A fermentation medium for E. coli production using naked plasmid pSFVK1-NMM

Abstract

This invention discloses an Escherichia coli fermentation medium for the production of naked plasmid pSFVK1-NMM. The fermentation medium comprises: seed culture medium, basal fermentation medium, fed fermentation medium, and micronutrient solution. Each liter of seed culture medium contains: 5-7g tryptone, 10-14g yeast extract, 10g glycerol, 10-11g Na2HPO4, and 2-3g KH2PO4; each liter of basal fermentation medium contains: 10-14g tryptone, 22-26g yeast extract, 8-12g glycerol, and 2g Na2HPO4. 4 The fermentation feed medium contains 5-6g of glucose, 1-1.5g of KH₂PO₄, and 1-3ml of MgSO₄ solution; each liter of feed medium contains 160-200g of glucose; each 100mL of micronutrient solution contains 2.5g of MgSO₄, 10g of NaCl, 5g of L-glutamine, 2g of glycine, and 5g of NaH₂PO₄. Using the engineered strain XL10-Gold / pNMM and this culture medium, by controlling the fermentation conditions, a high-specificity yield of the plasmid can be obtained, achieving the fermentation production of the naked NMM tumor fusion antigen plasmid pSFVK1-NMM.

Description

[0001] This application is a divisional application of Escherichia coli fermentation medium for the production of naked plasmid pSFVK1-NMM, filed on August 10, 2018, application number 201810908221.7. Technical Field

[0002] This invention belongs to the field of genetic engineering technology, specifically relating to an Escherichia coli fermentation medium and culture method for the production of naked plasmid pSFVK1-NMM tumor fusion antigen. Background Technology

[0003] Plasmids are a type of double-stranded circular DNA molecule that carries genetic information outside of chromosomes. Because plasmids have the ability to replicate themselves, a single cell can contain multiple copies. Over the past few decades, plasmids have become one of the most important tools in fields such as genetics and molecular biology, and especially in recent years, they have become indispensable tools in the fields of gene therapy and gene vaccines. Plasmids can be used to treat single-gene defects, such as cystic fibrosis, muscular dystrophy, and hemophilia, and can also be used to induce specific immune responses against infectious diseases such as hepatitis and even tumors.

[0004] Plasmid DNA vaccines are a novel type of vaccine that has been under research since the 1990s. After nearly 30 years of development, they possess characteristics such as high safety, vector-free immunogenicity, and ease of production, giving them unique advantages compared to virus-based vaccines. To date, four veterinary DNA vaccines are commercially available, and 45 other DNA vaccines for the prevention or treatment of human diseases have entered Phase I, II, and III clinical trials. With the development of DNA vaccines and gene therapy, the demand for plasmid DNA will increase, necessitating the development of simple, time-saving, and high-yield plasmid DNA production processes.

[0005] The research, development, and production of plasmid DNA vaccines basically involve three stages: upstream, midstream, and downstream. The upstream stage includes vector and recombinant plasmid construction, host bacteria, and engineered bacterial seed banks. The midstream stage mainly involves bacterial fermentation. The downstream stage focuses on the isolation and purification of plasmid DNA. The naked plasmid pSFVK1-NMM, a tumor fusion antigen, has a large molecular weight, reaching approximately 15K, which is a heavy burden on the host bacteria. During the growth of the host bacteria, it easily causes instability in the plasmid DNA, thus affecting the copy number of the plasmid in a single bacterium. Optimizing the parameters and conditions of the engineered host bacteria fermentation culture mainly aims to increase yield, obtaining as many cells as possible per liter of culture medium, and then extracting as much plasmid DNA as possible per gram of cells. Achieving high yields generally requires optimizing the composition of the culture medium and using special fermentation strategies (dissolved oxygen feedback fed-batch fermentation). There are many research reports on the optimization of fermentation conditions for recombinant proteins, but the fermentation conditions for plasmid DNA are different from those for recombinant proteins. There are very few studies on the design and optimization of fermentation media and conditions for plasmid DNA. Under these circumstances, it is essential to establish a scientific and reasonable method for optimizing plasmid fermentation culture. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide an Escherichia coli fermentation culture medium for the production of the naked plasmid pSFVK1-NMM, a tumor fusion antigen.

[0007] The present invention also aims to provide a method for constructing a seed bank of engineered bacteria for producing the naked plasmid pSFVK1-NMM tumor fusion antigen, as well as the seed bank product. The engineered bacteria used to produce the naked plasmid pSFVK1-NMM tumor fusion antigen is *Escherichia coli* XL10-Gold transformed with plasmid pSFVK1-NMM, named XL10-Gold / pNMM.

[0008] The present invention also aims to provide a high-density fermentation method based on the above-mentioned Escherichia coli fermentation medium. This method utilizes the aforementioned engineered bacteria and fermentation medium, and controls the fermentation conditions to obtain engineered XL10-Gold / pNMM with high specific yield.

[0009] An E. coli fermentation medium for the production of naked plasmid pSFVK1-NMM.

[0010] Each liter of seed culture medium contains: 5-7g tryptone, 10-14g yeast extract, 10g glycerol, 10-11g Na2HPO4, and 2-3g KH2PO4;

[0011] Each liter of fermentation basal medium contains: 10-14g tryptone, 22-26g yeast extract, 8-12g glycerol, 5-6g Na2HPO4, 1-1.5g KH2PO4, and 1-3ml MgSO4 solution;

[0012] Each liter of fermentation feed medium contains: 160-200g of glucose;

[0013] Each 100mL of micronutrient solution contains: 2.5g MgSO4, 10g NaCl, 5g L-glutamine, 2g glycine, and 5g NaH2PO4.

[0014] The preparation method of the Escherichia coli fermentation medium is as follows:

[0015] The preparation method for 1 liter of seed culture medium is as follows: Weigh 5-7g of tryptone, 10-14g of yeast extract, 10g of glycerol, 10-11g of Na2HPO4, and 2-3g of KH2PO4, add deionized water to dissolve, make up to 1000mL, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0016] The preparation method for 1 liter of fermentation basal medium is as follows: Weigh 10-14g of tryptone, 22-26g of yeast extract, 8-12g of glycerol, 10-11g of Na2HPO4, and 2-3g of KH2PO4, dissolve in deionized water, bring the volume to 1000mL, autoclave at 121℃ for 30min, and store at 4℃ for later use; Before the start of fermentation, add 1-3ml of MgSO4 solution to each liter of fermentation basal medium; The preparation method for MgSO4 solution is as follows: Weigh 50g of MgSO4 solid, dissolve in deionized water, bring the volume to 100mL, sterilize through a 0.22um filter membrane, and store at 4℃ for later use;

[0017] The preparation method for 1 liter of fermentation feed medium is as follows: Weigh 180g of glucose, add deionized water to dissolve, make up to 1000mL, autoclave at 115℃ for 30min, and store at 4℃ for later use;

[0018] The preparation method for 100mL of micronutrient solution is as follows: Weigh 2.5g of MgSO4, 10g of NaCl, 5g of L-glutamine, 2g of glycine, and 5g of NaH2PO4, add deionized water to dissolve, and make up to 100mL. After sterilization by filtration through a 0.22um filter membrane, store at 4℃ for later use.

[0019] The construction method of the XL10-Gold / pNMM engineered bacteria seed bank is as follows:

[0020] (1) Preparation of the original seed bank: The engineered bacteria XL10-Gold / pNMM was activated and cultured in LB solid plates containing kanamycin at 30°C. Single clones were picked and inoculated into test tubes containing 5 mL of LB medium and cultured overnight at 30°C for 12 h. Then, they were inoculated into centrifuge tubes containing 20 mL of LB medium and cultured overnight at 30°C for 12 h. Glycerol was added to a final concentration of 20%, and the mixture was dispensed into 10 tubes and frozen in a freezer at -80°C to obtain the original seed bank.

[0021] (2) Preparation of the master seed bank: Resuscitate one original seed and inoculate it into 8 tubes of LB liquid medium. After subculturing, extract the plasmid. Select the bacterial culture with the highest plasmid specific yield and expand it to 100 mL of LB medium. After culturing at 30℃ and 200 RPM for 12 h, add glycerol to the final concentration of 20%. Dispense into 50 tubes and freeze in a -80℃ freezer to obtain the master seed bank.

[0022] (3) Preparation of working seed bank: One master seed was revived and inoculated into 8 tubes of LB liquid medium. After subculturing, plasmids were extracted. The bacterial culture with the highest plasmid specificity yield was selected and expanded to 100 mL of LB medium. After culturing at 30℃ and 200 RPM for 12 h, glycerol was added to a final concentration of 20%. The culture was then aliquoted into 50 tubes and frozen at -80℃ to obtain the working seed bank. The bacterial strain in each working seed bank was limited to five generations for the production of the naked plasmid pSFVK1-NMM, which is the NMM tumor fusion antigen.

[0023] A method for fermenting and culturing *E. coli* for the production of naked plasmid pSFVK1-NMM tumor fusion antigen includes the following steps:

[0024] (1) Seed culture preparation: The engineered bacteria XL10-Gold / pNMM working seed was revived in a -80℃ refrigerator and inoculated into the seed culture medium at a ratio of 0.6%~1%. The Erlenmeyer flask containing the seed culture was placed in a constant temperature shaker at 30℃ and 180-220r / min for 12-14 hours. If the bioreactor volume is large, the preparation of secondary and tertiary seed cultures can be increased.

[0025] (2) Fermentation culture: The seed liquid was inoculated into the bioreactor at a ratio of 8% to 10%, and fermentation culture was started under the control of the equipment. The fermentation tank was filled with the basic fermentation culture medium. During the fermentation process, hydrochloric acid and ammonia water were automatically added through the bioreactor to keep the pH value at 7±0.1. The culture temperature was 30℃ for 0-8h of fermentation culture and 37℃ for 8-12h of fermentation culture. 20mL / L micronutrient solution was added after 8 hours of fermentation culture. The dissolved oxygen was controlled above 30% by increasing the rotation speed and the aeration rate in the tank until the rotation speed reached 600rpm and the aeration rate reached 500L / h. The culture medium was fed in the form of dissolved oxygen feedback gradient constant flow feed. The fermentation culture was ended after 12 hours. The obtained bacterial liquid was centrifuged at not less than 6000g for 20 minutes to collect the bacterial cells and measure the fermentation parameters.

[0026] The seed culture temperature was 30 degrees Celsius, the fermentation temperature was 30 degrees Celsius from 0 h to 8 h, and the fermentation temperature was 37 degrees Celsius from 8 h to 12 h.

[0027] The dissolved oxygen feedback type gradient constant rate feeding method includes the following steps:

[0028] When the dissolved oxygen level rises significantly for the first time due to insufficient nutrients in the fermenter, the feed medium is automatically added at a flow rate of 10 mL / min / L.

[0029] When the dissolved oxygen level rises significantly for the second time due to insufficient nutrients in the fermenter, the feeding medium is automatically added at a flow rate of 15 mL / min / L.

[0030] When the dissolved oxygen level rises significantly for the third time due to insufficient nutrients in the fermenter, the feed medium is automatically added at a flow rate of 20 mL / min / L.

[0031] When the dissolved oxygen level rises significantly for the fourth time due to insufficient nutrients in the fermenter, the feeding medium is automatically added at a flow rate of 25 mL / min / L.

[0032] The beneficial effects of this invention are as follows: The method for constructing an engineered bacterial seed bank for producing the NMM tumor fusion antigen naked plasmid pSFVK1-NMM provided by this invention uses *E. coli* XL10-Gold transformed with plasmid pSFVK1-NMM, named XL10-Gold / pNMM, as the engineered bacterial strain used to produce the NMM tumor fusion antigen naked plasmid pSFVK1-NMM. The *E. coli* fermentation medium of this invention is used for the production of the NMM tumor fusion antigen naked plasmid pSFVK1-NMM. This invention employs a high-density fermentation method using *E. coli* fermentation medium, which utilizes the aforementioned engineered bacteria and fermentation medium, and controls the fermentation conditions to obtain the engineered bacteria XL10-Gold / pNMM with high specific yield. Attached Figure Description

[0033] Figure 1 The image shows the effect of culturing E. coli carrying the pSFVK1-NMM plasmid at 37 degrees Celsius. In the image, 1 is the 15K DNA Marker, 2 is the first generation of engineered bacteria cultured at 37 degrees Celsius, 3 is the second generation of engineered bacteria cultured at 37 degrees Celsius, and 4 is the third generation of engineered bacteria cultured at 37 degrees Celsius.

[0034] Figure 2 The image shows the effect of culturing E. coli carrying the pSFVK1-NMM plasmid at 37 degrees Celsius for more than 10 hours. In the image, 1 is the 15K DNA Marker, 2 is the engineered bacteria cultured at 37 degrees Celsius for 6 hours, 3 is the engineered bacteria cultured at 37 degrees Celsius for 8 hours, 4 is the engineered bacteria cultured at 37 degrees Celsius for 10 hours, and 5 is the engineered bacteria cultured at 37 degrees Celsius for 12 hours.

[0035] Figure 3 The image shows the effect of culturing E. coli carrying the pSFVK1-NMM plasmid at 30 degrees Celsius. In the image, 1 is the 15K DNA Marker, 2 is the first generation of engineered bacteria cultured at 30 degrees Celsius, and 3 is the tenth generation of engineered bacteria cultured at 30 degrees Celsius.

[0036] Figure 4 The growth curves of engineered bacteria cultured under four different temperature conditions are shown.

[0037] Figure 5 The growth curves of engineered bacteria fermented under different feed media are shown.

[0038] Figure 6 The growth curves of engineered bacteria under fermentation culture for 0-14 hours are shown.

[0039] Figure 7 The growth curves are for three consecutive batches of engineered bacteria cultured on a pilot-scale basis. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0041] Example 1: Screening of Seed Culture Temperature

[0042] Because the naked plasmid pSFVK1-NMM, a tumor fusion antigen, has a large molecular weight of approximately 15K, it poses a significant burden on the host bacteria. During the growth and division of the engineered bacterium XL10-Gold / pNMM, plasmid DNA instability is highly likely. Previous studies have shown that excessively high culture temperatures can cause changes in plasmid DNA size, while excessively low temperatures result in slow bacterial growth and require long incubation periods for the seed culture.

[0043] First, the engineered bacteria were cultured for three generations at 37°C, 200 rpm, and LB medium, with each generation lasting 12 hours. Then, plasmids were extracted using a plasmid miniprep kit. Agarose gel electrophoresis showed that the plasmid pSFVK1-NMM shrank to varying degrees. Therefore, the engineered bacteria XL10-Gold / pNMM cannot be cultured at 37°C for extended periods.

[0044] Then, samples were taken after culturing the engineered bacteria at 37°C, 200 rpm, and LB medium for 6, 8, 10, and 12 hours. Plasmids were extracted using a plasmid miniprep kit, and agarose gel electrophoresis showed that when cultured at 37°C for more than 10 hours, the plasmid pSFVK1-NMM shrank to varying degrees. This indicates that the culture time for the engineered bacteria XL10-Gold / pNMM at 37°C should be controlled within 10 hours.

[0045] The final test was conducted at 30 degrees Celsius and 200 rpm, with 12 hours constituting one generation. The engineered bacteria were continuously cultured for 10 generations. Then, the plasmid was extracted using a plasmid miniprep kit. Agarose gel electrophoresis showed that the size of plasmid pSFVK1-NMM was consistent with the expectation, indicating that the engineered bacteria can be stably cultured at 30 degrees Celsius for a long period of time.

[0046] Example 2: Screening of HG seed medium (HG seed medium)

[0047] The purpose of seed culture is to obtain a bacterial culture with good growth status and sufficient concentration in a short period of time to prepare for subsequent fermenter culture. To screen the most suitable seed culture medium, HGSM, engineered E. coli XL10-Gold / pNMM were inoculated into the following media, and the culture results were compared.

[0048] LB medium – Each 1 liter of LB medium contains: 10g tryptone, 5g yeast extract, and 10g NaCl. Dissolve in deionized water, bring the volume to 1000mL, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0049] LBC medium – Each liter of LBC medium contains: 10g tryptone, 5g yeast extract, 10g NaCl, and 5g glucose. Dissolve in deionized water, bring the volume to 1000mL, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0050] TB medium – Each liter of TB medium contains: 12g tryptone, 24g yeast extract, 10.22g Na2HPO4, 2.32g KH2PO4, and 5g glycerol. Dissolve in deionized water, bring the volume to 1000mL, autoclave at 21°C for 20min, and store at 4°C for later use.

[0051] TB1 medium – Each liter of TB medium contains: 18g tryptone, 18g yeast extract, 10.22g Na2HPO4, 2.32g KH2PO4, and 5g glycerol. Dissolve in deionized water, bring the volume to 1000mL, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0052] TBC medium – Each liter of TBC medium contains: 6g tryptone, 12g yeast extract, 10.22g Na2HPO4, 2.32g KH2PO4, and 10g glycerol. Dissolve in deionized water, bring the volume to 1000mL, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0053] TBC1 medium—Each liter of TBC1 medium contains: 9g tryptone, 9g yeast extract, 10.22g Na2HPO4, 2.32g KH2PO4, and 10g glycerol. Dissolve in deionized water, bring the volume to 1000mL, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0054] To facilitate comparison and evaluation, the shake-flask culture conditions were standardized as follows: One XL10-Gold / pNMM working seed culture was revived and inoculated into 240 mL of culture medium at a ratio of 1:200. After incubation at 30℃ and 200 rpm for 12 hours, a sample was taken to measure OD600, and the wet culture was weighed after centrifugation. By comparison, the culture medium with the highest OD600 value and the highest bacterial yield was selected as the seed culture medium.

[0055] Table 1 shows the bacterial yield and OD600 value of the bacterial solution under different culture medium conditions.

[0056]

[0057] The data in the table above shows that, under the same culture conditions, the engineered strain XL10-Gold / pNMM exhibits the fastest growth rate and the highest OD600 value in TBC medium. Therefore, TBC medium was chosen as the seed culture medium (HGSM).

[0058] Example 3: Screening of HGBM (HG basic medium) for fermentation

[0059] Before fermentation in a fermenter, it is necessary to first cultivate the bacteria on a shaker to determine the most suitable fermentation medium, laying the foundation for subsequent research. This section uses shaker cultivation as the main method to preliminarily study the bacterial yield and specific yield of the engineered bacteria in different media. The optimal medium determined will be used as the "fermentation basal medium HGBM (HGbasic medium)". For ease of comparison and evaluation, we have uniformly defined the following conditions for shake flask cultivation:

[0060] Working seed inoculation ratio: 1:100

[0061] Culture medium volume: 250 mL culture medium / 1000 mL Erlenmeyer flask

[0062] Shaker parameters: 30℃, 200 rpm

[0063] Incubation time: 12 hours

[0064] For the cultivation of engineered bacteria, we focus on the following two core parameters:

[0065] 1) Bacterial yield (g / L): The amount of wet bacteria that can be harvested per liter of culture medium.

[0066] 2) Specific yield (mg / g): The mass of plasmid DNA contained in each gram of wet bacteria.

[0067] When comparing different fermenter culture conditions, the above two parameters can be used for analysis and evaluation. Priority should be given to those with higher specific yields, followed by bacterial yield.

[0068] (1) First, compare the conventional culture media LB and TB for Escherichia coli, and compare the bacterial yield and specific yield, as shown in Table 2:

[0069] Table 2

[0070]

[0071] TB medium is superior to LB medium in both bacterial yield and bacterial-specific yield, so we chose to continue screening based on TB medium.

[0072] (2) Based on TB medium, the ratio of peptone to yeast extract was changed, and the bacterial yield and specific yield were compared, as shown in Table 3:

[0073] Table 3

[0074]

[0075] Although TB1 medium had the lowest bacterial yield, it had the best bacterial-specific yield. We chose to continue screening based on TB1 medium.

[0076] (3) Based on TB1 medium, different concentrations of glycerol were adjusted, and the bacterial yield and specific yield were compared, as shown in Table 4:

[0077] Table 4

[0078]

[0079] Among them, TB8 medium showed the highest bacterial-specific yield. We chose to further optimize based on TB8 medium.

[0080] (4) Based on TB8 medium, the phosphate concentration was adjusted and MgSO4 was increased. The bacterial yield and specific yield were compared, as shown in Table 5:

[0081] Table 5

[0082]

[0083] TB10 medium had the highest bacterial specific yield, so the selected TB10 medium was used as "HGBM (HG basic medium)" for subsequent fermentation experiments.

[0084] Example 4: Screening of Fermentation Temperature

[0085] Because the plasmid pSFVK1-NMM is relatively large, it exhibits fission instability during host bacterial replication, resulting in plasmid quality that does not meet requirements. The inventors discovered through shake-flask experiments that the engineered bacteria XL10-Gold / pNMM grows slowly at 30℃, failing to reach sufficient bacterial concentration quickly, but exhibits good plasmid stability. While growth is rapid at 37℃, plasmid stability is poor; agarose gel electrophoresis revealed that the plasmid began to shrink after 6 hours of culture at 37℃. Therefore, this invention employs a variable-temperature fermentation mode, with an initial temperature of 30℃ and a later temperature of 37℃, to accelerate the growth of the engineered bacteria while ensuring the quality of plasmid pSFVK1-NMM. The timing of temperature transitions in this mode significantly impacts the overall fermentation results. To facilitate comparison, the effects of different temperature transition times on fermentation process parameters were explored (…). Figure 4 In the following comparative experiment, the total fermentation time was set at 12 hours, and the specific steps are as follows:

[0086] Shake-flask culture of seed culture: The working seed was inoculated into the seed culture medium at a ratio of 1:100 and cultured at 30 degrees Celsius and 200 rpm for 13 hours.

[0087] Fermenter culture: The seed culture was inoculated into 3L of fermentation basal medium at a ratio of 10%, and initially cultured at 30 degrees Celsius. At 6, 7, 8 and 9 hours after the start of fermentation, the temperature was increased from 30 degrees Celsius to 37 degrees Celsius. When dissolved oxygen began to rise rapidly due to insufficient nutrients in the fermenter, glycerol (180 g / L) solution was added by constant-rate gradient feeding using a dissolved oxygen feedback method. Fermentation was stopped after 12 hours, and the cells were collected by centrifugation to determine the cell yield and specific yield.

[0088] Table 6 compares the biomass and specific yield under different fermentation temperatures.

[0089]

[0090] The yield of bacteria did not differ much at different temperature transition times, but the engineered bacteria cultured at 30 degrees Celsius for 8 hours and then raised to 37 degrees Celsius for further culture had the highest specific yield.

[0091] Example 5: Effect of Feeding Components on Fermentation Culture

[0092] After the engineered bacteria were cultured in a fermenter at 30°C for 8 hours on a basic fermentation medium, the fermentation temperature was increased to 37°C for continued cultivation. At this point, due to the increased temperature, the engineered bacteria's reproduction and metabolism accelerated, causing the dissolved oxygen level to gradually decrease. After approximately 10 hours of fermentation, a rapid increase in dissolved oxygen was observed, indicating that the nutrients in the fermenter had been almost entirely consumed by the engineered bacteria, necessitating timely replenishment. This project employed a dissolved oxygen feedback-type gradient constant-rate feeding method to replenish nutrients until the end of fermentation. The specific feeding method is as follows:

[0093] When the dissolved oxygen level rises significantly for the first time due to insufficient nutrients in the fermenter, the feed medium is automatically added at a flow rate of 10 mL / min / L.

[0094] When the dissolved oxygen level rises significantly for the second time due to insufficient nutrients in the fermenter, the feeding medium is automatically added at a flow rate of 15 mL / min / L.

[0095] When the dissolved oxygen level rises significantly for the third time due to insufficient nutrients in the fermenter, the feed medium is automatically added at a flow rate of 20 mL / min / L.

[0096] When the dissolved oxygen level rises significantly for the fourth time due to insufficient nutrients in the fermenter, the feed medium is automatically added at a flow rate of 25 mL / min / L.

[0097] Different feed ingredients have a significant impact on bacterial growth in the later stages of fermentation. Figure 5 In this study, glucose or glycerol was selected as the fed culture medium, and the differences in various fermentation parameters were compared. For ease of comparison, the total fermentation time was set to 12 hours in the following comparative experiments, and the concentrations of glucose and glycerol were both 180 g / L.

[0098] Table 7

[0099]

[0100] As shown in Table 7, when glucose (180 g / L) or glycerol (180 g / L) is used as the supplemented culture medium, the bacterial yield is almost the same. However, the specific yield of engineered bacterial plasmids obtained by supplementing glucose is significantly higher than that obtained by supplementing glycerol.

[0101] Example 6: Screening of total fermentation time

[0102] Bacterial growth in fermenters is divided into three phases: lag phase, logarithmic growth phase, and plateau phase. High concentrations of seed culture and high inoculum ratios can shorten the lag phase. During the logarithmic growth phase, the bacterial population increases rapidly, but the plasmid content of individual bacteria may not be high. Generally, towards the end of the logarithmic growth phase or the plateau phase, when the bacterial population no longer grows rapidly, the plasmid content of individual bacteria gradually increases. However, because the fermentation medium does not contain antibiotics and there is no selective pressure, if bacteria remain in the plateau phase for too long, the number of bacteria without plasmids will increase, leading to a decrease in specific yield. Therefore, determining the appropriate fermentation termination time is crucial.

[0103] The engineered bacteria were cultured in a fermenter at 30°C for 8 hours on a basic fermentation medium, and then the fermentation temperature was increased to 37°C for continued cultivation. After approximately 10 hours of fermentation, due to nutrient deficiency in the fermenter, feed was added via a dissolved oxygen feedback gradient constant-rate feeding method. Since the 37°C incubation period could not exceed 6 hours, fermentation was terminated at 14 hours. The optimal fermentation termination time was determined by comparing the bacterial yield and bacterial-specific yield from 10 to 14 hours. Figure 6 (Table 8).

[0104] Table 8 compares the bacterial yield and specific yield after 10 to 14 hours of fermentation.

[0105]

[0106] During fermentation, bacterial growth is slow from 0 to 5 hours; from 5 to 10 hours, the bacterial count enters the logarithmic phase and grows rapidly; after 10 hours, a plateau phase occurs, and the rate of increase slows down. The highest bacterial-specific yield occurs at 12 hours of fermentation; after 12 hours, although bacterial yield continues to increase, the specific yield gradually decreases. This may be because the culture medium lacks antibiotics; after 12 hours of fermentation, the number of engineered bacteria containing plasmids gradually decreases, while the number of bacteria without plasmids gradually increases, ultimately leading to a decrease in the specific yield. Therefore, the optimal fermentation termination time is 12 hours.

[0107] Example 7: Production of plasmid pSFVK1-NMM from engineered strain XL10-Gold / pNMM in a 5L fermenter

[0108] Based on research into the culture medium and cultivation methods, the optimal cultivation steps for a 5L fermenter were determined as follows:

[0109] (1) Preparation of various culture media required for fermentation:

[0110] Prepare 300ml of seed culture medium HGSM: Weigh 1.8g of tryptone, 3.6g of yeast extract, 1.5g of glycerol, 3.07g of Na2HPO4, and 0.7g of KH2PO4, add deionized water to dissolve, and bring the volume to 300mL. Autoclave at 121℃ for 20min and store at 4℃ for later use.

[0111] To prepare 10 ml of MgSO4 solution: Weigh 5 g of solid MgSO4, add deionized water to dissolve it, make up to 10 mL, filter it through a 0.22 μm filter membrane for sterilization, and store it at 4 °C for later use.

[0112] Prepare 3L of HGBM fermentation basal medium: Weigh 36g tryptone, 72g yeast extract, 30g glycerol, 15.33g Na2HPO4, and 3.48g KH2PO4, dissolve in deionized water, and bring the volume to 3L. Autoclave at 121℃ for 30min and store at 4℃ for later use. Before starting fermentation, add 6ml of MgSO4 solution to the fermentation basal medium.

[0113] Prepare 300ml of HGAM fermentation feed medium: Weigh 54g of glucose, add deionized water to dissolve, bring the volume to 300mL, autoclave at 115℃ for 30min, and store at 4℃ for later use.

[0114] Prepare 100ml of micronutrient solution: Weigh 1g of MgSO4, 4g of NaCl, 2g of L-glutamine, 0.8g of glycine, and 2g of NaH2PO4. Dissolve in deionized water, bring the volume to 100mL, filter through a 0.22um filter membrane for sterilization, and store at 4℃ for later use.

[0115] (2) Preparation of seed solution:

[0116] Two engineered bacterial strains XL10-Gold / pNMM were revived at -80℃ and inoculated into 300ml of seed culture medium at an 8% ratio. The Erlenmeyer flask containing the seed culture was then incubated in a constant temperature shaker at 30℃ and 200r / min for 13 hours.

[0117] (3) 5L fermenter culture:

[0118] The seed culture was inoculated at a ratio of 10% into a 5L bioreactor, and fermentation was initiated under equipment control. The fermenter contained 3L of basal fermentation medium. During fermentation, hydrochloric acid and ammonia were automatically added to the bioreactor to maintain the pH at 7±0.1. The fermentation temperature was 30℃ for 0-8 hours. After 8 hours of fermentation, the temperature was increased to 37℃, and 20mL / L of micronutrient solution was added. Dissolved oxygen was maintained above 30% by increasing the rotation speed and aeration rate until the rotation speed reached 600rpm and the aeration rate reached 10L / min. When the dissolved oxygen level showed a significant upward trend for the first time due to insufficient nutrients in the fermenter, feed medium was automatically added at a flow rate of 10mL / min / L. When the dissolved oxygen level showed a second, third, and fourth significant upward trend, the corresponding feed flow rates were 15mL / min / L, 20mL / min / L, and 25mL / min / L, respectively. Fermentation was completed after 12 hours. The obtained bacterial culture was centrifuged at 8000g for 20 minutes to collect the bacterial cells, and the fermentation parameters were measured.

[0119] Following the above fermentation steps, three fermentations were carried out in a 5L fermenter (containing 3L of culture medium). Figure 7 The growth curves are for three consecutive batches of engineered bacteria cultured on a pilot-scale basis.

[0120] To confirm that the above culture medium and method are superior to conventional E. coli culture, TB medium was used as the control medium. The medium was cultured at 30°C using a standard single-feeding method until the nutrients in the fermenter were depleted. Other culture conditions were the same as the experimental group. Specific fermentation parameters are shown in Table 9.

[0121] Table 9

[0122]

[0123] Using the culture medium and special method obtained in this invention, the engineered bacteria XL10-Gold / pNMM were fermented to produce plasmid pSFVK1-NMM. The plasmid-specific yield was consistently above 1.15 mg / g, and the bacterial yield reached 60-65 g / L. In contrast, the control group had a plasmid-specific yield of only 0.71 mg / g and a bacterial yield of only 38.1 g / L. This demonstrates that the culture medium and method developed in this invention are superior to conventional E. coli fermentation methods.

Claims

1. An Escherichia coli fermentation medium for the production of naked plasmid pSFVK1-NMM, characterized in that: Each liter of seed culture medium contains: 5-7g tryptone, 10-14g yeast extract, 10g glycerol, 10-11g Na2HPO4, and 2-3g KH2PO4; Each liter of fermentation basal medium contains: 10-14g tryptone, 22-26g yeast extract, 8-12g glycerol, 5-6g Na2HPO4, 1-1.5g KH2PO4, and 1-3ml MgSO4 solution; Each liter of fermentation feed medium contains: 160-200g of glucose; Each 100mL of micronutrient solution contains: 2.5g MgSO4, 10g NaCl, 5g L-glutamine, 2g glycine, and 5g NaH2PO4.

2. The Escherichia coli fermentation medium for producing naked plasmid pSFVK1-NMM according to claim 1, characterized in that, The preparation method of the Escherichia coli fermentation medium is as follows: The preparation method for 1 liter of seed culture medium is as follows: Weigh 5-7g of tryptone, 10-14g of yeast extract, 10g of glycerol, 10-11g of Na2HPO4, and 2-3g of KH2PO4, add deionized water to dissolve, make up to 1000mL, autoclave at 121℃ for 20min, and store at 4℃ for later use. The preparation method for 1 liter of fermentation basal medium is as follows: Weigh 10-14g of tryptone, 22-26g of yeast extract, 8-12g of glycerol, 10-11g of Na2HPO4, and 2-3g of KH2PO4, dissolve in deionized water, bring the volume to 1000mL, autoclave at 121℃ for 30min, and store at 4℃ for later use; Before the start of fermentation, add 1-3ml of MgSO4 solution to each liter of fermentation basal medium; The preparation method for MgSO4 solution is as follows: Weigh 50g of MgSO4 solid, dissolve in deionized water, bring the volume to 100mL, sterilize through a 0.22um filter membrane, and store at 4℃ for later use; The preparation method for 1 liter of fermentation feed medium is as follows: Weigh 180g of glucose, add deionized water to dissolve, make up to 1000mL, autoclave at 115℃ for 30min, and store at 4℃ for later use; The preparation method for 100mL of micronutrient solution is as follows: Weigh 2.5g of MgSO4, 10g of NaCl, 5g of L-glutamine, 2g of glycine, and 5g of NaH2PO4, add deionized water to dissolve, and make up to 100mL. After sterilization by filtration through a 0.22um filter membrane, store at 4℃ for later use.

Images