Method for producing mRNA (messenger ribonucleic acid) template plasmid by fermenting escherichia coli

Abstract

The invention discloses a method for producing mRNA (messenger ribonucleic acid) template plasmids by fermenting escherichia coli. When the recombinant escherichia coli is fermented and cultured, a seed solution of the recombinant escherichia coli is inoculated into a basic culture medium to be fermented and cultured until the OD600 value of fermentation liquor is 18-22, then a fed-batch culture medium is continuously added at the feeding rate of 5-15 mL / L / h to be subjected to continuous fed-batch fermentation culture, the basic culture medium contains 0.05-0.2 mmoL / L of ferric salt compound and 0.05-0.2 mmoL / L of copper chloride dihydrate, and the basic culture medium contains 0.05-0.2 mmoL / L of ferric salt compound and 0.05-0.2 mmoL / L of copper chloride dihydrate. The basal culture medium contains 10-15 g / L of copper chloride dihydrate and 100-300 mg / L of p-hydroxybenzoic acid, the carbon source of the basal culture medium contains 0.1-0.3 mmoL / L of a ferric salt compound, 0.1-0.3 mmoL / L of copper chloride dihydrate and 100-300 mg / L of p-hydroxybenzoic acid, the carbon source of the fed-batch culture medium contains 360-480 g / L of glycerol, and the ferric salt compound is one or more of ferrous citrate, ferric ammonium citrate or ferrous sulfate heptahydrate. According to the method, the yield and superhelix content of the mRNA template plasmids are improved by optimizing the culture medium formula and culture conditions, and large-scale production of the high-quality target mRNA template plasmids is facilitated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and specifically relates to a method for fermenting Escherichia coli to produce mRNA template plasmid. BACKGROUND

[0002] mRNA template plasmid has a wide range of applications in biotechnology and medical fields, especially in gene therapy, vaccine development and protein production.

[0003] Escherichia coli is often used as a carrier microorganism for producing mRNA template plasmid due to its clear genetic background, rapid growth and low culture cost. When fermenting Escherichia coli to produce mRNA template plasmid, plasmid yield and plasmid monomer supercoiling content are key indicators, which directly affect the quality and efficiency of subsequent mRNA drugs. The existing technology mainly improves mRNA template plasmid from the following aspects: 1. Genetic engineering modification method, which is used for directional modification of host strains and optimization of plasmid backbone. This method has a long research and development cycle and the industrial application of genetically edited strains faces strict approval, so it is difficult to be used on a large scale in the short term. 2. Medium formula optimization, which is simple to operate, does not require equipment modification, has low cost and can be quickly applied. 3. Culture condition regulation, which can synergistically improve plasmid quality in combination with suitable culture medium. 4. Downstream process optimization, which improves the supercoiling ratio through different purification methods. The common method includes chromatography, which can only remove aggregated plasmids and open-loop plasmids in mRNA template plasmid extract, and cannot improve the yield of mRNA template plasmid.

[0004] Therefore, optimizing the yield and supercoiling content of mRNA template plasmid from the aspects of medium formula and culture conditions is the most cost-effective and easiest way to be applied on a large scale in the short term. SUMMARY

[0005] The purpose of the present application is to provide a method for fermenting Escherichia coli to produce mRNA template plasmid, which can simultaneously improve the yield and supercoiling ratio of mRNA template plasmid.

[0006] In order to achieve the above purpose, the present application provides the following technical scheme:

[0007] A method for fermenting Escherichia coli to produce mRNA template plasmid, wherein when fermenting the recombinant Escherichia coli, the seed liquid of the recombinant Escherichia coli is inoculated into the basic medium for fermentation culture until the OD 600when the value is 18-22, then continuously feeding the feed medium at a feeding rate of 5-15 mL / L / h to carry out continuous feeding fermentation culture, the recombinant E. coli is E. coli transformed by the mRNA template plasmid, the base medium contains iron salt compound 0.05-0.2 mmoL / L, copper chloride dihydrate 0.05-0.2 mmoL / L, p-hydroxybenzoic acid 50-200 mg / L, the carbon source of the base medium is glycerol 10-15 g / L, the feed medium contains iron salt compound 0.1-0.3 mmoL / L, copper chloride dihydrate 0.1-0.3 mmoL / L, p-hydroxybenzoic acid 100-300 mg / L, the carbon source of the feed medium is glycerol 360-480 g / L, and the iron salt compound is one or more of ferrous citrate, ferric ammonium citrate or ferrous sulfate heptahydrate.

[0008] In some embodiments, the formula of the base medium is: glycerol 10-15 g / L, yeast extract 10-30 g / L, yeast peptone 5-15 g / L, sodium chloride 0.3-0.7 g / L, ammonium sulfate 5-15 g / L, anhydrous magnesium sulfate 1-2 g / L, disodium hydrogen phosphate dodecahydrate 4-8 g / L, potassium hydrogen phosphate 2-5 g / L, kanamycin sulfate 0.03-0.07 g / L, antifoam agent 0.05-0.5 mL / L, iron salt compound 0.05-0.2 mmoL / L, copper chloride dihydrate 0.05-0.2 mmoL / L, p-hydroxybenzoic acid 50-200 mg / L, and the balance is deionized water.

[0009] In some embodiments, the formula of the feed medium is: glycerol 360-480 g / L, yeast extract 100-160 g / L, yeast peptone 40-80 g / L, ammonium sulfate 15-80 g / L, iron salt compound 0.1-0.3 mmoL / L, copper chloride dihydrate 0.1-0.3 mmoL / L, p-hydroxybenzoic acid 100-300 mg / L, and the balance is deionized water.

[0010] In some preferred embodiments, the formula of the base medium is: glycerol 10-15 g / L, yeast extract 15-25 g / L, yeast peptone 5-15 g / L, sodium chloride 0.3-0.7 g / L, ammonium sulfate 5-15 g / L, anhydrous magnesium sulfate 1-2 g / L, disodium hydrogen phosphate dodecahydrate 4-8 g / L, potassium hydrogen phosphate 2-5 g / L, kanamycin sulfate 0.03-0.07 g / L, antifoam agent 0.05-0.2 mL / L, iron salt compound 0.05-0.2 mmoL / L, copper chloride dihydrate 0.05-0.2 mmoL / L, p-hydroxybenzoic acid 50-200 mg / L, and the balance is deionized water.

[0011] In some preferred embodiments, the formula of the feed medium is: glycerol 360-480 g / L, yeast extract 150-160 g / L, yeast peptone 70-80 g / L, ammonium sulfate 15-30 g / L, iron salt compound 0.1-0.3 mmoL / L, copper chloride dihydrate 0.1-0.3 mmoL / L, p-hydroxybenzoic acid 100-300 mg / L, and the balance is deionized water.

[0012] In some preferred embodiments, the iron salt compound is ferrous citrate.

[0013] In some preferred embodiments, the E. coli is a Stable competent cell.

[0014] In some embodiments, the fermentation culture conditions are: inoculation volume ratio of 4%-6%, temperature of 36.5-37.5°C, pH of 6.8-7.0, dissolved oxygen of 30%-40%, aeration amount of 0.8-1.5 VVM, and rotation speed of 300-1000 r / min.

[0015] In some embodiments, the pH is adjusted using a phosphoric acid aqueous solution with a mass concentration of 15%-25% and a sodium hydroxide aqueous solution with a mass concentration of 300-500 g / L.

[0016] In some embodiments, the continuous feed fermentation culture lasts for 20-30 h.

[0017] In some preferred embodiments, when feeding, the feeding rate is first 5-5.5 mL / L / h for 8-10 h of fermentation, and then the feeding rate is sequentially 6.5-7.5 mL / L / h, 8.5-9.5 mL / L / h, 10.5-11.5 mL / L / h, 12.5-13.5 mL / L / h, and 14.5-15.5 mL / L / h for 2.5-3.5 h of fermentation, respectively.

[0018] According to some specific and preferred embodiments, the recombinant E. coli seed liquid is added to a glass fermentation tank at an inoculation volume ratio of 4%-6%, the fermentation temperature is set to 36.5-37.5°C, the pH is maintained at 6.8-7.0 by acid feeding and alkali feeding, the aeration amount is 0.8-1.5 VVM, the rotation speed is in the range of 300-1000 r / min, the dissolved oxygen is maintained at 30%-40% by association with the rotation speed, and the OD 600When the value is 18-22, first feed at a feed rate of 5-5.5 mL / L / h for 8-10 h, and then continue to feed at a feed rate of 6.5-7.5 mL / L / h, 8.5-9.5 mL / L / h, 10.5-11.5 mL / L / h, 12.5-13.5 mL / L / h, and 14.5-15.5 mL / L / h, respectively, for 2.5-3.5 h.

[0019] In some embodiments, the preparation method of the recombinant E. coli seed solution is as follows: inoculating the recombinant E. coli into a seed culture medium at a volume ratio of 0.05%-0.15%, and culturing at 36.5-37.5°C and 200-220 r / min for 12-16 hours to obtain the seed solution.

[0020] In some embodiments, the formula of the seed culture medium is as follows: yeast extract powder 3-7 g / L, yeast peptone 8-12 g / L, sodium chloride 8-12 g / L, kanamycin sulfate 0.03-0.07 g / L, and the rest is deionized water.

[0021] Due to the use of the above technical solutions, the present application has the following advantages compared with the prior art:

[0022] The present application optimizes the yield and supercoiling content of mRNA template plasmid from the aspects of culture medium formula and culture conditions, which is beneficial to large-scale production of high-quality target mRNA template plasmid. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure of mRNA template plasmid numbered as CT039. DETAILED DESCRIPTION

[0024] In order to realize high-density culture of E. coli to improve the yield and quality of mRNA template plasmid produced by E. coli (especially Stable strain) fermentation, the present inventors have conducted a large number of researches and found that during the fermentation of E. coli, the accumulation of organic acid (such as acetic acid) as a by-product due to the mismatch between the metabolism of E. coli and the fermentation formula and process, which leads to waste of carbon source and inhibits the growth of bacterial cells and the synthesis of the final product mRNA template plasmid. Therefore, it is tried to reduce the generation of acetic acid from the metabolic pathway of E. coli to improve the growth of bacterial cells and the generation of the final product plasmid.

[0025] According to the existing experimental results, glycerol as a slow-release carbon source has a lower rate of entering the glycolysis pathway than glucose, thus reducing the rate of generating pyruvate and reducing the generation of acetic acid. Adding appropriate amounts of iron salt compounds, copper chloride dihydrate and p-hydroxybenzoic acid to the medium with glycerol as the carbon source can improve the utilization rate of the carbon source, reduce the generation of acetic acid, and increase the yield of mRNA template plasmid produced by E. coli fermentation under the condition that the supercoiling ratio does not fluctuate significantly, so that more plasmids can be produced in a single batch, thereby reducing the overall production cost.

[0026] According to further experimental results, when the iron salt compound is ferrous citrate, it is more easily absorbed by E. coli, which is conducive to the maintenance of the enzyme activity of the E. coli respiratory electron transport chain-related enzyme system. In combination with other components in the medium, the fermentation by-products are further reduced to achieve high-density culture of E. coli, and the target plasmid supercoiling ratio is also improved.

[0027] Specifically, the method for fermenting mRNA template plasmid by E. coli in the present application is as follows: when fermenting the recombinant E. coli, the seed liquid of the recombinant E. coli is inoculated into the basic medium for fermentation until the OD 600 value of the fermentation broth is 18-22, and then the feed medium is continuously added at a feed rate of 5-15 mL / L / h for continuous fed-batch fermentation, = the recombinant E. coli is E. coli transformed by the mRNA template plasmid, the basic medium contains 0.05-0.2 mmol / L of iron salt compound, 0.05-0.2 mmol / L of copper chloride dihydrate, and 50-200 mg / L of p-hydroxybenzoic acid, the carbon source of the basic medium is 10-15 g / L of glycerol, the feed medium contains 0.1-0.3 mmol / L of iron salt compound, 0.1-0.3 mmol / L of copper chloride dihydrate, and 100-300 mg / L of p-hydroxybenzoic acid, the carbon source of the feed medium is 360-480 g / L of glycerol, and the iron salt compound is one or more of ferrous citrate, ferric ammonium citrate, or ferrous sulfate heptahydrate.

[0028] More specifically, the seed liquid of the recombinant E. coli is added to the glass fermenter at a volume ratio of 4%-6%, the fermentation temperature is set to 36.5-37.5°C, the pH is associated with acid and alkali feeding to maintain a stable pH of 6.8-7.0, the aeration rate is 0.8-1.5 VVM, the rotation speed is in the range of 300-1000 r / min, the dissolved oxygen content is associated with the rotation speed to maintain a stable dissolved oxygen content of 30%-40%, and the OD 600When the value is 18-22, feed at a feed rate of 5-5.5 mL / L / h for 8-10 h, and then continue to feed at a feed rate of 6.5-7.5 mL / L / h, 8.5-9.5 mL / L / h, 10.5-11.5 mL / L / h, 12.5-13.5 mL / L / h, 14.5-15.5 mL / L / h, respectively, for 2.5-3.5 h.

[0029] The application will be further described below in conjunction with examples. However, the application is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the application can be combined with each other as long as there is no conflict.

[0030] Some raw materials or reagents used in the examples and comparative examples of the application are as follows:

[0031] Yeast extract powder is from Angel Yeast Co., Ltd., item number FM885.

[0032] Yeast peptone is from Angel Yeast Co., Ltd., item number FP108.

[0033] Kanamycin sulfate is from Aladdin Reagent (Shanghai) Co., Ltd., item number K103024-25g.

[0034] Glucose is from National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 63005518.

[0035] Glycerol is from National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 10010618.

[0036] Ammonium sulfate is from National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 10002918.

[0037] Anhydrous magnesium sulfate is from National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 20025117.

[0038] Disodium hydrogen phosphate dodecahydrate is from National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 10020318.

[0039] Dipotassium hydrogen phosphate is from National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 51020460.

[0040] Defoaming agent is from Wuhan Ruimeite Chemical Co., Ltd., item number DOWFAX*DF104.

[0041] Copper chloride dihydrate is from National Pharmaceutical Group Chemical Reagent Co., Ltd., item number 10007818.

[0042] p-Hydroxybenzoic acid was from Shanghai Macklin Biochemical Technology Co., Ltd., item number H811078-100g.

[0043] Ammonium ferric citrate was from Sinopharm Chemical Reagent Co., Ltd., item number 30011428.

[0044] Ferrous citrate was fed in the form of aqueous solution, which was obtained by mixing ferrous sulfate heptahydrate solution and citric acid solution according to the molar ratio of ferrous sulfate heptahydrate to citric acid of 1:2. Before feeding, it was filtered through a 0.22 μm filter to remove bacteria. Ferrous sulfate heptahydrate was from Sinopharm Chemical Reagent Co., Ltd., item number 10012118. Citric acid was from Sinopharm Chemical Reagent Co., Ltd., item number 30196768.

[0045] Cuprous chloride dihydrate, p-hydroxybenzoic acid and karanjin sulfate were fed in the form of aqueous solution. Before feeding, they were filtered through a 0.22 μm filter to remove bacteria, and were added after the other components were mixed and sterilized at 121°C for 30 minutes.

[0046] The water used was deionized water prepared in the laboratory.

[0047] The recombinant Escherichia coli used in the following examples and comparative examples was a glycerol bacterium preserved in the laboratory, which was prepared by infecting Escherichia coli competent cells Stable cells with an mRNA template plasmid numbered CT039 with a size of 7327 bp.

[0048] The spectrum of the mRNA template plasmid numbered CT039 is shown in Figure 1 which is a pET series plasmid containing a T7 promoter.

[0049] The remaining raw materials or reagents were commercially available products unless otherwise specified.

[0050] In this application, the operation methods involved in each example and comparative example are conventional methods in the art unless otherwise specified.

[0051] Example 1: This example provides a method for fermenting Escherichia coli to produce an mRNA template plasmid, which is as follows:

[0052] Preparation of seed liquid: The glycerol bacterium was inoculated into 150 mL of seed culture medium at a volume ratio of 0.1%, and cultured at 37°C, 220 rpm for 16 hours to obtain the seed liquid.

[0053] Fermentation production: 3L base medium was added to a 5L glass fermenter, and the seed liquid was inoculated into the glass fermenter at a volume ratio of 5%, the fermentation temperature was set to 37±0.2℃, the pH was maintained at 6.9±0.1 by acid and alkali feeding, the aeration rate was 1VVM, and the rotation speed was in the range of 300-1000 rpm / min. The initial 100% dissolved oxygen calibration conditions were: temperature 37℃, aeration rate 1VVM, and rotation speed 300 rpm / min. The dissolved oxygen was maintained at 30%-40% by correlating the rotation speed. The feeding strategy was: when the OD of the fermentation broth reached 20, feeding was started at a feeding rate of 5mL / L / h for 9h (from inoculation, the fermentation time was calculated), then the feeding rate was sequentially increased to 7mL / L / h, 9mL / L / h, 11mL / L / h, 13mL / L / h, and 15mL / L / h for 3h respectively, after stopping the feeding, sampling was detected, and the total fermentation time was 24h. 600 The dissolved oxygen content in the fermentation stage was 30%-40%, which means that the dissolved oxygen (DO) concentration was controlled to reach 30%-40% air saturation during the fermentation process. The dissolved oxygen content in the fermentation stage was monitored in real time by an automatic control system, and the stirring speed was adjusted dynamically to maintain the dissolved oxygen within the preset range.

[0054] The dissolved oxygen content in the fermentation stage was 30%-40%, which means that the dissolved oxygen (DO) concentration was controlled to reach 30%-40% air saturation during the fermentation process. The dissolved oxygen content in the fermentation stage was monitored in real time by an automatic control system, and the stirring speed was adjusted dynamically to maintain the dissolved oxygen within the preset range.

[0055] The medium formula involved is as follows:

[0056] Seed medium: yeast extract powder 5g / L, yeast peptone 10g / L, sodium chloride 10g / L, kanamycin sulfate 0.05g / L, and the rest is deionized water.

[0057] Base medium: glycerol 12g / L, yeast extract powder 20g / L, yeast peptone 10g / L, sodium chloride 0.5g / L, ammonium sulfate 10g / L, anhydrous magnesium sulfate 1.5g / L, disodium hydrogen phosphate dodecahydrate 6g / L, potassium hydrogen phosphate 3g / L, kanamycin sulfate 0.05g / L, antifoam agent 0.1mL / L, ferrous citrate 0.1mmoL / L, copper chloride dihydrate 0.1mmoL / L, p-hydroxybenzoic acid 100mg / L, and the rest is deionized water.

[0058] Feeding medium: glycerol 422g / L, yeast extract powder 160g / L, yeast peptone 80g / L, ammonium sulfate 20g / L, ferrous citrate 0.2mmoL / L, copper chloride dihydrate 0.2mmoL / L, p-hydroxybenzoic acid 200mg / L, and the rest is deionized water.

[0059] The acid and alkali feeding for maintaining pH is as follows:

[0060] Acid feed: 20% phosphoric acid in water.

[0061] Base feed: 400 g / L sodium hydroxide in water.

[0062] Example 2: This example provides another method for fermenting E. coli to produce mRNA template plasmid, which is substantially the same as Example 1, except that the feed medium formulation is slightly different from Example 1. The feed medium formulation in this example is as follows:

[0063] Feed medium: glycerol 422 g / L, yeast extract 120 g / L, yeast peptone 60 g / L, ammonium sulfate 20 g / L, ferrous citrate 0.2 mmoL / L, copper chloride dihydrate 0.2 mmoL / L, p-hydroxybenzoic acid 200 mg / L, and the balance deionized water.

[0064] Example 3: This example provides another method for fermenting E. coli to produce mRNA template plasmid, which is substantially the same as Example 1, except that the base medium and feed medium formulations are slightly different from Example 1. The base medium and feed medium formulations in this example are as follows:

[0065] Base medium: glycerol 12 g / L, yeast extract 20 g / L, yeast peptone 10 g / L, sodium chloride 0.5 g / L, ammonium sulfate 10 g / L, magnesium sulfate anhydrous 1.5 g / L, disodium phosphate dodecahydrate 6 g / L, potassium phosphate dibasic 3 g / L, kanamycin sulfate 0.05 g / L, antifoam 0.1 mL / L, ferric ammonium citrate 0.1 mmoL / L, copper chloride dihydrate 0.1 mmoL / L, p-hydroxybenzoic acid 100 mg / L, and the balance deionized water.

[0066] Feed medium: glycerol 422 g / L, yeast extract 120 g / L, yeast peptone 60 g / L, ammonium sulfate 20 g / L, ferric ammonium citrate 0.2 mmoL / L, copper chloride dihydrate 0.2 mmoL / L, p-hydroxybenzoic acid 200 mg / L, and the balance deionized water.

[0067] Example 4: This example provides another method for fermenting E. coli to produce mRNA template plasmid, which is substantially the same as Example 1, except that the base medium and feed medium formulations are slightly different from Example 1. The base medium and feed medium formulations in this example are as follows:

[0068] Base medium: glycerol 12 g / L, yeast extract 20 g / L, yeast peptone 10 g / L, sodium chloride 0.5 g / L, ammonium sulfate 10 g / L, magnesium sulfate anhydrous 1.5 g / L, disodium phosphate 12H20 6 g / L, potassium phosphate dibasic 3 g / L, kanamycin sulfate 0.05 g / L, antifoam 0.1 mL / L, ferrous sulfate heptahydrate 0.1 mMOL / L, copper chloride dihydrate 0.1 mMOL / L, p-hydroxybenzoic acid 100 mg / L, the balance being deionized water.

[0069] Feed medium: glycerol 422 g / L, yeast extract 120 g / L, yeast peptone 60 / L, ammonium sulfate 20 g / L, ferrous sulfate heptahydrate 0.2 mMOL / L, copper chloride dihydrate 0.2 mMOL / L, p-hydroxybenzoic acid 200 mg / L, the balance being deionized water.

[0070] Example 5: This example provides another method for fermenting E. coli to produce mRNA template plasmid, which is substantially the same as Example 4, except that the feed medium formulation is slightly different from Example 4. The feed medium formulation in this example is as follows:

[0071] Feed medium: glycerol 422 g / L, yeast extract 120 g / L, yeast peptone 60 / L, ammonium sulfate 40 g / L, ferrous sulfate heptahydrate 0.2 mMOL / L, copper chloride dihydrate 0.2 mMOL / L, p-hydroxybenzoic acid 200 mg / L, the balance being deionized water.

[0072] Example 6: This example provides another method for fermenting E. coli to produce mRNA template plasmid, which is substantially the same as Example 4, except that the feed medium formulation is slightly different from Example 4. The feed medium formulation in this example is as follows:

[0073] Feed medium: glycerol 422 g / L, yeast extract 120 g / L, yeast peptone 60 / L, ammonium sulfate 60 g / L, ferrous sulfate heptahydrate 0.2 mMOL / L, copper chloride dihydrate 0.2 mMOL / L, p-hydroxybenzoic acid 200 mg / L, the balance being deionized water.

[0074] Example 7: This example provides another method for fermenting E. coli to produce mRNA template plasmid, which is substantially the same as Example 4, except that the feed medium formulation is slightly different from Example 4. The feed medium formulation in this example is as follows:

[0075] Feed medium: glycerol 422 g / L, yeast extract 120 g / L, yeast peptone 60 / L, ammonium sulfate 80 g / L, ferrous sulfate heptahydrate 0.2 mMOL / L, copper chloride dihydrate 0.2 mMOL / L, p-hydroxybenzoic acid 200 mg / L, the balance being deionized water.

[0076] Comparative Example 1: This comparative example provides another method for fermenting E. coli to produce mRNA template plasmid, which is basically the same as Example 1, the only difference is that the base medium and the feed medium formula are slightly different from Example 1. The base medium and the feed medium formula in this comparative example are as follows:

[0077] Base medium: glucose 12 g / L, yeast extract 20 g / L, yeast peptone 10 g / L, sodium chloride 0.5 g / L, ammonium sulfate 10 g / L, anhydrous magnesium sulfate 1.5 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 3 g / L, kanamycin sulfate 0.05 g / L, antifoam agent 0.1 mL / L, the balance is deionized water.

[0078] Feed medium: glucose 422 g / L, yeast extract 120 g / L, yeast peptone 60 / L, ammonium sulfate 20 g / L, the balance is deionized water.

[0079] Comparative Example 2: This comparative example provides another method for fermenting E. coli to produce mRNA template plasmid, which is basically the same as Example 1, the only difference is that the base medium and the feed medium formula are slightly different from Example 1. The base medium and the feed medium formula in this comparative example are as follows:

[0080] Base medium: glycerol 12 g / L, yeast extract 20 g / L, yeast peptone 10 g / L, sodium chloride 0.5 g / L, ammonium sulfate 10 g / L, anhydrous magnesium sulfate 1.5 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 3 g / L, kanamycin sulfate 0.05 g / L, antifoam agent 0.1 mL / L, the balance is deionized water.

[0081] Feed medium: glycerol 422 g / L, yeast extract 120 g / L, yeast peptone 60 / L, ammonium sulfate 20 g / L, the balance is deionized water.

[0082] The fermentation broth samples of each of the above examples and comparative examples are detected according to the following detection methods:

[0083] 1. OD 600 : Adjust the spectrophotometric detection value to 600, add the detection value of purified water as zero control in a cuvette, then dilute the fermentation broth sample several times and add it to the cuvette so that the detection value is between 0.2-0.8, and the OD value of the fermentation broth is calculated by dilution times. The bacterial cell density is positively correlated with OD 600 (when OD 600 =1, the concentration of E. coli is 10 8 cells / mL), and the OD 600 detection method is simple and fast.

[0084] 2. Plasmid expression (mg / L): The plasmid expression (i.e. plasmid yield) was detected by using the plasmid extraction kit (DP103) of Tiangen Biochemical Technology (Beijing) Co., Ltd. according to the instructions. 50 microliters of the fermentation broth was centrifuged to collect the bacterial cells, and then the plasmid was extracted by using the kit. The concentration was detected after elution with 200 microliters of ultrapure water. The 4-fold detection concentration was the volume expression (mg / L).

[0085] 3. Calculation of unit bacterial expression: unit bacterial expression = plasmid expression / OD 600 .

[0086] 4. Monomer supercoiling ratio: The plasmid monomer supercoiling content was detected by using agarose gel electrophoresis (AGE method).

[0087] The detection results are shown in Table 1.

[0088] Table 1

[0089]

[0090] The alkali feed amount (mL) of each example and each comparative example is shown in Table 2.

[0091] Table 2

[0092]

[0093] As can be seen from Table 1 and Table 2, compared with using glucose as the slow-release carbon source, the carbon source utilization rate is higher, the acidic substances produced are less, the bacterial growth is better, and the plasmid yield is higher when glycerol is used as the slow-release carbon source.

[0094] Further, after adding ferrous sulfate heptahydrate, copper chloride dihydrate, and p-hydroxybenzoic acid and the like in the fermentation medium with glycerol as the carbon source, the alkali feed is further reduced. The addition of these three substances is beneficial to the respiratory chain electron transfer in the fermentation process of Escherichia coli, and the effective utilization of the carbon source increases the generation of precursor substances required for plasmid synthesis, so that the plasmid yield is further increased. At the same time, an appropriate amount of ammonium sulfate in the feed is beneficial to the growth and metabolism of the bacteria, thereby improving the plasmid yield. The addition of high-concentration ammonium sulfate leads to an imbalance of the carbon-nitrogen ratio, resulting in an excess of nitrogen source, and the cells lack sufficient carbon skeleton to synthesize corresponding proteins and nucleic acid precursors and the like, causing the bacterial concentration and plasmid yield to decrease.

[0095] Further, the ferrous ion is more easily absorbed and utilized by Escherichia coli after being chelated with citric acid, and the absorption and utilization rate of ferrous ion is higher than that of ferric ion. With the significant increase in plasmid yield, the monomer supercoiling ratio is also increased to 90% or above.

[0096] The above embodiments are only the preferred embodiments of the present application, and are not used to limit the protection scope of the present application, and the modifications or improvements made without departing from the spirit of the present application shall fall within the scope of protection of the present application.

Claims

1. A method for fermentative production of mRNA template plasmid by Escherichia coli, characterized by, In the fermentation culture of the recombinant E. coli, the seed liquid of the recombinant E. coli is inoculated into the base medium to carry out the fermentation culture until the OD value of the fermentation liquid is 18-22, and then the continuous feeding medium is continuously added at a feeding rate of 5-15 mL / L / h to carry out the continuous feeding fermentation culture, 600 18-22, and then the continuous feeding medium is continuously added at a feeding rate of 5-15 mL / L / h to carry out the continuous feeding fermentation culture, The recombinant E. coli is E. coli transformed by mRNA template plasmid, The base medium contains 0.05-0.2 mmoL / L of iron salt compound, 0.05-0.2 mmoL / L of copper chloride dihydrate, 50-200 mg / L of p-hydroxybenzoic acid, and the carbon source of the base medium is 10-15 g / L of glycerol, The feeding medium contains 0.1-0.3 mmoL / L of iron salt compound, 0.1-0.3 mmoL / L of copper chloride dihydrate, 100-300 mg / L of p-hydroxybenzoic acid, and the carbon source of the feeding medium is 360-480 g / L of glycerol, The iron salt compound is one or more of ferrous citrate, ammonium iron citrate or ferrous sulfate heptahydrate.

2. The method of fermenting production of mRNA template plasmid by E. coli according to claim 1, characterized in that, The base medium is formulated as follows: 10-15 g / L of glycerol, 10-30 g / L of yeast extract powder, 5-15 g / L of yeast protein peptone, 0.3-0.7 g / L of sodium chloride, 5-15 g / L of ammonium sulfate, 1-2 g / L of anhydrous magnesium sulfate, 4-8 g / L of disodium hydrogen phosphate dodecahydrate, 2-5 g / L of potassium hydrogen phosphate, 0.03-0.07 g / L of kanamycin sulfate, 0.05-0.5 mL / L of antifoaming agent, 0.05-0.2 mmoL / L of iron salt compound, 0.05-0.2 mmoL / L of copper chloride dihydrate, 50-200 mg / L of p-hydroxybenzoic acid, and the rest is deionized water. The feeding medium is formulated as follows: 360-480 g / L of glycerol, 100-160 g / L of yeast extract powder, 40-80 g / L of yeast protein peptone, 15-80 g / L of ammonium sulfate, 0.1-0.3 mmoL / L of iron salt compound, 0.1-0.3 mmoL / L of copper chloride dihydrate, 100-300 mg / L of p-hydroxybenzoic acid, and the rest is deionized water.

3. The method of fermenting production of mRNA template plasmid by E. coli according to claim 1, characterized in that, The base medium is formulated as follows: 10-15 g / L of glycerol, 15-25 g / L of yeast extract powder, 5-15 g / L of yeast protein peptone, 0.3-0.7 g / L of sodium chloride, 5-15 g / L of ammonium sulfate, 1-2 g / L of anhydrous magnesium sulfate, 4-8 g / L of disodium hydrogen phosphate dodecahydrate, 2-5 g / L of potassium hydrogen phosphate, 0.03-0.07 g / L of kanamycin sulfate, 0.05-0.2 mL / L of antifoaming agent, 0.05-0.2 mmoL / L of iron salt compound, 0.05-0.2 mmoL / L of copper chloride dihydrate, 50-200 mg / L of p-hydroxybenzoic acid, and the rest is deionized water. The feeding medium is formulated as follows: 360-480 g / L of glycerol, 150-160 g / L of yeast extract powder, 70-80 g / L of yeast protein peptone, 15-30 g / L of ammonium sulfate, 0.1-0.3 mmoL / L of iron salt compound, 0.1-0.3 mmoL / L of copper chloride dihydrate, 100-300 mg / L of p-hydroxybenzoic acid, and the rest is deionized water.

4. The method of fermentative production of mRNA template plasmid by E. coli according to any one of claims 1 to 3, characterized in that, The iron salt compound is ferrous citrate; And / or, the E. coli is Stable competent cell.

5. The method of fermenting an mRNA template plasmid by E. coli according to claim 1, wherein, The fermentation culture conditions are as follows: inoculation volume ratio 4%-6%, temperature 36.5-37.5 DEG C, pH 6.8-7.0, dissolved oxygen 30%-40%, ventilation volume 0.8-1.5 VVM, rotation speed 300-1000 r / min.

6. The method of fermenting an mRNA template plasmid by E. coli according to claim 5, wherein, The pH is adjusted by using 15%-25% phosphoric acid aqueous solution and 300-500 g / L sodium hydroxide aqueous solution.

7. The method of fermenting production of mRNA template plasmid by E. coli according to claim 1, characterized in that, The continuous feeding fermentation culture time is 20-30 h.

8. The method of fermenting production of mRNA template plasmid by E. coli according to claim 1, characterized in that, During feeding, first, the feeding rate is 5-5.5 mL / L / h for 8-10 h, then the feeding rate is sequentially 6.5-7.5 mL / L / h, 8.5-9.5 mL / L / h, 10.5-11.5 mL / L / h, 12.5-13.5 mL / L / h, 14.5-15.5 mL / L / h for 2.5-3.5 h.

9. The method of fermenting production of mRNA template plasmid by E. coli according to claim 1, characterized in that, The recombinant E. coli seed liquid is added into the glass fermenter according to the inoculation amount of 4%-6% by volume, the fermentation temperature is set to 36.5-37.5℃, the pH is maintained at 6.8-7.0 by acid and alkali feeding, the aeration amount is 0.8-1.5 VVM, the rotation speed is in the range of 300-1000 r / min, the dissolved oxygen amount is associated with the rotation speed, and the dissolved oxygen amount is maintained at 30%-40%. When the OD value of the fermentation liquid is 18-22, the feeding rate is first 5-5.5 mL / L / h for 8-10 h, and then the feeding rate is sequentially 6.5-7.5 mL / L / h, 8.5-9.5 mL / L / h, 10.5-11.5 mL / L / h, 12.5-13.5 mL / L / h, and 14.5-15.5 mL / L / h for 2.5-3.5 h, respectively. 600 The recombinant E. coli seed liquid is added into the glass fermenter according to the inoculation amount of 4%-6% by volume, the fermentation temperature is set to 36.5-37.5℃, the pH is maintained at 6.8-7.0 by acid and alkali feeding, the aeration amount is 0.8-1.5 VVM, the rotation speed is in the range of 300-1000 r / min, the dissolved oxygen amount is associated with the rotation speed, and the dissolved oxygen amount is maintained at 30%-40%. When the OD value of the fermentation liquid is 18-22, the feeding rate is first 5-5.5 mL / L / h for 8-10 h, and then the feeding rate is sequentially 6.5-7.5 mL / L / h, 8.5-9.5 mL / L / h, 10.5-11.5 mL / L / h, 12.5-13.5 mL / L / h, and 14.5-15.5 mL / L / h for 2.5-3.5 h, respectively.

10. The method of fermentative production of mRNA template plasmid by E. coli according to claim 9, characterized in that, The preparation method of the recombinant E. coli seed liquid is as follows: the recombinant E. coli is inoculated into seed culture medium at a volume ratio of 0.05%-0.15%, and then cultured at 36.5-37.5 DEG C and 200-220 r / min for 12-16 hours to obtain the seed liquid. The formula of the seed culture medium is as follows: yeast extract powder 3-7 g / L, yeast peptone 8-12 g / L, sodium chloride 8-12 g / L, kanamycin sulfate 0.03-0.07 g / L, and the rest is deionized water.

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