A fermentation method of escherichia coli containing a plasmid and a method of producing a plasmid

Abstract

The application discloses a fermentation method of E. coli containing plasmid and a method for producing plasmid. The fermentation method comprises the following secondary fermentation steps: primary tank fermentation, constant temperature fermentation at 30-34 DEG C, and maintaining OD at 20-40; secondary tank fermentation, transferring the fermentation liquid in the primary tank into the secondary tank, constant temperature fermentation at 37 DEG C to 42 DEG C, and adopting the way of fed-batch fermentation for culture. The application adopts the way of two-stage fermentation, can continuously ferment for at least 50 hours or more, and the secondary tank can continuously produce at least 4 cycles. In one fermentation cycle, the plasmid yield per unit time is increased by at least 300% or more, and the improvement effect is obvious. The plasmid production cycle is greatly shortened, and the continuous cycle can continue, the seed culture and the pre-treatment and post-treatment time ratio in the whole fermentation cycle is greatly shortened, a large amount of fermentation time cost can be saved, and the purpose of high-yield plasmid in the same time can be achieved.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a fermentation method for Escherichia coli containing plasmids and a method for producing plasmids. Background Technology

[0002] Plasmid fermentation is a technique that involves inoculating a bacterial strain that produces plasmids into a suitable culture system, allowing the strain to replicate rapidly and in large quantities within the bacterial cells, simultaneously synthesizing plasmid DNA. Alternatively, it can be described as plasmid amplification, a chemical biological method for obtaining large quantities of plasmids. This typically involves transforming bacterial plasmids into *E. coli* and then harvesting large quantities of plasmids from the *E. coli*.

[0003] Existing technologies have explored continuous plasmid production. For example, US2008 / 0318283A1 explored a two-stage fermentation and continuous feed-and-discharge method, where the first stage fermentation temperature was 30℃ and the second stage fermentation temperature was 36-45℃. However, the fermentation process and bacterial culture harvesting were continuous and uninterrupted, yielding only 0.32L of bacterial culture per hour. The drawbacks are twofold: firstly, this continuous fermentation results in some bacterial culture entering the second stage tank without sufficient temperature induction, leading to low plasmid yields before harvesting as plasmid products; secondly, this continuous fermentation results in some feed-and-discharge medium entering the second stage tank and bacterial culture from the first stage being harvested as plasmid products before their nutrients are fully consumed, resulting in low material utilization and cost waste. Third, this continuous fermentation method for harvesting plasmid-rich bacterial solutions is difficult to implement continuously via centrifugation or hollow fiber methods. A third storage tank is needed to temporarily store the bacterial solution until a certain volume is reached before harvesting the cells. However, these bacteria will continue to grow in the storage tank, especially the earlier harvested solution which remains there for an extended period. To maintain low growth and metabolism, the temperature should ideally be lowered to below 15°C. On a production scale, this would be extremely costly. Fourth, the secondary tank's feed medium is added continuously as the bacterial solution enters the secondary tank. At this point, the bacterial solution has a low OD600 and abundant nutrients, which may lead to nutrient excess and an imbalance in the carbon-nitrogen ratio in the secondary tank, hindering bacterial growth.

[0004] Currently, the plasmid fermentation platform has undergone two generations of process iterations. The first-generation plasmid fermentation process was relatively crude. The bacterial strain was initially revived and cultured to obtain bacterial seeds, which were then inoculated into a fermenter. After 18-22 hours of fermentation, the fermentation was stopped and the bacterial cells were harvested after the nutrients in the culture medium were depleted. The fermentation type was a batch fermentation process. As fermentation progressed, metabolic waste in the culture medium also increased slowly, and the timing of bacterial growth and plasmid production was not perfectly matched. When fermentation ended, the nutrients were exhausted, but the bacterial cells had not reached their peak in producing plasmids.

[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 aforementioned shortcomings and considerations, a second-generation fermentation process was developed. This process is a fed-batch fermentation process. Building upon batch fermentation, in the later stages of fermentation, when nutrients are exhausted, a fed-batch process is used to increase the nutrients in the culture medium, extending the plasmid production time and achieving high plasmid yield. A comparison of plasmid yields between the two fermentation processes is provided. Figure 1 As shown, compared to batch fermentation, the fed-batch fermentation process can increase plasmid yield from approximately 550 mg / L before feeding to 770 mg / L. Simultaneously, the fermentation volume increases due to the addition of feed, ultimately increasing yield by over 50%. However, in existing fed-batch fermentation processes, the entire production cycle for plasmid production is approximately 3.5 days, but the time spent in the fermenter is only 28 hours. A significant amount of time is spent on seed culture, preliminary preparation, and post-processing, resulting in relatively low plasmid production efficiency. Therefore, considering how to extend the growth time in the fermenter can significantly shorten the time allocated to seed culture and preliminary and post-processing throughout the fermentation cycle, saving substantial fermentation time costs and achieving high plasmid yields in the same amount of time. Furthermore, the shortcomings of existing continuous fermentation processes should be fully considered and overcome.

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

[0008] This invention provides a fermentation method for Escherichia coli containing plasmids, which is a two-stage fermentation.

[0009] S1 primary tank fermentation uses constant temperature fermentation at 30-34℃ to maintain OD at 20-40;

[0010] S2 secondary fermentation involves transferring the fermentation broth from the primary tank to the secondary tank for heated fermentation, maintaining a constant temperature between 37°C and 42°C. Furthermore, S2 employs fed-batch fermentation, with feeding initiated when the carbon source is depleted and the dissolved oxygen (DO) level rapidly recovers to above 70%.

[0011] S1 and S2 use fermentation medium as the basic fermentation medium, which is a liquid medium containing dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, yeast extract powder, glycerol, antifoaming agent, kanamycin sulfate and magnesium sulfate heptahydrate.

[0012] Preferably, the formulation 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 glycerin; 0.05% (v / v) of defoamer; 0.05 g / L of kanamycin sulfate; and 0.50 g / L of magnesium sulfate heptahydrate.

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

[0014] In S1, the optimal culture temperature is 31-33℃, more preferably 32℃; the maintenance OD is 25-35, more preferably 28-32, and the optimal maintenance OD is 30.

[0015] In S2, DO feedback feeding is used, specifically feeding volume of 35-45 mL / time, feeding time of 3-5 hours, for 2-5 cycles of continuous fermentation. Preferably, feeding volume of 40 mL / time, feeding time of 4 hours, for 3-4 cycles of continuous fermentation.

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

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

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

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

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

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

[0022] The *Escherichia coli* can be any type of wild-type *E. coli* or its derivatives, such as mutant *E. coli*, for example, the *DH5α*, *DH10B*, *BL21*, *JM109*, *JM110*, *TOP10*, *HB101*, *XL0-Gold*, *M110*, *SCS10* and other series of *E. coli*.

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

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

[0025] This invention, through extensive research and exploration, has ultimately developed a two-stage fermentation process suitable for E. coli plasmid production. Specifically, this invention, through the exploration of isothermal fermentation and two-stage fermentation conditions, including temperature, maintained OD, elevated OD, feed composition, feed feeding methods, and seed retention ratio, found that the two-stage fermentation method can effectively achieve continuous fermentation, allowing for at least 50 hours of continuous fermentation. The secondary tank can continuously produce at least four cycles, while the plasmid quality remains largely unchanged. Within one fermentation cycle, the plasmid yield per unit time increases by at least 300%, demonstrating a significant improvement. Although two devices are used, the production cycle in the secondary tank is approximately 11 hours, greatly shortening the cycle for efficient plasmid production. Continuous cycling can continue, further enhancing subsequent yield data. Simultaneously, the growth time in the primary fermenter is extended, significantly reducing the time spent on seed culture and pre- and post-treatment processes throughout the fermentation cycle, saving substantial fermentation time costs and achieving high plasmid yields in the same amount of time. In particular, it has been confirmed in the embodiments of the present invention that intermittent feeding and replenishment are more beneficial to maintaining the vitality of the microorganisms. This may be because after intermittent feeding and replenishment, the proportion of metabolic waste in the tank will be lower than that of continuous fermentation, while the content of added nutrients will be higher than that of continuous feeding and replenishment. Maintaining a lower OD level (60-80) will also be beneficial to the level of microbial vitality. Attached Figure Description

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

[0027] Figure 2 Results of plasmid fermentation at constant temperatures of 35℃ and 37℃;

[0028] Figure 3 The results are from continuous fermentation at a constant temperature of 35℃;

[0029] Figure 4 To reduce the fermentation results at a constant temperature of 35℃ after maintaining OD;

[0030] Figure 5 To maintain a constant temperature of 32℃ and achieve fermentation results with an OD of 80-100;

[0031] Figure 6 The fermentation results were maintained at a constant temperature of 32℃ with an OD of 60-80.

[0032] Figure 7 The results were obtained from continuous fermentation at a constant temperature of 30℃, maintaining an OD of 60-80.

[0033] Figure 8 Results of unit OD plasmid yield for continuous fermentation at isothermal temperatures of 30℃ and 32℃;

[0034] Figure 9 This is a schematic diagram of two-stage fermentation;

[0035] Figure 10 A schematic diagram showing the volume change of the fermentation liquid in a two-stage fermenter;

[0036] Figure 11 Results of OD and feeding / replenishment for the primary tank in a two-stage fermentation process;

[0037] Figure 12 Results of unit OD plasmid yield in the secondary fermentation tank of a two-stage fermentation process;

[0038] Figure 13 For the results of plasmid fermentation under different maintenance OD levels;

[0039] Figure 14 The effects of adding trace elements and vitamins to the feed on plasmid fermentation were investigated. FE1 was a normal feed control, FE2 was fed with added trace elements, FE3 was fed with added vitamins, and FD was achieved when the temperature reached an OD of 80 or higher.

[0040] Figure 15 To maintain OD in the primary tank of the two-stage fermentation;

[0041] Figure 16 The results show the OD and plasmid yield of the secondary tank in the two-stage fermentation process. FE1 represents normal feed as control 1, FE2 represents feed with added trace elements as control 2, FE3 represents normal feed as control 2, and FD represents OD above 60 after temperature increase.

[0042] Figure 17 The results are from a continuous two-stage fermentation tank. FB-1 is cycle 1, FB-2 is cycle 2, FB-3 is cycle 3, and FB-4 is cycle 4. Detailed Implementation

[0043] The technical solution of the present invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are not all embodiments of the present invention. All other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0044] Example 1 explores the isothermal fermentation temperature on an existing platform-based batch fed-batch fermentation process.

[0045] The main parameters of the existing batch fed-batch fermentation process are shown in Table 1. This is a variable-temperature batch fed-batch fermentation process. During fermentation, the fermentation temperature is controlled based on the growth of OD (Organic Determination). When OD < 10, the fermentation temperature is controlled at 30℃ ± 0.5℃; when OD > 10, the fermentation temperature is controlled at 37℃ ± 0.5℃. For continuous fermentation, it is difficult to maintain a stable temperature within the tank, and temperature has a significant impact on the fermentation plasmids. Therefore, the main challenge in the development is temperature stability control, and the entire development process revolves around addressing this challenge.

[0046] The strain used in the process development was the 1101-B-04 strain constructed as described below, in which the production plasmid was pAD1016 (described in Chinese patent application CN202310087259.3), the plasmid vector was pVAX1 (ThermoFisher, catalog number: V26020), the size was 4250bp, and it contained a kanamycin resistance gene. Fermentation was carried out in a 5L fermenter to explore the conditions.

[0047] Construction of pAD1016 recombinant plasmid: The coronavirus-related DNA sequence was inserted between the BamHI and XhoI sites of the pVAX1 vector to obtain the recombinant plasmid, denoted as pAD1016.

[0048] Construction process of strain 1101-B-04: This strain was constructed using a purchased Thermo Fisher One Shot... TM MAXEfficiency TMDH10B T1 phage-tolerant cells (E. coli cells) were used as recipient cells. The recombinant plasmid pAD1016 (pVAX1 vector, 4250 bp in size) was introduced into competent cells. Since the recombinant plasmid carries a kanamycin resistance gene, transformants could be screened based on kanamycin resistance. If the recipient cells could grow on plates containing kanamycin resistance, it proved that the recipient cells had been introduced with the recombinant pVAX1 plasmid. Then, single recombinant plasmids were selected from the kanamycin resistance plates and cultured in LB medium at 30°C and 220 rpm until the OD600 reached 2-3. The recombinant bacteria were then cryopreserved at 1 ml / vial, with each vial containing 20% ​​glycerol as a cryoprotectant.

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

[0050]

[0051] Table 2 Fermentation Feeding Culture Medium

[0052]

[0053]

[0054] Since temperature is particularly important for plasmid fermentation production, and temperature parameters affect the development of continuous fermentation processes, continuous heating and cooling may affect cell viability and plasmid production, and increase operational difficulty and control costs. Therefore, we are exploring a pre-existing batch fed-batch fermentation platform process to conduct long-term constant temperature fermentation, and to explore the constant temperature fermentation temperature between the termination growth temperature of 30℃ and the fermentation temperature of 37℃.

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

[0056] Since 37℃ is the optimal temperature for high plasmid yield, in the early stages of development, to achieve continuous fermentation, preliminary explorations were conducted using constant temperature conditions of 37℃ and 35℃. Figure 2 As shown, the results showed that cell growth was inhibited at 37℃, with the highest OD value being around 30, which is only 1 / 3 of the normal fermentation level of the existing batch fed-batch fermentation platform process. However, under constant temperature conditions of 35℃, the OD value could grow to the required level. Therefore, 35℃ was selected as the stable temperature for constant temperature fermentation, and continuous fermentation was attempted.

[0057] When exploring the continuous fermentation temperature at 35℃, a method of continuous dispensing of culture medium (dispensing) and replenishment of culture medium (replenishment) was adopted. The replenishment consisted of supplementing the basal fermentation medium, with the formula shown in Table 3. The OD value in the fermenter was controlled between 80-100 by adjusting the dispensing and replenishment rates, ensuring a constant fermentation liquid volume (acid / alkali addition and evaporation consumption were negligible). Results showed that during continuous fermentation at 35℃, the dispensing and replenishment rates continuously decreased in the later stages of fermentation, indicating a slowing of cell growth and weakened cell activity. The OD value of 80-100 could only be maintained for about 8 hours, after which it rapidly declined. The plasmid yield reached approximately 420 mg / L. Figure 3 As shown.

[0058] By lowering the OD to 60-80 and employing intermittent feeding and replenishment every 1 hour to reduce the content of metabolic waste in the tank, decrease the competitive pressure between bacteria in the fermentation broth, lower the fermentation broth concentration, and increase the mass transfer rate of oxygen and nutrients, continuous fermentation could be extended to 11 hours. However, the feeding and replenishment rates then decreased rapidly, indicating that bacterial activity was difficult to maintain in the later stages. Figure 4 As shown.

[0059] In summary, constant temperature fermentation at 37℃ inhibits cell growth, resulting in slow cell growth and a low final OD. In contrast, constant temperature fermentation at 35℃ allows the OD to grow normally to around 100, reaching the cell concentration level of the existing batch-fed fermentation process. However, during continuous fermentation, the duration of this process is short, lasting only about 8-11 hours, and the plasmid yield can reach the plasmid concentration level of batch fermentation.

[0060] Table 3 Fermentation Basic Culture Medium

[0061]

[0062]

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

[0064] Based on the above results, further exploration of temperature conditions was conducted, attempting to control the temperature parameter at 32℃ for isothermal fermentation (continuous fermentation mode as in 1.1.1). The results showed that at 32℃, maintaining an OD of 80-100 with continuous feeding and discharging, the OD could be maintained for approximately 14 hours. Maintaining an OD of 60-80 with intermittent feeding and discharging every 1 hour, the OD could be maintained for approximately 45 hours. Figure 5 , Figure 6 As shown.

[0065] The above results indicate that continuous fermentation can be maintained for at least 14 hours at 32℃. Using intermittent feeding and replenishment at 1-hour intervals and maintaining an OD of 60-80, continuous fermentation can continue for at least 45 hours (the entire fermentation cycle is 60 hours). After 60 hours, switching to continuous feeding and replenishment, with the same feeding and replenishment rates, results in a decrease in OD, indicating that intermittent feeding and replenishment are more beneficial for maintaining cell viability. This may be because the proportion of metabolic waste in the tank is lower with intermittent feeding and replenishment compared to continuous fermentation, while the content of added nutrients is higher, maintaining a lower OD level (60-80) also benefits cell viability. Under constant temperature conditions of 32℃, the plasmid level is around 200 mg / L, lower than the plasmid yield in batch fermentation processes at 35℃, indicating that 32℃ is not conducive to plasmid production from cells.

[0066] Simultaneously, tests were conducted at 30℃ to maintain an OD of 60-80. Under intermittent feeding conditions with a 1-hour cycle, the OD could be maintained at at least 60-80 for approximately 43 hours (fermentation was manually terminated and then resumed). Furthermore, the feeding and discharging rates were found to be essentially consistent throughout the cycle, without significant fluctuations, indicating more stable cell viability. However, the plasmid yield was lower than under the 32℃ condition, approximately 2.0 mg / L / OD. Figure 7 and Figure 8 As shown.

[0067] Example 2 explores continuous fermentation conditions using a two-stage fermentation process.

[0068] As can be seen from Example 1 above, constant temperature fermentation at 30-32℃ can maintain continuous fermentation for a long time, initially achieving the time requirement for continuous fermentation. However, the low fermentation temperature results in low plasmid yield, with a unit OD plasmid yield of only 2.0-2.5 mg / L / OD and a plasmid yield in the fermentation broth of only 100-200 mg / L, which is far lower than that of batch fed fermentation.

[0069] Therefore, after considering the above results, the effect of a two-stage fermentation method on continuous fermentation was investigated. The strain used was 1101-B-04, and the production plasmid was pAD1016. The schematic diagram of the two-stage fermentation is shown below. Figure 9 As shown, two 2.5-liter fermenters are used for fermentation. The primary fermenter is maintained at a temperature of 30-32℃ to ensure continuous cell growth, while the secondary fermenter is maintained at 37℃ or higher to increase plasmid yield. The fermentation broth, having reached a certain OD in the primary fermenter, is transferred to the secondary fermenter for plasmid production. An equal volume of fresh basal culture medium is then added to the primary fermenter to allow for further cell growth. Once the plasmid yield in the secondary fermenter reaches its maximum, the fermentation broth is discharged. The cells in the primary fermenter are then transferred back to the secondary fermenter after reaching a certain OD, and this cycle is repeated multiple times to achieve continuous fermentation. The volume changes of the fermentation broth in the two fermenters are illustrated in the diagram. Figure 10 As shown. Specific optimization conditions will be described in detail below.

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

[0071] Two-stage fermentation was used for testing. The initial fermentation volume in the first-stage tank was 2.5L, and fermentation was carried out at a constant temperature of 30℃. When the OD reached 60-80, 1.2L was transferred to the second-stage tank for heated fermentation. Fresh basal culture medium was added to the first-stage tank for continuous fermentation, and intermittent feeding was used to maintain the OD at 60-80. The temperatures in the second-stage tank were 37℃, 40℃, and 42℃ to test the effect of different temperatures on plasmid fermentation. Feeding was initiated when the nutrient content in the culture medium was depleted and DO rebounded, using DO feedback feeding.

[0072] The results showed that the primary fermenter could ferment continuously for at least 48 hours without any decrease in OD, indicating stable cell viability. The plasmid yield in the secondary fermenter varied with temperature; the plasmid yield at 40℃ and 42℃ was higher than at 37℃, reaching maximums of 457.8 mg / L and 446.8 mg / L respectively. The highest plasmid yield per unit OD was approximately 4.5 mg / L / OD, about 50% higher than at 37℃. Figure 11 and 12 As shown.

[0073] 2) Primary tank OD optimization

[0074] During the two-stage fermentation process, a mismatch was observed between the time intervals in the primary and secondary fermenters. Specifically, the secondary fermenter had a longer growth time, while the OD growth rate in the primary fermenter was faster, resulting in wasted cells in the primary fermenter and incomplete conversion into plasmid production capacity. Therefore, an attempt was made to slow down the growth rate in the primary fermenter by adjusting the maintained OD, while also investigating the effect of different OD levels at varying temperatures on plasmid fermentation. Simultaneous testing was conducted in two primary fermenters: fermenter FD maintained an OD of 10-30, and fermenter FE maintained an OD of 30-50. Upon reaching the target OD, continuous feeding and feeding were performed for 3-4 hours for continuous fermentation before transferring to the secondary fermenter, where the fermentation temperature was 40℃. The results showed that maintaining OD10-30 (i.e., OD at 10-30 during temperature rise) resulted in a slightly lower cell concentration compared to maintaining OD30-50, but a higher plasmid concentration. Fermentation was artificially terminated after approximately 12 hours of continuous fermentation when OD reached its peak. The highest plasmid yield was 547 mg / L, about 12% higher than maintaining OD30-50. Therefore, maintaining OD10-30 can be chosen as the cell concentration level for the primary fermentation tank. Figure 13 As shown.

[0075] 3) Optimization of feed composition in secondary tanks

[0076] To improve plasmid yield in the secondary fermentation tank, we attempted to optimize the feed composition to enhance plasmid production during continuous fermentation. Previous batch-feeding experiments showed that yeast powders beneficial for plasmid production had higher levels of trace elements and vitamins. Therefore, we added vitamins and trace elements to the feed culture medium to investigate their effects on plasmid fermentation. Specific feed formulations and the formulas for trace element and vitamin stock solutions are shown in Tables 4-7. The initial fermentation volume in the primary tank was 2.5 L, maintaining an OD of 10-30. After reaching the target OD, 1.2-1.5 L was transferred to the secondary tank for fermentation at 40°C. After heating, fed-batch fermentation was used in the secondary tank. Feeding was initiated when the carbon source was depleted and the DO rapidly recovered to over 70%. The feeding method was DO feedback feeding (replenishing the feed culture medium with added vitamins or trace elements, as shown in Tables 4 or 5), with a feeding volume of 40 mL per feeding and a feeding time of 4 hours.

[0077] Table 4. Fermentation culture medium with added trace elements

[0078]

[0079]

[0080] Table 5. Fermentation culture medium with added vitamins

[0081] glycerin 300 yeast powder 250 Magnesium sulfate heptahydrate 1 Vitamin solution stock solution (100x dilution) 10ml / L

[0082] Table 6. Micronutrient Mother Liquor Formula

[0083] 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 Copper chloride dihydrate 0.256 Ammonium molybdate tetrahydrate 1.85 Sodium borate decahydrate 3.05 concentrated hydrochloric acid 25ml

[0084] Table 7 Vitamin Mother Liquor Formula

[0085] Thiamine hydrochloride (VB1) 4 Biotin 0.20 Nicotinamide (VB3) 4 Vitamin B12 4 D-Calcium Pantothenate (VB5) 2 Pyridoxine hydrochloride (VB6) 4

[0086] Fermentation results showed that the addition of trace elements and vitamins did not significantly increase plasmid yield, indicating that the nutrients limiting plasmid growth in the feed composition were sufficient. However, the OD of the cells did not decrease in the later stages of fermentation after the addition of trace elements, suggesting that the addition of trace elements is beneficial to cell growth and metabolism. Figure 14 As shown.

[0087] 4) Optimization of seed retention ratio

[0088] The optimized results show that in the two-stage fermentation, the highest yield in the secondary tank is approximately 500 mg / L, which is still some distance from the 700 mg / L yield of the existing batch fed-batch fermentation platform. Further analysis revealed that even with better parameters such as temperature rise OD, feed composition, and temperature compared to batch fed-batch fermentation, the secondary tank still failed to reach the plasmid yield level. This may be due to a higher number of cell passages or an excessive proportion of bacteria transferred from the primary tank to the secondary tank with longer growth periods. Therefore, we attempted to optimize the inoculum ratio in the primary tank.

[0089] In the aforementioned study, the initial fermentation volume in the primary tank was 2.5 L, with 1.2-1.5 L transferred to the secondary tank, leaving approximately 1 L as seed. Therefore, this experiment optimized the effect of different seed retention ratios on plasmid fermentation and also explored the unfavorable parameter range for seed retention in the primary tank, allowing for better control and integration between the primary and secondary tanks. This experiment used a seed retention ratio of 0.5%-1% and set the OD (difference temperature) in the fourth cycle of the secondary tank to be above 80°C to further verify the effect of high OD temperature rise on plasmid fermentation. Results Figure 15 The results show that maintaining an OD of 20-40 in the primary tank allows for good cell growth, which can be maintained for at least 50 hours, enabling at least four cycles of continuous fermentation. Figure 16 The results showed that when the inoculum retention ratio in the primary fermentation tank was 0.5%-1.0%, the plasmid yield reached 730 mg / L, with an average yield of approximately 650 mg / L. The plasmid yield per unit OD also reached 6.75 mg / L / OD, essentially reaching the plasmid yield level of batch-fed fermentation. This represents an increase of over 350% in plasmid yield compared to constant-temperature fermentation at 32℃, indicating that reducing the inoculum retention ratio can better improve the plasmid performance of the cells and maintain cell viability. Results with high OD (>80) temperature increases showed that although the final fermentation OD was high, the plasmid level was the lowest.

[0090] Simultaneously, cell lysis was performed in different cycles of the continuous fermentation secondary tank to examine the changes in plasmid quality at different fermentation times. HPLC was used to detect plasmid supercoil and impurities. The results showed that the proportion of supercoiled plasmids and impurities did not change significantly between different cycles of continuous fermentation, which was basically consistent with the batch-fed fermentation process, indicating that four cycles of continuous fermentation do not affect plasmid quality. (See Table 8 below.)

[0091] Table 8

[0092] Cycle 1-1 2.13 94.70 3.17 Cycle 1-2 2.16 94.98 2.86 Cycle 2-1 2.52 94.85 2.63 Cycle 2-2 2.62 94.91 2.48 Cycle 3-1 2.33 95.31 2.36 Cycle 3-2 2.31 95.33 2.35 Cycle 4-1 2.59 95.22 2.19 Cycle 4-2 2.48 95.53 1.99

[0093] Example 3: Confirmation of a two-stage continuous fermentation process

[0094] Based on the experimental conditions of Example 2 above, the fermentation batch was confirmed. The strain was 1101-B-04, and the plasmid for fermentation production was pAD1016. A two-stage fermentation process was adopted. The first-stage tank maintained an OD of 20-40, controlled at around 30, with an initial fermentation volume of 2.5L. After reaching the target OD, it was transferred to the second-stage tank for fermentation at 40℃. The inoculum ratio in the first-stage tank was 0.5%-1%, and an equal volume of fresh fermentation basal medium was added to the second-stage tank for continuous culture. After heating in the second-stage tank, fed-batch fermentation was used. The timing of feeding was when the carbon source was depleted and the DO rapidly recovered to more than 70%. The feeding method was DO feedback feeding (replenishing the fermentation feed medium, which included trace elements), with a feeding volume of 40mL / time and a feeding time of 4h. Then, the cells were collected. At least 4 cycles of continuous fermentation were performed. The growth OD and cycle in the second-stage tank were basically consistent. The OD at the end of fermentation was around 110, and the highest plasmid yield was 623.6-688.3mg / L. The plasmid production stage in the secondary tank consists of four cycles, with each cycle lasting approximately 10 hours. The initial volume of the secondary tank is 2.5L, with a feed increment of 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. Figure 17 As shown. Based on an initial fermentation volume of 2.5L, the total plasmid yield of this patented two-stage continuous fermentation process can reach 72mg / h / L. Compared with batch fed fermentation, the plasmid production capacity per unit time of this invention is increased by more than 320%, which is a very significant advantage.

[0095] Example 4 uses a two-stage continuous fermentation process to ferment plasmids.

[0096] Based on the experimental conditions confirmed by continuous fermentation in Example 3 above, recombinant plasmid pGX9501 (described in PCT patent application No. PCT / US2022 / 072135) was produced. The plasmid vector was pVAX1, and the construction method was the same as that of strain 1101-B-04 mentioned above. Specifically, the pGX9501 recombinant plasmid was constructed by inserting a SARS-CoV-2-related DNA sequence between the BamHI and XhoI sites of the pVAX1 vector, thus obtaining the recombinant plasmid, denoted as pGX9501. A two-stage fermentation process was adopted. In this fermentation, the OD of the first-stage tank was maintained at 20-40, controlled at around 30, with an initial fermentation volume of 2.5L. After reaching the target OD, the culture was transferred to the second-stage tank for fermentation at 40℃. The inoculum ratio in the first-stage tank was 0.5%-1%, and an equal volume of fresh fermentation basal medium was added to the second-stage tank for continuous culture. After the secondary tank is heated, fed-batch fermentation is used for cultivation. Feeding is initiated when the carbon source is depleted and the dissolved oxygen (DO) rapidly recovers to above 70%. The feeding method is DO feedback feeding (replenishing the fermentation medium with added trace elements), with a feeding volume of 40 mL per feeding and a feeding time of 4 hours. Cells are then collected. Four cycles of continuous fermentation are performed, each cycle lasting approximately 10 hours. The total plasmid yield is:

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

[0098] With an initial fermentation volume of 2.5L, the total plasmid yield can reach 115mg / h / L. Under the same conditions, scaling up to an initial fermentation volume of 10L, the yield can reach approximately 1190mg / h. Compared to batch fed-batch fermentation, the plasmid production capacity per unit time is increased by more than 300%.

Claims

1. A fermentation method for Escherichia coli containing plasmids, comprising two-stage fermentation: S1 primary tank fermentation uses constant temperature fermentation at 30-34℃ to maintain OD at 20-40; S2 secondary tank fermentation involves transferring the fermentation broth from the primary tank to the secondary tank for heated fermentation, maintaining a constant temperature between 37°C and 42°C. In S2, fed-batch fermentation is employed, with feeding occurring when the carbon source is depleted and the dissolved oxygen (DO) level rapidly recovers to over 70%. Each feeding cycle lasts 3-5 hours, and 2-5 consecutive fermentation cycles are performed. Furthermore, when transferring the fermentation broth from the primary tank to the secondary tank, the proportion of seed retained in the primary tank is 0.5%-1.0%, and an equal volume of fresh fermentation basal culture medium is added to the primary tank to allow continuous fermentation culture to continue in the primary tank.

2. The fermentation method as described in claim 1, characterized in that, S1 and S2 use a fermentation medium as the basic fermentation medium, which is a liquid medium containing dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, yeast extract powder, glycerol, antifoaming agent, kanamycin sulfate and magnesium sulfate heptahydrate.

3. The fermentation method as described in claim 2, characterized in that, The formula of the fermentation basal culture medium 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 defoamer; 0.05 g / L of kanamycin sulfate; and 0.50 g / L of magnesium sulfate heptahydrate.

4. The fermentation method as described in claim 1, characterized in that, In S1, the culture temperature is 31-33℃; the OD is maintained at 25-35.

5. The fermentation method as described in claim 4, characterized in that, In S1, the culture temperature is 32℃; the OD is maintained at 28-32.

6. The fermentation method as described in claim 4, characterized in that, In S2, the culture temperature is 39-41℃.

7. The fermentation method according to claim 1, characterized in that, In S2, the feeding method used is DO feedback feeding, that is, the fermentation feeding medium is replenished, the feeding volume is 40mL / time, the feeding time is 4h, and 3-4 cycles of continuous fermentation are carried out. The supplemental culture medium contains glycerol, yeast powder, magnesium sulfate heptahydrate, and trace element stock solution; wherein the trace element stock solution contains ferrous sulfate heptahydrate, calcium chloride dihydrate, manganese chloride tetrahydrate, zinc sulfate heptahydrate, cobalt chloride hexahydrate, copper chloride dihydrate, ammonium molybdate tetrahydrate, sodium borate decahydrate, and concentrated hydrochloric acid.

8. The fermentation method as described in claim 7, characterized in that, The feed culture medium formula is as follows: glycerol 300g / L; yeast powder 250g / L; magnesium sulfate heptahydrate 1g / L; trace element stock solution 10ml / L; wherein the trace element stock solution, per 1000ml, contains 6.95g ferrous sulfate heptahydrate; 1.47g calcium chloride dihydrate; 9.90g manganese chloride tetrahydrate; 2.88g zinc sulfate heptahydrate; 0.357g cobalt chloride hexahydrate; 0.256g copper chloride dihydrate; 1.85g ammonium molybdate tetrahydrate; 3.05g sodium borate decahydrate; and 25ml concentrated hydrochloric acid.

9. The fermentation method according to any one of claims 1 to 8, characterized in that, The Escherichia coli mentioned is wild-type Escherichia coli or its derivatives.

10. The fermentation method as described in claim 9, characterized in that, The Escherichia coli mentioned are the DH5α, DH10B, BL21, JM109, JM110, TOP10, HB101, XL0-Gold, M110, and SCS10 series of Escherichia coli.

11. The fermentation method according to any one of claims 1 to 8, characterized in that, The plasmid is suitable for Escherichia coli.

12. The fermentation method according to claim 11, characterized in that, The plasmid is a pUC series plasmid, a pGEX series vector, a pET series vector, a pPL series vector, a pINII series vector, a pKK223 series vector, or a shuttle vector.

13. The fermentation method according to claim 12, characterized in that, The plasmids are pUC19, pLysS, pRARE, pGEX-1λT, pGEX-2T, pGEX-3T, pET-28a-c(+), pPL-Lamda, pEZZ18, pINII-ompA1, pINII-comA1, pINII-comA2, pINII-comA3, pKK223-3, pVAX1, or pYES2 / NT / lacZ.

14. A method for producing plasmids, characterized in that, It includes the steps of obtaining Escherichia coli containing plasmids using the fermentation method as described in any one of claims 1 to 13, and the step of collecting said plasmids therefrom.

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