Mutagenesis, screening and application of high-yield cytidine strain

Abstract

This invention discloses a high-cytidine-producing bacterial strain and its mutagenesis screening method. Based on metabolic engineering and combined with chemical mutagenesis, this method selects mutant strains with high cytidine yield, high conversion rate, and few byproducts. Using this mutant strain to produce cytidine not only significantly improves the yield and conversion rate but also simplifies subsequent purification and separation steps, effectively reducing production costs.

Description

Technical Field

[0001] This invention belongs to the field of microbial and genetic engineering technology, specifically relating to the mutagenesis, screening, and application technology of a high-cytidine-producing strain. Background Technology

[0002] Cytidine, also known as cytidine, is a structural component of RNA, a genetic material. It participates in a variety of physiological and biochemical reactions in organisms and can also be used as a drug precursor for the synthesis of antiviral, antitumor, and leukemia treatment drugs. It can also be used as a dietary supplement, and its application prospects are very broad.

[0003] Cytidine production mainly includes chemical synthesis, biocatalytic directed synthesis, and microbial fermentation. Chemical synthesis uses cytosine and ribose as main raw materials to produce cytidine through a chemical catalytic process. This method suffers from problems such as multiple reaction steps, harsh reaction conditions, high impurity content in the product, and severe environmental pollution. While significant research and attempts have been made in the biocatalytic synthesis of nucleosides and analogues over the past decade, the high cost and limited recyclability of catalytic enzymes, coupled with the high price of substrates, have resulted in persistently high production costs, hindering large-scale production. In contrast, microbial fermentation can synthesize cytidine intracellularly using relatively inexpensive carbon sources such as glucose. This method is low-cost, simple, easy to control, yields high amounts, and is environmentally friendly, thus attracting widespread attention. Constructing efficient microbial cell factories for cytidine synthesis can effectively alleviate the environmental pollution and sustainable development problems caused by traditional production methods, significantly improving economic efficiency.

[0004] To achieve efficient cytidine synthesis, the most direct method is to rationally modify microbial cells, such as *Escherichia coli*, using metabolic engineering techniques. Yang K et al. (Multistep construction of metabolically engineered *Escherichia coli* for enhanced cytidine biosynthesis[J]. *Biochemical Engineering Journal*, 2019, 154:107433.) achieved a cytidine yield of 7.84 g / L by knocking out genes related to cytidine catabolism to block the bypass metabolism of cytidine to uridine, UMP, and cytosine. Although rational modification offers strong predictability and controllability, due to the high complexity of intracellular regulatory networks, the existence of unknown metabolic bypasses, and complex interactions between genes, rational design may not be able to replicate or achieve the optimal solution achievable through natural evolution, especially when multiple seemingly unrelated genes need to co-evolve. This makes it difficult to overcome bottlenecks in cytidine production.

[0005] Furthermore, the study found that even after knocking out the genes encoding the specific hydrolases that convert cytidine to cytosine in the cytidine biosynthesis pathway (rihA, rihB, and rihC), the produced cytidine product still contained a high amount of the byproduct cytosine. The similar physicochemical properties of the two products posed challenges for subsequent separation and purification. This result is speculated to be due to the existence of other unknown non-specific hydrolases within the cell that can hydrolyze cytidine to cytosine, partially converting cytidine into cytosine. These non-specific hydrolases have not been identified or annotated, thus rendering rational modification methods ineffective against them.

[0006] To address the issues of yield and byproducts, we attempted to combine rational metabolic engineering with irrational mutagenesis and selection techniques. First, we obtained strains with higher cytidine yields through rational modification. Then, we used chemical mutagenesis to screen out high-quality production strains with higher cytidine yields and lower byproduct content through the irrational optimization and adaptation of the cells themselves. Summary of the Invention

[0007] One of the objectives of this invention is to obtain mutant strains with high cytidine production and low byproduct levels by combining metabolic engineering with mutagenesis and selection.

[0008] To achieve the above objectives, the present invention selects *Escherichia coli* (E. coli) Escherichia coli Using a chassis strain as a base, its metabolic pathway was first rationally modified through genetic engineering techniques to enhance the cytidine synthesis pathway and block cytidine degradation, thus constructing a basic strain for cytidine production. Then, the basic strain was subjected to multiple mutagenesis, and mutant strain T5 with high cytidine production and low by-product levels was selected in batches, with strain number LWP019.

[0009] This invention provides a mutant strain of Escherichia coli with high cytidine production and low byproduct levels.

[0010] The mutant strain T5, strain number LWP019, was deposited on December 11, 2025, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and classified as: *Escherichia coli*. Escherichia coli (The accession number is CGMCC No.37036).

[0011] The present invention also provides a method for mutagenesis and selection of Escherichia coli with high cytidine production and low by-product levels.

[0012] The present invention also provides a mutagenesis and structural analog screening technique for strains with high cytidine production and low by-product levels.

[0013] The mutagenesis technique is N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis.

[0014] The screening technique is structural analog screening, and the analogs are 5-fluorouracil and 6-azauracil.

[0015] The present invention also provides the application of a strain with high cytidine production and low by-product levels in cytidine production.

[0016] The present invention also provides a method for producing cytidine, wherein the method uses an Escherichia coli strain that produces high cytidine and low levels of byproducts.

[0017] The method includes: culturing the Escherichia coli.

[0018] The method preferably includes: first inoculating the strain into a seed tank culture medium for seed culture, and then transferring it to a fermentation tank culture medium for fermentation culture.

[0019] The present invention also provides a culture medium for fermentation production of cytidine, the culture medium comprising a seed tank culture medium and a fermentation tank culture medium.

[0020] The seed tank culture medium consists of: glucose 20-50 g / L, yeast extract 2.5-10 g / L, peptone 1-3 g / L, potassium dihydrogen phosphate 2-7 g / L, magnesium sulfate 0.8-2.5 g / L, ammonium sulfate 1.5-5 g / L, citric acid 1.1-4.5 g / L, ferrous sulfate 5-35 mg / L, manganese sulfate 2-6 mg / L, trace element stock solution 5 mL / L, and biotin 1.2-4.6 mg / L.

[0021] The fermentation tank culture medium consists of: glucose 15-35 g / L, yeast powder 1-3 g / L, potassium dihydrogen phosphate 3-7 g / L, magnesium sulfate 3-6 g / L, ammonium sulfate 2.5-5 g / L, citric acid 1.8-5.6 g / L, ferrous sulfate 20-50 mg / L, manganese sulfate 0.3-1.5 mg / L, trace element stock solution 10 mL / L, and biotin 0.2-1 mg / L.

[0022] The trace element mother liquor consists of: sodium molybdate dihydrate 10-40 mg / L, aluminum sulfate octadechydrate 8-20 mg / L, nickel chloride hexahydrate 10-20 mg / L, calcium chloride dihydrate 50-150 mg / L, copper sulfate pentahydrate 2-8 mg / L, cobalt chloride hexahydrate 10-30 mg / L, zinc sulfate heptahydrate 2-10 mg / L, boric acid 0.5-2 mg / L, vitamin B1 1-5 mg / L, vitamin B3 1-5 mg / L, vitamin B5 1-5 mg / L, vitamin B12 1-5 mg / L, and vitamin B6 8-20 mg / L.

[0023] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0024] To increase cytidine yield, this invention combines rational metabolic engineering with irrational mutagenesis and selection techniques, resulting in a mutant strain with high cytidine production and low byproduct levels. Using this mutant strain, a cytidine yield of 110 g / L was achieved in a 7 L fermenter system, with a sugar-acid conversion rate of 36.7%, and the proportion of the byproduct cytosine was controlled below 1%. By improving yield and conversion rate and controlling byproducts, production costs were effectively reduced. This application also provides an efficient process and fermentation medium for mutagenesis and selection of high-yielding cytidine strains based on metabolic engineering, which can further optimize strain performance and provide guidance for the efficient screening of other nucleoside analogs. Attached Figure Description

[0025] Figure 1 Lethality curves induced by different NTG concentrations

[0026] Figure 2 Results of cytidine and cytosine production in shake flasks of T3-derived mutagenic strains

[0027] Figure 3 Results of cytidine and cytosine production in shake flasks of T4-derived mutagen strains

[0028] Figure 4 T5 genetic stability test

[0029] Figure 5 Results of cytidine yield and conversion rate in fermenters T3 and T5

[0030] Figure 6 HPLC chromatograms of T3 and T5 fermentation products Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Unless otherwise specified, all experimental conditions are conventional conditions well known to those skilled in the art.

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or as recommended by the manufacturer's instructions.

[0033] Example 1: Construction of a basic bacterial strain using metabolic engineering techniques

[0034] (1) Following the construction method described in Reference 1 (Yang K, Li Z. Multistep construction of metabolically engineered Escherichia coli for enhanced cytidine biosynthesis[J]. Biochemical Engineering Journal, 2019, 154:107433.DOI:10.1016 / j.bej.2019.107433.), using Escherichia coli W3110 as the starting strain, the enzyme genes cdd, udk, rihA, and rihC related to cytidine catabolism were sequentially knocked out, thereby blocking the metabolic decomposition pathway of cytidine to uridine, UMP, and cytosine, resulting in strain T0. Based on this, the ribonucleoside hydrolase gene rihB, which catalyzes the conversion of cytidine to cytosine, was further knocked out to obtain strain T1.

[0035] (2) Following the construction method in reference 2 (REAVES ML, YOUNG BD, HOSIOS AM, et al. Pyrimidine homeostasis is accomplished by directed overflow metabolism[J / OL]. Nature, 2013, 500(7461): 237-241. DOI:10.1038 / nature12445.), strain T2 was obtained by sequentially knocking out umpH and umpG, the main branch genes related to the cytidine synthesis pathway competing for UMP, in order to weaken the metabolic flux of the pyrimidine rescue pathway.

[0036] (3) Following the construction method in reference 3 (LI C, SHI T, FAN W, et al. High-level and -yield oroticacid production in Escherichia coli through systematic modular engineering and “Chaos to Order Cycles” fermentation[J / OL]. Bioresource Technology, 2024, 411: 131345. DOI:10.1016 / j.biortech.2024.131345.), with T2 as the starting strain, the Ptrc-pyrB-pyrC-pyrAA-pyrABE949*-pyrK-pyrD operon from Bacillus subtilis F126 was integrated into the yghX site to enhance the synthesis of precursor orotic acid, resulting in strain T3, which served as the basic strain for mutagenesis initiation.

[0037] Table 1. Strains used in this invention

[0038]

[0039] Example 2: Testing of the basic strain

[0040] (1) Using a sterile inoculation loop, take an appropriate amount of T3 strain from the culture tube, streak it on an LB solid medium plate, and incubate it overnight in a 37 ℃ constant temperature incubator.

[0041] (2) Select 3-5 single colonies, inoculate them into a shaker containing LB liquid medium, and culture overnight at 37 ℃ and 220 rpm to prepare seed culture.

[0042] (3) The seed culture was transferred to a shake flask containing 20 mL of fermentation medium at an inoculation rate of 5%~10% (v / v) and cultured at 37 ℃ and 220 rpm for 24 h.

[0043] (4) After the culture is completed, the fermentation broth is centrifuged and the supernatant is collected. The supernatant is diluted 5 times and the cytidine content in the diluted solution is determined by high performance liquid chromatography (HPLC).

[0044] The fermentation medium consists of the following components: glucose 10 g / L, yeast powder 2 g / L, potassium dihydrogen phosphate 3.5 g / L, magnesium sulfate 2.8 g / L, citric acid 1.2 g / L, ferrous sulfate 15 mg / L, manganese sulfate 5 mg / L, biotin 0.8 mg / L, and trace element stock solution 1 mL / L.

[0045] The trace element mother liquor consists of: sodium molybdate dihydrate 25 mg / L, aluminum sulfate octadecahydrate 13 mg / L, nickel chloride hexahydrate 16 mg / L, calcium chloride dihydrate 100 mg / L, copper sulfate pentahydrate 4 mg / L, cobalt chloride hexahydrate 18 mg / L, zinc sulfate heptahydrate 5 mg / L, boric acid 1 mg / L, vitamin B1 2 mg / L, vitamin B3 2 mg / L, vitamin B5 2 mg / L, vitamin B12 2 mg / L, and vitamin B6 13.4 mg / L.

[0046] The high-performance liquid chromatography (HPLC) conditions were as follows: column: ZORBAX Eclipse Plus C-18 (4.6 × 150 mm, 5 µm); mobile phase A: 50 mM potassium dihydrogen phosphate, mobile phase B: 50% methanol, A: B = (90:10, v / v); flow rate: 0.6 mL / min, column temperature: 30 ℃, detection wavelength: 270 nm; injection volume: 10 µL.

[0047] Shake-flask fermentation results showed that after 24 h of fermentation, the OD of the unmutated starting strain T3 was... 600 The yield was approximately 4 g / L, with cytidine production at approximately 1 g / L and cytosine production at approximately 0.35 g / L.

[0048] Example 3: Determination of NTG lethality

[0049] 1. Preparation of bacterial suspension

[0050] (1) Using a sterile inoculation loop, take an appropriate amount of T3 strain from the culture tube, perform three-zone streak separation on an LB solid medium plate, and incubate overnight in a 37 ℃ constant temperature incubator.

[0051] (2) Select a single colony on the plate, inoculate it into 5 mL of LB liquid medium for activation, and culture overnight with shaking at 37 ℃ and 220 rpm.

[0052] (3) The activated bacterial culture was transferred to 100 mL LB liquid medium at an inoculation rate of 1% (v / v) and cultured with shaking at 37 ℃ and 220 rpm until OD. 600 It reaches 0.8~1.5.

[0053] (4) Collect bacterial cells by centrifugation, wash three times with 0.9% (w / v) sterile physiological saline, and discard the supernatant after each wash. Finally, resuspend the bacterial cells in 0.9% (w / v) sterile physiological saline and adjust the bacterial suspension concentration to OD. 600 = 1.0, and the resulting bacterial suspension is reserved for subsequent mutagenesis treatment.

[0054] 2. Mutagenesis

[0055] Take an appropriate amount of NTG stock solution (10 g / L) and mix it with 10 mL of bacterial suspension in a 50 mL capped sterile centrifuge tube. Add 0.9% (w / v) sterile physiological saline to bring the total reaction volume to 20 mL, so that the final NTG concentrations are 0, 0.1, 0.5, 1.0, and 1.5 mg / mL (the treatment group with an NTG concentration of 0 serves as the mutagenesis control group). Incubate the reaction system at 37 ℃ and 220 rpm for 30 min with shaking.

[0056] 3. Termination of mutagenesis

[0057] The bacterial cells were collected from the centrifuged mutagenesis reaction system and washed three times with 0.9% (w / v) physiological saline to terminate the mutagenesis. The waste liquid containing NTG was placed in a special waste liquid container. All experimental equipment was soaked in NaOH overnight and then rinsed with plenty of water.

[0058] 4. Dilute the coating plate

[0059] After washing, resuspend the bacterial culture in 0.9% (w / v) physiological saline to OD. 600 = 1.0, serially diluted 1000 times in 1.5 mL sterile centrifuge tubes, take 100 μL and spread it on LB solid medium plates (three in parallel), and incubate overnight at 37°C.

[0060] 5. Calculation of mortality rate

[0061] Mortality rate (%) = (number of colonies in control group - number of colonies in treatment group) / number of colonies in control group.

[0062] The results showed that ( Figure 1 The survival rate of the starting strain T3 decreased with increasing final NTG concentration. When the NTG concentrations were 0, 0.1, 0.5, 1.0, and 1.5 mg / mL, the corresponding lethality rates were 0%, 33.5%, 78.1%, 98.0%, and 100%, respectively. According to mutagenesis breeding theory, a lethality rate of approximately 80% makes it easier to obtain effective positive mutants while ensuring a sufficient number of surviving strains for subsequent screening. Considering all these factors, the optimal mutagenesis conditions were determined to be: a final NTG concentration of 0.5 mg / mL and a mutagenesis treatment time of 30 min.

[0063] Example 4: Selection of high-cytidine-producing strains

[0064] 1. NTG mutagenesis

[0065] (1) The starting strain T3 was inoculated into 5 mL of LB liquid medium for activation and cultured overnight at 37 ℃ and 220 rpm with shaking.

[0066] (2) The activated bacterial culture was transferred to 100 mL LB liquid medium at an inoculation rate of 1% (v / v) and cultured with shaking at 37 ℃ and 220 rpm until OD. 600 It reaches 0.8~1.5.

[0067] (3) Collect the bacterial cells by centrifugation, resuspend the bacterial cells in 0.9% (w / v) physiological saline, add 10 g / L of NTG stock solution to make the final NTG concentration 0.5 mg / mL, and culture in a shaker at 37℃ and 220 rpm for 30 min.

[0068] (4) After the mutagenesis is complete, centrifuge to collect the bacterial cells and wash them three times with 0.9% (w / v) physiological saline to terminate the mutagenesis.

[0069] 2. Resistance plate screening

[0070] The induced bacterial cells were resuspended at OD using 0.9% (w / v) physiological saline. 600 = 1.0, and spread on LB solid medium containing 5 g / L 5-fluorouracil, and incubated in a 37 ℃ incubator for 2-5 days.

[0071] 3. Deep-hole plate primary screening

[0072] (1) The strains obtained by the resistance plate screening were transferred to a deep well plate containing 400 μL LB medium for activation and cultured at 37 ℃ and 1000 rpm for 24 h.

[0073] (2) The activated seed culture was transferred to a deep-well plate containing 400 μL of fermentation medium at an inoculation rate of 5-10% (v / v) for fermentation culture at 37 ℃ and 1000 rpm for 24 h. The composition of the fermentation medium is the same as that in Example 2.

[0074] (3) After the culture is completed, the supernatant is centrifuged and diluted 50 times. 100 μL of the diluted solution is added to a 96-well UV microplate and the absorbance at 270 nm is measured. The relative yield of cytidine for each strain is calculated.

[0075] Initial screening using plate culture revealed that over 200 mutant strains with higher cytidine production than the starting strain T3 were obtained from more than 1000 strains grown on resistance plates. Among them, eight mutant strains were particularly outstanding, with cytidine production more than twice that of the starting strain T3. These eight high-yielding mutant strains were then subjected to shake-flask fermentation for further screening and verification.

[0076] 4. Shake flask and screen again

[0077] (1) The strains obtained from the initial screening were inoculated into 5 mL of LB liquid medium for activation and cultured overnight at 37 ℃ and 220 rpm with shaking.

[0078] (2) The activated seed culture was transferred to a shake flask containing 20 mL of fermentation medium at an inoculation rate of 5-10% (v / v) and cultured at 37 °C and 220 rpm for 24 h. The composition of the fermentation medium is the same as that in Example 2.

[0079] (3) After the culture is completed, the supernatant is centrifuged and diluted 5 times. The contents of cytidine and cytosine in the diluted solution are then determined by high performance liquid chromatography.

[0080] The high-performance liquid chromatography (HPLC) conditions were as follows: column: ZORBAX Eclipse Plus C-18 (4.6 × 150 mm, 5 µm); mobile phase A: 50 mM potassium dihydrogen phosphate, mobile phase B: 50% methanol, A: B = (90:10, v / v); flow rate: 0.6 mL / min, column temperature: 30 ℃, detection wavelength: 270 nm; injection volume: 10 µL.

[0081] The results of the shake-flask re-screening showed that ( Figure 2 Among the eight high-yielding mutant strains, T3-2, T3-4, and T3-5 showed significantly increased cytidine production compared to the starting strain T3. Furthermore, despite the knockout of cytidine hydrolase genes rihA, rihB, and rihC, the byproduct cytosine content remained high in several strains, including the starting strain T3, accounting for approximately 10% of cytidine production. To further improve cytidine production and reduce the level of byproduct cytosine, the mutant strain T3-5, which had the highest cytidine production, was selected as the new starting strain and named T4 for a second round of mutagenesis screening.

[0082] Example 5: Selection of cytidine mutant strain T5

[0083] 1. NTG mutagenesis

[0084] Using T4 as the starting strain, the specific operation is the same as step 1 in Example 4.

[0085] 2. Resistance plate screening

[0086] The induced bacterial cells were resuspended at OD using 0.9% (w / v) physiological saline. 600 = 1.0, and spread on LB solid medium containing 1 g / L 6-azauracil, and incubated in a 37 ℃ incubator for 2-5 days.

[0087] 3. Deep-hole plate primary screening

[0088] The specific operation is the same as step 3 in Example 4.

[0089] Initial screening using microplates revealed that, based on the cytidine production of the starting strain T4, 186 mutant strains with higher cytidine production than the starting strain T4 were selected from approximately 2000 strains grown on resistance plates. These 186 mutant strains underwent shake-flask fermentation for further screening to determine their cytidine production and the content of the byproduct cytosine, and superior mutant strains with high cytidine production and low cytosine accumulation were selected.

[0090] 4. Shake flask and screen again

[0091] The specific operation and high-performance liquid chromatography conditions are the same as step 4 in Example 4.

[0092] The results of the shake-flask re-screening showed that ( Figure 3 Among the 186 mutant strains screened again, four strains were found to have significantly reduced cytosine content, far lower than the original strain T4. Among them, mutant strain T4-62 had the lowest cytosine content, while its cytidine production remained at a high level, showing potential for application as a strain for industrial cytidine production. This strain was named T5, with the strain number LWP019.

[0093] Example 6: Stability test of T5 strain

[0094] To assess the genetic stability of the mutant strain T5, a serial subculture experiment was conducted. T5 was subcultured five times on LB solid medium and fermented in shake flasks for 24 h. The cell density (OD) of the first and fifth generations was measured. 600 ), cytidine production and the content of the byproduct cytosine.

[0095] The results showed that ( Figure 4 The above indicators showed no significant differences between the 1st and 5th generations, demonstrating good consistency. This result indicates that the mutant strain T5 exhibits good genetic stability during continuous passage and is suitable for the industrial production of cytidine.

[0096] Example 7: T5 fermenter test

[0097] To further investigate the fermentation performance of the mutant strain T5, a fed-batch fermentation test was conducted in a 7L fermenter.

[0098] 1. Activation of the strain

[0099] Using an inoculation loop, collect T5 and T3 strains from the preservation tube, streak them in three zones on an LB agar plate, and incubate overnight at 37 °C. Pick a single colony from the plate and activate it in 5 mL of LB liquid medium, then incubate overnight at 37 °C and 220 rpm.

[0100] 2. Seed liquid preparation

[0101] The activated bacterial culture was transferred to 100 mL of LB liquid medium at an inoculation rate of 1% (v / v) and cultured for 4-6 hours.

[0102] 3. Fermentation in fermentation tanks

[0103] (1) Inoculate the seed culture into a seed tank containing 2L of seed tank culture medium for seed culture. The inoculation amount is 5-10‰, the culture temperature is 34-40℃, the pH is 6.6-7.2, the dissolved oxygen is controlled at 25-45%, the residual sugar is controlled at 1-3%, and the culture period is 12-20 h.

[0104] (2) After the seed culture is completed, the culture is transferred to a 7 L fermenter containing 2.5 L fermenter culture medium for cytidine fermentation culture. The inoculum size is 5-20%, the culture temperature is 35-40 ℃, the pH is 6.6-7.2, the dissolved oxygen is controlled at 10-30%, the residual sugar is controlled at 0.05-0.1%, and the fermentation cycle is 60 h.

[0105] (3) After fermentation, the fermentation broth was serially diluted 100 times, centrifuged to remove bacteria, and the supernatant was taken. The contents of cytidine and cytosine were determined by high performance liquid chromatography (HPLC). The HPLC conditions were the same as those in step 4 of Example 4.

[0106] The seed culture medium consists of: glucose 30 g / L, yeast extract 3 g / L, peptone 1 g / L, potassium dihydrogen phosphate 4 g / L, magnesium sulfate 1.6 g / L, ammonium sulfate 2.2 g / L, citric acid 2 g / L, ferrous sulfate 25 mg / L, manganese sulfate 5 mg / L, trace element stock solution 5 mL / L, and biotin 3.5 mg / L.

[0107] The fermentation tank culture medium consists of: glucose 25 g / L, yeast powder 2.5 g / L, potassium dihydrogen phosphate 6 g / L, magnesium sulfate 2.4 g / L, ammonium sulfate 3.5 g / L, citric acid 2.2 g / L, ferrous sulfate 35 mg / L, manganese sulfate 0.5 mg / L, trace element stock solution 10 mL / L, and biotin 0.5 mg / L.

[0108] The trace element mother liquor consists of: sodium molybdate dihydrate 25 mg / L, aluminum sulfate octadecahydrate 13 mg / L, nickel chloride hexahydrate 16 mg / L, calcium chloride dihydrate 100 mg / L, copper sulfate pentahydrate 4 mg / L, cobalt chloride hexahydrate 18 mg / L, zinc sulfate heptahydrate 5 mg / L, boric acid 1 mg / L, vitamin B1 2 mg / L, vitamin B3 2 mg / L, vitamin B5 2 mg / L, vitamin B12 2 mg / L, and vitamin B6 13.4 mg / L.

[0109] from Figure 5 The fermentation results in the fermenter showed that the highest cytidine yield of the unmutated starting strain T3 was 61 g / L, with a sugar-acid conversion rate of 25%. In contrast, the highest cytidine yield of the mutant strain T5 reached 110 g / L, with a sugar-acid conversion rate of 36.7%, representing increases of 80% and 47% respectively compared to T3.

[0110] from Figure 6 The HPLC chromatograms clearly show that a high level of the byproduct cytosine can be detected in the fermentation products of the unmutated T3 strain, while cytosine is almost undetectable in the fermentation products of the mutant T5 strain.

[0111] In summary, the mutant strain T5 obtained through mutagenesis not only exhibits a high level of cytidine production, but also produces a very low content of the byproduct cytosine in the fermentation product, accounting for less than 1%, which is almost undetectable. Using this mutant strain T5 for cytidine production can significantly improve cytidine yield and conversion rate, reduce byproduct levels, simplify operations, increase production efficiency, and effectively reduce production costs.

[0112] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A cytidine-producing Escherichia coli strain, characterized in that, Classified and named as Escherichia coli ( Escherichia coli (The accession number is CGMCC No. 37036).

2. Use of the strain as described in claim 1 in the production of cytidine.

3. A method for producing cytidine, characterized in that, Cultivate the strain as described in claim 1.

4. The method as described in claim 3, characterized in that, The strain described in claim 1 is first inoculated into the seed tank culture medium for seed culture, and then transferred to the fermentation tank culture medium for fermentation culture.

5. The method as described in claim 4, characterized in that, The seed tank culture medium consists of: glucose 20-50 g / L, yeast extract 2.5-10 g / L, peptone 1-3 g / L, potassium dihydrogen phosphate 2-7 g / L, magnesium sulfate 0.8-2.5 g / L, ammonium sulfate 1.5-5 g / L, citric acid 1.1-4.5 g / L, ferrous sulfate 5-35 mg / L, manganese sulfate 2-6 mg / L, trace element stock solution 5 mL / L, and biotin 1.2-4.6 mg / L.

6. The method as described in claim 4, characterized in that, The fermentation tank culture medium consists of: glucose 15-35 g / L, yeast powder 1-3 g / L, potassium dihydrogen phosphate 3-7 g / L, magnesium sulfate 3-6 g / L, ammonium sulfate 2.5-5 g / L, citric acid 1.8-5.6 g / L, ferrous sulfate 20-50 mg / L, manganese sulfate 0.3-1.5 mg / L, trace element stock solution 10 mL / L, and biotin 0.2-1 mg / L.

7. The method according to any one of claims 5-6, characterized in that, The trace element mother liquor consists of: sodium molybdate dihydrate 10-40 mg / L, aluminum sulfate octadechydrate 8-20 mg / L, nickel chloride hexahydrate 10-20 mg / L, calcium chloride dihydrate 50-150 mg / L, copper sulfate pentahydrate 2-8 mg / L, cobalt chloride hexahydrate 10-30 mg / L, zinc sulfate heptahydrate 2-10 mg / L, boric acid 0.5-2 mg / L, vitamin B1 1-5 mg / L, vitamin B3 1-5 mg / L, vitamin B5 1-5 mg / L, vitamin B12 1-5 mg / L, and vitamin B6 8-20 mg / L.

8. The method as described in claim 7, characterized in that, The seed culture medium consists of: glucose 30 g / L, yeast extract 3 g / L, peptone 1 g / L, potassium dihydrogen phosphate 4 g / L, magnesium sulfate 1.6 g / L, ammonium sulfate 2.2 g / L, citric acid 2 g / L, ferrous sulfate 25 mg / L, manganese sulfate 5 mg / L, trace element stock solution 5 mL / L, and biotin 3.5 mg / L.

9. The method as described in claim 7, characterized in that, The trace element mother liquor consists of: sodium molybdate dihydrate 25 mg / L, aluminum sulfate octadecahydrate 13 mg / L, nickel chloride hexahydrate 16 mg / L, calcium chloride dihydrate 100 mg / L, copper sulfate pentahydrate 4 mg / L, cobalt chloride hexahydrate 18 mg / L, zinc sulfate heptahydrate 5 mg / L, boric acid 1 mg / L, vitamin B1 2 mg / L, vitamin B3 2 mg / L, vitamin B5 2 mg / L, vitamin B12 2 mg / L, and vitamin B6 13.4 mg / L.

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