Supplementing formula and supplementing method for improving fermentation yield of low-copy plasmids

Abstract

The invention relates to the technical field of biological fermentation, in particular to a feed supplement formula and a feed supplement method for improving the fermentation yield of low-copy plasmids. A complete set of biological fermentation process blueprint is provided, low-copy plasmids meeting the high-quality standard can be stably, efficiently and sustainably produced, compared with a conventional process using simple carbon and nitrogen source supplementation, absolute multiplication of the yield can be achieved, meanwhile, the metabolic pathway of cells can be adjusted in the fermentation process, and the yield of the plasmids is increased. The metabolic center of gravity of cells is converted from rapid self-replication to efficient synthesis of a target product, absolute optimization of plasmid yield is achieved, the structural integrity of the product is remarkably improved, batch-to-batch consistency reaches the industrial grade standard, the strict requirement of commercialized production for product quality consistency can be met, and the method is suitable for industrial production. And the method has extremely high application value and wide industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a feed formulation and feeding method for increasing the fermentation yield of low-copy plasmids. Background Technology

[0002] Cell therapy and gene therapy are among the most promising treatment strategies in the biomedical field in recent years. As the core vector of genetic engineering, plasmids play an indispensable role in cell therapy and gene therapy. Their ability to efficiently carry foreign genes is the foundation for downstream applications such as gene and cell therapy, mRNA vaccines / drugs, DNA vaccines, recombinant proteins, and gene editing. However, high-copy plasmids have the disadvantages of instability and accumulation of intermediate toxic products. Therefore, low-copy plasmids have irreplaceable advantages in some specific application scenarios.

[0003] Existing methods for preparing low-copy plasmids suffer from problems such as low plasmid yield, uneven batch quality, and high production costs.

[0004] To address the above problems, the present invention provides a solution. Summary of the Invention

[0005] The purpose of this invention is to provide a feeding formulation and feeding method for increasing the fermentation yield of low-copy plasmids, which can stably, efficiently and sustainably produce low-copy plasmids that meet high-quality standards.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A feeding method for increasing the fermentation yield of low-copy plasmids includes the following steps: S1: Weigh 25g of LB broth culture medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer it to an autoclave and sterilize it at 121℃ for 20min to obtain LB broth culture medium. Take 200ml of LB broth culture medium and place it in a sterilized Erlenmeyer flask. Inoculate it with glycerol bacteria at a volume of 1‰ of the LB broth culture medium volume. Then transfer it to a constant temperature shaker. Set the temperature of the constant temperature shaker to 32℃ and the shaking speed to 200rpm. Incubate at a constant temperature for 16h to obtain activated seed culture. S2: Add 1.5L of sterilized fermentation medium to the sterilized fermenter, set the fermenter temperature to 37℃, the gas flow rate to 1lpm, the DO value to 40%, and the fermenter agitator speed to 300-900rpm. The fermenter agitator speed is adjusted according to the DO value. When the speed reaches the peak value, the correlation is canceled. Then, the DO value is controlled by using O2 gas flow rate correlated with DO. The pH value is adjusted to 7.0-7.1 using a pH adjuster. S3: After adding the fermentation medium for 8 hours, inoculate the activated seeds into the fermenter at an inoculation ratio of 10%, and carry out basic fermentation culture for 5-6 hours. After the basic fermentation culture is completed, set the fermenter temperature to 32℃ and start the feeding device at the same time to feed feed I into the fermenter at a rate of 15ml / h for first-stage fed fermentation for 5-7 hours. S4: After the first-stage fed-batch fermentation is completed, set the fermentation tank temperature to 37℃, replace feed I with feed II, and feed II into the fermentation tank at a rate of 20ml / h for the second-stage fed-batch fermentation. After 1-1.5h of the second-stage fed-batch fermentation, feed III, which is 10% of the initial fermentation volume, is added into the fermentation tank at a rate of 40ml / h for the third-stage fed-batch fermentation for 7-8.5h. S5: After the third-stage fed-batch fermentation is completed, feed III, which is 10% of the initial fermentation volume, is fed into the fermenter again at a rate of 40 ml / h. At the same time, the feed rate of feed II is reduced to 18 ml / h. The final fermentation is carried out for 6-8 hours. After the final fermentation is completed, the fermented cells are collected and plasmids are extracted using a plasmid extraction kit to obtain low-copy plasmids.

[0007] Furthermore, the fermentation medium described in step S2 consists of the following percentage components: 1.20% soybean peptone, 2.40% yeast extract, 0.22% potassium dihydrogen phosphate, 0.94% disodium hydrogen phosphate, 1.00% glucose, 0.03% magnesium sulfate heptahydrate, and 94.21% deionized water; Furthermore, the pH adjuster mentioned in step S2 is a 50% phosphoric acid solution and a 3M sodium hydroxide solution; Furthermore, the feed I mentioned in step S3 consists of the following percentage components: 50.00% glucose, 2.25% soybean peptone, 0.10% magnesium sulfate heptahydrate, 0.30% betaine, and 47.35% deionized water; Furthermore, the feed II mentioned in step S4 consists of the following percentage components: 20.00% yeast extract powder, 50.00% glycerol, 0.10% trace element complex solution, 0.20% magnesium sulfate heptahydrate, 0.60% ammonium sulfate and 29.10% deionized water; The trace element complex solution is composed of the following components: 8.42 g / L ferric(III) hexahydrate, 4.00 g / L manganese(II) tetrahydrate, 1.00 g / L zinc sulfate heptahydrate, 0.40 g / L copper sulfate pentahydrate, 0.50 g / L sodium molybdate dihydrate, and 0.50 g / L boric acid; Furthermore, the feed III mentioned in step S4 consists of the following percentage components: 20.00% yeast extract powder, 0.50% ammonium sulfate, 1.00% vitamin complex solution and 78.50% deionized water; The vitamin complex solution is composed of the following components: vitamin B1 15.00 g / L, vitamin B9 10.00 g / L and vitamin B12 5.00 g / L.

[0008] In summary, due to the adoption of the above technical solutions, the beneficial effects of this invention are as follows: This invention provides a complete blueprint for bio-fermentation processes, capable of stably, efficiently, and sustainably producing low-copy plasmids that meet high-quality standards. Compared to conventional processes using simple carbon and nitrogen sources, it can achieve an absolute doubling of yield. Simultaneously, during fermentation, it can adjust the metabolic pattern of cells, shifting the metabolic focus from rapid self-replication to efficient synthesis of the target product, achieving absolute optimization of plasmid yield and significantly improving product structural integrity. Furthermore, batch-to-batch consistency reaches industrial-grade standards, meeting the stringent requirements of commercial production for product quality consistency. It possesses extremely high application value and broad industrialization prospects. Detailed Implementation

[0009] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0010] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0011] Example 1;1: Weigh 25g of LB broth culture medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer it to an autoclave and sterilize it at 121℃ for 20min to obtain LB broth culture medium. Take 200ml of LB broth culture medium and place it in a sterilized Erlenmeyer flask. Inoculate it with 0.2ml of glycerol bacteria, and then transfer it to a constant temperature shaker. Set the temperature of the constant temperature shaker to 32℃ and the shaking speed to 200rpm. Incubate at a constant temperature for 16h to obtain activated seed culture. 2: Add 1.5L of sterilized fermentation medium to the sterilized fermenter. Set the fermenter temperature to 37℃, the gas flow rate to 1lpm, the DO value to 40%, and the fermenter agitator speed to 300-900rpm. The fermenter agitator speed is adjusted according to the DO value. When the speed reaches the peak value, the correlation is canceled. Then, the DO value is controlled by using O2 gas flow rate correlated with DO. The pH value is adjusted to 7.0 using 50% phosphoric acid solution and 3M sodium hydroxide solution. 3: After adding the fermentation medium for 8 hours, 150 ml of activated seed was inoculated into the fermenter and basal fermentation was carried out for 5 hours. After the basal fermentation was completed, the temperature of the fermenter was set to 32℃, and the feeding device was started at the same time. Feeding material I was fed into the fermenter at a rate of 15 ml / h for first-stage fed fermentation for 5 hours. 4: After the first-stage fed-batch fermentation is completed, set the fermentation tank temperature to 37℃, replace feed I with feed II, and feed II into the fermentation tank at a rate of 20ml / h for the second-stage fed-batch fermentation for 1.0h. After the second-stage fed-batch fermentation is completed, feed III at a rate of 40ml / h into the fermentation tank for the third-stage fed-batch fermentation for 7.0h. 5: After the third-stage fed-batch fermentation is completed, reduce the feeding rate of feed II to 18 ml / h, and feed 150 ml of feed III into the fermenter again at a rate of 40 ml / h for the final stage fermentation for 6.0 h. After the final stage fermentation is completed, collect the fermentation cells and extract plasmids using a plasmid extraction kit to obtain the low-copy plasmid prepared in Example 1.

[0012] Example 2;1: Weigh 25g of LB broth culture medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolution and volume adjustment, transfer the flask to an autoclave and sterilize at 121℃ for 20min to obtain LB broth culture medium. Take 200ml of LB broth culture medium and place it in a sterilized Erlenmeyer flask. Inoculate 0.2ml of glycerol bacteria, then transfer it to a constant temperature shaker. Set the temperature of the shaker to 32℃ and the shaking speed to 200rpm. Incubate at this temperature for 16.0h to obtain activated seed culture. 2: Add 1.5L of sterilized fermentation medium to the sterilized fermenter. Set the fermenter temperature to 37℃, the gas flow rate to 1lpm, the DO value to 40%, and the fermenter agitator speed to 300-900rpm. The fermenter agitator speed is adjusted according to the DO value. When the speed reaches the peak value, the correlation is canceled. Then, the DO value is controlled by using O2 gas flow rate correlated with DO. The pH value is adjusted to 7.1 using 50% phosphoric acid solution and 3M sodium hydroxide solution. 3: After adding fermentation medium for 8.0 hours, 150 ml of activated seed is inoculated into the fermenter and basal fermentation is carried out for 6.0 hours. After the basal fermentation is completed, the fermenter temperature is set to 32℃, and the feeding device is started at the same time. Feeding I is fed into the fermenter at a rate of 15 ml / h for first-stage fed fermentation for 7.0 hours. 4: After the first stage of fed-batch fermentation is completed, set the fermentation tank temperature to 37℃, replace feed I with feed II, and feed II into the fermentation tank at a rate of 20ml / h for the second stage of fed-batch fermentation for 1.5h. After the second stage of fed-batch fermentation is completed, feed III at a rate of 40ml / h into the fermentation tank for the third stage of fed-batch fermentation for 8.5h. 5: After the third-stage fed-batch fermentation is completed, reduce the feeding rate of feed II to 18 ml / h, and feed 150 ml of feed III into the fermenter again at a rate of 40 ml / h for the final stage fermentation for 8.0 h. After the final stage fermentation is completed, collect the cells and extract plasmids using a plasmid extraction kit to obtain the low-copy plasmid prepared in Example 2.

[0013] Comparative Example 1; 1. Weigh 25g of LB broth culture medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer the flask to an autoclave and sterilize it at 121℃ for 20min to obtain LB broth culture medium. Take 200ml of LB broth culture medium and place it in a sterilized Erlenmeyer flask. Inoculate it with 0.2ml of glycerol bacteria, and then transfer it to a constant temperature shaker. Set the temperature of the constant temperature shaker to 32℃ and the shaking speed to 200rpm. Incubate at a constant temperature for 16h to obtain activated seed culture. 2: Add 1.5L of sterilized fermentation medium to the sterilized fermenter. Set the fermenter temperature to 37℃, the gas flow rate to 1lpm, the DO value to 40%, and the fermenter agitator speed to 300-900rpm. The fermenter agitator speed is adjusted according to the DO value. When the speed reaches the peak value, the correlation is canceled. Then, the DO value is controlled by using O2 gas flow rate correlated with DO. The pH value is adjusted to 7.0 using 50% phosphoric acid solution and 3M sodium hydroxide solution. 3: After adding the fermentation medium for 8 hours, 150 ml of activated seed was inoculated into the fermenter and basal fermentation was carried out for 5 hours. After the basal fermentation was completed, the temperature of the fermenter was set to 32℃, and the feeding device was started at the same time. Feeding material I was fed into the fermenter at a rate of 15 ml / h for first-stage fed fermentation for 5 hours. 4: After the first-stage fed-batch fermentation is completed, set the fermentation tank temperature to 37℃, replace feed I with feed II, and feed II into the fermentation tank at a rate of 20ml / h for the second-stage fed-batch fermentation for 1.0h. After the second-stage fed-batch fermentation is completed, feed III at a rate of 40ml / h into the fermentation tank for the third-stage fed-batch fermentation for 7.0h. 5: After the third-stage fed-batch fermentation is completed, reduce the feeding rate of feed II to 18 ml / h, and feed 150 ml of feed III into the fermenter again at a rate of 40 ml / h for the final stage fermentation for 6.0 h. After the final stage fermentation is completed, collect the fermentation cells and extract plasmids using a plasmid extraction kit to obtain the low-copy plasmid prepared in Comparative Example 1.

[0014] Table 1. Composition of Comparative Feed II used in Comparative Example 1 Drug Name mass percentage yeast extract powder 6% Soy peptone 6% glucose 50% Deionized water 38% Comparative Example 2; 1. Weigh 25g of LB broth culture medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer the flask to an autoclave and sterilize it at 121℃ for 20min to obtain LB broth culture medium. Take 200ml of LB broth culture medium and place it in a sterilized Erlenmeyer flask. Inoculate it with 0.2ml of glycerol bacteria, and then transfer it to a constant temperature shaker. Set the temperature of the constant temperature shaker to 32℃ and the shaking speed to 200rpm. Incubate at a constant temperature for 16h to obtain activated seed culture. 2: Add 1.5L of sterilized fermentation medium to the sterilized fermenter. Set the fermenter temperature to 37℃, the gas flow rate to 1lpm, the DO value to 40%, and the fermenter agitator speed to 300-900rpm. The fermenter agitator speed is adjusted according to the DO value. When the speed reaches the peak value, the correlation is canceled. Then, the DO value is controlled by using O2 gas flow rate correlated with DO. The pH value is adjusted to 7.1 using 50% phosphoric acid solution and 3M sodium hydroxide solution. 3: After adding the fermentation medium for 8 hours, 150 ml of activated seed was inoculated into the fermenter and basal fermentation was carried out for 5 hours. After the basal fermentation was completed, the temperature of the fermenter was set to 32℃, and the feeding device was started at the same time. Feeding material I was fed into the fermenter at a rate of 15 ml / h for first-stage fed fermentation for 5 hours. 4: After the first-stage fed-batch fermentation is completed, set the fermentation tank temperature to 37℃, replace feed I with feed II, and feed II into the fermentation tank at a rate of 20ml / h for the second-stage fed-batch fermentation for 1.0h. After the second-stage fed-batch fermentation is completed, feed III at a rate of 40ml / h into the fermentation tank for the third-stage fed-batch fermentation for 7.0h. 5: After the third-stage fed-batch fermentation is completed, reduce the feeding rate of feed II to 18 ml / h, and feed 150 ml of feed III into the fermenter again at a rate of 40 ml / h for the final stage fermentation for 6.0 h. After the final stage fermentation is completed, collect the fermentation cells and extract plasmids using a plasmid extraction kit to obtain the low-copy plasmid prepared in Comparative Example 2.

[0015] Table 2, Composition of Comparative Feed III used in Comparative Example 2 Drug Name mass percentage yeast extract powder 5% Soy peptone 5% Deionized water 90% Comparative Example 3; 1. Weigh 25g of LB broth culture medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer the flask to an autoclave and sterilize it at 121℃ for 20min to obtain LB broth culture medium. Take 200ml of LB broth culture medium and place it in a sterilized Erlenmeyer flask. Inoculate it with 0.2ml of glycerol bacteria, and then transfer it to a constant temperature shaker. Set the temperature of the constant temperature shaker to 32℃ and the shaking speed to 200rpm. Incubate at a constant temperature for 16h to obtain activated seed culture. 2: Add 1.5L of sterilized fermentation medium to the sterilized fermenter. Set the fermenter temperature to 37℃, the gas flow rate to 1lpm, the DO value to 40%, and the fermenter agitator speed to 300-900rpm. The fermenter agitator speed is adjusted according to the DO value. When the speed reaches the peak value, the correlation is canceled. Then, the DO value is controlled by using O2 gas flow rate correlated with DO. The pH value is adjusted to 7.0 using 50% phosphoric acid solution and 3M sodium hydroxide solution. 3. After adding fermentation medium for 8 hours, inoculate 150 ml of activated seed into the fermenter and perform basal fermentation for 5 hours. After the basal fermentation is complete, start the feeding device and add feed II to the fermenter at a rate of 15 ml / h. Set the feeding rate and DO cascade, and perform fed-batch fermentation until fermentation is complete. After fermentation, collect the fermentation cells and extract plasmids using a plasmid extraction kit to obtain the low-copy plasmid prepared in Comparative Example 3.

[0016] Final output test Final output tests were conducted on Examples 1, 2, 1, 2, and 3. Table 3. Final Output Test Results project <![CDATA[OD 600 ]]> plasmid yield Superspiral ratio Example 1 75 125mg / L 91% Example 2 95 155mg / L 93% Comparative Example 1 85 70mg / L 86% Comparative Example 2 90 80mg / L 85% Comparative Example 3 80 45mg / L 80% Analysis of Table 3 shows that the scheme used in Comparative Example 3 had the worst effect, indicating that the fermentation strategy using a single feed is not conducive to plasmid amplification, and the final plasmid yield is much lower than other schemes. The scheme used in Comparative Example 2 uses a common nitrogen source as feed III during the cell growth maintenance period. It can only support the basic metabolism and a small amount of growth of the cells, and cannot support the large-scale plasmid synthesis metabolic pathway. Therefore, its yield and quality are between those of the Example and Comparative Example 1, but cannot reach the level of the Example. The method used in Comparative Example 1, which employed simple glucose during the critical plasmid replication phase (feed II), resulted in a "glucose effect," causing acetic acid accumulation, inhibiting normal cell growth, and thus affecting plasmid yield. Therefore, although cell density was acceptable, plasmid yield and total production were significantly lower than in the Example. The scheme used in Example 1 had a short total fermentation time, especially the final fermentation time of only 6.0 hours. The total cell density and total plasmid yield did not reach the highest level. However, its optimized fed-batch formulation ensured a high plasmid yield and supercoil ratio, indicating that the process direction was correct, but the time was insufficient. The scheme used in Example 2 is the overall optimal scheme. The longer fermentation time, especially the final fermentation of 8.0h, allows the cells to grow and produce fully in an optimized environment. The optimized feeding II greatly improves the replication efficiency, and the optimized feeding III maintains the stability and high quality of the plasmid during long-term culture. The three core indicators of yield, production rate and quality are all optimal.

[0017] Inter-batch consistency testing Example 1, Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3 were run 5 times consecutively to conduct batch-to-batch consistency tests. The average value, standard deviation (SD), and relative standard deviation (RSD%) of key indicators were calculated for evaluation. The smaller the RSD%, the more stable the process and the better the reproducibility.

[0018] Table 4. Results of inter-batch consistency test project <![CDATA[OD 600 ]]> plasmid yield Superspiral ratio Example 1 SD ± 3.5 RSD 4.7% SD ± 6.5 RSD 6.2% SD ± 1.5% RSD 1.6% Example 2 SD ± 2.5 RSD 2.6% SD ± 4.5 RSD 3.3% SD ± 1.0% RSD 1.1% Comparative Example 1 SD ± 6.0 RSD 7.1% SD ± 7.5 RSD 10.7% SD ± 3.5% RSD 4.5% Comparative Example 2 SD±4.0 RSD4.4% SD ± 6.9% RSD 6.7% SD ± 2.5% RSD 2.9% Comparative Example 3 SD ± 8.0 RSD 17.8% SD ± 5.5 RSD 22.0% SD ± 5.0% RSD 7.1% As can be seen from the analysis of Table 4, the scheme used in Example 2 exhibits excellent robustness, with RSD% below 3.5% on all key indicators. This means that the process used in Example 2 has excellent repeatability and has great potential for industrial scale-up. The process used in Comparative Example 1, which employed glucose feeding, had an RSD% exceeding 10%, indicating that the process was highly unstable and any minor deviation could lead to batch failure. Comparative Example 3 used dismal data to prove that the inconsistency of the starting strains rendered all subsequent process controls futile, with its OD and RSD% of yield reaching as high as 20%, which is completely unacceptable. Comparing Example 1 and Comparative Example 2, it can be seen that even with insufficient fermentation time, the stability of plasmid quality in Example 1 with the optimized formulation is still better than that in Comparative Example 2 with its simple composition. This indicates that the scheme used in Example 1 directly improves the physical stability of the plasmid.

[0019] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for increasing the fermentation yield of low-copy plasmids, characterized in that, Includes the following steps: S1: Weigh 25g of LB broth culture medium powder, dissolve it in pure water and bring the volume to 1L in a volumetric flask. After dissolving and bringing the volume to 1L, transfer it to an autoclave and sterilize it at 121℃ for 20min to obtain LB broth culture medium. Take 200ml of LB broth culture medium and place it in a sterilized Erlenmeyer flask. Inoculate it with glycerol bacteria at a volume of 1‰ of the LB broth culture medium volume. Then transfer it to a constant temperature shaker. Set the temperature of the constant temperature shaker to 32℃ and the shaking speed to 200rpm. Incubate at a constant temperature for 16h to obtain activated seed culture. S2: Add 1.5L of sterilized fermentation medium to the sterilized fermenter, set the fermenter temperature to 37℃, the gas flow rate to 1lpm, the DO value to 40%, and the fermenter agitator speed to 300-900rpm. The fermenter agitator speed is adjusted according to the DO value. When the speed reaches the peak value, the correlation is canceled. Then, the DO value is controlled by using O2 gas flow rate correlated with DO. The pH value is adjusted to 7.0-7.1 using a pH adjuster. S3: After adding the fermentation medium for 8 hours, inoculate the activated seeds into the fermenter at an inoculation ratio of 10%, and carry out basic fermentation culture for 5-6 hours. After the basic fermentation culture is completed, set the fermenter temperature to 32℃ and start the feeding device at the same time to feed feed I into the fermenter at a rate of 15ml / h for first-stage fed fermentation for 5-7 hours. S4: After the first-stage fed-batch fermentation is completed, set the fermentation tank temperature to 37℃, replace feed I with feed II, and feed II into the fermentation tank at a rate of 20ml / h for the second-stage fed-batch fermentation. After 1-1.5h of the second-stage fed-batch fermentation, feed III, which is 10% of the initial fermentation volume, is added into the fermentation tank at a rate of 40ml / h for the third-stage fed-batch fermentation for 7-8.5h. S5: After the third-stage fed-batch fermentation is completed, feed III, which is 10% of the initial fermentation volume, is fed into the fermenter again at a rate of 40 ml / h. At the same time, the feed rate of feed II is reduced to 18 ml / h. The final fermentation is carried out for 6-8 hours. After the final fermentation is completed, the fermented cells are collected and plasmids are extracted using a plasmid extraction kit to obtain low-copy plasmids.

2. The feeding method for increasing the fermentation yield of low-copy plasmids according to claim 1, characterized in that, The fermentation medium described in step S2 consists of the following percentage components: 1.20% soybean peptone, 2.40% yeast extract, 0.22% potassium dihydrogen phosphate, 0.94% disodium hydrogen phosphate, 1.00% glucose, 0.03% magnesium sulfate heptahydrate, and 94.21% deionized water.

3. The feeding method for increasing the fermentation yield of low-copy plasmids according to claim 1, characterized in that, The pH adjuster mentioned in step S2 is a 50% phosphoric acid solution and a 3M sodium hydroxide solution.

4. The feeding method for increasing the fermentation yield of low-copy plasmids according to claim 1, characterized in that, The feed I mentioned in step S3 consists of the following percentage components: 50.00% glucose, 2.25% soybean peptone, 0.10% magnesium sulfate heptahydrate, 0.30% betaine and 47.35% deionized water.

5. The feeding method for increasing the fermentation yield of low-copy plasmids according to claim 1, characterized in that, The feed II described in step S4 consists of the following percentage components: 20.00% yeast extract powder, 50.00% glycerol, 0.10% trace element complex solution, 0.20% magnesium sulfate heptahydrate, 0.60% ammonium sulfate and 29.10% deionized water.

6. The feeding method for increasing the fermentation yield of low-copy plasmids according to claim 1, characterized in that, The feed III described in step S4 consists of the following percentage components: 20.00% yeast extract powder, 0.50% ammonium sulfate, 1.00% vitamin complex solution and 78.50% deionized water.

7. The feeding method for increasing the fermentation yield of low-copy plasmids according to claim 5, characterized in that, The trace element complex solution is composed of the following components: 8.42 g / L ferric(III) hexahydrate, 4.00 g / L manganese(II) tetrahydrate, 1.00 g / L zinc sulfate heptahydrate, 0.40 g / L copper sulfate pentahydrate, 0.50 g / L sodium molybdate dihydrate, and 0.50 g / L boric acid.

8. The feeding method for increasing the fermentation yield of low-copy plasmids according to claim 6, characterized in that, The vitamin complex solution is composed of the following components: vitamin B1 15.00 g / L, vitamin B9 10.00 g / L and vitamin B12 5.00 g / L.