Mutagenesis, screening and application of cytidine high-yield strain

Abstract

The invention discloses a cytidine high-yield strain and a mutagenesis screening method thereof. According to the method, on the basis of metabolic engineering, a chemical mutagenesis means is combined, and a mutant strain which is high in cytidine yield, high in conversion rate and few in by-product is bred. When the mutant strain is used for producing cytidine, the yield and the conversion rate of the cytidine can be greatly improved, the subsequent purification and separation steps can be simplified, and the production cost is effectively reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms and genetic engineering, and particularly relates to a mutagenesis, screening and application technology of a high-yield cytidine strain. BACKGROUND

[0002] Cytidine, also known as cytosine nucleoside, is a structural component of genetic material RNA and participates in various physiological and biochemical reactions in the body. It can also be used as a drug precursor to synthesize antiviral, antitumor and leukemia treatment drugs, and can also be used as a dietary supplement, with a very wide application prospect.

[0003] The production of cytidine mainly includes chemical synthesis, biological catalytic directed synthesis and microbial fermentation. Chemical synthesis is a process of producing cytidine by chemical catalysis using cytosine and ribose as main raw materials. This method has problems such as multiple reaction steps, harsh reaction conditions, high impurity content in the product, and serious environmental pollution. In the past decade, although there have been many researches and attempts on the biological catalytic synthesis of nucleosides and analogs, due to the characteristics of catalytic enzyme inactivation in vitro and inability to recycle and reuse, combined with the relatively high price of substrates, the production cost is high, which is difficult to expand the production scale. In contrast, microbial fermentation can use relatively low-cost carbon sources such as glucose to synthesize cytidine in cells, with low cost, simple conditions, easy control, high yield and environmental friendliness, and therefore attracts widespread attention. By constructing a microbial cell factory for efficient synthesis of cytidine, the environmental pollution and sustainable development problems caused by traditional production methods can be effectively alleviated, and the economic benefits can be greatly improved.

[0004] In order to synthesize cytidine efficiently, the most direct method is to use metabolic engineering technology to rationally modify microorganisms such as Escherichia coli. Yang K et al. (Multistep construction of metabolically engineered Escherichia coli for enhanced cytidine biosynthesis [J]. Biochemical Engineering Journal, 2019, 154:107433.) blocked the bypass metabolism of cytidine to uridine, UMP and cytosine by knocking out the genes related to cytidine catabolism, and the cytidine yield reached 7.84 g / L. Although rational modification has strong predictability and controllability, due to the high complexity of the internal regulation network of the cell, the existence of unknown metabolic bypass and the complex interaction between genes, rational design may not be able to reproduce or achieve the optimal solution that natural evolution can achieve, especially when multiple surface-related genes need to be co-evolved, which makes it difficult to break through the bottleneck of cytidine production.

[0005] In addition, research has found that even if the specific hydrolase encoding genes rihA, rihB and rihC, which convert cytidine into cytosine in the cytidine synthesis metabolic pathway, are knocked out, a high content of cytosine, a byproduct, is still mixed in the produced cytidine product. The similar physicochemical properties of the two bring troubles to subsequent separation and purification. The reason for this result is presumably that there are other unknown non-specific hydrolases in the cell that can hydrolyze cytidine into cytosine, and can hydrolyze part of the cytidine into cytosine. These non-specific hydrolases have not been identified or annotated, so the method of rational modification is helpless.

[0006] In order to solve the problems of yield and byproducts, rational metabolic engineering is combined with non-rational mutagenesis breeding technology. First, a strain with high cytidine yield is obtained through rational modification, and then a high-quality production strain with higher cytidine yield and lower byproduct content is screened through non-rational optimization and adaptation of the cell itself in steps by using chemical mutagenesis technology. SUMMARY

[0007] One of the purposes of the present application is to obtain a mutant strain with high cytidine yield and low byproduct level based on metabolic engineering combined with mutagenesis breeding.

[0008] To achieve the above purpose, the present application selects Escherichia coli (E. coli) as the chassis strain, first performs rational modification on the metabolic pathway of the chassis strain through genetic engineering technology, enhances the cytidine synthesis pathway and blocks cytidine degradation, and then constructs a basic strain for cytidine production. Then, the basic strain is subjected to multiple mutagenesis, and a mutant strain T5 with high cytidine yield and low byproduct level is obtained through batch breeding, and the strain number is LWP019. Escherichia coli The present application provides an Escherichia coli mutant strain with high cytidine yield and low byproduct level.

[0009] The mutant strain T5 has a strain number of LWP019, and was preserved in the China General Microbiological Culture Collection Center (address: No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences) on December 11, 2025, and is classified and named as Escherichia coli (E. coli), and the preservation number is CGMCC No. 37036.

[0010] Escherichia coli The present application also provides a mutagenesis and breeding method of Escherichia coli with high cytidine yield and low byproduct level.

[0011] The present application also provides a mutagenesis and structure analogue screening technology of a strain with high cytidine yield and low byproduct level.

[0012] The present application also provides a mutagenesis and breeding method of Escherichia coli with high cytidine yield and low byproduct level.

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

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

[0015] The application also provides application of the strain with high cytidine yield and low by-product level in cytidine production.

[0016] The application also provides a cytidine production method, which uses the Escherichia coli strain with high cytidine yield and low by-product level.

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

[0018] Preferably, the method comprises: inoculating the strain into seed tank medium for seed culture, and then transferring to fermentation tank medium for fermentation culture.

[0019] The application also provides a fermentation medium for producing cytidine, which comprises seed tank medium and fermentation tank medium.

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

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

[0022] The microelement mother liquor comprises: sodium molybdate dihydrate 10-40 mg / L, aluminum sulfate octadecahydrate 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, VB1 1-5 mg / L, VB3 1-5 mg / L, VB5 1-5 mg / L, VB12 1-5 mg / L, and VB6 8-20 mg / L.

[0023] By the above technical solution, the present application has at least the following advantages and beneficial effects: In order to increase the yield of cytidine, the present application combines rational metabolic engineering with non-rational mutagenesis breeding technology, and screens a mutant strain with high cytidine yield and low by-product level. Using the mutant strain to produce cytidine, the cytidine yield in a 7 L fermenter system reaches 110 g / L, the sugar acid conversion rate reaches 36.7%, and the proportion of by-product cytosine is controlled below 1%. Through the improvement of yield and conversion rate and the control of by-products, the production cost is effectively reduced. The present application also provides an effective process for establishing a high-yield cytidine strain based on metabolic engineering and mutagenesis and breeding, which can further optimize the strain performance in this way and provide guidance for the efficient screening of other nucleoside substances. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 : Mortality curve of mutagenesis with different NTG concentrations Figure 2 : Cytidine and cytosine yield results of mutagenic strains starting from T3 in a shake flask Figure 3 : Cytidine and cytosine yield results of mutagenic strains starting from T4 in a shake flask Figure 4 : Genetic stability test of T5 Figure 5 : Cytidine yield and conversion rate results of T3 and T5 in a fermenter Figure 6 : HPLC chromatogram of fermentation products of T3 and T5 DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. If no specific experimental conditions are noted, it is according to the conventional conditions well known to those skilled in the art.

[0026] The following examples are intended to illustrate the present application but not to limit the scope of the present application. If not specifically indicated, the examples are performed according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning: A Laboratory Manual (2001) or the conditions suggested by the manufacturer's instructions.

[0027] Example 1: Construction of base strain by metabolic engineering (1) According to the construction method of 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.), the strain T0 was obtained by knocking out the enzyme genes cdd, udk, rihA and rihC related to cytidine catabolism in turn based on the Escherichia coli W3110 strain, so as to block the metabolic decomposition pathways of cytidine to uridine, UMP and cytosine. On this basis, the strain T1 was obtained by further knocking out the ribonucleoside hydrolase gene rihB which catalyzes cytidine to cytosine.

[0028] (2) According to the construction method of reference 2 (REAVES M L, YOUNG B D, HOSIOS A M, et al. Pyrimidine homeostasis is accomplished by directed overflow metabolism [J / OL]. Nature, 2013, 500(7461): 237-241. DOI: 10.1038 / nature12445.), the strain T2 was obtained by knocking out the genes umpH and umpG related to the main branch pathway of cytidine synthesis pathway competing for UMP in turn based on the strain T1, so as to weaken the metabolic flux of pyrimidine salvage pathway.

[0029] (3) According to the construction method of reference 3 (LI C, SHI T, FAN W, et al. High-level and -yield orotic acid 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.), T2 was used as the starting strain, and the Ptrc-pyrB-pyrC-pyrAA-pyrABE949*-pyrK-pyrD operon from Bacillus subtilis F126 was integrated into the yghX site to enhance the synthesis of the precursor orotic acid, and strain T3 was obtained as the basic strain for mutagenesis.

[0030] Table 1 Strains used in the present application

[0031] Example 2: Test of the basic strain (1) A sterile inoculation loop was used to dip an appropriate amount of T3 strain from the strain preservation tube, and streak isolation was performed on an LB solid culture medium plate, which was then placed in a 37 °C constant temperature incubator for overnight culture.

[0032] (2) 3-5 single colonies were selected and inoculated into a shaking tube containing LB liquid medium, and the seed solution was prepared by shaking culture at 37 °C and 220 rpm overnight.

[0033] (3) The seed solution was transferred to a shaking flask containing 20 mL of fermentation medium at an inoculation amount of 5%-10% (v / v), and the shaking culture was performed at 37 °C and 220 rpm for 24 h.

[0034] (4) After the culture was completed, the fermentation broth was centrifuged to collect the supernatant, and the obtained supernatant was diluted 5 times, and the cytidine content in the diluted solution was determined by high performance liquid chromatography (HPLC).

[0035] The composition of the fermentation medium is as follows: 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, trace element stock solution 1 mL / L.

[0036] The composition of the trace element mother liquor is: 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, VB1 2 mg / L, VB3 2 mg / L, VB5 2 mg / L, VB12 2 mg / L, VB6 13.4 mg / L.

[0037] The high performance liquid chromatography conditions are: chromatographic column: ZORBAX Eclipse Plus C-18 (4.6x150mm, 5µm); 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.

[0038] The shake flask fermentation results show that the OD 600 of the starting strain T3 without mutagenic treatment is about 4, the cytidine yield is about 1 g / L, and the cytosine yield is about 0.35 g / L.

[0039] Example 3: Determination of NTG lethality 1. Preparation of bacterial suspension (1) A sterile inoculation loop was used to take an appropriate amount of T3 strain from the strain preservation tube, and three-zone streak isolation was performed on an LB solid culture medium plate, which was then placed in a 37 ℃ constant temperature incubator for overnight culture.

[0040] (2) Single colonies on the plate were selected and inoculated into 5 mL of LB liquid medium for activation, and the culture was incubated at 37 ℃ and 220 rpm overnight.

[0041] (3) The activated bacterial solution was inoculated into 100 mL of LB liquid medium at a 1% (v / v) inoculation amount, and the culture was incubated at 37 ℃ and 220 rpm until the OD 600 reached 0.8-1.5.

[0042] (4) The bacterial cells were collected by centrifugation and washed with 0.9% (w / v) sterile physiological saline for 3 times, and the supernatant was discarded after each washing. Finally, the bacterial cells were resuspended with 0.9% (w / v) sterile physiological saline, and the concentration of the bacterial suspension was adjusted to OD 600 = 1.0, and the obtained bacterial suspension was prepared for subsequent mutagenic treatment.

[0043] 2. Mutagenesis Take the appropriate amount of NTG mother liquor (10 g / L) and 10 mL of bacterial suspension mixed in 50 mL sterile centrifuge tube with cover, using 0.9% (w / v) sterile physiological saline to make up to the total volume of the reaction system 20 mL, so that the final mass concentration of NTG is 0, 0.1, 0.5, 1.0, 1.5 mg / mL (NTG concentration of 0 treatment group is mutagenesis control group). The above reaction system is treated at 37 ℃, 220 rpm for 30 min.

[0044] 3, termination of mutagenesis Centrifugal mutagenesis reaction system to collect bacterial cells, washed three times with 0.9% (w / v) of physiological saline to terminate mutagenesis, with NTG waste liquid to the special waste tank, the experimental equipment involved are soaked with NaOH overnight, then washed with plenty of water.

[0045] 4, dilution and plating After washing, using 0.9% (w / v) of physiological saline to resuspend the bacterial liquid to OD 600 = 1.0, gradient dilution 1000 times in 1.5 mL sterile centrifuge tube, take 100 μL coated on LB solid medium plate (three parallel), at 37 ℃ incubator overnight culture.

[0046] 5, mortality calculation Mortality (%) = (control group of bacterial number - treatment group of bacterial number) / control group of bacterial number.

[0047] The results show that (0 Figure 1 ), the survival rate of the starting strain T3 decreases with the increase of the final concentration of NTG. When the NTG treatment concentration is 0, 0.1, 0.5, 1.0 and 1.5 mg / mL, the corresponding mortality is 0, 33.5%, 78.1%, 98.0% and 100% respectively. According to the theory of mutagenesis breeding, when the mortality is about 80%, it is easier to obtain effective positive mutant strains, and at the same time, the number of viable strains can meet the needs of subsequent screening. Considering the above factors, the optimal mutagenesis conditions are determined as follows: the final concentration of NTG is 0.5 mg / mL, and the mutagenesis treatment time is 30 min.

[0048] Example 4: selection of high cytidine-producing strain 1, NTG mutagenesis (1) the starting strain T3 was inoculated into 5 mL LB liquid medium for activation, and cultured at 37 ℃, 220 rpm for overnight.

[0049] (2) the activated bacterial liquid was inoculated into 100 mL LB liquid medium at 1% (v / v) inoculation amount, and cultured at 37 ℃, 220 rpm until OD 600 0.8~1.5.

[0050] (3) Centrifuge to collect the bacteria, resuspend the bacteria in 0.9% (w / v) normal saline, add 10 g / L of NTG mother liquor to make the final concentration of NTG 0.5 mg / mL, and culture at 37°C in a 220 rpm shaker for 30 min.

[0051] (4) After mutagenesis, centrifuge to collect the bacteria, wash with 0.9% (w / v) normal saline for 3 times to terminate mutagenesis.

[0052] 2. Resistance plate screening Resuspend the bacteria after termination of mutagenesis to OD 600 = 1.0 using 0.9% (w / v) normal saline, and spread on LB solid medium containing 5 g / L of 5-fluorouracil, and place in a 37°C incubator for 2-5 days.

[0053] 3. Deep well plate primary screening (1) The strains obtained by resistance plate screening are transferred to a deep well plate containing 400 μL of LB medium for activation, and cultured at 37°C in a 1000 rpm shaker for 24 h.

[0054] (2) The activated seed liquid is transferred to a deep well plate containing 400 μL of fermentation medium for fermentation culture at an inoculation amount of 5-10% (v / v), and cultured at 37°C in a 1000 rpm shaker for 24 h. The composition of the fermentation medium is shown in Example 2.

[0055] (3) After the culture is completed, centrifuge to obtain the supernatant, dilute 50 times, take 100 μL of the diluted solution to a 96-well ultraviolet enzyme-labeled plate, measure the absorbance at 270 nm, and calculate the relative yield of cytidine of each strain.

[0056] The results of the deep well plate primary screening show that, from more than 1000 strains grown on the resistance plate, more than 200 mutant strains with cytidine yield higher than that of the starting strain T3 are obtained. Among them, 8 mutant strains are particularly outstanding, with cytidine yield more than 2 times that of the starting strain T3. The above 8 high-yield mutant strains are subjected to shake flask fermentation rescreening verification.

[0057] 4. Shake flask rescreening (1) The strains obtained by primary screening are inoculated into 5 mL of LB liquid medium for activation, and cultured at 37°C and 220 rpm overnight.

[0058] (2) The activated seed liquid was inoculated into a shake flask containing 20 mL of fermentation medium at an inoculation amount of 5-10% (v / v) and cultured at 37 °C in a 220 rpm shaker for 24 h. The composition of the fermentation medium is shown in Example 2.

[0059] (3) After the culture, the supernatant was centrifuged and diluted 5 times, and then the content of cytidine and cytosine in the diluted solution was determined by high performance liquid chromatography.

[0060] The high performance liquid chromatography conditions were as follows: column: ZORBAX Eclipse Plus C-18 (4.6 x 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 °C, detection wavelength: 270 nm; injection volume: 10 µL.

[0061] The shake flask re-screening results showed that (Table 2) Figure 2 Among the 8 high-yield mutant strains, the cytidine yield of T3-2, T3-4 and T3-5 was significantly improved compared with the starting strain T3. In addition, although the cytidine hydrolase genes rihA, rihB and rihC were knocked out, the content of the by-product cytosine was still at a high level in many strains, including the starting strain T3, accounting for about 10% of the cytidine yield. To further improve the yield of cytidine and reduce the level of the by-product cytosine, the mutant strain T3-5 with the highest cytidine yield was selected as a new starting strain and named T4, which was used for the second round of mutagenesis screening.

[0062] Example 5: Breeding of Cytidine Mutant T5 1. NTG mutagenesis T4 was used as the starting strain, and the specific operation was the same as step 1 in Example 4.

[0063] 2. Resistance plate screening The bacteria after mutagenesis were resuspended in 0.9% (w / v) normal saline to OD 600 = 1.0, and then coated on LB solid medium containing 1 g / L 6-azauracil and placed in a 37 °C incubator for 2-5 days.

[0064] 3. Deep well plate primary screening The specific operation was the same as step 3 in Example 4.

[0065] The results of the well plate primary screening showed that, based on the cytidine yield of the starting strain T4, from about 2000 strains grown on the resistant plates, a total of 186 mutant strains with higher cytidine yield than the starting strain T4 were screened. The 186 mutant strains were subjected to shake flask fermentation re-screening to further determine their cytidine yield and the content of the by-product cytosine, and excellent mutant strains with high cytidine yield and low accumulation of the by-product cytosine were screened therefrom.

[0066] 4. Shake flask re-screening The specific operation and high performance liquid chromatography conditions were the same as step 4 in Example 4.

[0067] The results of the shake flask re-screening showed that (Table 2) Figure 3 ), among the 186 re-screened mutant strains, it was found that the content of the by-product cytosine of four strains was significantly reduced, far lower than that of the starting strain T4. Among them, the cytosine content of the mutant strain T4-62 was the lowest, and at the same time, its cytidine yield was still maintained at a high level, which had the application potential as a cytidine industrial production strain. The strain was named as T5, and the strain number was LWP019.

[0068] Example 6: Stability test of T5 strain To evaluate the genetic stability of the mutant strain T5, a continuous subculture experiment was carried out. T5 was subcultured on LB solid medium for 5 times, and the strain was subjected to shake flask fermentation for 24 h. The cell growth density (OD 600 ), cytidine yield and the content of the by-product cytosine of the strain of the 1st generation and the 5th generation were determined, respectively.

[0069] The results showed that (Table 3) Figure 4 ), there was no significant difference between the 1st generation and the 5th generation in each index, showing good consistency. The results showed that the mutant strain T5 had good genetic stability in the continuous subculture process, and was suitable for the industrial production of cytidine.

[0070] Example 7: T5 fermenter test To further investigate the fermentation production performance of the mutant strain T5, a fed-batch fermentation test was carried out in a 7L fermenter.

[0071] 1. Strain activation T5 and T3 strains were taken from the preservation tube using a inoculation loop, and three-zone streaking was carried out on LB plates, and overnight culture was carried out at 37°C. Single colonies on the plates were picked and inoculated in 5 mL of LB liquid medium for activation, and overnight culture was carried out at 37°C and 220 rpm.

[0072] 2. Seed liquid preparation The activated bacterial liquid was inoculated into 100 mL of LB liquid medium at an inoculation amount of 1% (v / v), and cultured for 4-6 h.

[0073] 3. Fermentation in a fermenter (1) Seed culture was carried out by inoculating seed liquid into a seed tank containing 2 L of seed tank medium, the inoculation amount was 5-10‰, the culture temperature was 34-40 ℃, the pH was 6.6-7.2, the dissolved oxygen was controlled at 25-45%, the residual sugar was controlled at 1-3%, and the culture period was 12-20 h.

[0074] (2) After the seed culture was completed, cytidine fermentation culture was carried out by transferring into a 7 L fermenter containing 2.5 L of fermenter medium, the inoculation amount was 5-20%, the culture temperature was 35-40 ℃, the pH was 6.6-7.2, the dissolved oxygen was controlled at 10-30%, the residual sugar was controlled at 0.05-0.1%, and the fermentation period was 60 h.

[0075] (3) After the fermentation was completed, the fermentation liquid was gradiently diluted by 100 times, the bacteria were removed by centrifugation, the supernatant was taken, and the contents of cytidine and cytosine therein were determined by high performance liquid chromatography (HPLC), the high performance liquid chromatography conditions were the same as step 4 in Example 4.

[0076] The seed tank medium was composed of: glucose 30 g / L, yeast powder 3 g / L, proteose 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 mother liquor 5 mL / L, and biotin 3.5 mg / L.

[0077] The fermenter medium was composed 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 mother liquor 10 mL / L, and biotin 0.5 mg / L.

[0078] The trace element mother liquor was composed 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, VB1 2 mg / L, VB3 2 mg / L, VB5 2 mg / L, VB12 2 mg / L, and VB6 13.4 mg / L.

[0079] From Figure 5The fermentation results of the fermenter can be seen that the highest yield of cytidine of the unmutated starting strain T3 is 61 g / L, and the sugar acid conversion rate is 25%. The highest yield of cytidine of the mutant strain T5 is 110 g / L, and the sugar acid conversion rate is 36.7%, which is increased by 80% and 47% respectively compared with T3.

[0080] From Figure 6 It can be clearly seen from the HPLC chromatogram of the fermentation product of the unmutated T3 strain that a high level of by-product cytosine can be detected, and the fermentation product of the mutant strain T5 hardly contains cytosine.

[0081] In summary, the mutant strain T5 obtained by mutagenesis not only has a high level of cytidine yield, but also has a very low content of by-product cytosine in the fermentation product, which accounts for less than 1%, and almost cannot be detected. Using the mutant strain T5 to ferment cytidine can greatly improve the yield and conversion rate of cytidine, reduce the level of by-products, simplify the operation, improve the production efficiency, and effectively reduce the production cost.

[0082] Although the present application has been described in detail by general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.

Claims

1. A cytidine high-producing Escherichia coli strain, characterized by, The classification name is Escherichia coli (E. coli) Escherichia coli , and the preservation number is CGMCC No. 37036.

2. Use of the strain according to claim 1 in the production of cytidine.

3. A method for producing cytidine, characterized by, Culturing the strain according to claim 1.

4. The method of claim 3, wherein, First inoculating the strain according to claim 1 into seed tank medium for seed culture, and then transferring to fermentation tank medium for fermentation culture.

5. The method of claim 4, wherein, The seed tank medium is composed of glucose 20-50 g / L, yeast powder 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 mother liquor 5 mL / L, and biotin 1.2-4.6 mg / L.

6. The method of claim 4, wherein, The fermentation tank medium is composed 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 mother liquor 10 mL / L, and biotin 0.2-1 mg / L.

7. The method according to any one of claims 5-6, wherein, The trace element mother liquor is composed of sodium molybdate dihydrate 10-40 mg / L, aluminum sulfate octadecahydrate 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, VB1 1-5 mg / L, VB3 1-5 mg / L, VB5 1-5 mg / L, VB12 1-5 mg / L, and VB6 8-20 mg / L.

8. The method of claim 7, wherein, The seed tank medium is composed of glucose 30 g / L, yeast powder 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 mother liquor 5 mL / L, and biotin 3.5 mg / L.

9. The method of claim 7, wherein, The fermentation tank medium is composed 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 mother liquor 10 mL / L, and biotin 0.5 mg / L.

10. The method of claim 7, wherein, The microelement 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, VB1 2 mg / L, VB3 2 mg / L, VB5 2 mg / L, VB12 2 mg / L, VB6 13.4 mg / L.

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