Culture medium composition and application thereof in fermentation of PDRN recombinant escherichia coli

Abstract

The invention belongs to the technical field of microbial culture, and particularly relates to a culture medium composition and application thereof in PDRN recombinant escherichia coli fermentation. The culture medium composition comprises a fermentation culture medium and a fed-batch culture medium, the fermentation culture medium consists of disodium hydrogen phosphate, monopotassium phosphate, ammonium sulfate, magnesium chloride, calcium nitrate tetrahydrate, glycerol and water; the supplemented culture medium is composed of dipotassium phosphate, sodium dihydrogen phosphate, sodium sulfate, ammonium sulfate, ammonium chloride, diammonium hydrogen citrate, glycerin and water, and the metabolic requirements of PDRN recombinant escherichia coli are accurately matched. Meanwhile, the invention provides a fermentation method using the composition, and the problems of unbalanced nutrition, low fermentation efficiency and the like of an existing culture medium are solved. Stable proliferation of thalli can be remarkably promoted, the yield of PDRN products is increased, the culture medium is easy and convenient to prepare, fermentation conditions are easy to control, and the culture medium is suitable for industrial large-scale production and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture technology, specifically relating to a culture medium composition and its application in the fermentation of PDRN recombinant Escherichia coli. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs), as bioactive nucleic acid substances, have broad application prospects in wound repair, anti-inflammation, and tissue regeneration, with particularly growing demand in the biopharmaceutical, cosmetic, and medical device industries. Currently, large-scale PDRN production mainly relies on recombinant E. coli fermentation technology, which offers advantages such as low cost, high efficiency, and easy control of product purity, making it the preferred production route in industry.

[0003] However, during the fermentation of recombinant *E. coli*, cell growth and PDRN synthesis are highly dependent on the nutrient composition of the culture medium, and the rationality of the medium formulation directly determines the fermentation efficiency and product yield. Existing technologies generally suffer from the following key problems with the culture media used for PDRN-recombinant *E. coli* fermentation: Imbalance in carbon and nitrogen source ratio: Carbon sources serve as the core substrates for microbial energy metabolism and substance synthesis, while nitrogen sources are essential components of biological macromolecules such as proteins and nucleic acids. An improper ratio between the two can lead to serious consequences. Insufficient carbon sources limit microbial growth and hinder PDRN synthesis; excessive carbon sources cause a rapid drop in pH and an increase in viscosity in the fermentation broth, thereby inhibiting dissolved oxygen transfer and leading to metabolic disorders. Insufficient nitrogen sources result in slow microbial proliferation, while excessive nitrogen sources increase the accumulation of metabolic byproducts, increasing the burden on downstream separation and purification processes.

[0004] Inorganic salts such as phosphates, magnesium sulfates, and calcium salts play crucial roles in fermentation, including regulating osmotic pressure, maintaining enzyme activity, and participating in nucleic acid synthesis. However, the concentration of inorganic salts in existing culture media often lacks targeted optimization. Too low a concentration fails to meet the physiological needs of cell growth and PDRN synthesis, leading to prolonged fermentation cycles and low product yields; too high a concentration, on the other hand, can cause high osmotic pressure stress, disrupting cell membrane stability, inhibiting normal cell metabolism, and even causing cell death.

[0005] In large-scale fermentation, the composition of the fed-batch culture medium needs to synergize with the basal fermentation medium to continuously provide nutrients for cell growth and product synthesis. In existing technologies, fed-batch culture media often suffer from problems such as inconsistent carbon-nitrogen source ratios with the basal medium and mismatches between nutrient release rates and cell metabolic needs. This leads to nutrient supply interruptions or excesses during fermentation, resulting in large fluctuations in cell growth curves and difficulty in stabilizing PDRN synthesis efficiency.

[0006] Therefore, developing a specialized culture medium composition that precisely optimizes the carbon-nitrogen source ratio, inorganic salt concentration, and feed formulation based on the metabolic characteristics of PDRN-recombinant Escherichia coli is of great practical significance and industrial application value for solving the problems of low fermentation efficiency and unstable product yield caused by nutrient imbalance in existing culture media. Summary of the Invention

[0007] To address the above shortcomings, the present invention provides a culture medium composition and its application in the fermentation of PDRN recombinant Escherichia coli.

[0008] The technical solution of this invention is as follows: On one hand, the present invention provides a culture medium composition, which is a fermentation medium and a fed culture medium; the fermentation medium is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium chloride, calcium nitrate tetrahydrate, glycerol and water; the fed culture medium is composed of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium sulfate, ammonium sulfate, ammonium chloride, diammonium hydrogen citrate, glycerol and water.

[0009] Specifically, the fermentation medium contains 2.7-2.8 or 2.8-2.9 g / L disodium hydrogen phosphate, 0.9-1.0 or 1.0-1.1 g / L potassium dihydrogen phosphate, and 7.5-8.0 or 8.0-8.5 g / L glycerol.

[0010] Preferably, the fermentation medium contains 2.8 g / L disodium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, and 8 g / L glycerol.

[0011] More preferably, the fermentation medium contains 2.8 g / L disodium hydrogen phosphate, 1.0 g / L potassium dihydrogen phosphate, 0.2-0.8 g / L ammonium sulfate, 0.05-0.06 g / L magnesium chloride, 0.04-0.06 g / L calcium nitrate tetrahydrate and 8 g / L glycerol; Specifically, the supplemental culture medium contains 145-150 g / L dipotassium hydrogen phosphate, 35-40 or 40-45 g / L sodium dihydrogen phosphate, and 380-390, 390-400, 400-410 or 410-420 g / L glycerol.

[0012] Preferably, the supplemental culture medium contains 146 g / L dipotassium hydrogen phosphate, 40 g / L sodium dihydrogen phosphate, and 400 g / L glycerol.

[0013] More preferably, the supplemental culture medium contains 146 g / L dipotassium hydrogen phosphate, 40 g / L sodium dihydrogen phosphate, 15-25 g / L sodium sulfate, 20-30 g / L ammonium sulfate, 2.5-7.5 g / L ammonium chloride, 8-12 g / L diammonium hydrogen citrate, and 400 g / L glycerol.

[0014] Specifically, the preparation method of the fermentation medium includes: weighing disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium chloride, calcium nitrate tetrahydrate and glycerol, adding water to obtain the fermentation medium; the preparation method of the fed medium includes: weighing dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium sulfate, ammonium sulfate, ammonium chloride, diammonium hydrogen citrate and glycerol, adding water to obtain the fed medium.

[0015] Specifically, the culture medium composition is used for fermenting recombinant Escherichia coli, wherein the recombinant Escherichia coli is a recombinant Escherichia coli expressing PDRN.

[0016] Preferably, the recombinant Escherichia coli is TA4-41, with the accession number CGMCC NO.33625.

[0017] In another aspect, the present invention provides the use of the culture medium composition described in any of the above claims in the fermentation of recombinant Escherichia coli.

[0018] Specifically, the recombinant Escherichia coli is a recombinant Escherichia coli expressing PDRN.

[0019] Preferably, the recombinant Escherichia coli is TA4-41, with the accession number CGMCC NO.33625.

[0020] In another aspect, the present invention provides a method for fermenting recombinant Escherichia coli, the method comprising using a culture medium composition as described in any of the preceding claims, wherein the recombinant Escherichia coli is a recombinant Escherichia coli expressing PDRN.

[0021] Preferably, the recombinant Escherichia coli is TA4-41, with the accession number CGMCC NO.33625.

[0022] Specifically, the method includes the following steps: S1. Add ampicillin sodium solution to fermentation medium to obtain fermentation broth; inoculate recombinant Escherichia coli seed culture into fermentation broth for fermentation culture; S2. After fermentation culture for 6 hours, add feeding medium and continue culture for 24-72 hours to obtain recombinant Escherichia coli fermentation broth.

[0023] Specifically, the amount of ampicillin sodium solution added in step S1 is 0.5‰-1.5‰ v / v of the fermentation medium volume.

[0024] Preferably, the amount of ampicillin sodium solution added in step S1 is 1‰ v / v of the fermentation medium volume.

[0025] Specifically, the inoculation amount of the seed liquid in step S1 is 0.5%-1.5% v / v of the fermentation liquid volume.

[0026] Preferably, the inoculation amount of the seed liquid in step S1 is 1% v / v of the fermentation liquid volume.

[0027] Specifically, the fermentation conditions described in step S1 are pH = 6.5-7.5, temperature 35-40℃, and rotation speed 200-400 rpm.

[0028] Preferably, the fermentation conditions in step S1 are pH = 7.0, temperature 37°C, and rotation speed 200 rpm.

[0029] Specifically, in step S2, the feeding medium is added once every 4 hours, and the volume ratio of the feeding medium to the fermentation medium is 1:800-850 each time.

[0030] Preferably, the volume ratio of the feeding medium to the fermentation medium added each time is 1:800-810, 810-820, 820-830, 830-840 or 840-850.

[0031] More preferably, the volume ratio of the feeding medium to the fermentation medium added each time is 1:820.

[0032] Specifically, the cultivation conditions described in step S2 are: maintaining dissolved oxygen ≥ 30%, rotation speed 200-400 rpm, and temperature 35-40℃.

[0033] Preferably, the cultivation conditions described in step S2 are: maintaining dissolved oxygen ≥ 30%, rotation speed 200 rpm, and temperature 37°C.

[0034] The beneficial effects of this invention are as follows: 1. Richer Nutrients: In addition to common MS medium components such as disodium hydrogen phosphate, potassium dihydrogen phosphate, and ammonium sulfate, the basal medium also contains magnesium chloride and calcium nitrate tetrahydrate. These components provide more mineral elements, which are beneficial to the growth and metabolism of E. coli. For example, magnesium ions are activators of many enzymes, while calcium ions participate in processes such as cell signal transduction.

[0035] 2. Optimization of carbon and nitrogen sources: The glycerol concentration in the basal medium is 6-10 g / L, while the concentration in the fed-batch medium is as high as 350-450 g / L. This design provides an adequate carbon source in the early stages of fermentation, and as fermentation progresses, more carbon source is gradually added through fed-batch feeding to meet the carbon source requirements of *E. coli* during high-density fermentation. Simultaneously, nitrogen sources such as sodium sulfate, ammonium chloride, and diammonium hydrogen citrate are added to the fed-batch medium, further optimizing the carbon-nitrogen ratio and contributing to improved cell growth and product accumulation.

[0036] 3. Enhanced buffering capacity: The high concentrations of dipotassium hydrogen phosphate and sodium dihydrogen phosphate in the fed-batch culture medium enhance its buffering capacity, better maintaining pH stability during fermentation. In E. coli fermentation, the accumulation of metabolites can lead to pH changes, and a stable pH is crucial for cell growth and metabolism.

[0037] 4. The culture medium combination of the present invention enables the efficient scale-up of recombinant Escherichia coli TA4-41 in a 5L fermenter, with an OD600 peak value that is significantly better than conventional formulations such as modified M9, LB, and TB, without the need for additional nitrogen sources or expensive organic nitrogen supplementation.

[0038] Preservation Instructions The deposited strain is recombinant *Escherichia coli* TA4-41, deposited on February 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 33625, and classified as *Escherichia coli*. Escherichia coli . Attached Figure Description

[0039] Figure 1 The growth curves of strain TA4-41 in Example 1, Comparative Examples 1-4 are shown.

[0040] Figure 2 The growth curves of strain TA4-41 in fermentation medium 1-fermentation medium 4 in Experiment Example 1 are shown.

[0041] Figure 3 The growth curves of strain TA4-41 in fermentation medium 1-fermentation medium 4 in Experiment Example 2 are shown.

[0042] Figure 4 The growth curves of strains TA4-41 from Comparative Examples 5 to 8 are shown. Detailed Implementation

[0043] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0044] Basic Example 1 1. Preparation of ampicillin sodium solution (100 mg / mL): Weigh 1 g of ampicillin sodium powder, dissolve it in 10 mL of ultrapure water, filter it through a 0.22 μm microporous membrane for sterilization, aliquot it into 1 mL portions, and store at -20℃. When using, add it to the corresponding cooled culture medium (temperature less than 60℃) at a volume ratio of 1‰.

[0045] 2. Sodium hydroxide solution (10 mol / L): Weigh 40 g of sodium hydroxide and dissolve it in 100 mL of ultrapure water. Pour the solution into the glass bottle that comes with the fermenter and sterilize it at high temperature along with the fermenter.

[0046] 3. Defoamer: Purchased from Wuxi Shengjiayi Construction Engineering Co., Ltd., product number SY-W650.

[0047] Example 1 1. Preparation of culture medium (1) Preparation of seed culture medium (LB liquid medium, g / L): 5 g of yeast extract, 10 g of trypsinized peptone, 10 g of sodium chloride, and double-distilled water were added to make up to 1 L to obtain seed culture medium.

[0048] (2) Preparation of fermentation medium (g / L): 2.8 g of disodium hydrogen phosphate, 1.0 g of potassium dihydrogen phosphate, 0.5 g of ammonium sulfate, 0.053 g of magnesium chloride, 0.05 g of calcium nitrate tetrahydrate, and 8 g of glycerol were added to double-distilled water to make up to 1 L to obtain the fermentation medium.

[0049] (3) Preparation of feed medium (g / L): 146 g of dipotassium hydrogen phosphate, 40 g of sodium dihydrogen phosphate, 20 g of sodium sulfate, 25 g of ammonium sulfate, 5 g of ammonium chloride, 10 g of diammonium hydrogen citrate, and 400 g of glycerol were added to double-distilled water to make up to 1 L to obtain feed medium.

[0050] 2. Preparation of seed solution Take 2 mL of Escherichia coli strain TA4-41 (CGMCC NO.33625) from a -80℃ cryopreservation box and quickly place it in a full-temperature metal bath at 37℃ to melt it rapidly. Add the entire volume of the melted bacterial solution to 38 mL of seed culture medium containing 1‰ v / v ampicillin sodium solution and incubate overnight (12 h) at 37℃ and 200 r / min to obtain the seed culture.

[0051] 3. Fermentation production of recombinant Escherichia coli in fermenters (1) Prepare 4.1 L of fermentation medium and pour it into the glass jar of the fermenter. After calibrating the pH probe, remove the excess wires and put the glass jar, along with the internal culture medium, the external defoaming solution and 10 mol / L sodium hydroxide solution, into a vertical automatic pressure steam sterilizer and sterilize at 115°C for 15 min.

[0052] (2) Connect the fermenter pipeline, open the vent valve, adjust the temperature to 37°C, and set the gas flow rate to 2 L / min.

[0053] (3) Ignite the outer flame inoculation ring at the inoculation port, open the inoculation port, add the prepared ampicillin sodium solution (4.1 mL) at 1‰ v / v, close the inoculation port, extinguish the flame inoculation ring, and the fermentation broth is ready. Open the discharge port, take out 100 mL of fermentation broth and store it at 4℃ for later use as a reference when determining the growth curve.

[0054] (4) Reignite the flame inoculation ring, open the inoculation port, add seed liquid to the fermentation broth at an inoculation rate of 1% v / v, pH = 7, 37℃, 200 rpm, start fermentation and start timing; ① Four hours after the start of fermentation, when the pH of the fermentation broth decreases to below pH 7, add 10 mol / L sodium hydroxide solution to maintain the pH of the fermentation broth at 7. ② During the first 0-4 hours after fermentation begins, the cells grow rapidly, and the stirring and aeration are at their peak, making it easy for foam to increase suddenly. Add 0.5 mL of defoamer 6-8 hours after fermentation begins to eliminate foam. If foaming continues during fermentation, add 100 μL of defoamer dropwise. ③ Six hours after the start of fermentation, add feed medium, with 50 mL of feed medium added every 4 hours; after feeding, maintain dissolved oxygen ≥ 30%, rotation speed 200–400 rpm, and temperature 37 ℃.

[0055] (5) After 10 hours, open the feeding port and take out 3 mL of fermentation broth to measure the light absorption value (OD600) at 600 nm of the bacterial solution three times to monitor cell growth.

[0056] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is: "1. Preparation of culture medium".

[0057] (1) Preparation of seed culture medium (LB liquid medium, g / L): Same as in Example 1.

[0058] (2) Preparation of fermentation medium (modified M9 medium, g / L): 6 g disodium hydrogen phosphate, 7 g potassium dihydrogen phosphate, 5 g glucose, 3 g ammonium sulfate, 2 g peptone, 5 g yeast extract, 2 g magnesium sulfate, 1 mL / L trace element solution SL-4, add double distilled water to make up to 1 L to obtain fermentation medium.

[0059] The trace element solution SL-4 was purchased from Shandong Top Biotechnology, product number S2231.

[0060] (3) Preparation of fed culture medium (g / L): 100 g of glucose, 20 g of yeast extract, and 10 g of casein were added to double-distilled water and brought to a final volume of 1 L to obtain fed culture medium.

[0061] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is: "1. Preparation of culture medium".

[0062] (1) Preparation of seed culture medium (LB liquid medium, g / L): Same as in Example 1.

[0063] (2) Preparation of fermentation medium (g / L): 40 g of glucose, 5 g of ammonium sulfate, 5 g of disodium hydrogen phosphate, 5 g of sodium dihydrogen phosphate, 1 g of magnesium sulfate, add double-distilled water to make up to 1L, adjust pH to 7.0, and obtain fermentation medium.

[0064] (3) Preparation of feed culture medium (g / L): 800 g of glucose was added to double-distilled water and the volume was adjusted to 1L to obtain feed culture medium.

[0065] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is: "1. Preparation of culture medium".

[0066] (1) Preparation of seed culture medium (LB liquid medium, g / L): Same as in Example 1.

[0067] (2) Preparation of fermentation medium (g / L): 2.8 g of disodium hydrogen phosphate, 1.0 g of potassium dihydrogen phosphate, 0.5 g of ammonium sulfate, 0.053 g of magnesium chloride, and 0.05 g of calcium nitrate tetrahydrate were added to double-distilled water and brought to a final volume of 1 L to obtain the fermentation medium.

[0068] (3) Preparation of feed medium (g / L): 146 g of dipotassium hydrogen phosphate, 40 g of sodium dihydrogen phosphate, 20 g of sodium sulfate, 25 g of ammonium sulfate, 5 g of ammonium chloride, and 10 g of diammonium hydrogen citrate were added to double-distilled water to make up to 1 L to obtain feed medium.

[0069] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is: "1. Preparation of culture medium".

[0070] (1) Preparation of seed culture medium (LB liquid medium, g / L): Same as in Example 1.

[0071] (2) Preparation of fermentation medium (g / L): 2.8 g of disodium hydrogen phosphate, 1.0 g of potassium dihydrogen phosphate, 0.5 g of ammonium sulfate, 0.053 g of magnesium chloride, 0.05 g of calcium nitrate tetrahydrate, and 5 g of glucose were added to double-distilled water to make up to 1 L to obtain the fermentation medium.

[0072] (3) Preparation of feed medium (g / L): 146 g of dipotassium hydrogen phosphate, 40 g of sodium dihydrogen phosphate, 20 g of sodium sulfate, 25 g of ammonium sulfate, 5 g of ammonium chloride, 10 g of diammonium hydrogen citrate, and 100 g of glucose were added to double-distilled water to make up to 1 L to obtain feed medium.

[0073] Figure 1 The growth curves of strain TA4-41 in Example 1 and Comparative Examples 1-4 are shown. The results indicate that the OD value of Example 1 is significantly better than that of the formulations in Comparative Examples 1-4, showing a marked improvement in fermentation efficiency.

[0074] Experimental Example 1 This experiment investigates the effects of different concentrations of carbon source (glycerol) on microbial growth or product synthesis.

[0075] 1. Preparation of fermentation culture medium Fermentation medium 1 (MSM medium, g / L): 2.8 g disodium hydrogen phosphate, 1.0 g potassium dihydrogen phosphate, 0.5 g ammonium sulfate, 0.053 g magnesium chloride, 0.05 g calcium nitrate tetrahydrate, add double-distilled water to make up to 1 L to obtain the fermentation medium.

[0076] Fermentation medium 2 (MSM medium, g / L): 2.8 g disodium hydrogen phosphate, 1.0 g potassium dihydrogen phosphate, 0.5 g ammonium sulfate, 0.053 g magnesium chloride, 0.05 g calcium nitrate tetrahydrate, 4 g glycerol, add double-distilled water to make up to 1 L to obtain the fermentation medium.

[0077] Fermentation medium 3 (MSM medium, g / L): 2.8 g disodium hydrogen phosphate, 1.0 g potassium dihydrogen phosphate, 0.5 g ammonium sulfate, 0.053 g magnesium chloride, 0.05 g calcium nitrate tetrahydrate, 8 g glycerol, add double-distilled water to make up to 1 L to obtain the fermentation medium.

[0078] Fermentation medium 4 (MSM medium, g / L): 2.8 g disodium hydrogen phosphate, 1.0 g potassium dihydrogen phosphate, 0.5 g ammonium sulfate, 0.053 g magnesium chloride, 0.05 g calcium nitrate tetrahydrate, 16 g glycerol, add double-distilled water to a final volume of 1 L to obtain the fermentation medium.

[0079] 2. Preparation of Seed Culture Medium Preparation of seed culture medium (LB liquid medium, g / L): 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, add double-distilled water to make up to 1 L to obtain seed culture medium.

[0080] 3. Preparation of seed solution Refer to Example 1.

[0081] 4. Fermentation production of recombinant Escherichia coli in fermenters (1) Prepare 4.1 L of fermentation medium and pour it into the glass jar of the fermenter. After calibrating the pH probe, remove the excess wires and put the glass jar, along with the internal culture medium, the external defoaming solution and 10 mol / L sodium hydroxide solution, into a vertical automatic pressure steam sterilizer and sterilize at 115°C for 15 min.

[0082] (2) Connect the fermenter pipeline, open the vent valve, adjust the temperature to 37°C, and set the gas flow rate to 2 L / min.

[0083] (3) Ignite the outer flame inoculation ring at the inoculation port, open the inoculation port, add the prepared ampicillin sodium solution (4.1 mL) at 1‰ v / v, close the inoculation port, extinguish the flame inoculation ring, and the fermentation broth is obtained.

[0084] (4) Reignite the flame inoculation ring, open the inoculation port, add seed liquid to the fermentation broth at an inoculation rate of 1% v / v, pH = 7, 37℃, 200 rpm, start fermentation and start timing; ① Four hours after the start of fermentation, when the pH of the fermentation broth decreases to below pH 7, add 10 mol / L sodium hydroxide solution to maintain the pH of the fermentation broth at 7. ② During the first 0-4 hours after fermentation begins, the cells grow rapidly, and the stirring and aeration are at their peak, which can easily lead to a sudden increase in foam. Add 0.5 mL of defoamer 6-8 hours after fermentation begins to eliminate the foam. If foaming continues during fermentation, 100 μL of defoamer can be added dropwise.

[0085] (5) After a 6-hour interval, open the feeding port and take out 3 mL of fermentation broth to measure the light absorption value (OD600) at 600 nm of the bacterial solution three times to monitor cell growth.

[0086] The results showed that the OD600 of fermentation medium 1 without carbon source was significantly lower. The OD600 of fermentation medium 2 with low carbon source was lower than that of fermentation medium 3 and fermentation medium 4. Fermentation medium 4 had a higher carbon source concentration, and its OD600 was higher than that of the third group in the first 36 hours. However, the excess carbon source would lead to a decrease in pH, an increase in viscosity, and a decrease in dissolved oxygen in the fermentation broth, which would inhibit the growth of the cells and result in a higher OD600 in fermentation medium 3 in the later stage, indicating a better fermentation process.

[0087] Experiment Example 2 This experiment investigates the effects of different concentrations of inorganic salts (phosphates) on microbial growth or product synthesis.

[0088] Fermentation medium 1 (MSM medium, g / L): 1.3 g disodium hydrogen phosphate, 0.5 g potassium dihydrogen phosphate, 0.5 g ammonium sulfate, 0.053 g magnesium chloride, 0.05 g calcium nitrate tetrahydrate, 8 g glycerol, add double-distilled water to make up to 1 L to obtain the fermentation medium.

[0089] Fermentation medium 2 (MSM medium, g / L): 2.8 g disodium hydrogen phosphate, 1.0 g potassium dihydrogen phosphate, 0.5 g ammonium sulfate, 0.053 g magnesium chloride, 0.05 g calcium nitrate tetrahydrate, 8 g glycerol, add double-distilled water to make up to 1 L to obtain the fermentation medium.

[0090] Fermentation medium 3 (MSM medium, g / L): 4.2 g disodium hydrogen phosphate, 1.5 g potassium dihydrogen phosphate, 0.5 g ammonium sulfate, 0.053 g magnesium chloride, 0.05 g calcium nitrate tetrahydrate, 8 g glycerol, add double-distilled water to a final volume of 1 L to obtain the fermentation medium.

[0091] Fermentation medium 4 (MSM medium, g / L): 5.6 g disodium hydrogen phosphate, 2.0 g potassium dihydrogen phosphate, 0.5 g ammonium sulfate, 0.053 g magnesium chloride, 0.05 g calcium nitrate tetrahydrate, 8 g glycerol, add double-distilled water to a final volume of 1 L to obtain the fermentation medium.

[0092] 2. Preparation of Seed Culture Medium Preparation of seed culture medium (LB liquid medium, g / L): 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, add double-distilled water to make up to 1 L to obtain seed culture medium.

[0093] 3. Preparation of seed solution Refer to Example 1.

[0094] 4. Fermentation production of recombinant Escherichia coli in fermenters (1) Prepare 4.1 L of fermentation medium and pour it into the glass jar of the fermenter. After calibrating the pH probe, remove the excess wires and put the glass jar, along with the internal culture medium, the external defoaming solution and 10 mol / L sodium hydroxide solution, into a vertical automatic pressure steam sterilizer and sterilize at 115°C for 15 min.

[0095] (2) Connect the fermenter pipeline, open the vent valve, adjust the temperature to 37°C, and set the gas flow rate to 2 L / min.

[0096] (3) Ignite the outer flame inoculation ring at the inoculation port, open the inoculation port, add the prepared ampicillin sodium solution (4.1 mL) at 1‰ v / v, close the inoculation port, extinguish the flame inoculation ring, and the fermentation broth is obtained.

[0097] (4) Reignite the flame inoculation ring, open the inoculation port, add seed liquid to the fermentation broth at an inoculation rate of 1% v / v, pH = 7, 37℃, 200 rpm, start fermentation and start timing; ① Four hours after the start of fermentation, when the pH of the fermentation broth decreases to below pH 7, add 10 mol / L sodium hydroxide solution to maintain the pH of the fermentation broth at 7. ② During the first 0-4 hours after fermentation begins, the cells grow rapidly, and the stirring and aeration are at their peak, which can easily lead to a sudden increase in foam. Add 0.5 mL of defoamer 6-8 hours after fermentation begins to eliminate the foam. If foaming continues during fermentation, 100 μL of defoamer can be added dropwise.

[0098] (5) After a 6-hour interval, open the feeding port and take out 3 mL of fermentation broth to measure the light absorption value (OD600) at 600 nm of the bacterial solution three times to monitor cell growth.

[0099] The measurement results are as follows Figure 3 As shown, fermentation medium 2 has a higher OD600, indicating better performance.

[0100] Comparative Example 5 The only difference between Comparative Example 5 and Example 1 is: "1. Preparation of culture medium".

[0101] 1. Preparation of culture medium (1) Preparation of seed culture medium (LB liquid medium, g / L): Refer to Example 1.

[0102] (2) Preparation of fermentation medium (g / L): Refer to Example 1.

[0103] (3) Preparation of feed medium (g / L): 40 g of dipotassium hydrogen phosphate, 146 g of sodium dihydrogen phosphate, 20 g of sodium sulfate, 25 g of ammonium sulfate, 5 g of ammonium chloride, 10 g of diammonium hydrogen citrate, and 400 g of glycerol were added to double-distilled water to make up to 1 L to obtain feed medium.

[0104] Comparative Example 6 The only difference between Comparative Example 6 and Example 1 is: "1. Preparation of culture medium".

[0105] 1. Preparation of culture medium (1) Preparation of seed culture medium (LB liquid medium, g / L): Refer to Example 1.

[0106] (2) Preparation of fermentation medium (g / L): Refer to Example 1.

[0107] (3) Preparation of feed medium (g / L): 146 g of dipotassium hydrogen phosphate, 40 g of sodium dihydrogen phosphate, 20 g of sodium sulfate, 25 g of ammonium sulfate, 5 g of ammonium chloride, 10 g of diammonium hydrogen citrate, and 500 g of glycerol were added to double-distilled water to make up to 1 L to obtain feed medium.

[0108] Comparative Example 7 The only difference between Comparative Example 7 and Example 1 is: "1. Preparation of culture medium".

[0109] 1. Preparation of culture medium (1) Preparation of seed culture medium (LB liquid medium, g / L): Refer to Example 1.

[0110] (2) Preparation of fermentation medium (g / L): 4.2 g of disodium hydrogen phosphate, 1.5 g of potassium dihydrogen phosphate, 0.5 g of ammonium sulfate, 0.053 g of magnesium chloride, 0.05 g of calcium nitrate tetrahydrate, and 16 g of glycerol were added to double-distilled water to make up to 1 L to obtain the fermentation medium.

[0111] (3) Preparation of supplemental culture medium (g / L): Refer to Example 1.

[0112] Comparative Example 8 The only difference between Comparative Example 8 and Example 1 is: "1. Preparation of culture medium".

[0113] 1. Preparation of culture medium (1) Preparation of seed culture medium (LB liquid medium, g / L): Refer to Example 1.

[0114] (2) Preparation of fermentation medium (g / L): 4.2 g of disodium hydrogen phosphate, 1.5 g of potassium dihydrogen phosphate, 0.5 g of ammonium sulfate, 0.053 g of magnesium chloride, 0.05 g of calcium nitrate tetrahydrate, and 16 g of glycerol were added to double-distilled water to make up to 1 L to obtain the fermentation medium.

[0115] (3) Preparation of feed medium (g / L): 40 g of dipotassium hydrogen phosphate, 146 g of sodium dihydrogen phosphate, 20 g of sodium sulfate, 25 g of ammonium sulfate, 5 g of ammonium chloride, 10 g of diammonium hydrogen citrate, and 500 g of glycerol were added to double-distilled water to make up to 1 L to obtain feed medium.

[0116] Figure 4 The growth curves of strains TA4-41 in Comparative Examples 5-8 are shown. The results indicate that the OD600 values ​​of Comparative Examples 5-8 all increased over time, but the OD values ​​decreased to varying degrees compared to Example 1. The fermentation effect of the formulation in Example 1 of this invention is the best.

[0117] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A culture medium composition, characterized in that, The culture medium composition is a fermentation medium and a fed culture medium; The fermentation medium is composed of disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium chloride, calcium nitrate tetrahydrate, glycerol, and water; the fermentation medium contains 2.7-2.9 g / L disodium hydrogen phosphate, 0.9-1.1 g / L potassium dihydrogen phosphate, and 7.5-8.5 g / L glycerol. The feed medium is composed of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium sulfate, ammonium sulfate, ammonium chloride, diammonium hydrogen citrate, glycerol, and water; the feed medium contains 145-150 g / L dipotassium hydrogen phosphate, 35-45 g / L sodium dihydrogen phosphate, and 380-420 g / L glycerol.

2. The culture medium composition according to claim 1, characterized in that, The fermentation medium contains 2.8 g / L disodium hydrogen phosphate, 1.0 g / L potassium dihydrogen phosphate, 0.2-0.8 g / L ammonium sulfate, 0.05-0.06 g / L magnesium chloride, 0.04-0.06 g / L calcium nitrate tetrahydrate and 8 g / L glycerol.

3. The culture medium composition according to claim 1, characterized in that, The feed medium contains 146 g / L dipotassium hydrogen phosphate, 40 g / L sodium dihydrogen phosphate, 15-25 g / L sodium sulfate, 20-30 g / L ammonium sulfate, 2.5-7.5 g / L ammonium chloride, 8-12 g / L diammonium hydrogen citrate, and 400 g / L glycerol.

4. The culture medium composition according to claim 1, characterized in that, The method for preparing the fermentation medium includes: weighing disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium chloride, calcium nitrate tetrahydrate and glycerol, adding water to obtain the fermentation medium; The method for preparing the fed culture medium includes: weighing dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium sulfate, ammonium sulfate, ammonium chloride, diammonium hydrogen citrate, and glycerol, adding water to obtain the fed culture medium.

5. The use of the culture medium composition according to any one of claims 1-4 in the fermentation of recombinant *Escherichia coli*, characterized in that, The recombinant Escherichia coli is a recombinant Escherichia coli expressing PDRN, and the recombinant Escherichia coli is TA4-41 with accession number CGMCC No. 33625.

6. A method for fermenting recombinant Escherichia coli, characterized in that, The method includes using the culture medium composition according to any one of claims 1-4, wherein the recombinant Escherichia coli is a recombinant Escherichia coli expressing PDRN, and the recombinant Escherichia coli is TA4-41 with accession number CGMCC No. 33625.

7. The method according to claim 6, characterized in that, The method includes the following steps: S1. Add ampicillin sodium solution to fermentation medium to obtain fermentation broth; inoculate recombinant Escherichia coli seed culture into fermentation broth for fermentation culture; S2. After fermentation culture for 6 hours, add feeding medium and continue culture for 24-72 hours to obtain recombinant Escherichia coli fermentation broth.

8. The method according to claim 7, characterized in that, The amount of ampicillin sodium solution added in step S1 is 0.5‰-1.5‰ v / v of the fermentation medium volume; the amount of seed liquid inoculated is 0.5%-1.5% v / v of the fermentation liquid volume.

9. The method according to claim 7, characterized in that, In step S2, the feeding medium is added once every 4 hours, and the volume ratio of the feeding medium to the fermentation medium is 1:800-850 each time.

10. The method according to claim 7, characterized in that, The fermentation conditions described in step S1 are pH = 6.5-7.5, temperature 35-40℃, and rotation speed 200-400 rpm. An antifoaming agent is added after 6-8 hours of fermentation. The cultivation conditions described in step S2 are to maintain dissolved oxygen ≥ 30%, rotation speed 200-400 rpm, and temperature 35-40℃.

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