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, which comprises the following steps: firstly, inoculating a recombinant escherichia coli seed solution into a basic culture medium, fermenting and culturing until the OD600 value is 18-22, and then continuously adding a fed-batch culture medium at the feeding rate of 11-23 mL / L / h, the seed culture medium contains 1-5 mmoL / L of metal ion chelating agent and 0.5-1.5 g / L of anhydrous magnesium sulfate, the carbon source of the basic culture medium is 15-25 g / L of glycerol, and the pH value of the basic culture medium is 7-8. The culture medium contains 3-21 mmoL / L of a metal ion chelating agent and 1.5-4.5 g / L of anhydrous magnesium sulfate, the carbon source of the fed-batch culture medium is 260-450 g / L of glycerol, and the metal ion chelating agent is preferably sodium citrate. The method optimizes and reduces the aggregate superhelix content of the mRNA template plasmid from the aspects of culture medium formula and culture condition, and is beneficial to large-scale production of high-quality target mRNA template plasmid.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for producing mRNA template plasmids by fermentation of Escherichia coli. Background Technology

[0002] mRNA template plasmids have wide applications in biotechnology and medicine, especially in gene therapy, vaccine development, and protein production. mRNA template plasmids are primarily produced through fermentation using recombinant bacterial strains.

[0003] Escherichia coli (E. coli) is frequently used as a vector microorganism for producing mRNA template plasmids due to its clear genetic background, rapid growth, and low culture cost. When producing template mRNA plasmids through E. coli fermentation, the main components of the plasmid extract obtained after lysis of the fermentation broth include open circular plasmids (ocDNA), supercoiled plasmid monomers (scDNA), and plasmid aggregates (such as dimers and trimers). Among these, supercoiled plasmid monomers represent the ideal conformation, exhibiting high transfection efficiency and stability; pharmaceutical plasmids typically require a content ≥80% of ocDNA. Open circular plasmids, formed from single-strand breaks, are less stable than scDNA. They can be removed by optimizing lysis conditions and using chromatographic purification techniques, which are relatively easy and mature, thus having minimal impact on the function of the mRNA template plasmid and being easily overcome. Plasmid aggregates, formed during fermentation due to homologous recombination or replication errors, are tandem repeats or multimers that significantly affect the function of the mRNA template plasmid. Furthermore, due to their similar conformation and charge characteristics to monomers, plasmid aggregates are difficult to separate during subsequent purification, placing significant pressure on downstream purification process development. Furthermore, the linearization stage of plasmids also leads to a significant increase in the amount of linearizing enzymes used, which increases the manufacturing cost of mRNA template plasmids.

[0004] Chromatographic packing materials used in the downstream purification stage are typically expensive, while fermentation media are relatively inexpensive. Optimizing the culture medium formulation and conditions during fermentation to reduce the proportion of plasmid aggregates in the fermentation lysate is the most cost-effective and readily applicable method for large-scale application in the short term. Therefore, reducing the proportion of plasmid aggregates during fermentation is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing mRNA template plasmids by Escherichia coli fermentation that can reduce aggregate production.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for producing mRNA template plasmids by fermentation of Escherichia coli involves first inoculating the seed culture of recombinant E. coli into a basal culture medium for fermentation until the OD of the fermentation broth reaches a certain level.600 The value was 18–22, and then the feed medium was continuously added at a feeding rate of 11–23 mL / L / h for continuous fed fermentation culture.

[0008] The recombinant Escherichia coli is Escherichia coli transformed by an mRNA template plasmid;

[0009] The basic culture medium is formulated as follows: glycerol 10-15 g / L, metal ion chelating agent 1-25 mmol / L, yeast extract 15-25 g / L, yeast peptone 5-15 g / L, sodium chloride 0.2-0.8 g / L, ammonium sulfate 5-15 g / L, anhydrous magnesium sulfate 1-5 g / L, disodium hydrogen phosphate dodecahydrate 4-8 g / L, dipotassium hydrogen phosphate 1-5 g / L, antifoaming agent 0.1 mL / L, ferrous sulfate heptahydrate 0.05-0.2 mmol / L, copper chloride dihydrate 0.05-0.2 mmol / L, p-hydroxybenzoic acid 50-200 mg / L, with the balance being deionized water. The metal ion chelating agent is one or more of sodium citrate, sodium gluconate, and disodium edetate.

[0010] The formula of the supplemental culture medium is as follows: glycerol 260-450 g / L, yeast extract 100-250 g / L, ammonium sulfate 15-25 g / L, disodium hydrogen phosphate dodecahydrate 4-8 g / L, dipotassium hydrogen phosphate 1-5 g / L, and the balance is deionized water.

[0011] This invention improves the culture medium formulation used in the fermentation of recombinant *E. coli* to reduce plasmid aggregate content during the fermentation stage. Extensive research and experimental verification have shown that, under the fermentation conditions employed in this invention, the optimized culture medium formulation not only provides sufficient precursor substances and energy for *E. coli* fermentation but also avoids metabolic overflow due to excessive amounts of certain components, preventing byproducts such as acetic acid from inhibiting cell growth and metabolism during fermentation and ensuring smooth plasmid replication.

[0012] Magnesium ions are an essential cofactor for DNA polymerase, which directly participates in catalyzing the formation of phosphodiester bonds from nucleotides, enabling plasmid replication. Magnesium ions are also a core cofactor for topoisomerase activity. Topoisomerase is responsible for introducing negative supercoiling, uncoiling, and decoiling during plasmid replication, playing a crucial role in maintaining a high level of supercoiled structure and reducing aggregate proportions. However, adding appropriate amounts of magnesium ions to the fermentation medium can affect the bioavailability of magnesium ions due to the use of phosphates and inorganic nitrogen sources such as ammonium sulfate or ammonium chloride to promote cell growth and metabolism, thus hindering the stability of the supercoiled structure of plasmid monomers. This invention addresses this issue by adding appropriate amounts of metal ion chelators to a specific fermentation medium to bind with magnesium ions. Sodium citrate, sodium gluconate, and disodium edetate are metal ion chelators that have been experimentally verified to improve the absorption rate of magnesium ions by *E. coli* when used in the specific fermentation medium of this invention.

[0013] The carbon and organic nitrogen source content in the supplemental culture medium can affect plasmid yield and the proportion of plasmid monomers forming helices. By optimizing the dosage of each component, the culture medium can maximize nutrient utilization while reducing the proportion of aggregates through the synergistic effect between the components.

[0014] According to some preferred embodiments, the metal ion chelating agent is sodium citrate, which has the best effect on promoting the absorption of magnesium ions by Escherichia coli.

[0015] According to some preferred embodiments, the content of the metal ion chelating agent in the basal culture medium is 3-5 mmol / L, and the content of anhydrous magnesium sulfate is 1-2 g / L. Higher contents of the metal ion chelating agent and anhydrous magnesium sulfate do not further reduce the proportion of plasmid aggregates; instead, they increase the cost of the culture medium.

[0016] According to some preferred embodiments, the Escherichia coli is a stable competent cell.

[0017] According to some preferred embodiments, the mRNA template plasmid is a pET series plasmid.

[0018] According to some preferred embodiments, the mRNA template plasmid is less than 10,000 bp. More preferably, the size of the mRNA template plasmid is 5,000–9,000 bp, and even more preferably 6,000–8,000 bp.

[0019] According to some preferred embodiments, the fermentation culture conditions are as follows: inoculum volume ratio of 4% to 6%, temperature of 36.5 to 37.5°C, pH of 6.8 to 7.0, dissolved oxygen of 30% to 40%, and aeration rate of 0.8 to 1.5 VVM.

[0020] According to some preferred embodiments, the duration of the continuous fed fermentation culture is 20 to 30 hours.

[0021] In some embodiments, during the fermentation culture of recombinant Escherichia coli, the pH of the fermentation system is controlled to be 6.8–7.0 by acid feeding and alkali feeding. The acid feed is a 15%–25% phosphoric acid aqueous solution, a 15%–25% phosphoric acid aqueous solution containing 150–250 g / L glycerol, or a 15%–25% phosphoric acid aqueous solution containing 150–250 g / L glucose. The alkali feed is a 15%–25% ammonia aqueous solution or a 15%–25% ammonia aqueous solution containing 80–150 g / L yeast extract.

[0022] According to some preferred embodiments, the acid feed is a phosphoric acid aqueous solution containing 150-250 g / L glycerol at a mass fraction of 15%-25%; the alkali feed is an ammonia aqueous solution containing 80-150 g / L yeast extract at a mass fraction of 15%-25%. By simultaneously adding carbon sources with the acid feed and organic nitrogen sources with the alkali feed, dynamic regulation of carbon and organic nitrogen sources during fermentation is achieved. Combined with the basal fermentation medium and the fed fermentation medium, this further increases the expression level per unit cell and further reduces plasmid aggregates.

[0023] According to some preferred embodiments, the feed culture medium is formulated as follows: glycerol 280-350 g / L, yeast extract 160-200 g / L, ammonium sulfate 15-25 g / L, disodium hydrogen phosphate dodecahydrate 4-8 g / L, dipotassium hydrogen phosphate 1-5 g / L, with the remainder being deionized water.

[0024] In some embodiments, the recombinant *E. coli* seed culture is prepared by inoculating 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 15–18 hours to obtain the seed culture. The seed culture medium has the following formula: 15–25 g / L glycerol, 15–25 g / L yeast extract, 2–4 mmol / L sodium citrate, 5–15 g / L yeast peptone, 0.2–0.8 g / L sodium chloride, 2–5 g / L ammonium sulfate, 0.5–1.5 g / L anhydrous magnesium sulfate, 4–8 g / L disodium hydrogen phosphate dodecahydrate, 1–5 g / L dipotassium hydrogen phosphate, and the remainder being deionized water. Existing experimental results show that the more nutrient-rich seed culture medium used in this invention is beneficial for enhancing seed activity, increasing the expression level per unit cell, and reducing the proportion of plasmid aggregates.

[0025] A second aspect of the present invention also provides a culture medium composition for the fermentation production of mRNA template plasmids by Escherichia coli, the culture medium composition comprising a basal culture medium and a feed medium, and optionally including a seed culture medium, wherein the basal culture medium is the basal culture medium described above, the feed medium is the feed medium described above, and the seed culture medium is the seed culture medium described above.

[0026] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0027] This invention optimizes the culture medium formulation and culture conditions to reduce the production of mRNA template plasmids and plasmid aggregates during the fermentation stage. This is beneficial for the large-scale production of high-quality target mRNA template plasmids, and can better improve the yield of subsequent purification steps and the amount of enzyme used during linearization digestion, thereby reducing the overall production cost. Attached Figure Description

[0028] Figure 1 This is a map of the mRNA template plasmid numbered CT039. Detailed Implementation

[0029] To increase the expression level of plasmid per unit cell and reduce the proportion of plasmid aggregates during the fermentation stage, thereby providing a higher quality fermentation broth containing the target mRNA template plasmid, the inventors of this application have conducted extensive research and optimization on the culture medium formulation and culture conditions used in the fermentation culture of recombinant *E. coli*. This provides a method for producing mRNA template plasmids through *E. coli* fermentation that reduces aggregate formation. Specifically, during the fermentation culture of recombinant *E. coli*, the seed culture of recombinant *E. coli* is first inoculated into a basal culture medium for fermentation culture until the OD of the fermentation broth reaches a certain level. 600The value was 18-22, and then fed culture medium was continuously added at a feeding rate of 11-23 mL / L / h for continuous fed fermentation culture. The recombinant *E. coli* was *E. coli* transformed with an mRNA template plasmid. The basal culture medium formula was: 10-15 g / L glycerol, 1-25 mmol / L metal ion chelating agent, 15-25 g / L yeast extract, 5-15 g / L yeast peptone, 0.2-0.8 g / L sodium chloride, 5-15 g / L ammonium sulfate, 1-5 g / L anhydrous magnesium sulfate, 4-8 g / L disodium hydrogen phosphate dodecahydrate, and 1-5 g / L dipotassium hydrogen phosphate. / L, defoamer 0.1mL / L, ferrous sulfate heptahydrate 0.05-0.2mmol / L, copper chloride dihydrate 0.05-0.2mmol / L, p-hydroxybenzoic acid 50-200mg / L, balance deionized water; the metal ion chelating agent is one or more of sodium citrate, sodium gluconate, and disodium edetate; the feed medium formula is: glycerol 260-450g / L, yeast extract 100-250g / L, ammonium sulfate 15-25g / L, disodium hydrogen phosphate dodecahydrate 4-8g / L, dipotassium hydrogen phosphate 1-5g / L, balance deionized water.

[0030] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0031] The sources of some of the raw materials or reagents used in the embodiments and comparative examples of this invention are as follows:

[0032] The yeast extract powder is from Angel Yeast Co., Ltd., product number FM885.

[0033] The yeast peptone is from Angel Yeast Co., Ltd., product number FP108.

[0034] The glucose is from Sinopharm Chemical Reagent Co., Ltd., product number 63005518.

[0035] Glycerin is from Sinopharm Chemical Reagent Co., Ltd., product number 10010618.

[0036] The ammonium sulfate is from Sinopharm Chemical Reagent Co., Ltd., product number 10002918.

[0037] Anhydrous magnesium sulfate is from Sinopharm Chemical Reagent Co., Ltd., product number 20025117.

[0038] The disodium hydrogen phosphate dodecahydrate is from Sinopharm Chemical Reagent Co., Ltd., product number 10020318.

[0039] The dipotassium hydrogen phosphate is from Sinopharm Chemical Reagent Co., Ltd., product number 51020460.

[0040] The defoamer is from Wuhan Ruimeite Chemical Co., Ltd., product number DOWFAX*DF104.

[0041] Copper chloride dihydrate is from Sinopharm Chemical Reagent Co., Ltd., product number 10007818.

[0042] p-Hydroxybenzoic acid is from Shanghai Maclean Biochemical Technology Co., Ltd., product number H811078.

[0043] Sodium citrate is from Hubei Gedian Renfu Pharmaceutical Co., Ltd., product number unknown.

[0044] Sodium gluconate is from Sinopharm Chemical Reagent Co., Ltd., product number 63010838.

[0045] Disodium edetate, Hunan Ercon Pharmaceutical Co., Ltd.

[0046] Ferrous sulfate heptahydrate is from Sinopharm Chemical Reagent Co., Ltd., product number 10012118.

[0047] Cuprous chloride dihydrate, ferrous sulfate heptahydrate, and p-hydroxybenzoic acid were added separately in aqueous solution form. The solutions were sterilized by filtration through a 0.22 μm filter before addition, and were added after mixing with other components and sterilizing at 121°C for 30 minutes.

[0048] The water used was all deionized water prepared in the laboratory.

[0049] The recombinant *E. coli* used in the following examples and comparative examples were laboratory-preserved glycerol-containing bacteria. These recombinant *E. coli* were prepared by infecting Stable competent *E. coli* cells with a 7327 bp mRNA template plasmid numbered CT039. The chromatogram is shown below. Figure 1 It is a pET series plasmid containing the T7 promoter.

[0050] Unless otherwise specified, all other raw materials or reagents are commercially available products.

[0051] Unless otherwise specified, the operating methods involved in the embodiments and comparative examples in this application are conventional methods in the art.

[0052] Example 1

[0053] This embodiment provides a method for producing mRNA template plasmids by Escherichia coli fermentation, as detailed below:

[0054] Preparation of seed culture: Glycerin bacteria were inoculated into 150 mL of seed culture medium at a volume ratio of 0.1% and cultured at 37℃ and 220 rpm for 16 hours to obtain seed culture.

[0055] Fermentation Production: 3L of basal culture medium was added to a 5L glass fermenter. Seed culture was added at a volume ratio of 5%. The fermentation temperature was set at 37±0.2℃. pH was maintained stable at 6.9±0.1 through acid and alkali feeding, correlated with pH. Aeration rate was 1 VVM, and the turbine rotation speed ranged from 300 to 1000 rpm. Initial 100% dissolved oxygen calibration conditions were: temperature 37℃, aeration rate 1 VVM, and turbine rotation speed 300 rpm. Dissolved oxygen was maintained stable at 30%–40% by turbine rotation speed. Feeding strategy: When the OD of the fermentation broth… 600 Feeding was started when the inoculum reached 20 mL / L / h. Feeding was initially done at a rate of 12 mL / L / h until fermentation lasted 9 hours (calculated from the time of inoculation). Then, feeding was continued at rates of 14 mL / L / h, 16 mL / L / h, 18 mL / L / h, 20 mL / L / h, and 22 mL / L / h for 3 hours each. After feeding was stopped, samples were taken for testing. The total fermentation time was 24 hours.

[0056] The dissolved oxygen level of 30% to 40% during the fermentation stage refers to the state in which the dissolved oxygen (DO) concentration is controlled to reach an air saturation level of 30% to 40% during the fermentation process. The dissolved oxygen level during the fermentation stage is monitored in real time by an automatic control system, and the dissolved oxygen is maintained within the preset range by dynamically adjusting the stirring speed.

[0057] The culture medium formulations involved are as follows:

[0058] Seed culture medium: glycerol 20 g / L, yeast extract 20 g / L, sodium citrate 3 mmol / L, yeast peptone 10 g / L, sodium chloride 0.5 g / L, ammonium sulfate 5 g / L, anhydrous magnesium sulfate 1 g / L, disodium hydrogen phosphate dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, with the remainder being deionized water.

[0059] Basic culture medium: glycerol 12 g / L, sodium citrate 7 mmol / 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 dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, antifoaming agent 0.1 mL / L, ferrous sulfate heptahydrate 0.1 mmol / L, copper chloride dihydrate 0.1 mmol / L, p-hydroxybenzoic acid 100 mg / L, balance deionized water.

[0060] Feeding medium: glycerol 281 g / L, yeast extract 180 g / L, ammonium sulfate 20 g / L, disodium hydrogen phosphate dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, balance deionized water.

[0061] The acid and alkali feeds used to maintain pH are as follows:

[0062] Acid feed: 20% phosphoric acid aqueous solution.

[0063] Alkali feed: Sodium hydroxide aqueous solution with a mass concentration of 400 g / L.

[0064] Example 2

[0065] This embodiment provides another method for producing mRNA template plasmids by Escherichia coli fermentation, which is basically the same as in Example 1, except that the acid-fed and alkali-fed feeds are slightly different from those in Example 1. The formulations of the acid-fed and alkali-fed feeds in this embodiment are as follows:

[0066] Acid supplement: A phosphoric acid aqueous solution containing 200 g / L glycerol at a mass fraction of 20%.

[0067] Alkali feed: an ammonia solution containing 100g / L yeast extract at a mass fraction of 20%.

[0068] Example 3

[0069] This embodiment provides another method for producing mRNA template plasmids by Escherichia coli fermentation, which is basically the same as in Example 1, except that the acid-fed and alkali-fed feeds are slightly different from those in Example 1. The formulations of the acid-fed and alkali-fed feeds in this embodiment are as follows:

[0070] Acid supplement: A phosphate solution containing 200 g / L glucose at a mass fraction of 20%.

[0071] Alkali feed: an ammonia solution containing 100g / L yeast extract at a mass fraction of 20%.

[0072] Example 4

[0073] This embodiment provides a method for producing mRNA template plasmids by fermentation of *E. coli*, which is basically the same as in Example 1, except that the seed culture medium and basal culture medium are slightly different from those in Example 1. The formulations of the seed culture medium and basal culture medium in this embodiment are as follows:

[0074] Seed culture medium: glycerol 20 g / L, yeast extract 20 g / L, sodium gluconate 3 mmol / L, yeast peptone 10 g / L, sodium chloride 0.5 g / L, ammonium sulfate 5 g / L, anhydrous magnesium sulfate 1 g / L, disodium hydrogen phosphate dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, with the remainder being deionized water.

[0075] Basic culture medium: glycerol 12 g / L, sodium gluconate 7 mmol / 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 dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, antifoaming agent 0.1 mL / L, ferrous sulfate heptahydrate 0.1 mmol / L, copper chloride dihydrate 0.1 mmol / L, p-hydroxybenzoic acid 100 mg / L, balance deionized water.

[0076] Example 5

[0077] This embodiment provides another method for producing mRNA template plasmids by Escherichia coli fermentation, which is basically the same as in Example 1, except that the seed culture medium and basal culture medium are slightly different from those in Example 1. The formulations of the seed culture medium and basal culture medium in this embodiment are as follows:

[0078] Seed culture medium: glycerol 20 g / L, yeast extract 20 g / L, disodium edetate 3 mmol / L, yeast peptone 10 g / L, sodium chloride 0.5 g / L, ammonium sulfate 5 g / L, anhydrous magnesium sulfate 1 g / L, disodium hydrogen phosphate dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, with the remainder being deionized water.

[0079] Basic culture medium: glycerol 12 g / L, disodium edetate 7 mmol / 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 dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, antifoaming agent 0.1 mL / L, ferrous sulfate heptahydrate 0.1 mmol / L, copper chloride dihydrate 0.1 mmol / L, p-hydroxybenzoic acid 100 mg / L, balance deionized water.

[0080] Example 6

[0081] This embodiment provides another method for producing mRNA template plasmids by Escherichia coli fermentation, which is basically the same as in Example 1, except that the basal culture medium is slightly different from that in Example 1. The basal culture medium formula in this embodiment is as follows: glycerol 12 g / L, sodium citrate 14 mmol / 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 3.0 g / L, disodium hydrogen phosphate dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, antifoaming agent 0.1 mL / L, ferrous sulfate heptahydrate 0.1 mmol / L, copper chloride dihydrate 0.1 mmol / L, p-hydroxybenzoic acid 100 mg / L, and the balance being deionized water.

[0082] Example 7

[0083] This embodiment provides another method for producing mRNA template plasmids by Escherichia coli fermentation, which is basically the same as in Example 1, except that the basal culture medium is slightly different from that in Example 1. The formulation of the basal culture medium in this embodiment is as follows: glycerol 12 g / L, sodium citrate 21 mmol / 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 4.5 g / L, disodium hydrogen phosphate dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, antifoaming agent 0.1 mL / L, ferrous sulfate heptahydrate 0.1 mmol / L, copper chloride dihydrate 0.1 mmol / L, p-hydroxybenzoic acid 100 mg / L, with the remainder being deionized water.

[0084] Comparative Example 1

[0085] This comparative example provides a method for producing mRNA template plasmids by fermentation of *E. coli*, which is basically the same as in Example 1, except that the seed culture medium and the acidity of the basal culture medium are slightly different from those in Example 1. The formulations of the seed culture medium and the basal culture medium in this comparative example are as follows:

[0086] Seed culture medium: 10 g / L yeast peptone, 5 g / L sodium chloride, 10 g / L yeast extract, with the remainder being deionized water.

[0087] Basic culture 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 dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, defoamer 0.1 mL / L, ferrous sulfate heptahydrate 0.1 mmol / L, copper chloride dihydrate 0.1 mmol / L, p-hydroxybenzoic acid 100 mg / L, balance deionized water.

[0088] Comparative Example 2

[0089] This comparative example provides another method for producing mRNA template plasmids by E. coli fermentation, which is basically the same as Comparative Example 1, except that the feed medium formulation is slightly different from that of Comparative Example 1. The feed medium formulation in this comparative example is: glycerol 281 g / L, yeast extract 90 g / L, ammonium sulfate 20 g / L, disodium hydrogen phosphate dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, with the remainder being deionized water.

[0090] Comparative Example 3

[0091] This comparative example provides another method for producing mRNA template plasmids by Escherichia coli fermentation, which is basically the same as Comparative Example 1, except that the basal culture medium formulation is slightly different from that of Comparative Example 1. The basal culture medium formulation in this comparative example is as follows: glycerol 12 g / L, sodium citrate 0.7 mmol / 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 dodecahydrate 6 g / L, dipotassium hydrogen phosphate 3 g / L, antifoaming agent 0.1 mL / L, ferrous sulfate heptahydrate 0.1 mmol / L, copper chloride dihydrate 0.1 mmol / L, p-hydroxybenzoic acid 100 mg / L, with the remainder being deionized water.

[0092] Comparative Example 4

[0093] This comparative example provides another method for producing mRNA template plasmids by E. coli fermentation, which is basically the same as Comparative Example 1, except that the feed medium formulation is slightly different from that of Comparative Example 1. The feed medium formulation in this comparative example is: 365 g / L glycerol, 180 g / L yeast extract, 20 g / L ammonium sulfate, 6 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L dipotassium hydrogen phosphate, with the remainder being deionized water.

[0094] Comparative Example 5

[0095] This comparative example provides another method for producing mRNA template plasmids by E. coli fermentation, which is basically the same as Comparative Example 1, except that the feed medium formulation is slightly different from that of Comparative Example 1. The feed medium formulation in this comparative example is: 422 g / L glycerol, 180 g / L yeast extract, 20 g / L ammonium sulfate, 6 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L dipotassium hydrogen phosphate, with the remainder being deionized water.

[0096] The fermentation broth samples from the above embodiments and comparative examples were tested according to the following detection methods:

[0097] 1.OD 600 The spectrophotometric detection value was adjusted to 600. Purified water was added to the cuvette as a zero-point control. The fermentation broth sample was then diluted several times and added to the cuvette to achieve a detection value between 0.2 and 0.8. The OD value of the fermentation broth was calculated by back-calculating the dilution factor. (Bacterial cell density and OD...) 600 Positive correlation (as recorded in the Concise Guide to Molecular Biology Experiments (4th Edition) for OD) 600 When the concentration of E. coli is 1, the concentration is 10. 8 cells / mL), and OD 600 The detection method is simple and quick.

[0098] 2. Plasmid expression level (mg / L): The target mRNA template plasmid was extracted from the fermentation broth using the plasmid mini-prep kit (DP103) from Tiangen Biotech (Beijing) Co., Ltd., following the manufacturer's instructions. The target plasmid expression level (i.e., plasmid yield) was detected by centrifuging 50 μL of fermentation broth to collect the cells, extracting the plasmid using the kit, eluting with 200 μL of ultrapure water, and then detecting the concentration. The concentration obtained by detection at 4 times the target value was the volumetric expression level (mg / L).

[0099] 3. Calculate the expression level per unit bacterial cell: Expression level per unit bacterial cell = Plasmid expression level / OD 600 .

[0100] 4. The content of supercoiled monomers and plasmid aggregates in the plasmid products extracted from the fermentation broth using the plasmid mini-extraction kit (DP103) was determined by high performance liquid chromatography (HPLC).

[0101] The test results are shown in Table 1.

[0102] Table 1

[0103]

[0104] In the process of producing mRNA template plasmids by E. coli fermentation, the plasmid yield and the ratio of supercoiled monomers can be affected by adjusting the culture medium and feed formulation at each stage. According to Table 1, adding appropriate amounts of metal ion chelating agents (sodium citrate, sodium gluconate, and disodium edetate) to the seed culture and basal culture medium helps reduce plasmid aggregate content, with sodium citrate showing the best effect in inhibiting plasmid aggregate formation. A seed culture medium richer in nutrients is beneficial for enhancing seed viability. The carbon source (glycerol) and organic nitrogen source (yeast extract) content in the feed culture medium can affect plasmid yield and the ratio of supercoiled monomers. Further dynamic regulation of carbon source (glycerol or glucose) and organic nitrogen source (yeast extract) during fermentation, combined with acid-fed and alkaline-fed methods, further reduces plasmid aggregates. In summary, Example 2 is the optimal culture scheme, with the highest expression level per unit cell and the highest content of supercoiled monomers among all examples and comparative examples, while the lowest content of plasmid aggregates among all examples and comparative examples.

[0105] The above embodiments are merely preferred examples of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications or improvements made without departing from the spirit of the present invention shall fall within the scope of protection claimed by the present invention.

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 fed-batch medium is continuously added at a feeding rate of 11-23 mL / L / h to carry out the continuous feeding fermentation culture, 600 18-22, and then the fed-batch medium is continuously added at a feeding rate of 11-23 mL / L / h to carry out the continuous feeding fermentation culture, The recombinant Escherichia coli is Escherichia coli transformed by an mRNA template plasmid; The basic culture medium is formulated as follows: glycerol 10-15 g / L, metal ion chelating agent 1-25 mmol / L, yeast extract 15-25 g / L, yeast peptone 5-15 g / L, sodium chloride 0.2-0.8 g / L, ammonium sulfate 5-15 g / L, anhydrous magnesium sulfate 1-5 g / L, disodium hydrogen phosphate dodecahydrate 4-8 g / L, dipotassium hydrogen phosphate 1-5 g / L, antifoaming agent 0.1 mL / L, ferrous sulfate heptahydrate 0.05-0.2 mmol / L, copper chloride dihydrate 0.05-0.2 mmol / L, p-hydroxybenzoic acid 50-200 mg / L, with the balance being deionized water. The metal ion chelating agent is one or more of sodium citrate, sodium gluconate, and disodium edetate. The formula of the supplemental culture medium is as follows: glycerol 260-450 g / L, yeast extract 100-250 g / L, ammonium sulfate 15-25 g / L, disodium hydrogen phosphate dodecahydrate 4-8 g / L, dipotassium hydrogen phosphate 1-5 g / L, and the balance is deionized water.

2. The method of fermenting production of mRNA template plasmid by E. coli according to claim 1, characterized in that, The metal ion chelating agent is sodium citrate; And / or, in the basal culture medium, the content of the metal ion chelating agent is 3-5 mmol / L, and the content of the anhydrous magnesium sulfate is 1-2 g / L.

3. The method of fermenting production of mRNA template plasmid by E. coli according to claim 1, characterized in that, The Escherichia coli is a stable competent cell; And / or, the mRNA template plasmid is a pET series plasmid; And / or, the mRNA template plasmid is less than 10000 bp.

4. The method of fermenting an mRNA template plasmid by E. coli according to claim 1, characterized in that, The fermentation conditions were as follows: inoculum volume ratio of 4%–6%, temperature of 36.5–37.5℃, pH of 6.8–7.0, dissolved oxygen of 30%–40%, and aeration rate of 0.8–1.5 VVM. And / or, the duration of the continuous fed fermentation culture is 20 to 30 hours.

5. The method of fermentative production of mRNA template plasmid by E. coli according to any one of claims 1 to 4, characterized in that, When fermenting recombinant E. coli, the pH of the fermentation system is controlled to be 6.8–7.0 through acid-feeding and alkali-feeding methods. The acid supplement is a phosphoric acid aqueous solution with a mass fraction of 15% to 25%, a phosphoric acid aqueous solution containing 150 to 250 g / L glycerol with a mass fraction of 15% to 25%, or a phosphoric acid aqueous solution containing 150 to 250 g / L glucose with a mass fraction of 15% to 25%. The alkali supplement is an ammonia solution with a mass fraction of 15% to 25% or an ammonia solution containing 80 to 150 g / L yeast extract with a mass fraction of 15% to 25%.

6. The method of fermentative production of mRNA template plasmid by E. coli according to claim 5, characterized in that, The acid feedstock is an aqueous solution of phosphoric acid containing 150-250 g / L glycerol and having a mass fraction of 15%-25%. The alkali supplement is an ammonia solution containing 80-150 g / L yeast extract powder at a mass fraction of 15%-25%.

7. The method of fermentative production of mRNA template plasmid by E. coli according to claim 6, characterized in that, The feed culture medium is formulated as follows: glycerol 280-350 g / L, yeast extract 160-200 g / L, ammonium sulfate 15-25 g / L, disodium hydrogen phosphate dodecahydrate 4-8 g / L, dipotassium hydrogen phosphate 1-5 g / L, with the remainder being deionized water.

8. The method of fermenting an mRNA template plasmid by E. coli according to claim 5, wherein, When the OD 600 value of the fermentation broth is 18-22, first feed at a feed rate of 11-13 mL / L / h for 8-10 h, then continue to feed at a feed rate of 13-15 mL / L / h, 15-17 mL / L / h, 17-19 mL / L / h, 19-21 mL / L / h, 21-23 mL / L / h, respectively, 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 preparation method of the recombinant E. coli seed solution is as follows: the recombinant E. coli is inoculated into a seed culture medium at a volume ratio of 0.05% to 0.15%, and then cultured at 36.5 to 37.5°C and 200 to 220 r / min for 15 to 18 hours to obtain the seed solution, The formula of the seed culture medium is as follows: glycerol 15 to 25 g / L, yeast extract powder 15 to 25 g / L, sodium citrate 2 to 4 mmol / L, yeast peptone 5 to 15 g / L, sodium chloride 0.2 to 0.8 g / L, ammonium sulfate 2 to 5 g / L, anhydrous magnesium sulfate 0.5 to 1.5 g / L, disodium hydrogen phosphate dodecahydrate 4 to 8 g / L, potassium hydrogen phosphate 1 to 5 g / L, and the rest is deionized water.

10. A medium composition for Escherichia coli fermentation production of mRNA template plasmid, characterized by, The medium composition comprises a base medium, a feed medium, and optionally a seed medium, wherein the base medium is the base medium described in any one of claims 1 or 2, the feed medium is the feed medium described in any one of claims 1 or 2, and the seed medium is the seed medium described in claim 9.

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