A fermentation method for improving the yield of escherichia coli recombinant plasmid

Abstract

The application provides a fermentation method for improving the yield of recombinant plasmid of Escherichia coli, and belongs to the field of biopharmacy and fermentation engineering. In the fermentation induction stage, the application adds polyamine synthesis precursor substances into the fermentation liquor, activates the endogenous arginine decarboxylase pathway (SpeA-SpeB) and the spermidine synthase pathway (SpeE) of Escherichia coli, greatly increases the intracellular putrescine and spermidine levels, thereby accelerates the plasmid DNA replication and improves the yield of recombinant plasmid. In the 5L fermenter scale, the yield of chicken coccidiosis DNA vaccine plasmid is increased from 400-500 mg / L in the conventional optimization process to 1412 mg / L, which is increased by about 180%-250%, and the supercoiled proportion is maintained above 96%. The fermentation method provided by the application is simple in operation, low in cost and easy to scale up, and is suitable for the industrial scale production of DNA vaccine plasmid.

Description

Technical Field

[0001] This invention relates to the fields of biopharmaceuticals and fermentation engineering, specifically to a fermentation method for increasing the yield of recombinant plasmids of Escherichia coli. Background Technology

[0002] Large-scale plasmid DNA production is a core component of the DNA vaccine industrialization process. *E. coli*, as the most commonly used host for plasmid amplification, possesses advantages such as a clear genetic background, rapid growth, and low culture cost, making it widely used for the industrial production of plasmid DNA. In *E. coli*, polyamines participate in various key physiological processes, including DNA replication, transcription, translation, and cell division. Studies have shown that intracellular polyamine levels are closely related to plasmid DNA replication efficiency. Polyamines mainly include putrescine, spermidine, and spermine. Among these, spermidine can promote the movement speed of the DNA replication fork, stabilize the binding of DNA polymerase to the template, and neutralize the negatively charged phosphate groups on the DNA strand, thus facilitating efficient plasmid DNA amplification.

[0003] Currently, two common strategies for increasing E. coli plasmid yield are exogenous polyamine addition and E. coli strain modification. Exogenous polyamine addition involves directly adding exogenous spermidine or putrescine to the culture medium. However, this method has several drawbacks: First, reagent-grade spermidine is expensive, making it difficult to meet the economic requirements of industrial-scale production. Second, exogenous polyamines enter cells via passive diffusion, resulting in a slow and linear increase in intracellular concentration, which cannot simulate the explosive polyamine response under natural stress conditions, thus affecting plasmid DNA amplification yield. Third, the accumulation of large amounts of exogenous polyamines may be toxic to cells, affecting bacterial growth and plasmid quality. Another strategy is to modify E. coli strains to oversynthesize spermidine. However, this method involves permanent modification of the host genome, posing biosafety risks, and the genetic stability of the engineered strains and plasmid compatibility issues remain unresolved.

[0004] Therefore, there is an urgent need for a method for fermenting Escherichia coli recombinant plasmids that does not rely on exogenous polyamines, does not involve strain gene modification, has high yield, is easy to operate, and is low in cost. Summary of the Invention

[0005] The purpose of this invention is to provide a fermentation method that increases the yield of recombinant plasmids of Escherichia coli, thereby overcoming the shortcomings of existing technologies, such as poor performance, low yield, high cost, and high safety risks associated with metabolically engineered strains due to the addition of exogenous polyamines.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A fermentation method for increasing the yield of recombinant plasmids of Escherichia coli involves adding a polyamine precursor to the fermentation broth during the fermentation process. The polyamine precursor is any one of L-arginine, L-ornithine, and guanidinobutylamine.

[0007] Furthermore, the amount of L-arginine, the polyamine anabolic precursor, added is preferably 10-20 mM.

[0008] Furthermore, the culture medium used in the fermentation method comprises: glycerol, yeast extract, soybean peptone, K2HPO4, KH2PO4, MgSO4·7H2O, (NH4)2SO4, citric acid, and trace element solution.

[0009] Furthermore, the culture medium used in the fermentation method comprises: 10-20 g / L glycerol, 15-30 g / L yeast extract, 10-30 g / L soybean peptone, 8-15 g / L K2HPO4, 2-5 g / L KH2PO4, 0.5-2.0 g / L MgSO4·7H2O, 2-6 g / L (NH4)2SO4, 0.5-2.0 g / L citric acid, and 1-5 mL / L trace element solution.

[0010] Furthermore, the fermentation method is divided into a seed culture stage, a fermenter culture stage, a fed-batch culture stage, an induction stage, and a cell collection stage, with the polyamine synthesis precursor added during the induction stage.

[0011] Furthermore, the induction stage employs a high-temperature induction method, that is, raising the fermentation temperature to 40±2℃.

[0012] Furthermore, the polyamine precursor is added in a pulsed manner, with the pulsed addition occurring 30 minutes before the induction phase heating begins and continues until the heating stops.

[0013] The application of the fermentation method described above for increasing the yield of recombinant plasmids of Escherichia coli in the preparation of Escherichia coli chicken coccidia DNA vaccine plasmids.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a fermentation method for increasing the yield of recombinant plasmids from *E. coli*. In a 5 L fermenter, this method increases the yield of chicken coccidia DNA vaccine plasmids from approximately 400-500 mg / L under conventional optimized processes (without arginine treatment and the same culture conditions) to 1412 mg / L, an increase of approximately 180%-250%. Simultaneously, the supercoil ratio of the fermentation product is maintained above 96%, and the plasmid specific yield reaches above 24 mg / g wet cells.

[0015] This invention provides a fermentation method to increase the yield of recombinant plasmids in Escherichia coli. By activating the endogenous SpeA-SpeB-SpeE pathway, a "pulse-like" burst of polyamines is achieved. Compared with the method of directly adding exogenous polyamines, the intracellular polyamine increase is faster, greater, lower in cost, and closer to the natural stress response mode of bacteria.

[0016] The present invention adopts a pulse addition method, which is simple to operate and easy to control. It does not require continuous addition equipment, and does not require the use of IPTG chemical inducer under high temperature induction conditions, which further reduces costs and reduces process safety risks.

[0017] The fermentation method used in this invention has good reproducibility and is easy to scale up: the process verified at the scale of a 5 L fermenter can be successfully scaled up to 50 L and above, and the plasmid yield remains basically stable, making it suitable for the industrial-scale production of DNA vaccine plasmids.

[0018] The fermentation method provided by this invention involves adding exogenous polyamine synthesis precursors to the fermentation broth during the induction phase. This activates the endogenous polyamine synthesis pathway in *E. coli*, thereby increasing intracellular polyamine levels and ultimately improving the yield of recombinant plasmids. After being taken into the cells via the *E. coli*-specific transport system, the exogenously added polyamine synthesis precursors do not directly function as polyamines. Instead, they act as substrates within the endogenous polyamine synthesis pathway of *E. coli*. Arginine is decarboxylated by arginine decarboxylase (SpeA) to generate guanidinobutylamine. Guanidinobutylamine is then hydrolyzed by guanidinobutylamine urease (SpeB) to generate putrescine. Putrescine is further catalyzed by spermidine synthase (SpeE) to accept aminopropyl groups from decarboxylated S-adenosylmethionine (dcSAM) to generate spermidine. In addition, L-arginine can also be converted into L-ornithine via the arginase pathway. Ornithine can also be decarboxylated by constitutive ornithine decarboxylase (SpeC) or inducible ornithine decarboxylase (SpeF) to generate putrescine, which is also incorporated into the above-mentioned polyamine synthesis pathway.

[0019] Unlike existing techniques that directly add exogenous spermidine or putrescine to the culture medium, the increase in intracellular polyamine levels in this invention is synergistically catalyzed by the endogenous arginine decarboxylase pathway (SpeA-SpeB) and / or ornithine decarboxylase pathway (SpeC / SpeF) and spermidine synthase pathway (SpeE) of *E. coli*, without relying on the direct addition of exogenous spermidine, putrescine, or spermine. This difference in mechanism constitutes the core distinguishing feature of this invention from existing techniques. The exogenous addition of precursor substances can trigger a "pulsating" polyamine burst similar to the bacteria's natural stress response, with the magnitude and rate of intracellular polyamine increase being superior to the passive diffusion method of directly adding exogenous polyamines. Detailed Implementation

[0020] The following specific descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The following descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0021] Strains and Plasmids: The recombinant plasmid is a eukaryotic expression plasmid containing the Eimeria coccidia 3-1E antigen gene (e.g., the 3-1E gene). The Escherichia coli host strain is selected from DH5α, DH10B, JM109, Stbl3, TOP10, or NEB 5α strains, all of which are commercially available and commonly used strains. The following examples use Escherichia coli DH5α carrying the Eimeria coccidia 3-1E recombinant plasmid as an example for illustration.

[0022] Seed culture medium (g / L): soybean peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0.

[0023] Plasmid extraction and quantification methods: After fermentation, a certain amount of fermentation broth was collected and centrifuged at 4 ℃ and 6000 g for 10 minutes to collect the cells. Plasmid DNA was extracted using the alkaline lysis method (SDS-alkaline lysis). After purification, the plasmid DNA concentration was determined by ultraviolet spectrophotometry (A260 / A280 ratio between 1.80 and 1.95 was considered acceptable for purity). The plasmid yield was calculated (unit: mg plasmid / L fermentation broth). The supercoil ratio was determined by agarose gel electrophoresis combined with optical density scanning.

[0024] Intracellular polyamine determination method: After washing the bacterial cell sample with PBS, the intracellular polyamines were extracted with 5% trichloroacetic acid (TCA) at 4 °C. The extract was derivatized in advance with dansyl chloride, and the concentrations of putrescine and spermidine were determined by high performance liquid chromatography (HPLC), expressed as μmol / g wet cells.

[0025] L-Arginine: The L-arginine used in the following examples is industrial-grade L-arginine or its hydrochloride form (content ≥98.5%), purchased from domestic amino acid manufacturers.

[0026] Example 1: Comparative Experiment on the Effects of Different Metabolic Precursors in the Polyamine Synthesis Pathway of Escherichia coli Fermentation conditions: A 5 L fermenter (3 L liquid volume) was used, employing *E. coli* DH5α carrying the *Eimeria tenella* 3-1E recombinant plasmid. The fermentation medium consisted of the following basic formula: 15 g / L glycerol, 20 g / L yeast extract, 15 g / L soybean peptone, 12 g / L K2HPO4, 3 g / L KH2PO4, 1.2 g / L MgSO4·7H2O, 4 g / L (NH4)2SO4, 1.0 g / L citric acid, and 3 mL / L trace element solution. When the OD600 reached 60, induction was performed at 40℃, with the precursor substances added in a pulsed manner during the temperature rise. L-arginine (5 mM), L-ornithine (5 mM), guanidinobutylamine (5 mM), and exogenous spermidine (1 mM) were added in pulses. A blank control without any added substances was also included. Each group had three replicates. The experimental results are shown in Table 1.

[0027] Table 1: Effects of different polyamine synthesis precursors on plasmid yield

[0028] The results showed that all three polyamine anabolic precursors effectively increased plasmid yield, with L-arginine showing the best effect (860 mg / L), followed by L-ornithine (750 mg / L). The exogenous spermidine group (1 mM) produced a plasmid yield of 820 mg / L, approximately 95% of that of the L-arginine group. Furthermore, all precursor groups maintained a high supercoiling rate (≥96.1%), superior to the exogenous spermidine group (95.0%). From a cost perspective, the raw material cost of L-arginine was approximately RMB 0.04 / g plasmid, only 1.6% of the cost of the exogenous spermidine approach (approximately RMB 2.50 / g plasmid). These results fully demonstrate that replacing the direct addition of exogenous polyamines with an endogenous pathway activation strategy offers comprehensive advantages in terms of yield, quality, and cost.

[0029] Example 2: Effect of different final concentrations of L-arginine pulses on plasmid yield Fermentation conditions: A 5 L fermenter (3 L liquid volume) was used, employing *E. coli* DH5α carrying the *Eimeria tenella* 3-1E recombinant plasmid. The fermentation medium used a basic formula (per liter: glycerol 15 g / L, yeast extract 20 g / L, soybean peptone 15 g / L, K2HPO4 12 g / L, KH2PO4 3 g / L, MgSO4·7H2O 1.2 g / L, (NH4)2SO4 4 g / L, citric acid 1.0 g / L, trace element solution 3 mL / L). The fed-batch medium used a basic formula (glycerol 500 g / L, yeast extract 120 g / L, soybean peptone 100 g / L, MgSO4·7H2O 15 g / L). When the OD600 reached 60, induction was performed at 40℃, and L-arginine was added in a pulsed manner to final concentrations of 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, and 50 mM during the temperature rise. A control group without L-arginine was also included. Induction lasted for 12 hours, with three replicates per group. The experimental results are shown in Table 2 below.

[0030] Table 2: Effect of different L-arginine pulse concentrations on plasmid yield

[0031] The results showed that when the final pulse concentration of L-arginine was 10 mM, the plasmid yield reached its maximum of 950 mg / L, an increase of approximately 103% compared to the control group (468 mg / L). Simultaneously, the intracellular spermidine concentration reached 2.85 μmol / g wet cells, an increase of approximately 2.4 times compared to the control group. Too low an arginine concentration (5 mM) resulted in insufficient substrate supply to maximize the activation of the polyamine synthesis pathway; too high a concentration (≥30 mM) could lead to excessive arginine or its metabolites potentially causing osmotic pressure burden or metabolic interference on the cells, resulting in a decrease in plasmid yield and supercoil ratio. Therefore, the optimal final pulse concentration of L-arginine was determined to be 10 mM.

[0032] Example 3: Effect of different timings of L-arginine pulse addition on plasmid yield The basic fermentation conditions were the same as in Example 2, with the final L-arginine concentration fixed at 10 mM. The timing of pulse addition was set as follows: 60 minutes before temperature induction, 30 minutes before temperature induction, simultaneously with temperature induction (0 minutes), 30 minutes after temperature induction, 60 minutes after temperature induction, and a control group without addition. Each group had three replicates. The experimental results are shown in Table 3 below.

[0033] Table 3. Effects of different L-arginine pulse timings on plasmid yield.

[0034] The results showed that the timing of pulse addition significantly affected plasmid yield. Adding L-arginine 30 minutes before temperature induction resulted in the highest plasmid yield (1100 mg / L); adding it 60 minutes before temperature induction or simultaneously with temperature induction yielded the next best results; adding it after temperature induction significantly reduced the yield. Analysis suggests that administering arginine 30 minutes before temperature induction ensures that the intracellular polyamine synthesis pathway is fully activated at the start of high-temperature induction, guaranteeing a sufficient supply of polyamines when the plasmid DNA enters the rapid replication phase. Adding arginine after temperature induction results in a lag in polyamine synthesis activation, missing the optimal window for plasmid replication. Therefore, the optimal timing for pulse addition was determined to be 30 minutes before temperature induction.

[0035] Example 4: Effect of different fermentation medium formulations on plasmid yield.

[0036] Experimental Design: Orthogonal optimization was performed on the concentrations of key carbon and nitrogen source components in the fermentation medium. Other components were used as the basic formulation concentrations. Three factors were selected: glycerol concentration (A), yeast extract concentration (B), and soybean peptone concentration (C). Each factor had three concentration levels, using an L9 (32) algorithm. 3 An orthogonal experimental design was used. The supplemented culture medium adopted the basic formula (glycerol 450 g / L, yeast extract 100 g / L, soybean peptone 80 g / L, MgSO4·7H2O 10 g / L). Each group was set up in triplicate, and plasmid yield was the main evaluation index.

[0037] Table 4. Factor Level Table for Orthogonal Experiment

[0038] Orthogonal Experiment Results Table (L9 (3)) 3 ): Table 5: Results of the Orthogonal Experiment

[0039] Range analysis results: Factor A (glycerol): K1=1096.7, K2=1136.7, K3=1146.7, R=50.0; Factor B (yeast extract): K1=1073.3, K2=1126.7, K3=1180.0, R=106.7; Factor C (soy peptone): K1=1080.0, K2=1153.3, K3=1146.7, R=73.3.

[0040] The results showed that the order of influence of the three factors on plasmid yield was B (yeast extract) > C (soybean peptone) > A (glycerol). The optimal combination was A2B3C2, i.e., glycerol 15 g / L, yeast extract 25 g / L, and soybean peptone 20 g / L. Validation experiments were conducted under this optimal formulation, achieving a plasmid yield of 1250 mg / L (n=3, RSD=3.2%) and a cell wet weight of 57.8 g / L.

[0041] Example 5: Optimization Experiment of Fed Culture Medium

[0042] Experimental Design: Single-factor optimization of glycerol and yeast extract concentrations in the fed culture medium was performed, with three replicates per group. First, 100 g / L of yeast extract was used, and glycerol concentrations of 400, 500, and 600 g / L were set. Then, 80 g / L of soybean peptone and 10 g / L of MgSO4·7H2O were added to screen for the optimal glycerol concentration. Next, the optimal glycerol concentration was used to set yeast extract concentrations of 80, 100, and 120 g / L, with 80 g / L of soybean peptone and 10 g / L of MgSO4·7H2O added, finally determining the optimal yeast extract concentration. The experimental results are shown in Table 6.

[0043] Table 6 Experimental Results

[0044] Single-factor optimization was performed on the concentrations of soybean peptone and MgSO4·7H2O in the fed culture medium. First, 80 g / L of soybean peptone was used, and the concentrations of MgSO4·7H2O were set at 10, 15, and 20 g / L. Then, 500 g / L of glycerol and 120 g / L of yeast extract were added to screen for the optimal MgSO4·7H2O concentration. Next, the optimal MgSO4·7H2O concentration was determined by setting soybean peptone concentrations at 80, 100, and 120 g / L, and adding 500 g / L of glycerol and 120 g / L of yeast extract. Finally, the optimal soybean peptone concentration was screened. The experimental design and results are shown in Table 7.

[0045] Table 7 Experimental Results

[0046] Based on the experimental results, the optimal formulation of the fed culture medium is: 500 g / L glycerol, 120 g / L yeast extract, 100 g / L soybean peptone, and 15 g / L MgSO4·7H2O, with a plasmid yield of 1380 mg / L.

[0047] Example 6: Verification of optimal conditions and process scale-up The optimal conditions were as follows: L-arginine pulse concentration of 10 mM, pulse timing 30 minutes before induction, induction method of 40℃ high temperature induction, and induction duration of 12 hours; the fermentation medium used the preferred formula (glycerol 15 g / L, yeast extract 25 g / L, soybean peptone 20 g / L, K2HPO4 12 g / L, KH2PO4 3 g / L, MgSO4·7H2O 1.2 g / L, (NH4)2SO4 4 g / L, citric acid 1.0 g / L, trace element solution 3 mL / L); the fed medium used the preferred formula (glycerol 500 g / L, yeast extract 120 g / L, soybean peptone 100 g / L, MgSO4·7H2O 15 g / L).

[0048] A control group (with the same culture conditions but without the addition of L-arginine) was also set up to evaluate the net contribution of the arginine pulse. Three batches of validation experiments were conducted at a 5 L fermenter scale. The results are shown in Table 8. Table 8. Results of the Experiment Verification under Optimal Conditions

[0049] The results showed that under optimal conditions, the plasmid yield consistently reached 1412 mg / L, an increase of approximately 202% compared to the control group without arginine (468 mg / L); the specific plasmid yield reached 25.1 mg / g wet cells; and the supercoil ratio remained at 96.3%. The intracellular spermidine concentration increased by approximately 4.3 times compared to the control group, confirming the causal chain of arginine pulse → activation of endogenous polyamine synthesis → increased plasmid yield.

[0050] The optimal conditions were further scaled up to a 50 L fermenter (35 L liquid volume) for validation. The inoculum size, culture medium formulation, feeding strategy, and induction conditions for the 50 L fermenter were all scaled up proportionally to those for the 5 L fermenter. Only the stirring speed and aeration rate were adjusted appropriately based on the tank geometry and oxygen mass transfer characteristics. The results are shown in Table 9. Table 9. Results of the scale-up verification experiment

[0051] The results showed that the plasmid yield in the 50 L fermenter reached 1360 mg / L, which was 96.3% of that in the 5 L scale. The cell wet weight, plasmid yield, and supercoil ratio were also basically consistent, indicating that the fermentation process of the present invention has good scale-up feasibility and is suitable for industrial-scale production.

Claims

1. A fermentation method for increasing the yield of recombinant plasmids from *Escherichia coli*, characterized in that, During fermentation, a polyamine precursor is added to the fermentation broth, wherein the polyamine precursor is any one of L-arginine, L-ornithine, or guanidinobutylamine.

2. The fermentation method for increasing the yield of recombinant plasmids of *E. coli* according to claim 1, characterized in that, The preferred amount of L-arginine, a precursor of polyamine synthesis, is 10-20 mM.

3. The fermentation method for increasing the yield of recombinant plasmids of *E. coli* according to claim 1, characterized in that, The culture medium used in the fermentation method includes: glycerol, yeast extract, soybean peptone, K2HPO4, KH2PO4, MgSO4·7H2O, (NH4)2SO4, citric acid, and trace element solution.

4. The fermentation method for increasing the yield of recombinant plasmids of *E. coli* according to claim 3, characterized in that, The culture medium used in the fermentation method comprises: 10-20 g / L glycerol, 15-30 g / L yeast extract, 10-30 g / L soybean peptone, 8-15 g / L K2HPO4, 2-5 g / L KH2PO4, 0.5-2.0 g / L MgSO4·7H2O, 2-6 g / L (NH4)2SO4, 0.5-2.0 g / L citric acid, and 1-5 mL / L trace element solution.

5. The fermentation method for increasing the yield of recombinant plasmids of *E. coli* according to claim 1, characterized in that, The fermentation method is divided into a seed culture stage, a fermenter culture stage, a fed-batch culture stage, an induction stage, and a cell collection stage. The polyamine synthesis precursor is added during the induction stage.

6. The fermentation method for increasing the yield of recombinant plasmids of *E. coli* according to claim 5, characterized in that, The induction stage employs a high-temperature induction method, which involves raising the fermentation temperature to 40±2℃.

7. The fermentation method for increasing the yield of recombinant plasmids of *E. coli* according to claim 1, characterized in that, The polyamine anabolic precursor was added in a pulsed manner, starting 30 minutes before the induction phase heating began and continuing until the heating was stopped.

8. The application of the fermentation method for increasing the yield of Escherichia coli recombinant plasmids as described in claim 1 in the preparation of Escherichia coli chicken coccidia DNA vaccine plasmids.

Images