Fermentation method of plasmid-containing escherichia coli and method for producing plasmid

Abstract

The invention discloses a fermentation method of plasmid-containing escherichia coli and a method for producing plasmids. The fermentation method comprises the following secondary fermentation steps: performing primary tank fermentation: performing constant-temperature fermentation at 30-34 DEG C, and maintaining OD at 20-40; and secondary tank fermentation: transferring the fermentation liquid in the primary tank into a secondary tank, fermenting at the constant temperature of 37-42 DEG C, and culturing by adopting a fed-batch fermentation mode. A two-stage fermentation mode is adopted, continuous fermentation can be conducted for at least 50 h or above, and at least four cycles of continuous production can be conducted through a two-stage tank. In one fermentation period, the plasmid yield per unit time is at least improved by more than 300%, and the improvement effect is obvious. The method has the advantages that the high-efficiency plasmid production period is greatly shortened, continuous circulation can be continuously carried out, the proportion time of seed culture and early-stage and post-treatment can be greatly shortened in the whole fermentation period, a large amount of fermentation time cost can be saved, and the purpose of high yield of plasmids within the same time can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, in particular to a fermentation method of Escherichia coli containing a plasmid and a method for producing the plasmid. Background Art

[0002] Plasmid fermentation is a technology that inoculates the bacteria that produce plasmids into a suitable culture system, replicates the bacteria in large quantities in the appropriate culture system, and synthesizes plasmid DNA in large quantities and rapidly in bacterial cells. Or it can be said to be plasmid amplification, which is a chemical and biological method for obtaining plasmids in large quantities, usually including the process of transforming bacterial plasmids into Escherichia coli and then harvesting a large number of plasmids from Escherichia coli.

[0003] Some explorations have also been made in the prior art for the continuous production of plasmids. For example, US2008 / 0318283A1 explored a two-stage fermentation and continuous feeding and discharging method, in which the temperature of the first stage fermentation was 30°C and the temperature of the second stage fermentation was 36-45°C. However, the fermentation process and the harvesting of bacterial liquid have been continuous and uninterrupted, and only 0.32L of bacterial liquid is harvested per hour. The defects are that, first, this kind of continuous fermentation will cause part of the bacterial liquid that has just entered the secondary tank from the primary tank to be harvested as a plasmid product without sufficient temperature induction and low plasmid yield. Second, this kind of continuous fermentation will cause part of the feed culture medium that has just entered the secondary tank and the bacterial liquid from the primary tank to be harvested as a plasmid product before the nutrients are consumed, resulting in low material utilization and cost waste. Third, this kind of continuous fermentation continuously harvests plasmid bacterial liquid, and it is difficult to continuously harvest bacterial bodies by centrifugation or hollow fiber. A third storage tank is needed to temporarily store the bacterial liquid. After reaching a certain volume, the bacterial bodies are harvested by centrifugation or hollow fiber. However, at the same time, these bacteria will still grow in the storage tank, especially the bacterial liquid harvested earlier will stay in the storage tank for a long time. If you want to maintain a low growth metabolism, it is best to cool it down to below 15°C. If you put it on a production scale, the cost is extremely high. Fourth, the secondary tank feed medium starts to be added at the same time as the bacterial liquid from the primary tank enters the secondary tank. The feeding method is continuous feeding. At this time, the bacterial liquid OD600 is low and the nutrients are sufficient, which may cause excess nutrients in the secondary tank and an imbalance in the carbon-nitrogen ratio, which is not conducive to the growth of the bacterial liquid.

[0004] At present, the plasmid fermentation platform has undergone two generations of process iterations. The first generation of plasmid fermentation process is relatively extensive. The bacterial strain is initially revived and cultured to obtain bacterial seeds, which are inoculated into the fermentation tank. After 18-22 hours of fermentation, the nutrients in the culture medium are consumed, and the fermentation is ended and the bacteria are harvested. The fermentation type belongs to a batch fermentation process. As the fermentation proceeds, the metabolic waste in the culture medium is also slowly increasing, and the timing of bacterial growth and plasmid production is not completely matched. At the end of the fermentation, the nutrients are exhausted, but the bacterial production of plasmids has not reached its peak.

[0005] Therefore, it is necessary to research and develop more efficient plasmid fermentation or plasmid production methods. Summary of the invention

[0006] Based on the above defects and considerations, the second generation fermentation process was developed. This process is a batch fed fermentation process. On the basis of batch fermentation, in the late fermentation period, when the nutrients are consumed, feeding is performed to increase the nutrients in the culture medium, prolong the plasmid production time, and achieve the purpose of high plasmid production. Figure 1 As shown, compared with batch fermentation, the plasmid yield of the batch fed fermentation process can be increased from about 550 mg / L before feeding to 770 mg / L. At the same time, due to the addition of feeding, the fermentation volume will also increase, and the final yield can be increased by more than 50%. However, in the existing batch fed fermentation process, the entire production cycle is about 3.5 days during the plasmid production process, but the time in the fermenter is only 28 hours. A lot of time is spent on seed culture, preliminary preparation and post-processing, and the efficiency of plasmid production is relatively low. Therefore, considering how to extend the growth time in the fermenter, the proportion of seed culture and preliminary and post-processing in the entire fermentation cycle will be greatly shortened, which can save a lot of fermentation time costs, and can achieve the purpose of high-yield plasmids in the same time. In addition, the defects of existing continuous fermentation are fully considered and overcome.

[0007] In view of this, the present invention is proposed.

[0008] The present invention provides a fermentation method of Escherichia coli containing a plasmid, which is a two-stage fermentation.

[0009] S1 first-stage tank fermentation, using constant temperature fermentation at 30-34℃, maintaining OD at 20-40;

[0010] In S2, the fermentation liquid in the primary tank is transferred to the secondary tank for fermentation at a constant temperature of 37° C. to 42° C. In S2, the culture is carried out by fed-batch fermentation, and the feeding time is when the C source is exhausted and the DO rises rapidly to more than 70%.

[0011] S1 and S2 use a fermentation medium as a fermentation basal medium, which contains a liquid culture medium containing dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, yeast extract powder, glycerol, a defoamer, kanamycin sulfate and magnesium sulfate heptahydrate;

[0012] Preferably, the formula is as follows: 6.55 g / L of dipotassium hydrogen phosphate trihydrate; 3.5 g / L of potassium dihydrogen phosphate; 80.0 g / L of yeast extract powder; 60.0 g / L of glycerol; 0.05% (v / v) of defoaming agent; 0.05 g / L of kanamycin sulfate; and 0.50 g / L of magnesium sulfate heptahydrate.

[0013] In S1, the seed retention ratio in the primary tank is 0.5%-1%, and fresh fermentation medium equal in volume to the fermentation liquid transferred to the secondary tank is added for continuous culture.

[0014] In the S1, the culture temperature is optimally 31-33°C, more preferably 32°C; the OD is maintained at 25-35, more preferably 28-32, and the optimal OD is 30.

[0015] In S2, DO feedback feeding is adopted, specifically, the feeding volume is 35-45 mL / time, the feeding time is 3-5 h, and 2-5 cycles of continuous fermentation are carried out, preferably the feeding volume is 40 mL / time, the feeding time is 4 h, and 3-4 cycles of continuous fermentation are carried out.

[0016] The feed medium contains glycerol, yeast powder, magnesium sulfate heptahydrate, and trace element mother solution; wherein the trace element mother solution contains ferrous sulfate heptahydrate, calcium chloride dihydrate, manganese chloride tetrahydrate, zinc sulfate heptahydrate, cobalt chloride hexahydrate, cupric chloride dihydrate, ammonium molybdate tetrahydrate, sodium borate decahydrate, and concentrated hydrochloric acid;

[0017] Alternatively, the feed medium formula is as follows: it contains glycerol, yeast powder, magnesium sulfate heptahydrate, and vitamin solution mother liquor; wherein the vitamin solution mother liquor contains thiamine hydrochloride (VB1), biotin, nicotinamide, vitamin B12, D-calcium pantothenate (VB5) and pyridoxine hydrochloride (VB6).

[0018] Preferably, the feed medium is formulated as follows: 300 g / L glycerol; 250 g / L yeast powder; 1 g / L magnesium sulfate heptahydrate; 10 ml / L trace element mother solution; wherein the trace element mother solution contains 6.95 g ferrous sulfate heptahydrate per 1000 ml; and calcium chloride dihydrate.

[0019] 1.47g; manganese chloride tetrahydrate 9.90g; zinc sulfate heptahydrate 2.88g; cobalt chloride hexahydrate 0.357g; cupric chloride dihydrate 0.256g; ammonium molybdate tetrahydrate 1.85g; sodium borate decahydrate 3.05g; concentrated hydrochloric acid 25ml.

[0020] Alternatively, the feed medium formula is as follows: 300 g / L glycerol; 250 g / L yeast powder; 1 g / L magnesium sulfate heptahydrate; 10 ml / L vitamin solution mother liquor; wherein the vitamin solution mother liquor, calculated based on 1000 ml, comprises 4 g / L thiamine hydrochloride (VB1); 0.20 g / L biotin; 4 g / L nicotinamide (VB3); 4 g / L vitamin B12; 2 g / L D-calcium pantothenate (VB5); and 4 g / L pyridoxine hydrochloride (VB6).

[0021] In S2, the optimal culture temperature is 39-41°C, more preferably 40°C.

[0022] The Escherichia coli can be any type of Escherichia coli, which can be a wild-type Escherichia coli or a derivative thereof, such as a mutant Escherichia coli, for example, DH5α, DH10B, BL21, JM109, JM110, TOP10, HB101, XL0-Gold, M110, SCS10 and other series of Escherichia coli.

[0023] The plasmid can be any plasmid suitable for Escherichia coli, for example, pUC series plasmids such as pUC19, pLysS, pRARE, pGEX series vectors such as pGEX-1λT, pGEX-2T, pGEX-3T, pET series vectors such as pET-28a-c(+), pPL series vectors such as pPL-Lamda, pEZZ18, pINII series vectors such as pINII-ompA1, pINII-comA1, pINII-comA2, pINII-comA3, pKK223 series vectors such as pKK223-3, pVAX1, etc., and shuttle vectors such as pYES2 / NT / lacZ, etc.

[0024] The present invention also provides a method for producing a plasmid, which comprises the steps of obtaining Escherichia coli containing the plasmid by fermentation through the above method, and collecting the plasmid therefrom.

[0025] The present invention has finally formed a secondary fermentation process suitable for Escherichia coli plasmid production through a large number of research and exploration. Specifically, the present invention has explored the conditions of constant temperature fermentation and two-stage fermentation, including temperature, maintaining OD, heating OD, feeding ingredients, feeding methods, seed retention ratio and other parameters, and found that the two-stage fermentation method can better achieve the purpose of continuous fermentation, and can be continuously fermented for at least 50 hours, and the secondary tank can continuously produce at least 4 cycles, while the quality of the plasmid has no obvious change. In a fermentation cycle, the yield of plasmid per unit time is at least increased by more than 300%, and the improvement effect is obvious. Although two devices are used, the production cycle on the secondary tank is about 11 hours, which greatly shortens the cycle of efficient production of plasmids, and the continuous cycle can continue, and the subsequent yield data advantage will be more obvious; at the same time, the growth time of the primary fermentation tank is extended, and the proportion of seed culture and pre-processing and post-processing in the entire fermentation cycle will be greatly shortened, which can save a lot of fermentation time cost, and can achieve the purpose of high yield of plasmids in the same time. In particular, it is also confirmed in the examples of the present invention that intermittent discharge and feeding are more beneficial to maintaining the vitality of the bacteria. This may be related to the fact that after intermittent discharge and feeding, the proportion of metabolic waste in the tank will be lower than that in continuous fermentation, and the content of added nutrients will be higher than that in continuous discharge and feeding. Maintaining a lower OD level (60-80) will also be beneficial to the vitality level of the bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Comparison of plasmid yields between batch fermentation and fed-batch fermentation;

[0027] Figure 2 The results of plasmid fermentation at constant temperature of 35℃ and 37℃;

[0028] Figure 3 It is the result of continuous fermentation at a constant temperature of 35℃;

[0029] Figure 4 To reduce the fermentation results at 35°C after maintaining OD;

[0030] Figure 5 The fermentation result was maintained at a constant temperature of 32°C at OD80-100;

[0031] Figure 6 The fermentation results were maintained at OD60-80 at a constant temperature of 32°C;

[0032] Figure 7 The continuous fermentation results were maintained at OD60-80 at a constant temperature of 30°C;

[0033] Figure 8 The results of the plasmid yield per OD in the continuous fermentation at 30℃ and 32℃;

[0034] Fig. 9 It is a schematic diagram of two-stage fermentation;

[0035] Fig.10 It is a schematic diagram of the volume change of the fermentation liquid in the two-stage fermentation tank;

[0036] Fig.11 The OD and feeding results of the first tank of two-stage fermentation;

[0037] Fig.12 This is the result of the plasmid yield per unit OD in the secondary tank of two-stage fermentation;

[0038] Fig.13 The results of plasmid fermentation at different maintenance OD;

[0039] Fig.14 The effects of adding trace elements and vitamins to the feed on plasmid fermentation. Among them, FE1 is a normal feed as a control, FE2 is the addition of trace elements, FE3 is the addition of vitamins, and FD is the temperature increase above OD80;

[0040] Fig.15 Maintain OD for the first tank of the two-stage fermentation;

[0041] Fig.16 The results of OD and plasmid yield in the secondary tank in two-stage fermentation. Among them, FE1 is normal feed as control 1, FE2 is the addition of trace elements, FE3 is normal feed as control 2, and FD is the temperature increase OD60 or above;

[0042] Fig.17 The results of continuous fermentation in two-stage tanks are shown in Figure 1. FB-1 is cycle 1, FB-2 is cycle 2, FB-3 is cycle 3, and FB-4 is cycle 4. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be clearly described below in conjunction with the accompanying drawings. Obviously, the described embodiments are not all embodiments of the present invention, and all other embodiments obtained by ordinary technicians in the field without making creative work are within the protection scope of the present invention.

[0044] Example 1 Exploration of constant temperature fermentation temperature on the existing platform batch fed fermentation process

[0045] The main parameters of the batch fed-batch fermentation process of the existing process platform are shown in Table 1. It is a variable temperature batch fed-batch fermentation process. During fermentation, the fermentation temperature is controlled according to the growth of OD. When OD<10, the fermentation temperature is controlled to 30℃±0.5. When OD>10, the fermentation temperature is controlled to 37℃±0.5. For continuous fermentation, it is difficult to stably control the temperature in the tank at a constant value, and the temperature has a greater impact on the fermentation plasmid. Therefore, the difficulty of development is temperature stability control, and the entire development process is mainly developed around the difficulty of temperature stability control.

[0046] The strain used in process development is the following constructed 1101-B-04 strain, in which the production plasmid is pAD1016 (recorded in Chinese patent application with application number CN202310087259.3), the plasmid vector is pVAX1 (ThermoFisher, product number: V26020), the size is 4250bp, and it contains a kanamycin resistance gene. The fermentation conditions are explored in a 5L fermenter.

[0047] Construction of pAD1016 recombinant plasmid: The coronavirus-related DNA sequence was inserted between the BamH I and Xho I sites of the pVAX1 vector to obtain a recombinant plasmid, which was recorded as pAD1016.

[0048] 1101-B-04 strain construction process: This strain was purchased from Thermo Fisher One Shot TM MAXEfficiency TMDH10B T1 phage tolerant cells are used as recipient cells (E. coli cells), and pAD1016 recombinant plasmid (plasmid vector is pVAX1, size is 4250bp) is introduced into competent cells. Since the recombinant plasmid carries a kanamycin resistance gene, transformants can be screened by kanamycin resistance. If the recipient cells can grow on a plate containing kanamycin resistance, it proves that the recipient cells have been introduced with the recombinant pVAX1 plasmid. Then, a single recombinant plasmid is selected on a plate containing kanamycin resistance and placed in LB medium. When the bacteria OD600 is cultured at 30°C and 220rpm to 2-3, the recombinant bacteria are frozen. The freezing specification is 1ml / tube, and each frozen strain contains 20% glycerol as a protective agent.

[0049] Table 1 Main parameters of batch fed-batch fermentation process

[0050]

[0051] Table 2 Fermentation feed medium

[0052]

[0053]

[0054] Since temperature is particularly important for plasmid fermentation production, and temperature parameters affect the continuous fermentation process development process, continuous heating and cooling in the whole process may affect the viability of the bacteria and plasmid production, and increase the difficulty of operation and control costs. Therefore, a preliminary exploration of the constant temperature fermentation process is conducted on the basis of the existing batch fed fermentation platform process to try to carry out long-term constant temperature fermentation, and explore the constant temperature fermentation temperature between the termination growth temperature of 30°C and the fermentation temperature of 37°C.

[0055] 1) Exploration of constant temperature fermentation at 35℃ and 37℃

[0056] Since 37°C is the temperature parameter for high-yield plasmids, in the early stages of development, in order to achieve the purpose of continuous fermentation, we tried to conduct preliminary exploration at constant temperatures of 37°C and 35°C, such as Figure 2 As shown in the results, it was found that under 37°C conditions, bacterial growth was inhibited and the maximum OD was about 30, which was only 1 / 3 of the normal fermentation level of the existing batch fed fermentation platform process. Under the constant temperature condition of 35°C, the OD could grow to the same level. Therefore, 35°C was selected as the stable temperature for constant temperature fermentation, and continuous fermentation was attempted.

[0057] When using 35°C for continuous fermentation temperature exploration, the method of continuously releasing culture medium (discharging) and supplementing feed (feeding) was adopted. The feed was to supplement the fermentation basal culture medium, and the formula is shown in Table 3; that is, by adjusting the speed of feeding and discharging, the OD in the fermenter was controlled to be between 80-100, so that the discharge and feeding speeds were the same to keep the volume of fermentation liquid in the fermenter constant (acid and alkali addition and evaporation consumption were not taken into account). The results showed that under continuous fermentation conditions at 35°C, the feeding and discharging speeds continued to decrease in the later stage of fermentation, indicating that the growth rate of the bacteria in the tank continued to slow down, the vitality of the bacteria weakened, and the OD maintained at 80-100 could only last for about 8 hours, and then dropped rapidly. The plasmid yield can reach about 420 mg / L. Figure 3 shown.

[0058] The OD was maintained at 60-80, and the feeding and replenishing method was adopted with a 1h period to reduce the content of metabolic waste in the tank, reduce the competition pressure between bacteria in the fermentation broth, reduce the concentration of the fermentation broth, and increase the mass transfer rate of oxygen and nutrients. The results showed that the continuous fermentation could be increased to 11h, but the feeding and discharging speed dropped rapidly afterwards, indicating that the bacterial activity was difficult to maintain in the later period. Figure 4 shown.

[0059] In summary, during constant temperature fermentation at 37°C, bacterial growth is inhibited, bacterial growth is slow, and the final OD is low. However, during constant temperature fermentation at 35°C, the OD can grow normally to about 100, reaching the bacterial concentration level of the existing platform batch fed fermentation process. However, during continuous fermentation, the maintenance time is shorter, only about 8-11 hours, and the plasmid yield can reach the batch fermentation plasmid concentration level.

[0060] Table 3 Fermentation basal medium

[0061]

[0062]

[0063] 2) Exploration of constant temperature fermentation at 32℃ and 30℃

[0064] Based on the above results, we continued to explore the temperature conditions and tried to control the temperature parameters to 32℃ for constant temperature fermentation (the continuous fermentation mode is the same as 1.1.1). The results showed that under 32℃, OD80-100 was maintained, and the OD could be maintained for about 14 hours with continuous feeding and unloading. Under the condition of maintaining OD60-80, the OD could be maintained for about 45 hours with intermittent feeding and unloading for 1 hour. Figure 5 , Figure 6 shown.

[0065] The above results show that under 32℃, continuous fermentation can be maintained for at least 14h. With 1h period intermittent feeding and feeding and maintaining OD60-80, continuous fermentation can be maintained for at least 45h (the whole fermentation cycle is 60h). After 60h, continuous feeding and feeding are switched. Under the same feeding and feeding speed, OD will decrease, indicating that intermittent feeding and feeding are more beneficial to the maintenance of bacterial vitality. It may be related to the fact that after intermittent feeding and feeding, the proportion of metabolic waste in the tank will be lower than that of continuous fermentation, and the content of added nutrients will be higher than that of continuous feeding and feeding. Maintaining a lower OD level (60-80) will also be beneficial to the bacterial vitality level. Under the constant temperature of 32℃, the plasmid level is about 200mg / L, which is lower than the plasmid yield of 35℃ and batch fermentation process, indicating that 32℃ conditions are not conducive to bacterial production of plasmids.

[0066] At the same time, the test was carried out at 30℃, maintaining OD60-80. Under the condition of 1h periodic intermittent feeding, OD can be maintained at 60-80 for at least 43h (fermentation is terminated artificially), and it was found that the rates of feeding and feeding were basically the same within the cycle without large fluctuations, indicating that the bacterial activity is more stable, but the plasmid yield is lower than that at 32℃, about 2.0mg / L / OD. Figure 7 and Figure 8 shown.

[0067] Example 2 Exploration of continuous fermentation conditions using a two-stage fermentation process

[0068] From the above Example 1, it can be seen that constant temperature fermentation at 30-32°C can maintain long-term continuous fermentation, and the time requirement of continuous fermentation is preliminarily achieved. However, the lower fermentation temperature results in a lower plasmid yield, with a unit OD plasmid yield of only 2.0-2.5 mg / L / OD, and the plasmid yield in the fermentation broth is only 100-200 mg / L, which is much lower than batch fed fermentation.

[0069] Therefore, after summarizing the above results, we tried to investigate the effect of two-stage fermentation on continuous fermentation. The strain was 1101-B-04 and the production plasmid was pAD1016. The schematic diagram of two-stage fermentation is shown in the figure. Fig. 9 As shown, two 2.5-liter fermentation tanks are used for fermentation, that is, the temperature of the primary fermentation tank is controlled at 30-32°C to maintain the continuity of bacterial growth, and the temperature of the secondary fermentation tank is controlled at 37°C or higher to increase the yield level of the plasmid. The fermentation liquid in the primary tank that reaches a certain OD is transferred to the secondary tank for heating to produce plasmids, and the primary tank is supplemented with an equal volume of fresh fermentation basal medium transferred to the secondary tank for bacterial growth again. After the plasmid yield in the secondary tank reaches the highest, the fermentation liquid is released, and the bacteria in the primary tank grows to a certain OD before being transferred to the secondary tank again. This cycle is repeated many times to achieve continuous fermentation. In the two-stage tank, the volume change of the fermentation liquid in the fermentation tank is shown in the schematic diagram Fig.10 The specific optimization conditions will be described in detail below.

[0070] 1) Two-stage fermentation test and secondary tank temperature optimization

[0071] The test was conducted using two-stage fermentation. The initial fermentation volume of the first tank was 2.5L. The first tank was kept at a constant temperature of 30°C. When the OD grew to 60-80, 1.2L was transferred to the second tank for fermentation at a higher temperature. Fresh fermentation basal medium was added to the first tank to continue continuous fermentation and culture. Intermittent feeding was used to maintain the OD at 60-80. The temperature in the second tank was 37°C, 40°C and 42°C respectively to test the effect of different temperatures in the second tank on plasmid fermentation. After the nutrients in the culture medium were exhausted, feeding began when DO rebounded. The feeding method was DO feedback feeding.

[0072] The results showed that the first tank could ferment continuously for at least 48 hours without OD decline, indicating that the bacterial activity was stable and did not decline. The plasmid yield of the second tank was different at different temperatures. The plasmid yield at 40℃ and 42℃ was higher than that at 37℃, with the highest yields of 457.8mg / L and 446.8mg / L respectively. The highest yield per unit OD was about 4.5mg / L / OD, which was about 50% higher than that at 37℃. Fig.11 and 12 shown.

[0073] 2) OD optimization of primary tank

[0074] During the two-stage fermentation process, it was shown that the time of the primary tank and the secondary tank could not match, that is, the growth time of the secondary tank was longer, and the OD growth rate of the primary tank was faster, resulting in the waste of the primary tank bacteria, which could not be fully converted into the production capacity of the plasmid. Therefore, an attempt was made to slow down the growth rate of the primary tank by adjusting the maintenance OD, and at the same time, to investigate the effect of different temperature rise OD on the fermentation of the plasmid. The test was carried out simultaneously in two primary fermentation tanks, with the OD of the primary fermentation tank numbered FD maintained at 10-30, and the OD of the primary fermentation tank numbered FE maintained at 30-50. When the target OD was grown, the continuous discharge and feeding were carried out for 3-4 hours for continuous fermentation, and then transferred to the secondary fermentation tank, and the fermentation temperature of the secondary fermentation tank was 40°C. The results showed that the bacterial concentration was slightly lower when maintaining OD10-30 (i.e., the temperature was increased to OD10-30) than when maintaining OD30-50, but the plasmid concentration was higher than when maintaining OD30-50. When the OD grew to the highest, the fermentation was terminated after about 12 hours of continuous fermentation. The highest plasmid yield was 547 mg / L, which was about 12% higher than the OD30-50. Therefore, maintaining OD10-30 can be selected as the bacterial concentration level of the primary tank. The results are shown in Fig.13 shown.

[0075] 3) Optimization of secondary tank feed composition

[0076] In order to increase the plasmid yield of the secondary tank, the secondary tank feed composition was optimized to improve the continuous fermentation plasmid production level. Since the yeast powder composition differences were compared in previous batch feeding experiments, the trace elements and vitamins in the yeast powder models that were beneficial to plasmid production were higher, vitamins and trace elements were added to the fermentation feed medium to investigate their effects on plasmid fermentation. The specific feed formula and trace element and vitamin mother liquor formula are shown in Tables 4 to 7. The initial fermentation volume of the primary tank was 2.5L, and the OD was maintained at 10-30. After reaching the target OD, 1.2-1.5L was transferred to the secondary tank for 40°C temperature-raising fermentation. After the secondary tank was heated, it was cultured by fed-batch fermentation. The feeding time was when the C source was exhausted and the DO rose rapidly to more than 70%. The feeding method was DO feedback feeding (supplementing the fermentation feed medium, in which vitamins or trace elements were added, as shown in Table 4 or Table 5), the feeding volume was 40mL / time, and the feeding time was 4h.

[0077] Table 4 Fermentation feed medium with trace elements added

[0078]

[0079]

[0080] Table 5 Fermentation feed medium with vitamin addition

[0081] Material Name Concentration (g / L) glycerin 300 Yeast powder 250 Magnesium sulfate heptahydrate 1 Vitamin solution stock solution (100 times) 10ml / L

[0082] Table 6 Trace element mother solution formula

[0083] Material Name Concentration (g / L) Ferrous Sulfate Heptahydrate 6.95 Calcium chloride dihydrate 1.47 Manganese chloride tetrahydrate 9.90 Zinc sulfate heptahydrate 2.88 Cobalt chloride hexahydrate 0.357 Cupric chloride dihydrate 0.256 Ammonium molybdate tetrahydrate 1.85 Sodium borate decahydrate 3.05 Concentrated hydrochloric acid 25ml

[0084] Table 7 Vitamin mother solution formula

[0085] Material Name Concentration (g / L) Thiamine Hydrochloride (VB1) 4 Biotin 0.20 Niacinamide (VB3) 4 Vitamin B12 4 D-Calcium Pantothenate (VB5) 2 Pyridoxine Hydrochloride (VB6) 4

[0086] The fermentation results showed that the addition of trace elements and vitamins did not significantly increase the plasmid yield, indicating that the nutrients in the feed ingredients that limit the increase of plasmids are sufficient, but the OD of the bacteria did not decrease in the late fermentation after adding trace elements, indicating that the addition of trace elements is beneficial to the growth and metabolism of the bacteria. Fig.14 shown.

[0087] 4) Optimization of seed retention ratio

[0088] The above optimization results show that in the two-stage fermentation, the highest yield of the secondary tank is about 500 mg / L, which is still a certain distance from the 700 mg / L yield of the existing platform batch feed fermentation. After analysis, it was found that the secondary tank did not reach the level of plasmid yield of batch feed fermentation when the parameters such as temperature rise OD, feed composition, and temperature were better than those of batch feed fermentation. This may be due to the large number of bacterial cell passages or the excessive proportion of bacteria transferred from the primary tank to the secondary tank for a long growth time. Therefore, we tried to optimize the seed retention ratio of the primary tank.

[0089] In the above study, the initial fermentation volume of the first tank was 2.5L, 1.2-1.5L was transferred to the second tank, and the volume reserved for seeds was about 1L. Therefore, this optimization test was conducted to test the effects of different seed retention ratios on plasmid fermentation, and also to explore the unfavorable parameter range of seed retention in the first tank, which can further control the better connection between the first tank and the second tank. This experiment set a seed retention ratio of 0.5%-1%, and also set the OD of the fourth cycle of the second tank to be higher than 80, to further verify the effect of high OD temperature increase on plasmid fermentation. Results Fig.15 It shows that maintaining OD20-40 in the first-level tank can achieve good bacterial growth, which can be maintained for at least 50 hours and achieve at least 4 cycles of continuous fermentation. Fig.16 The results showed that when the seed retention ratio of the first-level tank was 0.5%-1.0%, the plasmid yield could reach 730mg / L, the average yield was about 650mg / L, and the unit OD plasmid yield also reached 6.75mg / L / OD, which basically reached the level of plasmid yield of batch fed fermentation, and the plasmid yield increased by more than 350% compared with the constant temperature 32℃ fermentation. This shows that reducing the seed retention ratio can better improve the production plasmid performance of the bacteria and maintain the production vitality of the bacteria. The results of high OD (>80) heating showed that although the final fermentation OD was high, the plasmid level was the lowest.

[0090] At the same time, the bacterial cells were lysed in different cycles of the continuous fermentation secondary tank to investigate the changes in the quality of the plasmid at different times of continuous fermentation. The supercoiling and impurities of the plasmid were detected by HPLC. The results showed that there was no significant change in the supercoiling ratio and impurities of the plasmid between different cycles of continuous fermentation, which was basically consistent with the batch fed fermentation process, indicating that 4 cycles of continuous fermentation would not affect the quality of the plasmid. As shown in Table 8 below.

[0091] Table 8

[0092] cycle Open loop ratio% Supercoil ratio% Impurities% Loop 1-1 2.13 94.70 3.17 Loop 1-2 2.16 94.98 2.86 Loop 2-1 2.52 94.85 2.63 Loop 2-2 2.62 94.91 2.48 Loop 3-1 2.33 95.31 2.36 Loop 3-2 2.31 95.33 2.35 Loop 4-1 2.59 95.22 2.19 Loop 4-2 2.48 95.53 1.99

[0093] Example 3 Continuous fermentation process confirmation of two-stage fermentation

[0094] Based on the experimental conditions of Example 2 above, the fermentation batch was confirmed. The strain was 1101-B-04 strain, and the fermentation production plasmid was pAD1016. A two-stage fermentation process was adopted. The primary tank maintained an OD of 20-40, which was controlled at about 30. The initial fermentation volume was 2.5L. After reaching the target OD, it was transferred to the secondary tank for 40°C heating fermentation. The seed ratio of the primary tank was 0.5%-1%, and an equal volume of fresh fermentation basal medium was added to the secondary tank for continuous culture. After the secondary tank was heated, it was cultured by feeding fermentation. The feeding time was when the C source was exhausted and the DO rose rapidly to more than 70%. The feeding method was DO feedback feeding (supplementing the fermentation feeding medium, in which trace elements were added), the feeding volume was 40mL / time, the feeding time was 4h, and then the bacteria were collected; at least 4 cycles of continuous fermentation were performed. The growth OD and cycle of the secondary tank were basically the same. The fermentation ended at about OD110, and the highest yield of the plasmid was 623.6-688.3mg / L. There are four cycles in the secondary tank for plasmid production. The total time for each cycle is about 10 hours. The initial volume of the secondary tank is 2.5L, and the feed is 0.3L. The total plasmid yield is 605.27mg / L*2.8L+661.79mg / L*2.8L+618.2mg / L*2.8L+688.29mg / L*2.8L=7206mg. Fig.17 According to the conversion of 2.5L initial fermentation volume, the total plasmid yield of the continuous fermentation process of the two-stage fermentation of the present invention can reach 72mg / h / L. Compared with the batch fed fermentation, the plasmid production capacity per unit time of the present invention is increased by more than 320%, which is a significant advantage.

[0095] Example 4: Fermentation of plasmid using a two-stage continuous fermentation process

[0096] Based on the experimental conditions confirmed by continuous fermentation in Example 3 above, the recombinant plasmid pGX9501 (recorded in PCT patent application with application number PCT / US2022 / 072135) was produced. The plasmid vector was pVAX1, and the construction method was the same as the above-mentioned 1101-B-04 strain; wherein, the pGX9501 recombinant plasmid was constructed by inserting the novel coronavirus-related DNA sequence between the BamH I and XhoI sites of the pVAX1 vector to obtain a recombinant plasmid, recorded as pGX9501. A two-stage fermentation process was adopted. The OD of the first-stage tank for this fermentation was maintained at 20-40 and controlled at about 30. The initial fermentation volume was 2.5L. After reaching the target OD, it was transferred to the second-stage tank for fermentation at 40°C. The seed retention ratio of the first-stage tank was 0.5%-1%, and an equal volume of fresh fermentation basal medium was added and transferred to the second-stage tank for continuous culture. After the temperature of the secondary tank is raised, the culture is carried out by feeding fermentation. The feeding time is when the C source is exhausted and the DO rises rapidly to more than 70%. The feeding method is DO feedback feeding (supplementing the fermentation feeding medium, in which trace elements are added). The feeding volume is 40mL / time, the feeding time is 4h, and then the bacteria are collected; 4 cycles of continuous fermentation are carried out, and each cycle is about 10h. The total yield of plasmid is:

[0097] 1029mg / L*2.8L+1056mg / L*2.8L+980mg / L*2.8L+1072mg / L*2.8L=11583mg.

[0098] According to the initial fermentation volume of 2.5L, the total plasmid yield can reach 115mg / h / L. Under the same conditions, the yield can reach about 1190mg / h when the initial volume fermentation scale is expanded to 10L. Compared with batch fed fermentation, the plasmid production capacity per unit time is increased by more than 300%.

Claims

1. A fermentation method of Escherichia coli containing a plasmid, comprising two-stage fermentation: S1 first-stage tank fermentation, using constant temperature fermentation at 30-34℃, maintaining OD at 20-40; In S2, the fermentation liquid in the primary tank is transferred to the secondary tank for fermentation at a temperature of 37°C to 42°C. In S2, the culture is carried out by fed-batch fermentation. Preferably, the feeding time is when the C source is exhausted and the DO rises rapidly to more than 70%.

2. The fermentation method according to claim 1, characterized in that S1 and S2 use a fermentation medium as a fermentation basal medium, which contains a liquid culture medium containing dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, yeast extract powder, glycerol, a defoamer, kanamycin sulfate and magnesium sulfate heptahydrate; Preferably, the formula is as follows: 6.55 g / L of dipotassium hydrogen phosphate trihydrate; 3.5 g / L of potassium dihydrogen phosphate; 80.0 g / L of yeast extract powder; 60.0 g / L of glycerol; 0.05% (v / v) defoaming agent; 0.05 g / L of kanamycin sulfate; and 0.50 g / L of magnesium sulfate heptahydrate.

3. The fermentation method according to claim 1, characterized in that In S1, the seed retention ratio in the primary tank is 0.5%-1%, and fresh fermentation basal medium equal in volume to the fermentation liquid transferred to the secondary tank is supplemented for continuous culture.

4. The fermentation method according to claim 1, characterized in that In the S1, the culture temperature is optimally 31-33°C, more preferably 32°C; the OD is maintained at 25-35, more preferably 28-32, and the optimal OD is 30.

5. The fermentation method according to claim 4, characterized in that In S2, the optimal culture temperature is 39-41°C, more preferably 40°C.

6. The fermentation method according to claim 1, characterized in that In S2, the feeding method adopted is DO feedback feeding, that is, supplementing the fermentation feed medium, specifically, the feeding volume is 35-45 mL / time, the feeding time is 3-5 h, and 2-5 cycles of continuous fermentation are performed, preferably, the feeding volume is 40 mL / time, the feeding time is 4 h, and 3-4 cycles of continuous fermentation are performed; Preferably, the feed medium contains glycerol, yeast powder, magnesium sulfate heptahydrate, and trace element mother solution; wherein the trace element mother solution contains ferrous sulfate heptahydrate, calcium chloride dihydrate, manganese chloride tetrahydrate, zinc sulfate heptahydrate, cobalt chloride hexahydrate, cupric chloride dihydrate, ammonium molybdate tetrahydrate, sodium borate decahydrate, and concentrated hydrochloric acid; Alternatively, the feed medium formula is as follows: it contains glycerol, yeast powder, magnesium sulfate heptahydrate, and vitamin solution mother liquor; wherein the vitamin solution mother liquor contains thiamine hydrochloride, biotin, nicotinamide, vitamin B12, D-calcium pantothenate, and pyridoxine hydrochloride.

7. The fermentation method according to claim 6, characterized in that The feed medium formula is as follows: 300 g / L glycerol; 250 g / L yeast powder; 1 g / L magnesium sulfate heptahydrate; 10 ml / L trace element mother solution; wherein the trace element mother solution, calculated as 1000 ml, contains 6.95 g ferrous sulfate heptahydrate; 1.47 g calcium chloride dihydrate; 9.90 g manganese chloride tetrahydrate; 2.88 g zinc sulfate heptahydrate; 0.357 g cobalt chloride hexahydrate; 0.256 g cupric chloride dihydrate; 1.85 g ammonium molybdate tetrahydrate; 3.05 g sodium borate decahydrate; and 25 ml concentrated hydrochloric acid; Alternatively, the feed medium formula is as follows: 300 g / L glycerol; 250 g / L yeast powder; 1 g / L magnesium sulfate heptahydrate; 10 ml / L vitamin solution mother liquor; wherein the vitamin solution mother liquor, based on 1000 ml, comprises 4 g / L thiamine hydrochloride; 0.20 g / L biotin; 4 g / L nicotinamide; 4 g / L vitamin B12; 2 g / L D-calcium pantothenate; and 4 g / L pyridoxine hydrochloride.

8. The fermentation method according to any one of claims 1 to 7, characterized in that The Escherichia coli is a wild-type Escherichia coli or an Escherichia coli derived therefrom, such as a mutant Escherichia coli, for example, DH5α, DH10B, BL21, JM109, JM110, TOP10, HB101, XL0-Gold, M110, SCS10 and other series of Escherichia coli.

9. The fermentation method according to any one of claims 1 to 7, characterized in that: The plasmid is a plasmid suitable for Escherichia coli, for example, pUC series plasmids such as pUC19, pLysS, pRARE, pGEX series vectors such as pGEX-1λT, pGEX-2T, pGEX-3T, pET series vectors such as pET-28a-c (+), pPL series vectors such as pPL-Lamda, pEZZ18, pINII series vectors such as pINII-ompA1, pINII-comA1, pINII-comA2, pINII-comA3, pKK223 series vectors such as pKK223-3, pVAX1, or shuttle vectors such as pYES2 / NT / lacZ.

10. A method for producing a plasmid, characterized in that The method comprises the steps of obtaining Escherichia coli containing a plasmid by using the fermentation method according to any one of claims 1 to 9, and collecting the plasmid therefrom.

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