High-density fermentation process of salmonella bacteriophage RNA polymerase

Abstract

The invention discloses a high-density fermentation process of salmonella bacteriophage RNA polymerase, and belongs to the technical field of salmonella. The invention relates to a high-density fermentation process of salmonella phage RNA polymerase. The high-density fermentation process comprises the following steps: S1, preparing a seed solution; s2, seed amplification culture; s3, performing high-density fermentation; s4, performing induced expression; s5, enzyme extraction and purification. The method solves the problems that in the prior art, the culture period completely depends on the splitting speed of the host bacteria, if the period needs to be shortened, the initial bacteriophage infection complex number can only be increased, however, the host bacteria are split too early, and the titer is further reduced. By optimizing a culture medium and a material supplementing strategy, the wet weight of thalli is greatly increased, the enzyme yield per unit fermentation volume is remarkably increased, the enzyme activity ratio and purity of the thalli are increased, the fermentation period is effectively shortened, the production efficiency is improved, and a short-period medium yield mode or a long-period high yield mode can be flexibly switched to meet diversified production requirements.

Description

Technical Field

[0001] This invention relates to the field of Salmonella phage technology, specifically a high-density fermentation process for Salmonella phage RNA polymerase. Background Technology

[0002] Salmonella phage RNA polymerase is a highly specific RNA polymerase that recognizes only promoter sequences in the Salmonella phage genome. It can efficiently synthesize specific RNA molecules in vitro and is widely used in in vitro transcription systems, RNA vaccine development, gene editing, and other fields. Currently, this enzyme is mainly produced through fermentation using recombinant engineered Escherichia coli.

[0003] Chinese patent CN112063594A discloses a thermoresistant Salmonella phage RDP-SA-18056 and its microcapsule preparation process. The host strain is Salmonella S7. This phage can form plaques with a diameter of 2-4 mm on biplates. Electron microscopy reveals a polyhedral, three-dimensionally symmetrical head encapsulating nucleic acid, approximately 50 nm in diameter, and a tail approximately 200 nm long with a tail sheath. The neck connects the head and tail. It belongs to the order Tailvirales, family Long-tailed Viriviruses, and has the accession number CGMCC No. 19293. This phage exhibits strong lytic activity against Salmonella, providing a phage source for industrial production and for the prevention and control of avian salmonellosis and environmental disinfection. The phage also demonstrates good temperature and acid / alkali tolerance.

[0004] In practical use, the aforementioned patents directly place the bacteriophages into the culture medium in the fermenter for cultivation. The cultivation cycle depends entirely on the lysis rate of the host bacteria. If the cycle needs to be shortened, the initial phage infection multiple can only be increased, but this will cause the host bacteria to lyse prematurely, further reducing the titer. Therefore, it does not meet the current needs. In response, we have proposed a high-density fermentation process for Salmonella bacteriophage RNA polymerase. Summary of the Invention

[0005] The purpose of this invention is to provide a high-density fermentation process for Salmonella phage RNA polymerase. By optimizing the culture medium and feeding strategy, the wet weight of the cells is significantly increased, the enzyme yield per unit fermentation volume is significantly improved, the induction timing and conditions are precisely controlled, the cell enzyme activity ratio and purity are improved, the industrial application needs are met, the fermentation cycle is effectively shortened, production efficiency is improved, energy consumption and costs are reduced, and through batch feeding fermentation, the short-cycle medium-yield or long-cycle high-yield modes can be flexibly switched to adapt to diverse production needs, thus solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a high-density fermentation process for Salmonella bacteriophage RNA polymerase, comprising the following steps:

[0007] S1: Seed culture preparation: Recombinant Escherichia coli containing the Salmonella phage RNA polymerase encoding gene was inoculated into LB seed medium and cultured for 8-10 hours at a temperature of 35-37℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.8-1.2 vvm to obtain primary seed culture;

[0008] S2: Seed expansion culture: Transfer the primary seed culture to the seed expansion culture medium at an inoculation rate of 5% to 8%, and culture at a temperature of 35 to 37°C and a rotation speed of 180 to 220 rpm for 6 to 8 hours to obtain the secondary seed culture;

[0009] S3: High-density fermentation: The secondary seed culture is transferred to the fermentation medium at an inoculation rate of 3% to 5% for batch fermentation;

[0010] S4: Induction of expression: Control the fermentation process parameters. When the cell density of the engineered bacteria reaches 25-30, add an inducer to induce expression. The induction temperature is 30-37℃. Continue to culture for 8-10 hours and then end the fermentation.

[0011] S5: Enzyme extraction and purification: Collect bacterial cells by centrifugation, and obtain Salmonella phage RNA polymerase after crushing and purification.

[0012] Preferably, the formulation of LB seed culture medium is as follows: tryptone, 10-12 g / L; yeast extract, 5-6 g / L; NaCl, 8-10 g / L; glucose, 2-3 g / L; and the formulation of seed expansion culture medium is as follows: tryptone, 10-12 g / L; yeast extract, 5-6 g / L; NaCl, 8-10 g / L; glucose, 7-8 g / L; KH2PO4, 1 g / L.

[0013] Preferably, the fermentation medium is formulated as follows: glucose, 15-20 g / L; yeast extract, 10-15 g / L; tryptone, 8-12 g / L; KH2PO4, 2-3 g / L; K2HPO4·3H2O, 5-7 g / L; MgSO4·7H2O, 0.8-1.2 g / L; and trace element solution, 1-2 mL / L. The trace element solution is formulated as follows: FeSO4·7H2O, 20-30 g / L; ZnSO4·7H2O, 2-3 g / L; MnSO4·H2O, 1-2 g / L; and CuSO4·5H2O, 0.5-1 g / L, with deionized water as the solvent.

[0014] The preferred conditions for fed-batch fermentation are: temperature of 36–37°C, pH of 6.8–7.2, and dissolved oxygen of 40%–60%.

[0015] Preferably, the feed broth composition for fed-batch fermentation is: glucose, 400-500 g / L; yeast extract, 80-100 g / L; tryptone, 50-60 g / L; the feeding rate is 5-8 mL / (L·h), and the feeding rate is adjusted in real time according to the glucose concentration in the fermentation broth.

[0016] Preferably, the feeding strategy for fed-batch fermentation is as follows: no feeding is performed for the first 8-10 hours of fermentation. When the glycerol concentration in the fermentation broth drops to 1-2 g / L, the feeding solution is started to be added. The initial flow rate is 5-8 mL / (L·h), and it is adjusted according to the dissolved oxygen content and cell density in the fermentation broth. When the cell density reaches 30-35, the flow rate is increased to 10-12 mL / (L·h).

[0017] Preferably, the inducing agent is IPTG, the induction concentration is 0.4-0.6 mmol / L, and after induction, the temperature is lowered to 30-32°C, the pH value is maintained at 7.0-7.2, and the dissolved oxygen content is controlled at 30%-40%.

[0018] Preferably, the centrifugation conditions are: centrifugation at 8000–10000 rpm and 4°C for 15–20 min; high-pressure homogenization is used for disruption at 80–100 MPa, with 2–3 disruptions; and purification is performed using… Affinity chromatography column, with Tris-HCl buffer containing 200–300 mmol / L imidazole as the elution buffer.

[0019] Preferably, during the induction expression process, the feed flow rate is adjusted to 8-10 mL / (L·h), and L-methionine with a final concentration of 0.1-0.2 g / L is added to improve enzyme activity.

[0020] Preferably, during the fermentation process, the glucose concentration, ammonia nitrogen concentration, and cell density in the fermentation broth are monitored online. When the ammonia nitrogen concentration is lower than 0.5 g / L, 0.5 to 1 g / L of ammonium sulfate is added.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention significantly increases the wet weight of the cells and the enzyme yield per unit fermentation volume by optimizing the culture medium and feeding strategy. It also precisely controls the induction timing and conditions, improves the enzyme activity ratio and purity of the cells, meets the needs of industrial applications, effectively shortens the fermentation cycle, improves production efficiency, and reduces energy consumption and costs. Through batch feeding fermentation, it can flexibly switch between short-cycle medium-yield or long-cycle high-yield modes to adapt to diverse production needs. Attached Figure Description

[0023] Figure 1This is a flowchart of an embodiment of the high-density fermentation process for Salmonella bacteriophage RNA polymerase of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1

[0026] To address the issue that existing technologies directly place bacteriophages into the culture medium in fermenters for cultivation, where the cultivation cycle depends entirely on the host bacterium's lysis rate, and shortening the cycle can only be achieved by increasing the initial phage multiplicity of infection, this leads to premature host bacterium lysis and further reduction in titer. Please refer to [link to relevant documentation]. Figure 1 This embodiment provides the following technical solution:

[0027] The high-density fermentation process for Salmonella bacteriophage RNA polymerase includes the following steps:

[0028] S1: Seed culture preparation: Recombinant Escherichia coli containing the Salmonella phage RNA polymerase encoding gene was inoculated into LB seed medium and cultured for 9 h at a temperature of 36℃, a rotation speed of 200 rpm and an aeration rate of 1:1 vvm to obtain primary seed culture. The cell density of the primary seed culture reached 2.0.

[0029] S2: Seed expansion culture: The primary seed culture was transferred to the seed expansion medium at an inoculation rate of 6% and cultured at 36℃ and 200 rpm for 7 hours to obtain the secondary seed culture. The cell density of the secondary seed culture reached 5.5.

[0030] S3: High-density fermentation: The secondary seed culture is transferred to the fermentation medium at an inoculation rate of 4% for batch fermentation;

[0031] S4: Induction of expression: Control the fermentation process parameters. When the cell density of the engineered bacteria reaches 28, add an inducer to induce expression. The induction temperature is 31℃. Continue to culture for 9 hours and then end the fermentation.

[0032] S5: Enzyme extraction and purification: Collect bacterial cells by centrifugation, and obtain Salmonella phage RNA polymerase after crushing and purification.

[0033] The LB seed culture medium formula is as follows: tryptone, 11 g / L; yeast extract, 5.5 g / L; NaCl, 9 g / L; glucose, 2.5 g / L, pH 7.0; the seed expansion culture medium formula is as follows: tryptone, 11 g / L; yeast extract, 5.5 g / L; NaCl, 9 g / L; glucose, 7.5 g / L; KH2PO4, 1 g / L, to increase seed density and lay the foundation for subsequent fermentation.

[0034] The fermentation medium was formulated as follows: glucose, 18 g / L; yeast extract, 13 g / L; tryptone, 10 g / L; KH2PO4, 2.5 g / L; K2HPO4·3H2O, 6 g / L; MgSO4·7H2O, 1 g / L; and trace element solution, 1.5 mL / L. The trace element solution was formulated as follows: FeSO4·7H2O, 25 g / L; ZnSO4·7H2O, 2.5 g / L; MnSO4·H2O, 1.5 g / L; and CuSO4·5H2O, 0.8 g / L. The solvent was deionized water.

[0035] The conditions for fed-batch fermentation were: temperature 36.5℃, pH 7 (pH was adjusted by automatically adding 28% ammonia), dissolved oxygen 50% (achieved by adjusting the stirring speed and aeration rate, with the initial stirring speed at 2.5 rpm and the initial aeration rate at 1.3 vvm).

[0036] The feed broth for fed-batch fermentation consisted of: glucose, 450 g / L; yeast extract, 90 g / L; and tryptone, 60 g / L. The feeding rate was 6 mL / (L·h), and the feeding rate was adjusted in real time according to the glucose concentration in the fermentation broth, maintaining the glucose concentration at 0.8 g / L.

[0037] The feeding strategy for fed-batch fermentation is as follows: no feeding is performed for 9 hours of fermentation. When the glycerol concentration in the fermentation broth drops to 1.5 g / L, the feeding solution is started to be fed in. The initial flow rate is 6 mL / (L·h), and it is adjusted according to the dissolved oxygen content and cell density in the fermentation broth. When the cell density reaches 33, the flow rate is increased to 11 mL / (L·h).

[0038] The inducing agent was IPTG at a concentration of 0.5 mmol / L. After induction, the temperature was lowered to 31°C, the pH was maintained at 7, and the dissolved oxygen was controlled at 35%.

[0039] Centrifugation conditions were 9000 rpm and 4℃ for 18 min. High-pressure homogenization was used for disruption at 90 MPa, repeated three times. Purification was performed using… Affinity chromatography column, elution buffer was Tris-HCl containing 250 mmol / L imidazole.

[0040] During the induction process, the feed flow rate was adjusted to 9 mL / (L·h), and L-methionine was added to a final concentration of 0.2 g / L to improve enzyme activity.

[0041] During fermentation, the concentrations of glucose, ammonia nitrogen, and cell density in the fermentation broth were monitored online. When the ammonia nitrogen concentration was below 0.5 g / L, 0.8 g / L of ammonium sulfate was added.

[0042] Example 2

[0043] The high-density fermentation process for Salmonella bacteriophage RNA polymerase includes the following steps:

[0044] S1: Seed culture preparation: Recombinant Escherichia coli containing the Salmonella phage RNA polymerase encoding gene was inoculated into LB seed medium and cultured for 8 hours at a temperature of 35℃, a rotation speed of 180 rpm and an aeration rate of 1:0.8 vvm to obtain primary seed culture;

[0045] S2: Seed expansion culture: The primary seed culture was transferred to the seed expansion medium at an inoculation rate of 5% and cultured at 35℃ and 180 rpm for 6 hours to obtain the secondary seed culture. The cell density of the secondary seed culture reached 5.0.

[0046] S3: High-density fermentation: The secondary seed culture is transferred to the fermentation medium at an inoculation rate of 3% for batch fermentation;

[0047] S4: Induction of expression: Control the fermentation process parameters. When the cell density of the engineered bacteria reaches 25, add an inducer to induce expression. The induction temperature is 30℃. Continue to culture for 8 hours and then end the fermentation.

[0048] S5: Enzyme extraction and purification: Collect bacterial cells by centrifugation, and obtain Salmonella phage RNA polymerase after crushing and purification.

[0049] The LB seed culture medium formula is as follows: tryptone, 10 g / L; yeast extract, 5 g / L; NaCl, 8 g / L; glucose, 2 g / L. The seed expansion culture medium formula is as follows: tryptone, 10 g / L; yeast extract, 5 g / L; NaCl, 8 g / L; glucose, 7 g / L; KH2PO4, 1 g / L. This increases the seed density and lays the foundation for subsequent fermentation.

[0050] The fermentation medium was formulated as follows: glucose, 15 g / L; yeast extract, 10 g / L; tryptone, 8 g / L; KH2PO4, 2 g / L; K2HPO4·3H2O, 5 g / L; MgSO4·7H2O, 0.8 g / L; and trace element solution, 1 mL / L. The trace element solution was formulated as follows: FeSO4·7H2O, 20 g / L; ZnSO4·7H2O, 2 g / L; MnSO4·H2O, 1 g / L; and CuSO4·5H2O, 0.5 g / L. The solvent was deionized water.

[0051] The conditions for fed-batch fermentation are: temperature 36℃, pH 6.8 (pH is adjusted by automatically adding 25% ammonia), dissolved oxygen 40% (achieved by adjusting the stirring speed and aeration rate, with the initial stirring speed being 200-300 rpm and the initial aeration rate being 1 vvm).

[0052] The feed broth for fed-batch fermentation consisted of: glucose, 400 g / L; yeast extract, 80 g / L; and tryptone, 50 g / L. The feeding rate was 5 mL / (L·h), and the feeding rate was adjusted in real time according to the glucose concentration in the fermentation broth, with the glucose concentration maintained at 0.5 g / L.

[0053] The feeding strategy for fed-batch fermentation is as follows: no feeding is performed for the first 8 hours of fermentation. When the glycerol concentration in the fermentation broth drops to 1 g / L, the feeding solution is started to be fed. The initial flow rate is 5 mL / (L·h), and it is adjusted according to the dissolved oxygen content and cell density in the fermentation broth. When the cell density reaches 30, the flow rate is increased to 10 mL / (L·h).

[0054] The inducing agent was IPTG at a concentration of 0.4 mmol / L. After induction, the temperature was lowered to 30°C, the pH was maintained at 7.0, and the dissolved oxygen was controlled at 30%.

[0055] Centrifugation conditions were 8000 rpm and 4℃ for 15 min. High-pressure homogenization was used for disruption at 80 MPa, repeated twice. Purification was performed using… Affinity chromatography column, elution buffer is Tris-HCl buffer containing 200 mmol / L imidazole.

[0056] During the induction expression process, the feed flow rate was adjusted to 8 mL / (L·h), and L-methionine was added to a final concentration of 0.1 g / L to improve enzyme activity.

[0057] During fermentation, the concentrations of glucose, ammonia nitrogen, and cell density in the fermentation broth are monitored online. When the ammonia nitrogen concentration is below 0.5 g / L, 0.5 g / L of ammonium sulfate is added.

[0058] The same fermentation method as in Example 1 was used, but the difference between Example 1 and Example 2 was the feeding rate and the induction temperature.

[0059] Example 3

[0060] The high-density fermentation process for Salmonella bacteriophage RNA polymerase includes the following steps:

[0061] S1: Seed culture preparation: Recombinant Escherichia coli containing the Salmonella phage RNA polymerase encoding gene was inoculated into LB seed medium and cultured for 8 hours at a temperature of 35℃, a rotation speed of 180 rpm and an aeration rate of 1:0.8 vvm to obtain primary seed culture;

[0062] S2: Seed expansion culture: The primary seed culture was transferred to the seed expansion medium at an inoculation rate of 8% and cultured at 35℃ and 180 rpm for 6 hours to obtain the secondary seed culture. The cell density of the secondary seed culture reached 5.0.

[0063] S3: High-density fermentation: The secondary seed culture is transferred to the fermentation medium at an inoculation rate of 3% for batch fermentation;

[0064] S4: Induction of expression: Control the fermentation process parameters. When the cell density of the engineered bacteria reaches 25, add an inducer to induce expression. The induction temperature is 37℃. Continue to culture for 8 hours and then end the fermentation.

[0065] S5: Enzyme extraction and purification: Collect bacterial cells by centrifugation, and obtain Salmonella phage RNA polymerase after crushing and purification.

[0066] The LB seed culture medium formula is as follows: tryptone, 10 g / L; yeast extract, 6 g / L; NaCl, 8 g / L; glucose, 2 g / L. The seed expansion culture medium formula is as follows: tryptone, 10 g / L; yeast extract, 5 g / L; NaCl, 8 g / L; glucose, 7 g / L; KH2PO4, 1 g / L. This increases the seed density and lays the foundation for subsequent fermentation.

[0067] The fermentation medium was formulated as follows: glucose, 15 g / L; yeast extract, 10 g / L; tryptone, 8 g / L; KH2PO4, 2 g / L; K2HPO4·3H2O, 5 g / L; MgSO4·7H2O, 0.8 g / L; and trace element solution, 2 mL / L. The trace element solution was formulated as follows: FeSO4·7H2O, 30 g / L; ZnSO4·7H2O, 2 g / L; MnSO4·H2O, 1 g / L; and CuSO4·5H2O, 0.5 g / L. The solvent was deionized water.

[0068] The conditions for fed-batch fermentation were: temperature 36℃, pH 6.8 (pH adjusted by automatic addition of 25% ammonia), and dissolved oxygen 40% (achieved by adjusting the stirring speed and aeration rate). The initial stirring speed was 200–300 rpm, and the initial aeration rate was 1 vvm.

[0069] The feed broth for fed-batch fermentation consisted of: glucose, 400 g / L; yeast extract, 80 g / L; and tryptone, 50 g / L. The feeding rate was 8 mL / (L·h), and the feeding rate was adjusted in real time according to the glucose concentration in the fermentation broth, with the glucose concentration maintained at 0.5 g / L.

[0070] The feeding strategy for fed-batch fermentation is as follows: no feeding is performed for the first 8 hours of fermentation. When the glycerol concentration in the fermentation broth drops to 1 g / L, the feeding solution is started to be fed. The initial flow rate is 5 mL / (L·h), and it is adjusted according to the dissolved oxygen content and cell density in the fermentation broth. When the cell density reaches 30, the flow rate is increased to 10 mL / (L·h).

[0071] The inducing agent was IPTG at a concentration of 0.4 mmol / L. After induction, the temperature was lowered to 30°C, the pH was maintained at 7.0, and the dissolved oxygen was controlled at 30%.

[0072] Centrifugation conditions were 8000 rpm and 4℃ for 15 min. High-pressure homogenization was used for disruption at 80 MPa, repeated twice. Purification was performed using… Affinity chromatography column, elution buffer is Tris-HCl buffer containing 200 mmol / L imidazole.

[0073] During the induction expression process, the feed flow rate was adjusted to 8 mL / (L·h), and L-methionine was added to a final concentration of 0.1 g / L to improve enzyme activity.

[0074] During fermentation, the concentrations of glucose, ammonia nitrogen, and cell density in the fermentation broth are monitored online. When the ammonia nitrogen concentration is below 0.5 g / L, 0.5 g / L of ammonium sulfate is added.

[0075] The same fermentation method as in Example 2 was used, but the difference between Example 2 and Example 2 was the feeding rate and the induction temperature.

[0076] Comparative Example 1

[0077] The high-density fermentation process for Salmonella bacteriophage RNA polymerase includes the following steps:

[0078] S1: Seed culture preparation: Recombinant Escherichia coli containing the Salmonella phage RNA polymerase encoding gene was inoculated into LB seed medium and cultured for 9 h at a temperature of 36℃, a rotation speed of 200 rpm and an aeration rate of 1:1 vvm to obtain primary seed culture. The cell density of the primary seed culture reached 2.0.

[0079] S2: Seed expansion culture: The primary seed culture was transferred to the seed expansion medium at an inoculation rate of 6% and cultured at 36℃ and 200 rpm for 7 hours to obtain the secondary seed culture. The cell density of the secondary seed culture reached 5.5.

[0080] S3: High-density fermentation: The secondary seed culture is transferred to the fermentation medium at an inoculum rate of 4% for fermentation.

[0081] S4: Induction of expression: Control the fermentation process parameters. When the cell density of the engineered bacteria reaches 28, add an inducer to induce expression. The induction temperature is 31℃. Continue to culture for 9 hours and then end the fermentation.

[0082] S5: Enzyme extraction and purification: Collect bacterial cells by centrifugation, and obtain Salmonella phage RNA polymerase after crushing and purification.

[0083] The LB seed culture medium formula is as follows: tryptone, 11 g / L; yeast extract, 5.5 g / L; NaCl, 9 g / L; glucose, 2.5 g / L, pH 7.0; the seed expansion culture medium formula is as follows: tryptone, 11 g / L; yeast extract, 5.5 g / L; NaCl, 9 g / L; glucose, 7.5 g / L; KH2PO4, 1 g / L, to increase seed density and lay the foundation for subsequent fermentation.

[0084] The fermentation medium was formulated as follows: glucose, 18 g / L; yeast extract, 13 g / L; tryptone, 10 g / L; KH2PO4, 2.5 g / L; K2HPO4·3H2O, 6 g / L; MgSO4·7H2O, 1 g / L; and trace element solution, 1.5 mL / L. The trace element solution was formulated as follows: FeSO4·7H2O, 25 g / L; ZnSO4·7H2O, 2.5 g / L; MnSO4·H2O, 1.5 g / L; and CuSO4·5H2O, 0.8 g / L. The solvent was deionized water.

[0085] The inducing agent was IPTG at a concentration of 0.5 mmol / L. After induction, the temperature was lowered to 31°C, the pH was maintained at 7, and the dissolved oxygen was controlled at 35%.

[0086] Centrifugation conditions were 9000 rpm and 4℃ for 18 min. High-pressure homogenization was used for disruption at 90 MPa, repeated three times. Purification was performed using… Affinity chromatography column, elution buffer was Tris-HCl containing 250 mmol / L imidazole.

[0087] During the induction process, the feed flow rate was adjusted to 9 mL / (L·h), and L-methionine was added to a final concentration of 0.2 g / L to improve enzyme activity.

[0088] During fermentation, the concentrations of glucose, ammonia nitrogen, and cell density in the fermentation broth were monitored online. When the ammonia nitrogen concentration was below 0.5 g / L, 0.8 g / L of ammonium sulfate was added.

[0089] The fermentation method used in this case was the same as in Example 1. The difference was that the secondary seed liquid was transferred to the fermentation medium at an inoculation rate of 4% for direct fermentation.

[0090] The Salmonella phages fermented in Examples 1, 2, 3, and Comparative Example 1 were tested, and the results are as follows:

[0091] Cell wet weight (g / L) Enzyme specific activity (U / mg) Fermentation cycle (h) Example 1 92 1280 29 Example 2 86 1220 31 Example 3 88 1150 28 Comparative Example 1 48 980 24

[0092] As can be seen from the table above, the Salmonella phage fermented using the method of Example 1 has the best cell density and enzyme activity, which can ensure normal cell metabolism and maximize enzyme activity. The Salmonella phage fermented using the method of Example 2 will limit cell growth and prolong the fermentation cycle. The Salmonella phage fermented using the method of Example 3 will lead to glucose accumulation and the generation of acetic acid to inhibit enzyme expression.

[0093] Compared with Comparative Example 1, Example 1 showed a significant increase in cell wet weight, enzyme activity ratio, and fermentation cycle, indicating that the wet weight, enzyme activity ratio, and fermentation cycle of the resulting Salmonella phage were effectively improved by transferring the secondary seed culture at an inoculation rate of 4% to the fermentation medium and fermenting it through batch feeding.

[0094] In summary, the high-density fermentation process for Salmonella phage RNA polymerase of this invention significantly increases cell wet weight and enzyme yield per fermentation volume by optimizing the culture medium and feeding strategy. Precise control of induction timing and conditions improves cell enzyme activity ratio and purity, meeting industrial application requirements. It effectively shortens the fermentation cycle, increases production efficiency, and reduces energy consumption and costs. Through batch feeding fermentation, the feeding rate can be dynamically adjusted based on real-time enzyme activity data, reducing activity loss. It can flexibly switch between short-cycle medium-yield and long-cycle high-yield modes within 24–48 hours, adapting to diverse production needs.

[0095] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0096] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.

Claims

1. A high-density fermentation process for Salmonella bacteriophage RNA polymerase, characterized in that, Includes the following steps: S1: Seed culture preparation: Recombinant Escherichia coli containing the Salmonella phage RNA polymerase encoding gene was inoculated into LB seed medium and cultured for 8-10 hours at a temperature of 35-37℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.8-1.2 vvm to obtain primary seed culture; S2: Seed expansion culture: Transfer the primary seed culture to the seed expansion culture medium at an inoculation rate of 5% to 8%, and culture at a temperature of 35 to 37°C and a rotation speed of 180 to 220 rpm for 6 to 8 hours to obtain the secondary seed culture; S3: High-density fermentation: The secondary seed culture is transferred to the fermentation medium at an inoculation rate of 3% to 5% for batch fermentation; S4: Induction of expression: Control the fermentation process parameters. When the cell density of the engineered bacteria reaches 25-30, add an inducer to induce expression. The induction temperature is 30-37℃. Continue to culture for 8-10 hours and then end the fermentation. S5: Enzyme extraction and purification: Collect bacterial cells by centrifugation, and obtain Salmonella phage RNA polymerase after crushing and purification.

2. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, The formulation of the LB seed culture medium is as follows: tryptone, 10-12 g / L; yeast extract, 5-6 g / L; NaCl, 8-10 g / L; glucose, 2-3 g / L; and the formulation of the seed expansion culture medium is as follows: tryptone, 10-12 g / L; yeast extract, 5-6 g / L; NaCl, 8-10 g / L; glucose, 7-8 g / L; KH2PO4, 1 g / L.

3. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, The fermentation medium is formulated as follows: glucose, 15-20 g / L; yeast extract, 10-15 g / L; tryptone, 8-12 g / L; KH2PO4, 2-3 g / L; K2HPO4·3H2O, 5-7 g / L; MgSO4·7H2O, 0.8-1.2 g / L; and trace element solution, 1-2 mL / L. The trace element solution is formulated as follows: FeSO4·7H2O, 20-30 g / L; ZnSO4·7H2O, 2-3 g / L; MnSO4·H2O, 1-2 g / L; and CuSO4·5H2O, 0.5-1 g / L, with deionized water as the solvent.

4. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, The conditions for the batch-fed fermentation are: temperature 36-37℃, pH 6.8-7.2, and dissolved oxygen 40%-60%.

5. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, The feed broth for the fed-batch fermentation consists of: glucose, 400–500 g / L; yeast extract, 80–100 g / L; and tryptone, 50–60 g / L. The feeding rate is 5–8 mL / (L·h), and the feeding rate is adjusted in real time according to the glucose concentration in the fermentation broth.

6. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, The feeding strategy for fed-batch fermentation is as follows: no feeding is performed for the first 8-10 hours of fermentation. When the glycerol concentration in the fermentation broth drops to 1-2 g / L, the feeding solution is started to be added. The initial flow rate is 5-8 mL / (L·h), and it is adjusted according to the dissolved oxygen content and cell density in the fermentation broth. When the cell density reaches 30-35, the flow rate is increased to 10-12 mL / (L·h).

7. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, The inducing agent is IPTG, with an induction concentration of 0.4–0.6 mmol / L. After induction, the temperature is lowered to 30–32°C, the pH is maintained at 7.0–7.2, and the dissolved oxygen content is controlled at 30%–40%.

8. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, The centrifugation conditions were 8000–10000 rpm and 4°C for 15–20 min. High-pressure homogenization was used for disruption at 80–100 MPa, repeated 2–3 times. Purification was performed using… Affinity chromatography column, with Tris-HCl buffer containing 200–300 mmol / L imidazole as the elution buffer.

9. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, During the induction expression process, the feed flow rate was adjusted to 8–10 mL / (L·h), and L-methionine was added at a final concentration of 0.1–0.2 g / L to enhance enzyme activity.

10. The high-density fermentation process for Salmonella bacteriophage RNA polymerase according to claim 1, characterized in that, During the fermentation process, the concentrations of glucose, ammonia nitrogen, and cell density in the fermentation broth are monitored online. When the ammonia nitrogen concentration is below 0.5 g / L, 0.5–1 g / L of ammonium sulfate is added.

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