Chemical component limited culture medium for escherichia coli fermentation culture and plasmid expression

Abstract

The invention belongs to the field of microbial culture media. The invention provides a chemical component limited culture medium for escherichia coli fermentation culture and plasmid expression, which comprises the following components: glucose, thiamine hydrochloride, diammonium hydrogen phosphate, anhydrous magnesium sulfate, sodium chloride, manganese sulfate monohydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, sodium molybdate dihydrate, boric acid, L-glutamic acid, calcium chloride dihydrate and citric acid. The culture medium is clear in chemical component, free of serum and protein and free of plant hydrolysate, nutrient supply of escherichia coli in the whole fermentation process is guaranteed through the components of the addition amount, normal metabolism and growth of escherichia coli are promoted through synergistic interaction, and the yield of escherichia coli is increased. The amount OD600 of thalli cultured for 24 hours can reach and exceed the level of an imported culture medium of a contrast; the yield of the expressed plasmid can reach 52.58 [mu] g / mL, which is obviously higher than the level of 29.38 [mu] g / mL of a control culture medium, and the method has obvious advantages.

Description

Technical Field

[0001] The invention belongs to the field of microbial culture medium, and in particular relates to a chemically defined culture medium suitable for Escherichia coli fermentation culture and subsequent plasmid expression. Background Art

[0002] mRNA vaccines break the immune activation model of traditional inactivated and attenuated vaccines. They innovatively use the human body's own cells to produce antigens to activate specific immunity, reflecting the technical advantages of mRNA vaccines.

[0003] mRNA vaccines have a very high effective protection rate. Compared with other innovative vaccines (such as DNA vaccines and viral vector vaccines), they are safer. In addition, the production process and cycle are shorter than other vaccines, taking only more than 60 days. In addition, the application prospects of mRNA are very broad. In addition to being used to prevent infectious diseases, mRNA vaccines also bring new hope for the treatment of tumors and immune diseases. At present, the three major international mRNA vaccine giants are BioNTech, CureVac, and Moderna, all of which have laid out multiple pipelines for tumor treatment. In addition to infectious diseases and tumor immune diseases, mRNA vaccines have development potential in many gene-related diseases, so mRNA vaccines have broad market prospects and growth potential.

[0004] The production process of mRNA vaccines can be roughly divided into the following three stages:

[0005] 1) Preparation of DNA stock solution: Plasmid DNA amplification is usually carried out by E. coli fermentation. Starting from the recovery of the strain, the production of plasmid DNA mainly goes through the steps of fermentation culture, harvest clarification, refining and purification, linearization, packaging and storage.

[0006] 2) Preparation of mRNA stock solution: This stage mainly uses in vitro transcription (IVT) technology to prepare mRNA using the linear DNA from the previous stage as a template. The main process steps include transcribing linearized plasmid DNA into mRNA, chemical modification, separation and purification, and packaging and freezing of mRNA stock solution.

[0007] 3) Preparation of mRNA preparations: Key breakthroughs in formulation technology have solved the problem of the drugability of mRNA.

[0008] The main process steps of mRNA preparations include mRNA encapsulation / loading, complex purification, sterile filtration, aseptic filling, etc.; among them, the basis of the encapsulation / loading process is the design and development of the delivery system.

[0009] Under the wave of mRNA vaccines, orders for culture media have increased significantly. On the one hand, vaccines are aimed at healthy people and have extremely high requirements for culture media. However, most of the culture media currently suitable for E. coli microbial culture and fermentation contain different hydrolyzates or peptone components, which cannot guarantee the batch-to-batch stability of culture media products, which will affect the quality control of plasmid production in the later stage.

[0010] Therefore, it is a foreseeable trend to develop culture media with obvious functional characterization and clear ingredients, shift the selection of culture medium raw materials from animal-derived products to animal-free products, stabilize the supply chain, localize raw materials, and improve the supply chain.

[0011] On the other hand, there is currently only one commercial fermentation medium in the world, Gibco Bacto CD Supreme, which is a chemically defined medium. There is no chemically defined medium suitable for the cultivation and fermentation of Escherichia coli microorganisms in China. Therefore, it is urgent to develop a chemically defined medium with domestic raw materials to meet domestic and foreign market demand. Summary of the invention

[0012] In order to solve the above problems, the present invention provides a chemically defined medium for Escherichia coli fermentation culture and plasmid expression and a preparation method thereof. The medium has clear chemical composition, is serum-free and protein-free, and does not add plant hydrolysates. Some small molecule compounds with known structures and functions are used as substitutes. Compared with the existing CD medium (chemically defined medium), it is more conducive to the culture of Escherichia coli and plasmid expression, has obvious functional characterization, and is more stable, safe and effective.

[0013] The object of the present invention is to provide a chemically defined medium for Escherichia coli fermentation and plasmid expression, comprising the following components and their contents:

[0014] Glucose 5000-25000mg / L;

[0015] Thiamine hydrochloride 5-50mg / L;

[0016] Diammonium hydrogen phosphate 7000-10000mg / L;

[0017] Anhydrous magnesium sulfate 800-1500mg / L;

[0018] Sodium chloride 5000-7000mg / L;

[0019] Manganese sulfate monohydrate 5-15 mg / L;

[0020] Zinc sulfate heptahydrate 5-15 mg / L;

[0021] Copper sulfate pentahydrate 2-6 mg / L;

[0022] Sodium molybdate dihydrate 2-6 mg / L;

[0023] Boric acid 0.5-3 mg / L;

[0024] L-glutamic acid 300-1000mg / L;

[0025] Calcium chloride dihydrate 20-150mg / L;

[0026] Citric acid 4000-6500mg / L.

[0027] The nutritional carbon source (energy substance) in the formula of the present invention is glucose, and the nutritional nitrogen source is provided in the form of inorganic nitrogen and organic nitrogen: including diammonium hydrogen phosphate and glutamic acid. Thiamine hydrochloride has the function of maintaining normal sugar metabolism, and an appropriate amount of metal elements is added: copper deficiency can lead to the downregulation of lactate dehydrogenase and other mitochondrial oxidases, thereby leading to tissue toxic hypoxia, and the addition of copper at an appropriate concentration can convert the lactic acid metabolism of cells from net lactic acid production to net lactic acid consumption, thereby promoting cell growth and titer; the addition of appropriate zinc ions can reduce the process of cell apoptosis; the addition of appropriate manganese ions can improve galactosylation and reduce the sialylation of products; the addition of molybdenum ions can increase the production of biologically active substances in cells; calcium chloride dihydrate can provide necessary ions, participate in the ion exchange process in cells, maintain the normal physiological function of cells, and can be used to adjust the pH value of the solution to provide a suitable growth environment for cells.

[0028] Citric acid in the formula of the present invention is an important intermediate product in the tricarboxylic acid cycle (TCA cycle), which connects glycolysis and tricarboxylic acid cycle, participates in the energy metabolism and fatty acid synthesis of cells, and is of great significance to the process optimization of cell culture. Magnesium ion is an indispensable trace element for cell growth, which is crucial for maintaining the normal metabolism and growth of microbial cells. During the fermentation process, the addition of magnesium ions can significantly improve the growth rate and biomass of cells. By adjusting the addition amount of magnesium sulfate, the fermentation cycle of microorganisms can be effectively shortened and the fermentation efficiency can be improved. The addition of magnesium sulfate can also improve the quality of fermentation products. Magnesium sulfate also plays an important role in protecting microbial cells, stabilizing cell membranes, reducing cell damage, and maintaining the integrity of cells, which is crucial for maintaining the efficient operation of the fermentation process.

[0029] The culture medium of the present invention has clear chemical composition, is serum-free and protein-free, and does not add plant hydrolyzates. Through the above-mentioned added amounts of various components, the supply of nutrients to Escherichia coli in the entire fermentation process is guaranteed, and the normal metabolism and growth of Escherichia coli are promoted synergistically. The bacterial cell volume (OD 600) can reach and exceed the level of the imported culture medium of the control example; the plasmid yield expressed can reach 52.58μg / mL, which is significantly higher than the level of 29.38μg / mL of the control culture medium, and has a significant advantage.

[0030] Furthermore, the chemical composition-defined culture medium includes the following components and their contents:

[0031] Glucose 16000-22000mg / L;

[0032] Thiamine hydrochloride 20-30 mg / L;

[0033] Diammonium hydrogen phosphate 9000-10000mg / L;

[0034] Anhydrous magnesium sulfate 900-1100 mg / L;

[0035] Sodium chloride 5000-6000mg / L;

[0036] Manganese sulfate monohydrate 5-15 mg / L;

[0037] Zinc sulfate heptahydrate 5-15 mg / L;

[0038] Copper sulfate pentahydrate 2-6 mg / L;

[0039] Sodium molybdate dihydrate 2-6 mg / L;

[0040] Boric acid 0.5-3 mg / L;

[0041] L-glutamic acid 800-1000 mg / L;

[0042] Calcium chloride dihydrate 90-110 mg / L;

[0043] Citric acid 4000-5000mg / L.

[0044] Furthermore, the above-mentioned Escherichia coli is Escherichia coli stabl3.

[0045] The method for preparing a chemically defined medium for Escherichia coli fermentation culture and plasmid expression of the present invention comprises weighing various raw materials according to the raw material ratio of the chemically defined medium, mixing the raw materials with deionized water, adjusting the pH to 6.8-7.2, and fixing the volume with deionized water to obtain the chemically defined medium.

[0046] The method for culturing Escherichia coli using the chemically defined medium of the present invention comprises the following steps:

[0047] (1) E. coli recovery: Thaw the strain in a water bath, inoculate the thawed bacterial solution onto solid LB medium, and culture under appropriate conditions for 24-48 hours;

[0048] (2) Activation of E. coli: Pick a single colony and transfer it to LB liquid culture medium. When the seeds are activated to the exponential growth phase, prepare to transfer to the chemically defined culture medium of the present invention;

[0049] (3) Fermentation culture of Escherichia coli: The bacterial solution is centrifuged at an inoculation density of 5-15% and then inoculated into the chemically defined culture medium of the present invention for fermentation culture.

[0050] Furthermore, the exponential growth phase is specifically 10-14h of shake flask culture, OD 600 The value is between 2-3.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The present invention is a completely chemically defined formula, does not contain protein or protein hydrolysate and other substances, has high product safety and stability, and is suitable for Escherichia coli microbial culture;

[0053] (2) The chemically defined culture medium of the present invention is at OD 600 The density of microorganism Escherichia coli and the plasmid yield exceeded those of the control group CD medium, and the plasmid expression level (52.58 μg / mL) was significantly higher than that of the control group (29.38 μg / mL);

[0054] (3) The present invention makes it easy to control the batch difference in the large-scale production process, and the culture medium is stable, and the transportation and storage conditions are relatively loose;

[0055] (4) The present invention does not contain substances such as protein or protein hydrolysate, and does not introduce non-target proteins during the production process, which simplifies separation and purification, and is beneficial to the time and cost control of the downstream purification process;

[0056] (5) The present invention can be produced by grinding with a full formula, has simple components, no additives, and is easy to prepare for client application. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a comparison of the growth curves of Escherichia coli in the chemically defined medium and the control group;

[0058] Figure 2 This is a comparison chart of 24h plasmid production of Escherichia coli in chemically defined medium and control group. DETAILED DESCRIPTION

[0059] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] Example 1-3 Chemically Defined Medium

[0061] The chemically defined medium for Escherichia coli microbial culture and plasmid expression of the present invention is composed of thiamine hydrochloride, anhydrous magnesium sulfate, glucose, sodium chloride, manganese sulfate monohydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, sodium molybdate dihydrate, boric acid, L-glutamic acid, citric acid, calcium chloride dihydrate, ammonium sulfate and deionized water. The above raw materials are all commercially available products.

[0062] The Cas numbers and reference manufacturer information of the components of the chemically defined medium of Example 1-3 are as follows:

[0063] Serial number name Cas Reference Manufacturers 1 glucose 50-99-7 Xiwang Pharmaceutical 2 Thiamine Hydrochloride 67-03-8 Maclean 3 Diammonium phosphate 7783-28-0 Sinopharm 4 Anhydrous magnesium sulfate 7487-88-9 Maclean 5 Sodium chloride 7647-14-5 Maclean 6 Manganese sulfate monohydrate 10034-96-5 Maclean 7 Zinc sulfate heptahydrate 7446-20-0 Maclean 8 Copper sulfate pentahydrate 7758-99-8 Maclean 9 Sodium molybdate dihydrate 10102-40-6 Maclean 10 Boric acid 10043-35-3 Ron 11 L-Glutamic Acid 56-86-0 Maclean 12 Calcium chloride dihydrate 10035-04-8 Maclean 13 Citric Acid 77-92-9 Sinopharm

[0064] The chemically defined culture media of Examples 1-3 are collectively referred to as "CDM01, CDM02, and CDM03" hereinafter.

[0065] The formulations of Examples 1-3 are shown in the following table:

[0066]

[0067] Preparation method of chemically defined medium: prepare the raw material components according to the above specific contents, and fully dissolve them in sterile deionized water, adjust the pH to 6.8-7.2, filter the volume to 1L with a filter membrane with a pore size of 0.22μm, and store at 4°C for use.

[0068] Comparative Example 1

[0069] The control culture medium used was Bacto CD Supreme FPM culture medium of Gibco brand.

[0070] Application example: Escherichia coli fermentation culture

[0071] The strain used in this application example is stabl3, and the stabl3 strain is used to express plasmid DNA. The culture mode used is batch fermentation culture.

[0072] The CDM culture media "CDM01, CDM02, CDM03" prepared in the above Examples 1-3 and the control culture media of Control Example 1 were used to carry out E. coli fermentation culture. The specific steps of the E. coli fermentation culture are as follows:

[0073] Step 1: E. coli recovery

[0074] (1) Thaw the stored bacteria in a 30°C water bath.

[0075] (2) Inoculation: After the E. coli glycerol tube is thawed, the bacterial solution is inoculated onto solid LB medium and cultured under appropriate conditions for 24-48 hours to restore the activity of the strain.

[0076] Step 2: E. coli activation

[0077] Pick a single colony on the LB solid culture medium plate and transfer it to the LB liquid culture medium. When the seeds are activated to the exponential growth phase (about 13-14 hours of shake flask culture, OD value between 2-3), you can consider transferring to the fermentation medium.

[0078] Step 3: E. coli fermentation culture

[0079] After the E. coli seed liquid is activated, the bacterial liquid is centrifuged at an inoculation density of 10%, resuspended in a fermentation medium and inoculated into a fermentation medium. The fermentation medium used includes commercial CD medium, EcoliCDM01, Ecoli CDM02 and Ecoli CDM03 medium.

[0080] OD of E. coli growth process 600 Determination of value

[0081] Growth curve data of Escherichia coli stabl3 (OD 600 According to the recorded data shown in Table 1 (the recorded data are shown in Table 1), the growth curve of Escherichia coli is formed, as shown in Figure 1 shown.

[0082] Table 1 Growth curve data of Escherichia coli stabl3 (OD 600 )

[0083]

[0084]

[0085] from Figure 1 The growth curve of Stab13 in the experiment shows that the amount of Stab13 bacteria (OD 600 ) is not much different from the amount of bacteria cultured using imported culture medium, OD 600 The value can reach about 12.6; wherein Example 1 uses Stabl3 cultured with Ecoli CDM01, OD 600 The value was 13.5, which was higher than the amount of bacteria cultured in the imported culture medium of the control example.

[0086] Determination of plasmid content expressed in E. coli

[0087] The plasmid content expressed by E. coli stabl3 was determined by using an endotoxin-free plasmid mini-preparation kit (TIANGEN brand, item number DP118) (refer to the kit instructions for operation). The 24h plasmid production data of E. coli are shown in Table 2, and the expression level results of E. coli plasmids are shown in Table 2. Figure 2 shown.

[0088] Table 2 E. coli 24h plasmid production data (unit: μg / mL)

[0089] Culture medium CD medium CDM01 CDM02 CDM03 24h plasmid yield (μg / ml) 29.38 52.58 48.82 45.75

[0090] from Figure 2 It can be seen from the results of plasmid yields expressed by stabl3 strains cultured in different culture media that the plasmid expression level of the stabl3 strain cultured using the Ecoli CDM culture medium of this embodiment is significantly higher than that of the control group. The plasmid expression level of the control group is 29.38 μg / mL, while the plasmid expression level of the Ecoli CDM01 culture medium is 52.58 μg / mL, which has a significant advantage.

[0091] In summary, the present invention provides a chemically defined medium for Escherichia coli fermentation and plasmid expression, wherein the bacterial cell volume (OD 600 ) can reach and exceed the level of the control example imported culture medium, OD 600 The value is about 12.6; secondly, in terms of the yield of the expressed plasmid, the yield of the plasmid expressed by the culture medium of the present invention can reach 52.58 μg / mL, which is significantly higher than the level of the control culture medium (29.38 μg / mL), and has a significant advantage.

[0092] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those skilled in the relevant technical field may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also belong to the scope of the present invention and should be defined by the claims.

Claims

1. A chemically defined medium for Escherichia coli fermentation and plasmid expression, characterized in that: Includes the following components and their contents: Glucose 5000-25000mg / L; Thiamine hydrochloride 5-50mg / L; Diammonium hydrogen phosphate 7000-10000mg / L; Anhydrous magnesium sulfate 800-1500mg / L; Sodium chloride 5000-7000mg / L; Manganese sulfate monohydrate 5-15 mg / L; Zinc sulfate heptahydrate 5-15 mg / L; Copper sulfate pentahydrate 2-6 mg / L; Sodium molybdate dihydrate 2-6 mg / L; Boric acid 0.5-3 mg / L; L-glutamic acid 300-1000mg / L; Calcium chloride dihydrate 20-150mg / L; Citric acid 4000-6500mg / L.

2. The chemically defined medium for Escherichia coli fermentation and plasmid expression according to claim 1, characterized in that: The components and contents of the chemically defined medium are preferably as follows: Glucose 16000-22000mg / L; Thiamine hydrochloride 20-30 mg / L; Diammonium hydrogen phosphate 9000-10000mg / L; Anhydrous magnesium sulfate 900-1100 mg / L; Sodium chloride 5000-6000mg / L; Manganese sulfate monohydrate 5-15 mg / L; Zinc sulfate heptahydrate 5-15 mg / L; Copper sulfate pentahydrate 2-6 mg / L; Sodium molybdate dihydrate 2-6 mg / L; Boric acid 0.5-3 mg / L; L-glutamic acid 800-1000 mg / L; Calcium chloride dihydrate 90-110 mg / L; Citric acid 4000-5000mg / L.

3. The chemically defined medium for Escherichia coli fermentation and plasmid expression according to any one of claims 1 to 2, characterized in that: The Escherichia coli is Escherichia coli stabl3.

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