A high-density fermentation process, amplification method and fermentation device for high oxygen consumption and high heat production of Pichia pastoris

Abstract

The present invention relates to a high-oxygen-consuming, high-heat-producing, high-density fermentation process, amplification method, and fermentation apparatus for Pichia pastoris. The fermentation process comprises: inoculating activated bacterial strains into a seed culture medium; inoculating the expanded bacterial strains into a fermentation starter culture medium; when the dissolved oxygen value rebounds, starting to add a glycerol solution, and then adjusting the glycerol addition rate according to a gradient flow addition strategy; after reaching a certain bacterial concentration, stopping the glycerol addition and starting to add a methanol solution to induce expression of the target product. The present invention uses the fermentation of Pichia methanolophilus as the research object, optimizes the process, and designs a production-scale fermentation apparatus, thereby achieving fermentation scale expansion.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a high-oxygen-consuming and high-heat-producing Pichia pastoris high-density fermentation process, an amplification method and a fermentation device. Background Art

[0002] With the development of biological technology, large-scale fermentation technology using genetically engineered bacteria to produce biological products is now widely adopted. Among genetically engineered bacteria, Pichia pastoris is an expression system that can efficiently express foreign proteins. It boasts many advantages, including genetic stability, high expression levels, post-translational protein processing, product secretion, and high-density fermentation. It is widely used, and hundreds of foreign proteins have been expressed in this system.

[0003] High-density fermentation is an important strategy for genetically engineered bacteria to increase the expression level of exogenous proteins. To date, many protein products have achieved high product expression levels through high-density fermentation. However, for the high-density fermentation process of recombinant Pichia pastoris, due to the large-scale growth of the bacteria, the oxygen consumption rate increases and the oxygen demand is very high. At the same time, the metabolism of methanol by Pichia pastoris' alcohol oxidases (AOX1 and AOX2) requires a large amount of oxygen, making dissolved oxygen a limiting factor for high-density fermentation of Pichia pastoris. In addition, when Pichia pastoris consumes methanol, it also generates a large amount of heat. If this heat cannot be discharged in time, it will have a significant impact on bacterial growth and product expression.

[0004] The current high-density fermentation process for Pichia methanolica is complex and difficult to control. The scaled-up fermentation tank design fails to achieve a balance between oxygen supply and consumption, and heat generation and cooling. Many industries commonly use pure oxygen to achieve sufficient oxygen supply and add a refrigerant (such as ethylene glycol) to the cooling water for rapid cooling. Both designs result in high investment and maintenance costs, and also introduce significant safety risks. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide a high-density, high-expression fermentation process, an amplification method, and a fermentation tank design for Pichia methanolica, so as to overcome the problems of unstable fermentation results between batches, difficulty in amplifying and reproducing the process, and reduced fermentation yield due to insufficient oxygen supply from the equipment and inability to remove metabolic heat in a timely manner during the current high-density fermentation of Pichia methanolica.

[0006] In order to achieve the purpose of the present invention, the technical solution of the present invention is as follows:

[0007] A high-density yeast culture device for high-density fermentation processes, comprising a fermentation tank, includes an exhaust condenser, a stirring device, a ventilation device, a cooling annular inner coil, an air distributor, and a tank body. The stirring device comprises a stirring shaft located within the fermentation tank, driven by a stirring motor at the bottom of the tank, and equipped with a stirring paddle assembly.

[0008] Preferably, the air distributor is an annular air distributor with a plurality of small holes evenly distributed.

[0009] Preferably, the air distributor is an annular air distributor having 425-435 small holes with a diameter of 4-8 mm evenly distributed therein.

[0010] Preferably, the stirring blades on the stirring paddle group are axial mixing inclined blades and radial mixing semicircular blades arranged alternately.

[0011] Further preferably, the stirring paddle group consists of four groups of stirring paddles, the first and third layers of blades are pitched paddles, and the second and fourth layers of blades are semicircular paddles.

[0012] A high-density fermentation process for Pichia methanolica, comprising the following steps:

[0013] (1) Strain expansion: The activated strain was inoculated into the seed culture medium, cultured at 28-32°C with shaking for 10-14 hours, and then transferred to the secondary seed culture medium at a volume ratio of 0.5%-1.5%, and cultured at 28-30°C with shaking for 20-24 hours;

[0014] (2) Fermentation culture:

[0015] ① Initial growth stage: inoculate the expanded strain into the fermentation starting medium at a concentration of 2.0-6.0×10 5 cfu / mL, the culture temperature was controlled at 28-32°C, the ventilation ratio was 1.0-1.5vvm, as the bacterial concentration gradually increased, the carbon source in the initial culture medium was gradually consumed, and the dissolved oxygen value (DO) gradually decreased. The stirring speed was adjusted to control the dissolved oxygen value to ≥50%, and the culture was terminated after 14 hours;

[0016] ② Glycerol addition stage: After 14 hours of fermentation, glycerol solution was added. The glycerol addition rate was adjusted according to the gradient addition strategy to enable the exponential growth of the living cells. Urea solution was added as a nitrogen source. The temperature was controlled at 28-32°C and the ventilation ratio was 1.0-1.5 vvm. The culture was continued until OD 600 Stop adding glycerin when the temperature is between 150 and 300;

[0017] ③ Methanol induction stage: 30 to 50 minutes after stopping the addition of glycerol, start adding methanol solution for induction, and set the C / N (methanol to urea solution mass ratio) at different stages as follows: (8-12):1 from the start of induction to 70 hours, (14-16):1 from 70 hours to 90 hours, 30:1 from 90 hours to 120 hours, and (14-16):1 from 120 hours to the end of fermentation; the culture temperature is controlled at 25 to 28°C, the ventilation ratio is 1.5 to 2.5 vvm, and the culture is ended at 140 to 180 hours.

[0018] Preferably, in the methanol induction stage, 30 to 50 minutes after stopping the addition of glycerol, the methanol solution is added for induction, and the C / N (methanol to urea solution mass ratio) at different stages is set to 10:1 from the start of induction to 70 hours, 15:1 from 70 hours to 90 hours, 30:1 from 90 hours to 120 hours, and 15:1 from 120 hours to the end of fermentation. The culture temperature is controlled at 25 to 28° C., the ventilation ratio is 1.5 to 2.5 vvm, and the culture is ended at 140 to 180 hours.

[0019] Preferably, in step (2), the inoculation amount of the bacterial strain inoculated into the culture medium during the initial growth stage is: the volume of the bacterial strain is 1.0% to 5.0% of the volume of the fermentation culture medium.

[0020] Preferably, in step (2), the components in the fermentation starting medium include: 20-40 g / L of glycerol, 5-8 g / L of urea, 0.4-1.0 g / L of CaSO4·2H2O, 5-8 g / L of MgSO4·7H2O, 20-30 g / L of KOH, 20-30 ml / L of H3PO4, 2-8 ml / L of PTM1 solution, and 1-3 ml / L of defoaming agent.

[0021] Further preferably, the composition of the PTM1 solution is: CuSO4·5H2O 6g / L, KI 0.08g / L, MnSO4·H2O 3g / L, Na2MoO4·2H2O 0.2g / L, H3BO3 0.02g / L, ZnSO4·7H2O 20g / L, FeSO4·7H2O 65g / L, CoCl2·6H2O 0.5g / L, H2SO4 5ml / L, and Biotin 0.2g / L.

[0022] Preferably, in step (2), the composition of the urea solution is: 0.2-0.6 g / L urea solution.

[0023] Preferably, in the ② glycerol supplementation stage: glycerol solution is added after 14 hours of culture at a rate of 8 to 12 L / h / m 3 The glycerol solution is added at an initial rate of 0.3 to 0.7 g / L;

[0024] In the step 2), the glycerol addition stage (2) is as follows: the glycerol addition rate is: no glycerol is added during the 0-14h period, and the rate is set at 8-12 L / h / m during the 14-15h period. 3 , then the rate of increase is 2-4L / h / m per hour 3 , until the glycerol addition is completed.

[0025] Preferably, in the ③ methanol induction stage: the methanol solution is prepared by dissolving 8-14 mL of PTM1 in 1 L of methanol solution.

[0026] Preferably, in the methanol induction stage (3): after adding methanol solution, the temperature is first lowered to 25-28°C and cultured for 80-100 hours, and then the temperature is lowered to 25°C.

[0027] Further preferably, in the ③ methanol induction stage: after adding methanol solution, the temperature is first lowered to 28°C and cultured for 80-100 hours, and then the temperature is further lowered to 25°C.

[0028] This study uses the fermentation of Pichia methanolica as a research subject. Targeting the strain's physiological characteristics, such as high-density growth, high oxygen consumption, and high heat production, the study uses CFD computational fluid dynamics to study the flow field characteristics within the reactor. By comparing pilot studies (to obtain predetermined values) with the macro-metabolic characteristic parameters of industrial-scale fermenters, the study identified mixing and transfer defects in the industrial-scale fermenters. This led to the design of a production-scale fermentation apparatus and the implementation of appropriate process measures to achieve scale-up of the fermentation process. This apparatus is particularly suitable for Pichia methanolica.

[0029] The beneficial effects of the present invention are:

[0030] (1) Pichia pastoris fermentation usually uses BMXX series culture media (BMGY, BMMY, BMGT, etc.). However, the BMXX series culture media contain a high proportion of complex organic components (such as yeast extract and peptone, etc.). The composition of these complex organic components is unclear, the manufacturing process is complex, and there are differences between batches, which makes it difficult to reproduce the fermentation batches. In addition, the culture medium with unclear composition and differences between batches will also make the fermentation process difficult to control, making it difficult to ensure the amplification effect. The fermentation culture medium of the present invention is composed of a single chemical substance. At the same time, during the fermentation culture process, the feed rate is controlled by controlling the C / N (methanol to urea solution mass ratio) in stages according to the cell metabolism and product synthesis. The process control is simple, and both the fermentation repeatability and the amplification success rate can be effectively guaranteed. While ensuring the quality of the target product, the yield is increased by more than 50%. The comparison of 5000L and 12000L fermentation data is shown in Table 1. For the same expression system, the yield achieved by the present invention is at a relatively high level both domestically and internationally.

[0031] Table 1 Comparison of 5000L and 12000L batch production data (taking insulin glargine as an example)

[0032]

[0033] (2) After extensive and in-depth research, the present invention takes the fermentation of Pichia methanolica as the object, and uses the computational fluid dynamics (CFD) method to study the flow field characteristics in the reactor in view of the physiological characteristics of the strain, such as high-density growth, high oxygen consumption, and high heat production. By comparing the macro-metabolic characteristic parameters of the small-scale study (to obtain the predetermined values) with the industrial-scale fermentation tank, the mixing and transfer defects in the industrial-scale fermentation tank were discovered, the production-scale fermentation equipment was designed, and corresponding process measures were taken to achieve the amplification of the fermentation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the structure of a 12000L fermenter according to the present invention, including: an exhaust condensing device 1, a stirring device 2, a ventilation device 3, a stirring motor 4, a cooling annular inner coil 5, a tank body 6, a stirring shaft 7, a stirring paddle group 8, and an air distributor 9.

[0035] Figure 2 This is a detailed structural diagram of the exhaust condensation device 1 for a 12000L fermenter.

[0036] Figure 3 Design diagram of the 12000L fermenter jacket (a) and cooling ring tube (b).

[0037] Figure 4 CFD simulation diagram for the design of the jacket and cooling annular pipe of a 12000L fermenter.

[0038] a. CFD simulation of jacket cooling effect on a 12000L fermenter.

[0039] b. CFD simulation of the cooling effect of the annular inner coil of a 12000L fermenter.

[0040] Figure 5 Optimized design diagram for the stirring combination of a 12000L fermenter:

[0041] a. 12000L fermenter agitator assembly 1 (the first layer of blades is a pitched blade paddle, the second, third, and fourth layers of blades are semicircular paddles, from top to bottom);

[0042] b. 12000L fermenter stirring combination 2 (the first and third layers of blades are inclined blades, the second and fourth layers of blades are semicircular blades, from top to bottom).

[0043] Figure 6 This is the design diagram of the air distributor and the fourth-layer stirring paddle structure for a 12000L fermenter:

[0044] a. 12000L fermenter air distributor structure design drawing (d1 is the air distributor ring diameter, and the two end points are measured from the ring tube center);

[0045] b. Design diagram of the fourth-layer stirring paddle structure of a 12000L fermenter (d2 is the diameter of the fourth-layer stirring paddle, with the two end points measured from the farthest end of the stirring paddle).

[0046] Figure 7 CFD simulation results for the optimized design of different blade types + air distributor for a 12,000L fermenter:

[0047] a. Distribution of oxygen mass transfer coefficient (KLa) after CFD simulation of stirring combination 1 for a 12000 L fermenter;

[0048] b. Distribution diagram of oxygen mass transfer coefficient (KLa) after CFD simulation of 12000L fermenter stirring combination 2. DETAILED DESCRIPTION

[0049] The present invention will be further described below with reference to the examples, but the scope of protection claimed in the present invention is not limited to the scope described in the examples.

[0050] Example 1

[0051] A high-density yeast culture device for a high-density fermentation process, the culture device being a fermentation tank, the fermentation tank including an exhaust condensation device 1, a stirring device 2, a ventilation device 3, a cooling annular inner coil 5, an air distributor 9 and a tank body 6; the stirring device 2 includes: a stirring shaft 7 located in the fermentation tank and driven by a stirring motor 4 at the bottom of the tank, and a stirring paddle group 8 is provided on the stirring shaft 7.

[0052] The condenser of the industrial scale (12000L) fermentation tank is designed to reduce the evaporation during the fermentation process and thus reduce the loss of liquid volume. Figure 1 and Figure 2 As shown, the exhaust condensation device of the present invention is located at the tail gas outlet position on the top of the fermentation tank and adopts a shell and tube heat exchanger with a large cooling area and easy to clean (see exhaust condensation device 1 and Figure 2 ), the condensation effect is better, avoiding large amounts of evaporation of the fermentation liquid.

[0053] The fermentation process comprises the following steps:

[0054] (1) Strain expansion: The activated strain was inoculated into the seed culture medium, cultured at 30°C with shaking for 12 h, and then transferred to the secondary seed culture medium at a volume ratio of 1.2%, and cultured at 30°C with shaking for 22 h;

[0055] (2) Fermentation culture:

[0056] ① Initial growth stage: inoculate the expanded strain into the fermentation starting medium at a concentration of 4.0×10 5 cfu / mL, the culture temperature was controlled at 30°C, the ventilation ratio was 1.0vvm, and as the bacterial concentration gradually increased, the carbon source in the initial culture medium was gradually consumed, and the dissolved oxygen value (DO) also gradually decreased. The stirring speed was adjusted and the culture was terminated after 14 hours.

[0057] ② Glycerol addition stage: After 14 hours of fermentation, glycerol solution was added. The glycerol addition rate was adjusted according to the gradient addition strategy to enable the exponential growth of the living cells. Urea solution was added as a nitrogen source. The temperature was controlled at 30°C and the ventilation ratio was 1.2 vvm. The culture was continued until OD 600 When the temperature reaches 200, stop adding glycerol;

[0058] ③ Methanol induction stage: 30 minutes after stopping glycerol addition, methanol induction was resumed. The C / N ratio (methanol to urea mass ratio) at different stages was set as shown in Table 5 for the 12,000 L (industrial scale) culture conditions. The culture temperature was controlled at 28°C, the aeration ratio was 2.2 v / m, and the culture was terminated after 160 h.

[0059] Preferably, in step (2): the inoculation amount of the bacterial strain into the culture medium during the initial growth stage is: the volume of the bacterial strain is 3.0% of the volume of the fermentation culture medium.

[0060] Preferably, in step (2), the components in the fermentation starting medium include: 30 g / L glycerol, 7 g / L urea, 0.8 g / L CaSO4·2H2O, 7 g / L MgSO4·7H2O, 25 g / L KOH, 25 ml / L H3PO4, 6 ml / L PTM1 solution, and 2 ml / L defoaming agent.

[0061] Further preferably, the composition of the PTM1 solution is: CuSO4·5H2O 6g / L, KI 0.08g / L, MnSO4·H2O 3g / L, Na2MoO4·2H2O 0.2g / L, H3BO3 0.02g / L, ZnSO4·7H2O 20g / L, FeSO4·7H2O 65g / L, CoCl2·6H2O 0.5g / L, H2SO4 5ml / L, and Biotin 0.2g / L.

[0062] Preferably, in step (2), the composition of the urea solution is: 0.45 g / L urea solution.

[0063] Preferably, in the ② glycerol addition stage: after culturing for 14 hours, glycerol solution is added at a rate of 10 L / h / m 3The glycerol solution was added at an initial rate of 0.5 g / L;

[0064] In the step 2), the glycerol addition stage (2) is as follows: the glycerol addition rate is: no glycerol is added during 0-14 hours, and the rate is set at 10 L / h / m during 14-15 hours. 3 , then the rate of increase is 3L / h / m 3 After 21 hours, stop increasing the glycerol supplement rate and maintain it at 26 L / h / m 3 Until the glycerol addition is completed.

[0065] Preferably, in the ③ methanol induction stage: the methanol solution is prepared by dissolving 12 mL of PTM1 in 1 L of methanol solution.

[0066] Preferably, in the methanol induction stage (3): after adding methanol solution, the temperature is first lowered to 28°C and cultured for 90 hours, and then lowered to 25°C.

[0067] Table 2 Effect of condenser on evaporation of 12000L fermentation liquid

[0068]

[0069] According to Table 2, under the same production process conditions, the evaporation volume of the batch after using the condenser is significantly lower than that of the batch without using the condenser. The condensation effect is better, which avoids large-scale evaporation of the fermentation liquid.

[0070] Example 2

[0071] Design the temperature control system for industrial scale (12000L) fermentation tanks to quickly remove the heat generated during the fermentation process and improve the stability of temperature control. Figure 3 As shown, the present invention adopts two methods for temperature control:

[0072] (1) According to the gradual increase of the fermentation volume and liquid level during the fermentation process, a three-layer independent jacket temperature control system was designed to achieve temperature control while avoiding the waste of cooling resources caused by the small fermentation volume in the early stage.

[0073] (2) Use radial ring tube temperature control (2 groups of Φ38mm×2mm stainless steel pipes) with large heat exchange area and easy to clean.

[0074] The above two temperature control devices are both circulated with 5-7℃ chilled circulating water to achieve the purpose of temperature control. Through CFD simulation, the results are shown in Figure 4 The results show that under the simulation conditions, the temperature control capability of the design can meet the process requirements and the temperature is controlled uniformly.

[0075] The effects of different scales and different temperature control designs on temperature control during the fermentation process are shown in Table 3. The results show that although the fermentation scale becomes larger, the stability of temperature control can be further improved by further increasing the heat exchange area per unit volume through a good temperature control design strategy.

[0076] Table 3 Effects of different temperature control designs on temperature control during fermentation

[0077]

[0078] Example 3

[0079] The stirring and aeration system of industrial-scale (12000L) fermentation tanks is designed to improve the mixing effect and oxygen mass transfer effect under large-scale conditions, thereby ensuring the dissolved oxygen (DO) demand of Pichia methanolica under high-density fermentation conditions. Figure 5 As shown in the figure, four groups of stirring paddles with different stirring paddle types and positions fixed on a stirring shaft are optimized and designed. The optimization results show that the first and third layers of blades are pitched blades, which are axial flow type stirring paddles, and the second and fourth layers of blades are semicircular blades, which are radial flow type stirring paddles. This design can achieve better oxygen mass transfer effect.

[0080] like Figure 6 As shown, the aeration device 3 of the present invention is an annular air distributor with 431 evenly distributed outlet holes approximately 6.3 mm in diameter. The diameter (d1) of the annular distributor is the same as the diameter (d2) of the lowest stirring blade. Air enters the fermentation broth through the outlet holes and is quickly dispersed by the stirring blades, increasing the gas-liquid contact surface. The combination of axial and radial flow in the stirring process also delays the residence time of oxygen in the fermentation broth, promoting mixing of air and broth and improving oxygen mass transfer efficiency.

[0081] The oxygen mass transfer coefficient (KLa) was calculated by CFD simulation, as shown in the following example: Figure 7 The results show that compared with the pilot scale (5000L) fermenter design, the bubble dispersion and mixing effect of the industrial scale (12000L) fermenter is significantly improved after further design optimization ( Figure 6 ), there is no obvious mass transfer "dead zone" in the tank, and the gas holdup and oxygen mass transfer coefficient (KLa value) are significantly improved (Table 4).

[0082] Table 4 Comparison of CFD simulation results for stirring and air distributor design

[0083]

[0084] Example 4

[0085] The high-density fermentation process of Pichia methanolica was carried out using the 12000 L fermentor device optimized in Example 3 and a 5000 L (pilot scale) device. The fermentation process included the following steps:

[0086] (1) Strain expansion: The activated strain was inoculated into the seed culture medium, cultured at 30°C with shaking for 12 h, and then transferred to the secondary seed culture medium at a volume ratio of 1.2%, and cultured at 30°C with shaking for 22 h;

[0087] (2) Fermentation culture:

[0088] ① Initial growth stage: inoculate the expanded strain into the fermentation starting medium at a concentration of 4.0×10 5 cfu / mL, the culture temperature was controlled at 30°C, the ventilation ratio was 1.0 vvm, and as the bacterial concentration gradually increased, the carbon source in the initial culture medium was gradually consumed, and the dissolved oxygen value (DO) gradually decreased. The stirring speed was adjusted to control the dissolved oxygen value to ≥50%, and the culture was terminated after 14 hours;

[0089] ② Glycerol addition stage: After 14 hours of fermentation, glycerol solution was added. The glycerol addition rate was adjusted according to the gradient addition strategy to enable the exponential growth of the living cells. Urea solution was added as a nitrogen source. The temperature was controlled at 30°C and the ventilation ratio was 1.2 vvm. The culture was continued until OD 600 Stop adding glycerin when the temperature reaches 180;

[0090] ③ Methanol induction stage: After 40 minutes of glycerol addition, methanol induction was resumed. The C / N ratio (methanol to urea mass ratio) at different stages was set as shown in Table 5. The culture temperature was controlled at 28°C, the ventilation ratio was 2.2 v / m, and the culture was terminated after 160 hours.

[0091] Preferably, in step (2): the inoculation amount of the bacterial strain into the culture medium during the initial growth stage is: the volume of the bacterial strain is 3.0% of the volume of the fermentation culture medium.

[0092] Preferably, in step (2), the components in the fermentation starting medium include: 30 g / L glycerol, 7 g / L urea, 0.8 g / L CaSO4·2H2O, 7 g / L MgSO4·7H2O, 25 g / L KOH, 25 ml / L H3PO4, 6 ml / L PTM1 solution, and 2 ml / L defoaming agent.

[0093] Further preferably, the composition of the PTM1 solution is: CuSO4·5H2O 6g / L, KI 0.08g / L, MnSO4·H2O 3g / L, Na2MoO4·2H2O 0.2g / L, H3BO3 0.02g / L, ZnSO4·7H2O 20g / L, FeSO4·7H2O 65g / L, CoCl2·6H2O 0.5g / L, H2SO4 5ml / L, and Biotin 0.2g / L.

[0094] Preferably, in step (2), the composition of the urea solution is: 0.45 g / L urea solution.

[0095] Preferably, in the ② glycerol addition stage: glycerol solution is added after 14 hours of culture at a rate of 10 L / h / m 3 The glycerol solution was added at an initial rate of 0.5 g / L;

[0096] In the step 2), the glycerol addition stage (2) is as follows: the glycerol addition rate is: no glycerol is added during 0-14 hours, and the rate is set at 10 L / h / m during 14-15 hours. 3 , then the rate of increase is 3L / h / m 3 , until the glycerol addition is completed.

[0097] Preferably, in the ③ methanol induction stage: the methanol solution is prepared by dissolving 12 mL of PTM1 in 1 L of methanol solution.

[0098] Preferably, in the methanol induction stage (3): after adding methanol solution, the temperature is first lowered to 28°C and cultured for 90 hours, and then lowered to 25°C.

[0099] Table 5 C / N (methanol to urea solution mass ratio) in the induction stage

[0100]

[0101] As shown in Table 5, after the fermentation tank device was designed and the fermentation process was appropriately adjusted (the C / N ratio in the induction stage was adjusted from a constant value to a gradient adjustment based on metabolic changes), the expression level of the target protein product increased by 140% after the fermentation scale was scaled up. This indicates that the optimized design of the fermentation device and the fermentation process are not only conducive to scale-up but also can further improve the expression level of the target protein product.

[0102] Example 5

[0103] The process conditions of 12000 L (industrial scale) in Table 5 of Example 4 were adopted. The speed of glycerol addition was changed in this example. The specific differences were as follows: ② Glycerol addition stage: glycerol solution was added after 14 h of culture at a rate of 6 L / h / m3 or 10L / h / m 3 or 20L / h / m 3 The glycerol solution was added at an initial rate of 0.5 g / L. Table 6 shows the effect of different glycerol rates on the fermentation tank performance.

[0104] Table 6 Effect of different glycerol rates on fermentation tank performance

[0105]

[0106] As shown in Table 6, a too high glycerol rate will increase the solid content of the bacteria, thereby reducing the total amount of target protein secreted outside the cell; while a too low glycerol rate will lead to insufficient bacterial concentration, affecting the expression of the target protein, and at the same time affecting the expression of the target protein per unit volume.

[0107] Example 6

[0108] Adopting the process conditions of 12000 L (industrial scale) in Table 5 of Example 4, this example changes the induction temperature of the methanol induction stage ③. The specific differences are:

[0109] The methanol induction stage (3): After adding methanol solution, the temperature is first lowered to 28°C / 25°C / 20°C and cultured for 90 hours, specifically:

[0110] After adding methanol solution, the temperature was first lowered to 28°C and cultured for 90 hours, and then lowered to 25°C.

[0111] After adding methanol solution, the temperature was lowered to 25℃ / 20℃ and incubated for 90 hours until the fermentation was completed. The results are shown in Table 7.

[0112] Table 7 Effect of different induction temperatures on fermentation tank performance

[0113]

[0114] As shown in Table 7, a fermentation temperature that is too high will increase the solid content of the bacteria, thereby reducing the total amount of target protein secreted outside the cell, while a temperature that is too low will affect the growth of the bacteria and also affect the expression amount of the target protein per unit volume.

[0115] The above embodiments are merely preferred technical solutions of the present invention and should not be construed as limiting the present invention. The embodiments and features in the embodiments of this application may be arbitrarily combined with each other unless they conflict. The scope of protection of the present invention shall be the technical solutions described in the claims, including equivalent alternatives to the technical features of the technical solutions described in the claims. Equivalent alternatives and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A high-density yeast culture device for a high-density fermentation process for a high-density fermentation process of Pichia methanolophilus, characterized in that: The culture equipment is a fermentation tank with a scale of 12000L. The fermentation tank includes an exhaust condensation device (1), a stirring device (2), a ventilation device (3), a cooling annular pipe (5), an air distributor (9) and a tank body (6); the stirring device (2) includes: a stirring shaft (7) located in the fermentation tank and driven by a stirring motor (4) at the bottom of the tank, and a stirring paddle group (8) is provided on the stirring shaft (7); the air distributor (9) is an annular air distributor; a plurality of holes are evenly distributed; the stirring blades on the stirring paddle group (8) are axial mixing oblique blades and radial mixing semicircular blades arranged alternately; the stirring paddle group (8) includes four groups of stirring paddles, the first and third layers of paddles are oblique blades, and the second and fourth layers of paddles are semicircular paddles, the cooling annular pipe is located below the third and fourth layers of stirring paddles from bottom to top, and support rods are symmetrically provided on both sides of the cooling annular pipe; The ventilation device (3) is an annular air distributor with 431 6.3 mm air outlet holes evenly distributed. The diameter of the annular distributor is the same as the diameter of the bottom stirring blade. The fermentation tank body is equipped with three independent jackets, and the cooling ring pipe adopts radial ring pipe; Both the jacket and radial ring tube types circulate 5-7℃ chilled circulating water; The oxygen mass transfer capacity of the fermenter is KLa = 501h -1 ; The fermentation process comprises the following steps: (1) Strain expansion: The activated strain was inoculated into the seed culture medium, cultured at 30°C with shaking for 12 h, then transferred to the secondary seed culture medium at a volume ratio of 1.2%, and cultured at 30°C with shaking for 22 h; (2) Fermentation culture: ① Initial growth stage: inoculate the expanded strain into the fermentation starting medium at a concentration of 2.0-6.0×10 5 cfu / mL, the culture temperature was controlled at 28-32°C, the ventilation ratio was 1.0-1.5 vvm; the stirring speed was adjusted to control the dissolved oxygen value to ≥50%, and the culture was terminated after about 14 hours. ② Glycerol addition stage: No glycerol was added during the fermentation period of 0-14 hours. After 14 hours, glycerol solution was added and the rate was set at 10 L / h / m for 14-15 hours. 3 Glycerol solution was added at an initial rate of 3 L / h / m 3 After 21 hours, stop increasing the glycerol supplement rate and maintain it at 26 L / h / m 3 Until the addition of glycerol is completed, the composition of the glycerol solution is: 0.5 g / L glycerol solution; At the same time, urea solution was added as a nitrogen source, the temperature was controlled at 28-32°C, the ventilation ratio was 1.0-1.5 vvm, and the culture was continued until the bacterial concentration OD 600 When the temperature is between 150 and 300, stop adding glycerol; ③ Methanol induction stage: 30-50 minutes after stopping the addition of glycerol, methanol solution was added for induction. The C / N (methanol to urea solution mass ratio) at different stages was set as follows: 10:1 from the start of induction to 70 hours, 15:1 from 70 hours to 90 hours; 30:1 from 90 hours to 120 hours, and 15:1 from 120 hours to the end of fermentation. After adding methanol solution, the temperature was first lowered to 28°C for 90 hours, and then lowered to 25°C for the end of fermentation. The ventilation ratio was 1.5-2.5 vvm, and the culture was terminated at 140-180 hours. The concentration of the urea solution is 0.45 g / L.

2. The high-density fermentation process of Pichia methanolica according to claim 1, characterized in that: Said step (2): in the initial growth stage, the inoculation amount of the bacterial strain into the culture medium is: the volume of the bacterial strain is 1.0-5.0% of the volume of the fermentation culture medium.

3. The high-density fermentation process of Pichia methanolica according to claim 1, characterized in that: Step (2): the components in the fermentation starting medium include: 20-40 g / L of glycerol, 5-8 g / L of urea, 0.4-1.0 g / L of CaSO4·2H2O, 5-8 g / L of MgSO4·7H2O, 20-30 g / L of KOH, 20-30 ml / L of H3PO4, 2-8 ml / L of PTM1 solution, and 1-3 ml / L of defoaming agent.

4. The high-density fermentation process of Pichia methanolica according to claim 3, characterized in that: The composition of the PTM1 solution is: CuSO4·5H2O 6g / L, KI 0.08g / L, MnSO4·H2O 3g / L, Na2MoO4·2H2O 0.2g / L, H3BO3 0.02g / L, ZnSO4·7H2O 20g / L, FeSO4·7H2O 65g / L, CoCl2·6H2O 0.5g / L, H2SO4 5ml / L, and Biotin 0.2g / L.

5. The high-density fermentation process of Pichia methanolica according to claim 1, wherein: The methanol induction stage (3): the methanol solution is prepared by dissolving 8-14 mL of PTM1 in 1 L of methanol solution.

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