumors, many of which are widely used as clinically effective and first-line drugs. For example, zalcitabine (2',3'-dideoxycytosine nucleoside) competitively inhibits reverse transcriptase activity and is widely used to treat HIV infection and other retroviral infections. Capecitabine (5'-deoxy-5-fluoro-N-[(pentoxy)carbonyl]cytidine), also a cytidine analog, is used clinically to treat various cancers, including colorectal cancer, gastric cancer, liver cancer, and breast cancer. These cytidine structural analogs are important clinical drugs, all produced through chemical synthesis from cytidine. Therefore, cytidine is an important pharmaceutical intermediate.

[0003] Currently, there are three main industrial methods for producing cytidine: RNA hydrolysis, chemical synthesis, and microbial fermentation. While RNA hydrolysis offers advantages such as simplicity, ease of operation, and control, it suffers from high raw material requirements, complex processes, low product yield, and excessively high production costs, and has been largely superseded by other methods. Dozens of chemical synthesis technologies for cytidine production have been developed. The latest process uses bis(p-nitrophenol phosphate) as a catalyst to catalyze the reaction of N4-acetylcytosine and tetra-O-acetyl-β-D-furanose, followed by deacetylation to produce cytidine. The advancements in chemical synthesis have significantly reduced the price of cytidine. However, the inherent limitations of chemical synthesis—long reaction routes, demanding reaction conditions, expensive catalysts, numerous byproducts, and potential environmental pollution—make further cost reduction nearly impossible. These cost constraints limit its application in the pharmaceutical field and other uses.

[0004] Microbial fermentation for cytidine production includes precursor-added fermentation and direct fermentation. Precursor-added fermentation primarily utilizes the salvage pathway of cytidine synthesis. This pathway involves the addition of uracil to the culture medium and the reaction of 5-phosphate ribose pyrophosphate (PRPP) within the microbial cells, catalyzed by uracil ribose phosphoryltransferase (pyrR / upp), directly generating UMP. Cytidine then proceeds through UDP, UTP, and CTP to form cytidine. Direct fermentation of cytidine primarily utilizes the "from-situ" generation of UMP by microorganisms, followed by UDP, UTP, and CTP to produce cytidine. The salvage pathway requires uracil as a fermentation precursor, resulting in high raw material costs and low production efficiency, which is not a viable technological direction. In contrast, direct fermentation can utilize microbial genetic breeding technology to select high-yield cytidine-producing fermentation strains. Using glucose as the main raw material, under mild conditions and through a relatively simple process, high-quality cytidine can be produced. Direct fermentation can further reduce cytidine production costs, is relatively environmentally friendly, and has significant potential for technological improvement.

[0005] Patent CN106754602A discloses a recombinant microorganism for producing cytidine and a method for producing cytidine. The method involves knocking out cytidine deaminase, ribonucleoside hydrolase, cytidine / uridine kinase, and nucleoside transporter, while overexpressing cytidine triphosphate pyrophosphorylase and cytidine monophosphate phosphorylase to relieve feedback inhibition of the pyrimidine nucleoside pathway. This resulted in the construction of a recombinant *E. coli* strain that can achieve a cytidine production level of 20 g / L in a 5L fermenter. However, the strain constructed using this method requires the addition of antibiotics during fermentation, and the inducing agent IPTG is needed for cytidine production, with low yields.

[0006] In our previously published patent CN116555139B, we disclosed a method and application for enhancing cytidine production. The strain we constructed achieved a cytidine yield of 61.9 g / L in a 30 L fermenter. Further increasing the accumulation concentration of cytidine will further promote its industrial production and reduce its production costs. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by providing a method and application for improving cytidine production. In this invention, different linkers—KKAAEK, AEKKAAEKKA, SGGGSSGGG, GGGSSG, and GGGGG—are used to fuse and express the glucose-6-phosphate dehydrogenase gene. zwf and 6-phosphoglucose dehydrogenase gene gnd The sequence of these steps was optimized. This strategy significantly improved the synthesis of cytidine precursors (promoting the conversion of glucose-6-phosphate to ribulose-5-phosphate), facilitated the synthesis of the precursor PRPP, and further increased the yield of the target product cytidine.

[0008] The technical solution of this invention is as follows: Firstly, a method for improving cytidine production levels is provided, which uses recombinant Escherichia coli fermentation to produce cytidine.

[0009] In one embodiment, recombinant Escherichia coli is fermented in a glucose-containing medium. Fermentation is carried out by inoculating the recombinant Escherichia coli strain into the medium and culturing at 35-40°C and 100-300 rpm for 5-15 hours. For shake flask fermentation, 0.1-10% of the inoculum is inoculated into an Erlenmeyer flask containing 50-150 mL of LB medium and culturing at 35-40°C and 100-300 rpm for 40-75 hours.

[0010] In one embodiment, the culture medium used 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 extract 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.

[0011] In one embodiment, recombinant Escherichia coli is fermented in a glucose-containing medium. Fermentation is carried out by transferring the seed culture in the medium to the fermentation medium at an inoculation rate of 0.5-15%, and fermenting at 35-40°C and 100-300 rpm. During fermentation, the glucose concentration is controlled at 13-17 g / L and the dissolved oxygen is controlled at 20-30%. When the dissolved oxygen is lower than 25%, the stirring speed, aeration rate and tank pressure are increased. Ammonia water is used to control the pH at 6-8 during the fermentation process.

[0012] Furthermore, the fermentation process also involves feeding; the feeding medium contains: glucose 350-820 g / L, peptone 0.5-20 g / L, and yeast extract 0.5-20 g / L.

[0013] Secondly, a recombinant *E. coli* strain that can be applied to the aforementioned method for improving cytidine production overexpresses the glucose-6-phosphate dehydrogenase gene. zwf and 6-phosphoglucose dehydrogenase gene gnd .

[0014] In one embodiment, the glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd The expression is fused using Linkers of different properties.

[0015] In one implementation, a fusion expression is performed using linkers with different properties, including KKAAEK, AEKKAAEKKA, SGGGSSGGG, GGGSSG, and GGGGG.

[0016] In one embodiment, the glucose-6-phosphate dehydrogenase gene zwf The nucleotide sequence is shown in SEQ ID NO.1, glucose-6-phosphate dehydrogenase gene. zwf The amino acid sequence is shown in SEQ ID NO.2; 6-phosphoglucose dehydrogenase gene gnd The nucleotide sequence is shown in SEQ ID NO.3, 6-phosphoglucose dehydrogenase gene. gnd The amino acid sequence is shown in SEQ ID NO.4.

[0017] In one embodiment, the nucleotide sequences of KKAAEK, AEKKAAEKKA, SGGGSSGGG, GGGSSG, and GGGGG are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

[0018] In one embodiment, the glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd The fusion expression sequence is glucose-6-phosphate dehydrogenase gene zwf Previously, the 6-phosphoglucose dehydrogenase gene gnd The Linker is in the middle, with the Linker at the back.

[0019] More specifically, a method for preparing recombinant Escherichia coli includes the following steps: Using the Escherichia coli MG1655 genome as a template, the zwf and gnd gene fragments were amplified by PCR, and fusion PCR was used to link zwf, linker, and gnd into a fusion gene. The fusion gene was cloned into the expression plasmid pEtac to obtain the intermediate plasmid. PCR amplification of the complete expression cassette from the intermediate plasmid The expression cassette was connected to the upstream and downstream homologous arms of the ilvG gene by fusion PCR to form a fusion expression cassette; Construct the sgRNA plasmid pTargetF-ilvG targeting the ilvG site; Transform the pCas plasmid into the host bacteria E. coli Electrocompetent cells were prepared in Cr09. pTargetF-ilvG and the fusion expression cassette were co-electrotransduced into competent cells; Positive clones were screened and validated. pTargetF-ilvG was eliminated by IPTG induction, followed by elimination of pCas plasmid to obtain plasmid-free recombinant E. coli.

[0020] Compared with the prior art, the beneficial effects of the present invention are: This invention utilizes metabolic engineering techniques to construct a gene that overexpresses a fusion glucose-6-phosphate dehydrogenase. zwf and 6-phosphoglucose dehydrogenase gene gnd Recombinant Escherichia coli can be used for efficient fermentation to produce cytidine.

[0021] The glucose-6-phosphate dehydrogenase gene was expressed by fusing different linkers: KKAAEK, AEKKAAEKKA, SGGGSSGGG, GGGSSG, and GGGGG. zwf and 6-phosphoglucose dehydrogenase gene gnd The sequence of the steps was optimized. This strategy significantly improved the synthesis of cytidine precursors (promoting the conversion of glucose-6-phosphate to ribulose-5-phosphate) by enhancing key steps in the pentose phosphate pathway (PPP), thereby increasing the supply of the precursor PRPP and directly promoting cytidine biosynthesis.

[0022] This invention uses CRISPR-Cas9 technology to stably integrate fusion expression cassettes into the genome. The constructed engineered bacteria do not require the addition of IPTG inducers or antibiotics during fermentation to maintain plasmid stability, reducing raw material costs and simplifying subsequent product separation and purification processes.

[0023] The optimal strain of this invention in a 30L fermenter E. coli The cytidine yield of Cr10fd-3 can reach 82.1 g / L. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0025] In the attached diagram: Figure 1 This is a schematic diagram of the cytidine metabolic pathway; Figure 2 This is a schematic diagram illustrating the fusion process; Figure 3 Cytidine production levels (shake flask) of different recombinant strains. Figure 4 Recombinant strain E. coliCytosine production curve of Cr10fd-3 (30L fermenter); Figure 5 Recombinant strain E. coli HPLC chromatogram of cytidine produced by Cr10fd-3 (CR, cytidine). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Unless otherwise specified, the experimental methods used below are all conventional methods. Unless otherwise specified, the equipment, materials, reagents, etc. used can all be obtained commercially. The principles or mechanisms of equipment that can be purchased commercially are well known, so they will not be elaborated further.

[0028] Special note: Some materials and testing information are as follows: glucose-6-phosphate dehydrogenase gene zwf The nucleotide sequence is shown in SEQ ID NO.1, glucose-6-phosphate dehydrogenase gene. zwf The amino acid sequence is shown in SEQ ID NO.2; 6-phosphoglucose dehydrogenase gene gnd The nucleotide sequence is shown in SEQ ID NO.3, 6-phosphoglucose dehydrogenase gene. gnd The amino acid sequence is shown in SEQ ID NO.4.

[0029] The nucleotide sequences of KKAAEK (Linker1), AEKKAAEKKA (Linker2), SGGGSSGGG (Linker3), GGGSSG (Linker4), and GGGGG (Linker5) are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

[0030] Table 1. Strains involved in the specific implementation methods.

[0031] HPLC detection conditions for cytidine: Shimadzu 10A liquid chromatograph.

[0032] Column: INERTSIL ODS-SP 5 μm 4.6×250 mm.

[0033] Mobile phase: phosphate buffer: acetonitrile = 97:3, wherein phosphate buffer consists of 1.884 g disodium hydrogen phosphate, 0.726 g sodium dihydrogen phosphate, 1 L water, and 30.9 mL acetonitrile.

[0034] Column temperature: 35℃, wavelength: 260nm, flow rate: 1.0mL / min.

[0035] Primer sequences required for constructing recombinant strains: Table 2 Primer sequences used

[0036] Among them, the integration site is ilvG The plasmid used for the integrated expression of the key enzyme is pEtac, and the sgRNA is GTCACCGGAGATACAGACAA.

[0037] The ligase used for enzyme ligation is T4 DNA ligase.

[0038] Example 1 Construction of a fusion expression cassette for glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd Constructing a fusion expression box using Linker1:KKAAEK Using the Escherichia coli MG1655 genome as a template, primers were used. zwf -FW and zwf -RS1 was used for PCR amplification to obtain gene fragments. zwf Using the Escherichia coli MG1655 genome as a template, primers were used... gnd -FW1 and gnd Gene fragments were obtained by PCR amplification using RS. gnd Using primers zwf -FW and gnd -RS, add the obtained gene fragment zwf and gnd As a template, fusion PCR amplification was performed to obtain the gene fragment. zwf -Linker1- gnd Using restriction endonucleases EcoR I and Xho I respectively targeted the plasmid pEtac and the gene fragment. zwf -Linker1- gnd Enzyme digestion and purification of the digestion products were performed, and the digested plasmid pEtac and gene fragments were separated. zwf -Linker1- gnd Enzyme ligation was performed to obtain plasmid pEtac-P tac - zwf -Linker1- gnd- T.

[0039] With plasmid pEtac-P tac - zwf -Linker1- gnd- T is the template, and primers are used. zwf-gnd- FW and zwf-gnd- RS was used for PCR amplification to obtain gene fragment P. tac - zwf -Linker1- gnd- T; Using the Escherichia coli MG1655 genome as a template, primer Δ ilvG- up-FW and Δ ilvG- Gene fragments were obtained by up-RS PCR amplification. ilvG Upstream homologous arm; using the E. coli MG1655 genome as a template, primer Δ ilvG- down-FW and Δ ilvG- Down-RS PCR amplification was performed to obtain gene fragments. ilvG Downstream homologous arm; using primer Δ ilvG- up-FW and Δ ilvG- down-RS, add the obtained gene fragment P tac - zwf -Linker1- gnd- T, ilvG Upstream homologous arm and ilvG Using the downstream homologous arm as a template, fusion PCR amplification was performed to obtain the glucose-6-phosphate dehydrogenase gene. zwf and 6-phosphoglucose dehydrogenase gene gnd The fusion expression box 1 (Linker1: KKAAEK).

[0040] Using Linker2: AEKKAAEKKA to construct a fusion expression box Using the Escherichia coli MG1655 genome as a template, primers were used. zwf -FW and zwf -RS2 was used for PCR amplification to obtain gene fragments. zwf Using the Escherichia coli MG1655 genome as a template, primers were used... gnd -FW2 and gnd Gene fragments were obtained by PCR amplification using RS. gnd Using primers zwf -FW and gnd -RS, add the obtained gene fragment zwf and gnd As a template, fusion PCR amplification was performed to obtain the gene fragment. zwf -Linker2- gnd Using restriction endonucleases EcoR I andXho I respectively targeted the plasmid pEtac and the gene fragment. zwf -Linker2- gnd Enzyme digestion and purification of the digestion products were performed, and the digested plasmid pEtac and gene fragments were separated. zwf -Linker2- gnd Enzyme ligation was performed to obtain plasmid pEtac-P tac - zwf -Linker2- gnd- T.

[0041] With plasmid pEtac-P tac - zwf -Linker2- gnd- T is the template, and primers are used. zwf-gnd- FW and zwf-gnd- RS was used for PCR amplification to obtain gene fragment P. tac - zwf -Linker2- gnd- T; Using the Escherichia coli MG1655 genome as a template, primer Δ ilvG- up-FW and Δ ilvG- Gene fragments were obtained by up-RS PCR amplification. ilvG Upstream homologous arm; using the E. coli MG1655 genome as a template, primer Δ ilvG- down-FW and Δ ilvG- Down-RS PCR amplification was performed to obtain gene fragments. ilvG Downstream homologous arm; using primer Δ ilvG- up-FW and Δ ilvG- down-RS, add the obtained gene fragment P tac - zwf -Linker2- gnd- T, ilvG Upstream homologous arm and ilvG Using the downstream homologous arm as a template, fusion PCR amplification was performed to obtain the glucose-6-phosphate dehydrogenase gene. zwf and 6-phosphoglucose dehydrogenase gene gnd The fusion expression box 2 (Linker2: AEKKAAEKKA).

[0042] Using Linker3:SGGGSSGGG to construct a fusion expression box Using the Escherichia coli MG1655 genome as a template, primers were used. zwf -FW and zwf Gene fragments were obtained by PCR amplification using RS3. zwf Using the Escherichia coli MG1655 genome as a template, primers were used... gnd -FW3 andgnd Gene fragments were obtained by PCR amplification using RS. gnd Using primers zwf -FW and gnd -RS, add the obtained gene fragment zwf and gnd As a template, fusion PCR amplification was performed to obtain the gene fragment. zwf -Linker3- gnd Using restriction endonucleases EcoR I and Xho I respectively targeted the plasmid pEtac and the gene fragment. zwf -Linker3- gnd Enzyme digestion and purification of the digestion products were performed, and the digested plasmid pEtac and gene fragments were separated. zwf -Linker3- gnd Enzyme ligation was performed to obtain plasmid pEtac-P tac - zwf -Linker3- gnd- T.

[0043] With plasmid pEtac-P tac - zwf -Linker3- gnd- T is the template, and primers are used. zwf-gnd- FW and zwf-gnd- RS was used for PCR amplification to obtain gene fragment P. tac - zwf -Linker3- gnd- T; Using the Escherichia coli MG1655 genome as a template, primer Δ ilvG- up-FW and Δ ilvG- Gene fragments were obtained by up-RS PCR amplification. ilvG Upstream homologous arm; using the E. coli MG1655 genome as a template, primer Δ ilvG- down-FW and Δ ilvG- Down-RS PCR amplification was performed to obtain gene fragments. ilvG Downstream homologous arm; using primer Δ ilvG- up-FW and Δ ilvG- down-RS, add the obtained gene fragment P tac - zwf -Linker3- gnd- T, ilvG Upstream homologous arm and ilvG Using the downstream homologous arm as a template, fusion PCR amplification was performed to obtain the glucose-6-phosphate dehydrogenase gene. zwf and 6-phosphoglucose dehydrogenase gene gndThe fusion expression box 3 (Linker3: SGGGSSGGG).

[0044] Using Linker4:GGGSSG to build a fused expression box Using the Escherichia coli MG1655 genome as a template, primers were used. zwf -FW and zwf Gene fragments were obtained by PCR amplification using RS4. zwf Using the Escherichia coli MG1655 genome as a template, primers were used... gnd -FW4 and gnd Gene fragments were obtained by PCR amplification using RS. gnd Using primers zwf -FW and gnd -RS, add the obtained gene fragment zwf and gnd As a template, fusion PCR amplification was performed to obtain the gene fragment. zwf -Linker4- gnd Using restriction endonucleases EcoR I and Xho I respectively targeted the plasmid pEtac and the gene fragment. zwf -Linker4- gnd Enzyme digestion and purification of the digestion products were performed, and the digested plasmid pEtac and gene fragments were separated. zwf -Linker4- gnd Enzyme ligation was performed to obtain plasmid pEtac-P tac - zwf -Linker4- gnd- T.

[0045] With plasmid pEtac-P tac - zwf -Linker4- gnd- T is the template, and primers are used. zwf-gnd- FW and zwf-gnd- RS was used for PCR amplification to obtain gene fragment P. tac - zwf -Linker4- gnd- T; Using the Escherichia coli MG1655 genome as a template, primer Δ ilvG- up-FW and Δ ilvG- Gene fragments were obtained by up-RS PCR amplification. ilvG Upstream homologous arm; using the E. coli MG1655 genome as a template, primer Δ [[ID=18l]]ilvG- down-FW and Δ ilvG- Down-RS PCR amplification was performed to obtain gene fragments. ilvG Downstream homologous arm; using primer Δ ilvG-up-FW and Δ ilvG- down-RS, add the obtained gene fragment P tac - zwf -Linker4- gnd- T, ilvG Upstream homologous arm and ilvG Using the downstream homologous arm as a template, fusion PCR amplification was performed to obtain the glucose-6-phosphate dehydrogenase gene. zwf and 6-phosphoglucose dehydrogenase gene gnd The fusion expression box 4 (Linker4: GGGSSG).

[0046] Using Linker5: GGGGG to build a fusion expression box Using the Escherichia coli MG1655 genome as a template, primers were used. zwf -FW and zwf Gene fragments were obtained by PCR amplification using RS5. zwf Using the Escherichia coli MG1655 genome as a template, primers were used... gnd -FW5 and gnd Gene fragments were obtained by PCR amplification using RS. gnd Using primers zwf -FW and gnd -RS, add the obtained gene fragment zwf and gnd As a template, fusion PCR amplification was performed to obtain the gene fragment. zwf -Linker5- gnd Using restriction endonucleases EcoR I and Xho I respectively targeted the plasmid pEtac and the gene fragment. zwf -Linker5- gnd Enzyme digestion and purification of the digestion products were performed, and the digested plasmid pEtac and gene fragments were separated. zwf -Linker5- gnd Enzyme ligation was performed to obtain plasmid pEtac-P tac - zwf -Linker5- gnd- T.

[0047] With plasmid pEtac-P tac - zwf -Linker5- gnd- T is the template, and primers are used. zwf-gnd- FW and zwf-gnd- RS was used for PCR amplification to obtain gene fragment P. tac - zwf -Linker5- gnd-T; Using the Escherichia coli MG1655 genome as a template, primer Δ ilvG- up-FW and Δ ilvG- Gene fragments were obtained by up-RS PCR amplification. ilvG Upstream homologous arm; using the E. coli MG1655 genome as a template, primer Δ ilvG- down-FW and Δ ilvG- Down-RS PCR amplification was performed to obtain gene fragments. ilvG Downstream homologous arm; using primer Δ ilvG- up-FW and Δ ilvG- down-RS, add the obtained gene fragment P tac - zwf -Linker5- gnd- T, ilvG Upstream homologous arm and ilvG Using the downstream homologous arm as a template, fusion PCR amplification was performed to obtain the glucose-6-phosphate dehydrogenase gene. zwf and 6-phosphoglucose dehydrogenase gene gnd The fusion expression box 5 (Linker5: GGGGG).

[0048] Example 2: Contains the glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd Construction of recombinant bacteria with fusion expression cassette Using plasmid pTargetF as a template, primers were used. sg-ilvG -FW and sg-ilvG -RS was used for PCR amplification to obtain the linearized plasmid pTargetF- ilvG The PCR product was digested with restriction endonucleases to remove the original template, and then transformed into E. coli JM109 competent cells. The cells were plated on LB agar plates containing 100 mg / L spectinomycin and incubated at 37°C for 12 h. Transformants were selected, and plasmids were extracted to obtain the desired plasmid pTargetF-. ilvG .

[0049] Transform pCas plasmid into E. coli Cr09 competent cells were spread onto LB agar plates containing 50 mg / L kanamycin resistance and incubated at 30°C for 12 h. Transformants were selected and inoculated into 20 mL of liquid LB medium containing 50 mg / L kanamycin resistance, and incubated at 30°C for 12 h to prepare seed culture. The seed culture was then inoculated at a 1% (v:v) inoculation rate into 50 mL of liquid LB medium containing 50 mg / L kanamycin resistance and incubated at 30°C and 200 rpm until OD500 was reached. 600=0.2-0.4, add 1 mL of 30 mM L-arabinose, and continue culturing at 30℃ and 200 rpm until OD. 600 =0.6-0.8, place the bacterial culture on ice for 30 min. Centrifuge the bacterial culture at 4℃, 5000 rpm for 5 min. Discard the supernatant and resuspend in 30 mL of ice-cold deionized water, centrifuge at 4℃, 5000 rpm for 5 min, repeat this operation 3 times. Resuspend in 600 μL of 10% glycerol to obtain... E. coli Cr09 pCas electrocompetent state.

[0050] Recombinant bacteria E. coli Construction of Cr10fd-1 Plasmid pTargetF- ilvG and glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd The fusion expression box 1 (Linker1: KKAAEK) is electrically transferred. E. coli Cr09 pCas electrotransfer competent cells were plated on LB agar plates containing 50 mg / L kanamycin resistance and 100 mg / L spectinomycin resistance, and incubated at 30°C until single colonies appeared. Single colonies were selected for primer transfer. ilvG -PCR-FW and ilvG -PCR-RS was used to verify colony PCR and select positive single colonies. Each selected single colony was inoculated into 1 mL of liquid LB medium, and 10 μL of 100 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added. The culture was incubated at 30°C and 200 rpm for 2 h to eliminate plasmid pTargetF-. ilvG Then eliminate plasmid pTargetF- ilvG The recombinant bacteria were cultured at 42°C to eliminate the pCas plasmid, resulting in the recombinant strain. E. coli Cr10fd-1.

[0051] Recombinant bacteria E. coli Construction of Cr10fd-2 Plasmid pTargetF- ilvG and glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd Linker2 (AEKKAAEKKA) is transferred into the fusion expression box 2. E. coli Cr09 pCas electrotransfer competent cells were plated on LB agar plates containing 50 mg / L kanamycin resistance and 100 mg / L spectinomycin resistance, and incubated at 30°C until single colonies appeared. Single colonies were selected for primer transfer. ilvG -PCR-FW and ilvG-PCR-RS was used to verify colony PCR and select positive single colonies. Each selected single colony was inoculated into 1 mL of liquid LB medium, and 10 μL of 100 mM IPTG was added. The culture was incubated at 30°C and 200 rpm for 2 h to eliminate plasmid pTargetF-. ilvG Then eliminate plasmid pTargetF- ilvG The recombinant bacteria were cultured at 42°C to eliminate the pCas plasmid, resulting in the recombinant strain. E. coli Cr10fd-2.

[0052] Recombinant bacteria E. coli Construction of Cr10fd-3 Plasmid pTargetF- ilvG and glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd Linker3 (SGGGSSGGG) is transferred into the fusion expression box 3. E. coli Cr09 pCas electrotransfer competent cells were plated on LB agar plates containing 50 mg / L kanamycin resistance and 100 mg / L spectinomycin resistance, and incubated at 30°C until single colonies appeared. Single colonies were selected for primer transfer. ilvG -PCR-FW and ilvG -PCR-RS was used to verify colony PCR and select positive single colonies. Each selected single colony was inoculated into 1 mL of liquid LB medium, and 10 μL of 100 mM IPTG was added. The culture was incubated at 30°C and 200 rpm for 2 h to eliminate plasmid pTargetF-. ilvG Then eliminate plasmid pTargetF- ilvG The recombinant bacteria were cultured at 42°C to eliminate the pCas plasmid, resulting in the recombinant strain. E. coli Cr10fd-3.

[0053] Recombinant bacteria E. coli Construction of Cr10fd-4 Plasmid pTargetF- ilvG and glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd Linker4 (GGGSSG) is electrically transferred into the fusion expression box. E. coli Cr09 pCas electrotransfer competent cells were plated on LB agar plates containing 50 mg / L kanamycin resistance and 100 mg / L spectinomycin resistance, and incubated at 30°C until single colonies appeared. Single colonies were selected for primer transfer. ilvG -PCR-FW and ilvG-PCR-RS was used to verify colony PCR and select positive single colonies. Each selected single colony was inoculated into 1 mL of liquid LB medium, and 10 μL of 100 mM IPTG was added. The culture was incubated at 30°C and 200 rpm for 2 h to eliminate plasmid pTargetF-. ilvG Then eliminate plasmid pTargetF- ilvG The recombinant bacteria were cultured at 42°C to eliminate the pCas plasmid, resulting in the recombinant strain. E. coli Cr10fd-4.

[0054] Recombinant bacteria E. coli Construction of Cr10fd-5 Plasmid pTargetF- ilvG and glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd Linker5 (GGGGG) is the fusion expression box 5 (linker5: GGGGG) for electrical transfer. E. coli Cr09 pCas electrotransfer competent cells were plated on LB agar plates containing 50 mg / L kanamycin resistance and 100 mg / L spectinomycin resistance, and incubated at 30°C until single colonies appeared. Single colonies were selected for primer transfer. ilvG -PCR-FW and ilvG -PCR-RS was used to verify colony PCR and select positive single colonies. Each selected single colony was inoculated into 1 mL of liquid LB medium, and 10 μL of 100 mM IPTG was added. The culture was incubated at 30°C and 200 rpm for 2 h to eliminate plasmid pTargetF-. ilvG Then eliminate plasmid pTargetF- ilvG The recombinant bacteria were cultured at 42°C to eliminate the pCas plasmid, resulting in the recombinant strain. E. coli Cr10fd-5.

[0055] Example 3: Production of cytidine by recombinant bacteria through fermentation The recombinant cytidine-producing strain constructed above was inoculated into LB medium and cultured at 37°C and 200 rpm for 8 h. For shake-flask fermentation, 1% inoculum was added to 500 mL Erlenmeyer flasks containing 100 mL of LB medium and cultured at 37°C and 200 rpm for 60 h. After 60 h of horizontal shake-flask fermentation... E. coli Cr10fd-1 E. coli Cr10fd-2 E. coli Cr10fd-3 、E. coli Cr10fd-4 E. coli The cytidine yields of Cr10fd-5 reached 0.82 g / L, 0.87 g / L, 1.03 g / L, 0.59 g / L, and 0.51 g / L, respectively. However, the original strain... E. coliThe cytidine production of Cr09 was only 0.68 g / L. This is compared to the control strain. E. coli Cr09, E. coli Cr10fd-1 E. coli Cr10fd-2 E. coli Cr10fd-3 showed a significant increase in cytidine production (refer to...). Figure 3 The above results demonstrate that expressing the glucose-6-phosphate dehydrogenase gene using linker (KKAAEK, AEKKAAEKKA, SGGGSSGGG) fusion is effective. zwf and 6-phosphoglucose dehydrogenase gene gnd This enhanced the supply of the precursor PRPP, further increasing the accumulation of cytidine in cells.

[0056] The fermentation medium for the 30L fermenter consisted of: 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 extract 7g / L, peptone 13g / L, copper chloride 76mg / L, zinc sulfate 2mg / L, and sodium molybdate 5mg / L. The fed-batch medium consisted of: glucose 730g / L, peptone 11g / L, and yeast extract 15g / L. The fermentation conditions in the 30L fermenter were as follows: the strain was inoculated into LB medium and cultured at 37℃ and 200 rpm for 10 hours. The seed culture from the LB medium was then transferred to the fermentation medium (30L fermenter, initial volume 12L) at an 8% inoculation rate, and fermented at 35℃ and 200 rpm. The glucose concentration was controlled to be 15±2 g / L by measuring the glucose in the fermentation broth; dissolved oxygen was controlled to 25%; if it fell below 25%, the stirring speed, aeration rate, and tank pressure were increased. Ammonia was used to control the pH at 6.7. After fermentation, the bacterial cells were removed by centrifugation, and the supernatant was collected. The cytidine content was determined by HPLC. When using a 30L fermenter for high-density fermentation, after 48 hours of fermentation… E. coli Cr10fd-3 can efficiently produce cytidine, with a yield of 82.1 g / L, and the content of impurities such as uracil is very low (refer to...). Figure 4 , Figure 5 ).

[0057] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A recombinant Escherichia coli, characterized in that, Overexpression of glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd .

2. The recombinant Escherichia coli according to claim 1, characterized in that, glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd The expression is fused using Linkers of different properties.

3. The recombinant Escherichia coli according to claim 1, characterized in that, The fusion representation is achieved using linkers with different properties, including KKAAEK, AEKKAAEKKA, SGGGSSGGG, GGGSSG, and GGGGG.

4. The recombinant Escherichia coli according to claim 1, characterized in that, glucose-6-phosphate dehydrogenase gene zwf The nucleotide sequence is shown in SEQ ID NO.1, glucose-6-phosphate dehydrogenase gene. zwf The amino acid sequence is shown in SEQ ID NO.2; 6-phosphoglucose dehydrogenase gene gnd The nucleotide sequence is shown in SEQ ID NO.3, 6-phosphoglucose dehydrogenase gene. gnd The amino acid sequence is shown in SEQ ID NO.

4.

5. The recombinant Escherichia coli according to claim 3, characterized in that, The nucleotide sequences of KKAAEK, AEKKAAEKKA, SGGGSSGGG, GGGSSG, and GGGGG are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

6. The recombinant Escherichia coli according to claim 2, characterized in that, glucose-6-phosphate dehydrogenase gene zwf and 6-phosphoglucose dehydrogenase gene gnd The fusion expression sequence is glucose-6-phosphate dehydrogenase gene zwf Previously, the 6-phosphoglucose dehydrogenase gene gnd The Linker is in the middle, with the Linker at the back.

7. A method for improving cytidine production levels, characterized in that, Cytidine is produced by fermentation using the recombinant Escherichia coli according to any one of claims 1-6.

8. The method for improving cytidine production level according to claim 7, characterized in that, Recombinant Escherichia coli was fermented in a glucose-containing medium. Fermentation was carried out by inoculating the recombinant Escherichia coli strain into the medium and culturing at 35-40℃ and 100-300 rpm for 5-15 hours. For shake flask fermentation, 0.1-10% of the inoculum was inoculated into an Erlenmeyer flask containing 50-150 mL of LB medium and culturing at 35-40℃ and 100-300 rpm for 40-75 hours.

9. A method for improving cytidine production levels according to claim 7, characterized in that, Recombinant Escherichia coli was fermented in a glucose-containing medium. The fermentation was carried out by transferring the seed culture in the medium to the fermentation medium at an inoculation rate of 0.5-15%, and the fermentation was carried out at 35-40℃ and 100-300 rpm. The glucose concentration was controlled at 13-17 g / L and the dissolved oxygen was controlled at 20-30% during the fermentation process. When the dissolved oxygen was lower than 25%, the stirring speed, aeration rate and tank pressure were increased. Ammonia water was used to control the pH at 6-8 during the fermentation process.

10. A method for improving cytidine production level according to claim 9, characterized in that, The fermentation process also involves feeding; the feeding medium contains 350-820 g / L glucose, 0.5-20 g / L peptone, and 0.5-20 g / L yeast extract.

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