Continuous fermentation process of pichia pastoris

Abstract

The invention provides a continuous fermentation process suitable for pichia pastoris, and belongs to the technical field of bioengineering. According to the method, the overall salt concentration in a first-stage seed tank culture medium and a fermentation tank culture medium is reduced, and the pichia pastoris production speed is increased, so that the culture time in a seed tank and the time used before induction of a fermentation tank in the process of producing exogenous products through pichia pastoris fermentation are shortened, and the fermentation production period is effectively shortened; according to the continuous fermentation process disclosed by the invention, through continuous induction culture, the yield of a single batch is greatly improved, the productivity is greatly improved, and huge economic benefits are brought to large-scale production; according to the continuous fermentation process, manpower and energy required under the same yield are reduced, excessive operation is reduced, the production cost can be reduced, the bacterial contamination risk can be reduced, and the continuous fermentation process is suitable for large-scale production of exogenous products through pichia pastoris continuous fermentation and has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a continuous fermentation process suitable for Pichia pastoris. Background Art

[0002] As an efficient expression system, Pichia pastoris has significant advantages in producing exogenous products: (1) Pichia pastoris has a high expression level and can be expressed both intracellularly and secretedly, which can better meet the needs of large-scale production; (2) Pichia pastoris has a unique alcohol oxidase gene (AOX) promoter that can be strictly regulated by methanol. This precise regulatory mechanism is conducive to controlling the timing and yield of protein expression; (3) Pichia pastoris is a safe microorganism. When producing biological products, there is no need to worry about the introduction of harmful impurities that may harm human health, thereby improving the safety and quality of the product; (4) Pichia pastoris engineered bacteria have good genetic stability. During multiple subcultures and fermentations, exogenous genes can stably exist and continuously express, ensuring the stability of product quality and yield during the production process.

[0003] Although the Pichia pastoris expression system has the above advantages, it still has some disadvantages, such as: the Pichia pastoris expression system relies on methanol as an inducer to start the expression of exogenous genes, but methanol is toxic, and the precise control of its concentration is crucial for cell growth and product expression; in addition to carbon sources, Pichia pastoris has strict requirements on the types and concentrations of nutrients such as nitrogen sources, phosphorus sources, trace elements and vitamins; Pichia pastoris is sensitive to environmental factors such as temperature, pH value, dissolved oxygen content, etc. during the fermentation process, and requires professional fermentation equipment and advanced fermentation technology to accurately control these parameters; at the same time, the raw material cost of Pichia pastoris is high and the fermentation cycle is long during large-scale production. These increase the investment in Pichia pastoris fermentation equipment and the production and operation costs.

[0004] High-density fermentation of Pichia pastoris is of great significance in the field of bioengineering. It can improve production and reduce costs through strain selection, culture medium optimization, fermentation process control, and product separation and purification. In the prior art, there are many fermentation processes for Pichia pastoris to produce exogenous products, which can be roughly divided into four parts: production preparation, shake flask seed liquid culture, multi-stage seed culture, and fermentation culture. However, high-density fermentation of Pichia pastoris has the disadvantages of high cost and long cycle, especially for large-scale production, such as: (1) Production preparation work, such as material weighing, preparation and sterilization, and cleaning, disinfection and sterilization of fermentation equipment and pipelines, these operations consume a lot of manpower and energy; (2) The seed culture stage of Pichia pastoris is time-consuming, resulting in low production capacity and high cost; (3) Basic culture in fermentation tanks requires a long time to reach sufficient cell density, which is time-consuming; (4) There is a long methanol adaptation period at the beginning of induction culture. During this stage, the growth and metabolic rate of cells is greatly slowed down, and some cells may even die, prolonging the entire fermentation cycle; (5) From shake flask culture, multi-stage seed culture, fermentation growth culture, to fermentation induction culture, there are many processes and the risk of contamination is high. For large-scale production, contamination will bring huge economic losses.

[0005] Therefore, it is necessary to develop a continuous fermentation process suitable for Pichia pastoris that is suitable for large-scale production, shortens the production cycle and can reduce costs. Summary of the invention

[0006] In view of some deficiencies in the prior art, the present invention provides a continuous fermentation process suitable for Pichia pastoris; the present invention accelerates the production speed of Pichia pastoris by reducing the overall salt concentration in the primary seed tank culture medium and the fermentation tank culture medium, thereby reducing the culture time in the seed tank and the time before the fermentation tank induction during the Pichia pastoris fermentation to produce exogenous products, and effectively shortens the fermentation production cycle; the continuous fermentation process of the present invention greatly improves the output of a single batch through continuous induction culture, greatly improves the production capacity, and brings huge economic benefits to large-scale production; the continuous fermentation process of the present invention reduces the manpower and energy required at the same output, reduces excessive operations, can reduce both production costs and the risk of contamination, is suitable for large-scale continuous fermentation of Pichia pastoris to produce exogenous products, and has good practicality.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0008] The present invention provides a continuous fermentation process applicable to Pichia pastoris, the continuous fermentation process comprising:

[0009] (1) Shake flask seed culture:

[0010] Inoculating the fermentation bacteria species Pichia pastoris into a shake flask seed culture medium, culturing the culture flask, and obtaining a seed solution after the culture is completed;

[0011] (2) Primary seed tank culture:

[0012] The seed liquid is inoculated into the first-level seed tank culture medium for cultivation. When the basic glycerol in the first-level seed tank culture medium is consumed and the DO rebounds quickly, the cultivation is terminated to obtain the first-level seed tank fermentation liquid;

[0013] (3) Basic fermentation culture:

[0014] Transfer the fermentation liquid from the first-stage seed tank to the fermentation tank, and when the basic glycerol in the fermentation tank is consumed and the DO rebounds quickly, the cultivation is terminated;

[0015] (4) Growth and cultivation:

[0016] Add feed medium to the fermenter, and then feed growth medium according to the DO positive regulation feed strategy. When the carbon source in the tank is consumed, DO rebounds quickly and the growth culture ends;

[0017] (5) Induction culture:

[0018] The DO positive control feeding strategy is adopted to feed the induction medium to the fermentation tank after the growth culture is completed, and the induction culture is carried out after the feeding is completed until the growth of Pichia pastoris and the expression of exogenous protein enter the plateau phase to obtain the fermentation liquid;

[0019] (6) Continuous induction culture:

[0020] Discharging part of the fermentation liquid in the fermentation tank, adding continuous culture medium into the fermentation tank, and continuing induction culture until bacterial growth and exogenous protein expression enter a plateau phase;

[0021] (7) Repeat the continuous induction culture steps multiple times until the cell density and protein expression level show a significant decrease and the viability of the Pichia pastoris cells decreases, and the fermentation is terminated.

[0022] Preferably, in step (1), the shake flask seed culture medium is sterilized and the shake flask seed culture medium is Pichia pastoris YPD shake flask seed culture medium.

[0023] Preferably, in step (1), the shaking flask culture conditions are: culturing at 30.0±1.0°C and 220±10 rpm until the OD600 value of the fermentation bacteria Pichia pastoris in the seed liquid reaches 2-8.

[0024] Preferably, in step (2), before inoculating the seed solution, the rotation speed of the primary seed tank culture medium is adjusted to 300 rpm, the ventilation volume is 12 L / min, and the temperature is 30° C., and the pH of the primary seed tank culture medium is controlled to 5.0 with concentrated ammonia water.

[0025] Preferably, in step (2), the inoculation amount of the seed solution is 10%.

[0026] Preferably, in step (2), the composition of the primary seed tank culture medium is: 85wt% H3PO4 13.35mL / L, CaSO4·2H2O 0.465g / L, K2SO4 9.1g / L, MgSO4·7H2O 7.45g / L, KOH 2.065g / L, glycerol 20g / L, PTM1 solution 2mL / L, and purified water to volume;

[0027] After the basic glycerol is consumed, DO rebounds quickly, OD 600 It is 30±5.

[0028] Preferably, in step (3), a sterilized fermentation tank medium is added to the fermentation tank, and the components of the fermentation tank medium include:

[0029] 85wt% H3PO4 17.8mL / L, CaSO4·2H2O 0.62g / L, K2SO4 12.13g / L, MgSO4·7H2O9.33g / L, KOH 0.53g / L, glycerol 26.67g / L, PTM1 solution 2.67mL / L, purified water to volume;

[0030] Before inoculation, the speed in the fermenter was adjusted to 200 rpm, the ventilation volume to 90 L / min, and the temperature to 30 °C;

[0031] After the basic glycerol is consumed, DO rebounds quickly, OD 600 It is 40±5.

[0032] Preferably, in step (4), before adding the feed medium, the ventilation volume in the fermenter is controlled to be 120 L / min, the temperature is 30° C., and the fermentation pH value is controlled to be 5.0.

[0033] Preferably, in step (4), the requirement for adding the feed medium is: adding the feed medium until the components in the fermentation tank are restored to the same salt concentration as the inorganic salt in the conventional BSM medium;

[0034] The ingredients of the feed medium are:

[0035] 85wt% H3PO4 53.4mL / L, CaSO4·2H2O 1.86g / L, K2SO4 36.4g / L, MgSO4·7H2O29.8g / L, KOH 1.59g / L, and make up to volume with purified water.

[0036] Preferably, in step (4), the DO positive regulation feeding strategy is: when DO ≥ 30%, start feeding the growth medium, and when DO < 30%, stop feeding the growth medium;

[0037] After the carbon source is consumed, DO rebounds quickly and OD 600 It is 120±10.

[0038] Preferably, the composition of the growth medium is: 50% glycerol (mass to volume ratio), and 12 mL of PTM1 is added per liter of glycerol.

[0039] Preferably, in step (5), the DO positive regulation feeding strategy is: when DO ≥ 20%, start feeding the induction medium, and when DO < 20%, stop feeding the induction medium;

[0040] The components of the induction medium include: methanol, with 12 mL of PTM1 added per liter of methanol;

[0041] The induction culture conditions are: temperature 30° C., pH 5.0.

[0042] Preferably, in step (6), part of the fermentation liquid in the fermenter is released, and continuous culture medium is added to the fermenter, so that the OD600 of the fermentation liquid after the continuous culture medium is added is 120±10; and the total volume of the fermentation liquid after the continuous culture medium is added is 65-75% of the total volume of the fermenter;

[0043] The components of the continuous culture medium are:

[0044] 85% H3PO4 26.7mL / L, CaSO4·2H2O 0.93g / L, K2SO4 18.2g / L, MgSO4·7H2O 14.9g / L, KOH 4.13g / L, make up to volume with purified water.

[0045] Preferably, in step (7), the continuous induction culture step is repeated multiple times until the cell density and protein expression level of the fermentation bacteria Pichia pastoris are 10% or more lower than the detection results during the induction fermentation in step (5). At this time, the viability of the Pichia pastoris cells decreases and the fermentation is terminated.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] (1) The present invention reduces the overall salt concentration in the primary seed tank culture medium and the fermentation tank culture medium to accelerate the production speed of Pichia pastoris, thereby reducing the culture time in the seed tank and the time before induction in the fermentation tank during the Pichia pastoris fermentation process for producing exogenous products, which is shortened by nearly 8 hours compared with the existing process, effectively shortening the fermentation production cycle.

[0048] (2) The continuous fermentation process of the present invention greatly improves the yield of a single batch through continuous induction culture, greatly improving the production capacity. Based on a 100L fermentation scale and full-load production within one year, the yield of the continuous fermentation process can reach 68.64kg, while the yield of the conventional process is only 47.34kg, an increase of nearly 45%, which brings huge economic benefits to large-scale production.

[0049] (3) The continuous fermentation process of the present invention reduces the manpower and energy required for the same output and reduces excessive operations, which can reduce both production costs and the risk of contamination. It is suitable for large-scale continuous fermentation of Pichia pastoris to produce exogenous products and has good practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a flow chart of the 100L continuous fermentation process of the present invention.

[0051] Figure 2 This is the SDS-PAGE protein gel image of the fermentation broth after continuous induction fermentation for different times.

[0052] Figure 3 This is the SDS-PAGE protein gel image of the fermentation broth after different times of conventional fermentation induction. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. The technical scheme of the embodiment of the present invention is clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0054] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0055] In the following examples, a 100L fermentation tank was used as an example to carry out continuous fermentation of Pichia pastoris. The culture medium used in the fermentation process was as follows:

[0056] Pichia pastoris YPD shake flask seed culture medium: yeast powder 10g / L, peptone 20g / L, glucose 10g / L.

[0057] Primary seed tank culture medium: 85wt% H3PO4 13.35mL / L, CaSO4·2H2O 0.465g / L, K2SO4 9.1g / L, MgSO4·7H2O 7.45g / L, KOH 2.065g / L, glycerol 20g / L, PTM1 solution 2mL / L, and purified water to volume.

[0058] Fermentation tank culture medium: 85wt% H3PO4 17.8mL / L, CaSO4·2H2O 0.62g / L, K2SO4 12.13g / L, MgSO4·7H2O 9.33g / L, KOH 0.53g / L, glycerol 26.67g / L, PTM1 solution 2.67mL / L, and purified water to volume.

[0059] PTM1: CuSO4·5H2O 6.0g / L, NaI 0.08g / L, MnSO4·H2O 3.0g / L, NaMoO4·2H2O0.2g / L, H3BO3 0.02g / L, CoCl2 0.5g / L, ZnCl2 20.0g / L, FeSO4·7H2O 65.0g / L, biotin 0.2g / L, H2SO4 5.0mL / L, sterilize by filtration with 0.22μm filter membrane and store at 4℃.

[0060] Feed medium: 85wt% H3PO4 53.4mL / L, CaSO4·2H2O 1.86g / L, K2SO4 36.4g / L, MgSO4·7H2O 29.8g / L, KOH 1.59g / L, made up to volume with purified water.

[0061] Growth medium: 50% glycerol (mass-to-volume ratio), 12 mL of PTM1 was added per liter of glycerol.

[0062] Induction medium: methanol, add 12 mL of PTM1 per liter of methanol.

[0063] Continuous culture medium: 85% H3PO4 26.7mL / L, CaSO4·2H2O 0.93g / L, K2SO4 18.2g / L, MgSO4·7H2O14.9g / L, KOH 4.13g / L, made up to volume with purified water.

[0064] The following examples illustrate the continuous fermentation process of Pichia pastoris of the present invention for producing and expressing collagen using Pichia pastoris stored in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 24688 and a host strain of GS115. However, the scope of protection of the present invention is not limited thereto, and other Pichia pastoris expressing other exogenous products are also applicable to the continuous fermentation process of the present invention.

[0065] Protein concentration (g / L) = 0.144 (A215-A225) (A215 and A225 are the absorbance values ​​of the protein solution measured at 215 nm and 225 nm, respectively).

[0066] Embodiment 1:

[0067] This embodiment takes a 100L fermentation tank as an example to carry out continuous fermentation of Pichia yeast. The fermentation process is as follows: Figure 1 As shown, the specific steps are as follows:

[0068] (1) Shake flask seed culture:

[0069] In a 2L shake flask, 600mL of Pichia pastoris YPD shake flask seed culture medium was sterilized, and after cooling, 600μL of the fermentation strain Pichia pastoris (accession number CGMCC No.24688) was transferred from the frozen glycerol tube, and cultured at a temperature of 30.0±1.0℃ and a shaker speed of 220±10rpm for about 24 hours. At this time, no foreign bacteria were found under microscopic examination, and the OD600 value of the Pichia pastoris liquid reached 2-8, thus obtaining the seed liquid.

[0070] (2) Primary seed tank culture:

[0071] Sterilize 6L of the first-stage seed tank culture medium in a 10L stainless steel tank. Before inoculation, adjust the speed in the tank to 300rpm, the ventilation volume to 12L / min, and the temperature to 30°C. Then adjust the pH value in the tank to 5.0 with concentrated ammonia water. 600 The value is 2-8, and the seed liquid is added into the tank at 10% (fire ring inoculation). After the inoculation is completed, click the DO electrode to start fermentation until the basic glycerol in the culture medium of the first seed tank is consumed and DO rebounds quickly. At this time, OD 600 The temperature is 30±5, and the time is 17±3h to obtain the first-level seed tank fermentation liquid.

[0072] (3) Basic fermentation culture:

[0073] Sterilize 45L of fermentation medium in a 100L fermentation tank. Adjust the speed in the tank to 200rpm, ventilation volume to 90L / min, and temperature to 30℃ before inoculation. Then adjust the pH value in the tank to 5.0 with concentrated ammonia. Transfer the fermentation liquid from the first-stage seed tank to the fermentation tank, click the DO electrode to 100 and start fermentation until the basic glycerol in the fermentation medium is consumed and the fermentation stops. At this time, DO rebounds quickly and OD 600 It is 40±5 and takes 10±2h.

[0074] (4) Growth and cultivation:

[0075] The ventilation volume in the fermenter was controlled to be 120L / min, the temperature to be 30℃, and the pH value to be 5.0. Then, 15L of feed medium was added to the fermenter at one time. Then, the growth medium was added according to the DO positive control feeding strategy, that is, when DO ≥ 30%, the growth medium was added, and when DO < 30%, the growth medium was stopped. In this step, the feed was stopped after 3.4L of growth medium was added, and then the growth culture was stopped after the carbon source in the fermenter was consumed. At this time, DO quickly rebounded to more than 60%, and OD 600 It is 120±10 and takes 5h.

[0076] (5) Induction culture:

[0077] The temperature in the fermenter was controlled at 30°C and the pH value was 5.0. Then, the DO positive control feeding strategy was adopted to feed the induction medium. That is, feeding was started when DO ≥ 20%, and feeding was stopped when DO < 20%. The induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase. In this step, after 72 hours of induction culture, the expression of Pichia pastoris and collagen in the fermenter entered the plateau phase, and the fermentation liquid 1 was obtained. At this time, the volume of the fermentation liquid 1 was 70L. The OD was detected. 600 =280, wet weight of bacteria =420g / L, collagen UV quantification =10.9g / L.

[0078] (6) Continuous induction culture 1

[0079] 40 L of fermentation liquid 1 in the fermenter was discharged. At this time, 30 L of fermentation liquid 1 was contained in the tank. 40 L of continuous culture medium was added thereto, and then the induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase.

[0080] In this step, after 24 hours of induction culture, the expression of Pichia yeast and collagen in the fermenter entered a plateau phase, and fermentation broth 2 was obtained. At this time, the volume of fermentation broth 2 in the fermenter was 75 L, and the OD was detected. 600 =265, wet weight of bacteria =313g / L, collagen UV quantification =9.8g / L.

[0081] (7) Continuous induction culture 2:

[0082] 43 L of fermentation liquid 2 in the fermenter was discharged. At this time, there were 32 L of fermentation liquid 2 in the tank. 38 L of continuous culture medium was added thereto, and then the induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase.

[0083] In this step, after 24 hours of induction culture, the expression of Pichia yeast and collagen in the fermenter entered a plateau phase, and fermentation broth 3 was obtained. At this time, the volume of fermentation broth 3 in the fermenter was 75 L, and the OD was detected. 600=215, wet weight of bacteria =247g / L, collagen UV quantification =6.7g / L.

[0084] At this time, the cell density and collagen expression of the continuously induced cultured Pichia pastoris fermentation bacteria decreased by more than 10%, which was a significant decrease. This indicated that the activity of the Pichia pastoris cells decreased and the fermentation was terminated.

[0085] Samples were taken to detect the protein expression in the fermentation broth after 48h and 72h of induction culture, 24h of fermentation in continuous induction culture 1, and 24h of fermentation in continuous induction culture 2. The test results are as follows: Figure 2 As shown in the figure, it can be seen that the collagen expression level in continuous induction culture 1 after 24h fermentation is similar to that in 72h induction culture, and the protein expression level in continuous induction culture 2 has decreased significantly. In the continuous induction culture stage 1, the yeast cell growth and expression are normal, and in the continuous induction culture stage 2, the yeast cell growth and expression are all decreased, and continuous culture cannot be continued, so the entire fermentation cycle is terminated.

[0086] In summary, the entire continuous fermentation cycle from shake flask seed liquid culture to the end of fermentation in this example took a total of 176 hours, and 160 L of fermentation liquid was obtained. According to the quantitative calculation of V, 1379.5 g of collagen was obtained.

[0087] Embodiment 2:

[0088] (1) Shake flask seed culture:

[0089] In a 2L shake flask, 600mL of Pichia pastoris YPD shake flask seed culture medium was sterilized, and after cooling, 600μL of the fermentation strain Pichia pastoris (accession number CGMCC No.24688) was transferred from the frozen glycerol tube, and cultured at a temperature of 30.0±1.0℃ and a shaker speed of 220±10rpm for about 24 hours. At this time, no foreign bacteria were found under microscopic examination, and the OD600 value of the Pichia pastoris liquid reached 2-8, thus obtaining the seed liquid.

[0090] (2) Primary seed tank culture:

[0091] Sterilize 6L of the first-stage seed tank culture medium in a 10L stainless steel tank. Before inoculation, adjust the speed in the tank to 300rpm, the ventilation volume to 12L / min, and the temperature to 30°C. Then adjust the pH value in the tank to 5.0 with concentrated ammonia water. 600 The value is 2-8, and the seed liquid is added into the tank at 10% (fire ring inoculation). After the inoculation is completed, click the DO electrode to start fermentation until the basic glycerol in the culture medium of the first seed tank is consumed and DO rebounds quickly. At this time, OD 600 The temperature is 30±5, and the time is 17±3h to obtain the first-level seed tank fermentation liquid.

[0092] (3) Basic fermentation culture:

[0093] Sterilize 45L of fermentation medium in a 100L fermentation tank. Adjust the speed in the tank to 200rpm, ventilation volume to 90L / min, and temperature to 30℃ before inoculation. Then adjust the pH value in the tank to 5.0 with concentrated ammonia. Transfer the fermentation liquid from the first-stage seed tank to the fermentation tank, click the DO electrode to 100 and start fermentation until the basic glycerol in the fermentation medium is consumed and the fermentation stops. At this time, DO rebounds quickly and OD 600 It is 40±5 and takes 10±2h.

[0094] (4) Growth and cultivation:

[0095] The ventilation volume in the fermenter was controlled to be 120L / min, the temperature to be 30℃, and the pH value to be 5.0. Then, 15L of feed medium was added to the fermenter at one time. Then, the growth medium was added according to the DO positive control feeding strategy, that is, when DO ≥ 30%, the growth medium was added, and when DO < 30%, the growth medium was stopped. In this step, the feed was stopped after 3.4L of growth medium was added, and then the growth culture was stopped after the carbon source in the fermenter was consumed. At this time, DO quickly rebounded to more than 60%, and OD 600 It is 120±10 and takes 5h.

[0096] (5) Induction culture:

[0097] The temperature in the fermenter was controlled at 30°C and the pH value was 5.0. Then, the DO positive control feeding strategy was adopted to feed the induction medium. That is, feeding was started when DO ≥ 20%, and feeding was stopped when DO < 20%. The induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase. In this step, after 72 hours of induction culture, the expression of Pichia pastoris and collagen in the fermenter entered the plateau phase, and the fermentation liquid 1 was obtained. At this time, the volume of the fermentation liquid 1 was 70L. The OD was detected. 600 =281, wet weight of bacteria =423g / L, collagen UV quantification =11.0g / L.

[0098] (6) Continuous induction culture 1

[0099] 40 L of fermentation liquid 1 in the fermenter was discharged. At this time, 30 L of fermentation liquid 1 was contained in the tank. 40 L of continuous culture medium was added thereto, and then the induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase.

[0100] In this step, after 24 hours of induction culture, the expression of Pichia pastoris and collagen in the fermenter entered a plateau phase, and fermentation broth 2 was obtained. At this time, the volume of fermentation broth 2 in the fermenter was 75 L, and the OD 600 =264, wet weight of bacteria =310g / L, collagen UV quantification =9.7g / L.

[0101] (7) Continuous induction culture 2:

[0102] 43 L of fermentation liquid 2 in the fermenter was discharged. At this time, there were 32 L of fermentation liquid 2 in the tank. 38 L of continuous culture medium was added thereto, and then the induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase.

[0103] In this step, after 24 hours of induction culture, the expression of Pichia pastoris and collagen in the fermenter entered a plateau phase, and fermentation liquid 3 was obtained. At this time, the volume of fermentation liquid 3 in the fermenter was 75 L. The OD 600 =214, wet weight of bacteria =245g / L, collagen UV quantification =6.6g / L.

[0104] At this time, the cell density and collagen expression of Pichia pastoris fermentation bacteria decreased significantly, indicating that the activity of Pichia pastoris cells decreased and continuous induction culture was continued.

[0105] (8) Continuous induction culture 3:

[0106] 36 L of fermentation liquid 3 in the fermenter was discharged. At this time, there were 39 L of fermentation liquid 3 in the tank. 31 L of continuous culture medium was added thereto, and then the induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase.

[0107] In this step, after 16 hours of induction culture, the expression of Pichia yeast and collagen in the fermenter entered a plateau phase, and fermentation liquid 4 was obtained. At this time, the volume of fermentation liquid 4 in the fermenter was 73 L. The OD 600 =165, wet weight of bacteria =198g / L, collagen UV quantification =4.2g / L.

[0108] This example shows that after the cell density and collagen expression of the Pichia yeast in the continuous culture show a significant decrease, the cell concentration and collagen expression of the Pichia yeast in the continuous induction culture show a significant decrease, and the collagen expression almost stops increasing. Therefore, during the continuous induction culture process, the fermentation can be stopped when the yeast cell growth expression shows a significant decrease of more than 10% for the first time.

[0109] Embodiment 3:

[0110] (1) Shake flask seed culture:

[0111] In a 2L shake flask, 600mL of Pichia pastoris YPD shake flask seed culture medium was sterilized, and after cooling, 600μL of the fermentation strain Pichia pastoris (accession number CGMCC No.24688) was transferred from the frozen glycerol tube, and cultured at a temperature of 30.0±1.0℃ and a shaker speed of 220±10rpm for about 24 hours. At this time, no foreign bacteria were found under microscopic examination, and the OD600 value of the Pichia pastoris liquid reached 2-8, thus obtaining the seed liquid.

[0112] (2) Primary seed tank culture:

[0113] Sterilize 6L of the first-stage seed tank culture medium in a 10L stainless steel tank. Before inoculation, adjust the speed in the tank to 300rpm, the ventilation volume to 12L / min, and the temperature to 30°C. Then adjust the pH value in the tank to 5.0 with concentrated ammonia water. 600 The value is 2-8, and the seed liquid is added into the tank at 10% (fire ring inoculation). After the inoculation is completed, click the DO electrode to start fermentation until the basic glycerol in the culture medium of the first seed tank is consumed and DO rebounds quickly. At this time, OD 600 The temperature is 30±5, and the time is 17±3h to obtain the first-level seed tank fermentation liquid.

[0114] (3) Basic fermentation culture:

[0115] Sterilize 45L of fermentation medium in a 100L fermentation tank. Adjust the speed in the tank to 200rpm, ventilation volume to 90L / min, and temperature to 30℃ before inoculation. Then adjust the pH value in the tank to 5.0 with concentrated ammonia. Transfer the fermentation liquid from the first-stage seed tank to the fermentation tank, click the DO electrode to 100 and start fermentation until the basic glycerol in the fermentation medium is consumed and the fermentation stops. At this time, DO rebounds quickly and OD 600 It is 40±5 and takes 10±2h.

[0116] (4) Growth and cultivation:

[0117] The ventilation volume in the fermenter was controlled to be 120L / min, the temperature to be 30℃, and the pH value to be 5.0. Then, 15L of feed medium was added to the fermenter at one time. Then, the growth medium was added according to the DO positive control feeding strategy, that is, when DO ≥ 30%, the growth medium was added, and when DO < 30%, the growth medium was stopped. In this step, the feed was stopped after 3.4L of growth medium was added, and then the growth culture was stopped after the carbon source in the fermenter was consumed. At this time, DO quickly rebounded to more than 60%, and OD 600 It is 120±10 and takes 5 hours.

[0118] (5) Induction culture:

[0119] The temperature in the fermenter was controlled at 30°C and the pH value was 5.0. Then, the DO positive control feeding strategy was adopted to feed the induction medium. That is, feeding was started when DO ≥ 20%, and feeding was stopped when DO < 20%. The induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase. In this step, after 72 hours of induction culture, the expression of Pichia pastoris and collagen in the fermenter entered the plateau phase, and the fermentation liquid 1 was obtained. At this time, the volume of the fermentation liquid 1 was 70L. The OD was detected. 600 =279, wet weight of bacteria =417g / L, collagen UV quantification =10.8g / L.

[0120] (6) Continuous induction culture 1

[0121] 40 L of fermentation liquid 1 in the fermenter was discharged. At this time, 30 L of fermentation liquid 1 was contained in the tank. 40 L of continuous culture medium was added thereto, and then the induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase.

[0122] In this step, after 24 hours of induction culture, the expression of Pichia pastoris and collagen in the fermenter entered a plateau phase, and fermentation broth 2 was obtained. At this time, the volume of fermentation broth 2 in the fermenter was 75 L, and the OD 600 =266, wet weight of bacteria =317g / L, collagen UV quantification =9.8g / L.

[0123] (7) Continuous induction culture 2:

[0124] 43 L of fermentation liquid 2 in the fermenter was discharged. At this time, there were 32 L of fermentation liquid 2 in the tank. 38 L of continuous culture medium was added thereto, and then the induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase.

[0125] In this step, after 24 hours of induction culture, the expression of Pichia pastoris and collagen in the fermenter entered a plateau phase, and fermentation liquid 3 was obtained. At this time, the volume of fermentation liquid 3 in the fermenter was 75 L. The OD 600 =212, wet weight of bacteria =245g / L, collagen UV quantification =6.6g / L. Continue induction culture for 28h and take samples for testing, OD 600 =211, wet weight of bacteria = 242g / L, collagen UV quantification = 6.7g / L. Continue induction culture for 32h and take samples for testing, OD 600 =205, wet weight of bacteria =240g / L, UV quantitative collagen =6.4g / L. The cell density and collagen expression of Pichia pastoris fermentation bacteria showed a downward trend, and the fermentation was terminated.

[0126] This example shows that if the Pichia yeast continues to be induced and cultured after entering the stationary phase, it will enter a decline phase, and both the cell density and the collagen expression level will show a downward trend. Therefore, in a single induction culture process, when the bacterial growth and the exogenous protein expression enter the stationary phase, the next step of processing can be carried out.

[0127] Comparative Example 1:

[0128] This comparative example is compared with the conventional fermentation process of Pichia pastoris. In the fermentation process, the primary seed tank culture medium and the fermentation tank culture medium are BSM culture medium, and its components include: 85% H3PO4 26.7mL / L, CaSO4·2H2O 0.93g / L, K2SO418.2g / L, MgSO4·7H2O 14.9g / L, KOH 4.13g / L, glycerol 20g / L and PTM1 solution 4mL / L; and the growth culture step in the fermentation process is different from the continuous fermentation process of the present invention, and there is no continuous induction culture step. The specific steps are as follows:

[0129] (1) Shake flask seed culture:

[0130] Place 600 mL of Pichia YPD shake flask seed culture medium in a 2 L shake flask, sterilize it, and after cooling, transfer 600 μL of the fermentation strain Pichia from the frozen glycerol tube, and culture it at a temperature of 30.0±1.0°C and a shaker speed of 220±10 rpm for about 24 hours. At this time, no foreign bacteria are found under microscopic examination, and the OD600 value of the Pichia culture liquid reaches 2 to 8, thereby obtaining the seed liquid.

[0131] (2) Primary seed tank culture:

[0132] Sterilize 6L of BSM medium in a 10L stainless steel tank. Before inoculation, adjust the speed in the tank to 300rpm, the ventilation volume to 12L / min, and the temperature to 30°C. Then adjust the pH value in the tank to 5.0 with concentrated ammonia water. 600 The value is 2-8, and the seed liquid is added into the tank at 10% (fire ring inoculation). After the inoculation is completed, click the DO electrode to start fermentation until the basic glycerol in the culture medium of the first seed tank is consumed and DO rebounds quickly. At this time, OD 600 The temperature is 30±5, and the time is 20±3h to obtain the first-level seed tank fermentation liquid.

[0133] (3) Basic fermentation culture:

[0134] Sterilize 60L of BSM medium in a 100L fermenter. Adjust the speed in the tank to 200rpm, ventilation volume to 120L / min, and temperature to 30℃ before inoculation. Then adjust the pH value in the tank to 5.0 with concentrated ammonia. Transfer the fermentation liquid from the first-stage seed tank to the fermenter, click the DO electrode to 100 and start fermentation until the basic glycerol in the fermentation medium is consumed and the fermentation stops. At this time, DO rebounds quickly and OD 600 The time is 30±5 and the time taken is 15±2h.

[0135] (4) Growth and cultivation:

[0136] The ventilation volume in the fermenter was controlled at 120 L / min, the temperature was 30 °C, and the pH value was 5.0. The growth medium was added according to the DO positive control feeding strategy, that is, when DO ≥ 30%, the growth medium was started to be added, and when DO < 30%, the growth medium was stopped. In this step, the feeding was stopped after 3.4 L of growth medium was added, and then the growth culture was stopped after the carbon source in the fermenter was consumed. At this time, DO quickly rebounded to more than 60%, and OD 600 It is 120±10 and takes 5h.

[0137] (5) Induction culture:

[0138] The temperature in the fermenter was controlled at 30°C and the pH value was 5.0. Then, the DO positive control feeding strategy was adopted to feed the induction medium. That is, feeding was started when DO ≥ 20%, and feeding was stopped when DO < 20%. The induction culture was continued until the bacterial growth and the expression of the exogenous protein entered the plateau phase. In this step, after 72 hours of induction culture, the expression of Pichia pastoris and collagen in the fermenter entered the plateau phase, and the fermentation broth was obtained. At this time, the volume of the fermentation broth was 70L. The OD was detected. 600 =275, wet weight of bacteria =404g / L, collagen UV quantification =10.5g / L.

[0139] The fermentation broths of the sample comparison induction culture stage at 48h and 72h were taken respectively, and the expression of collagen was compared with the fermentation broth 1 of the continuous fermentation process of the present invention at 72h. The comparison results are as follows: Figure 3 As shown in the figure, the expression and purity of the exogenous protein in the comparative fermentation for 72 hours were similar to those in the continuous fermentation for 72 hours. It can be seen that reducing the overall salt concentration in the primary seed tank culture medium and the fermentation tank basal culture medium and shortening the fermentation cycle had no effect on the expression and purity of the exogenous protein.

[0140] The entire production cycle from shaking the bottle to putting it in the tank took 136 hours, and a total of 70L of fermentation liquid was obtained. According to UV quantitative calculation, 735g of collagen was obtained. It can be seen that under the premise of producing the same amount of collagen at a fermentation scale of 100L, the continuous fermentation production cycle is nearly 31% shorter than that of conventional fermentation. The larger the production scale, the more obvious the advantages of this process in reducing production costs and shortening production cycles.

[0141] In summary, the present invention reduces the salt concentration in the culture medium and accelerates the production speed of Pichia pastoris, thereby reducing the culture time in the seed tank and the time before induction in the fermenter during the fermentation of Pichia pastoris to produce exogenous products, and effectively shortens the fermentation production cycle; the continuous fermentation process of the present invention greatly improves the output of a single batch through continuous induction culture, greatly improves the production capacity, and brings huge economic benefits to large-scale production; the continuous fermentation process of the present invention reduces the manpower and energy required at the same output, reduces excessive operations, can reduce both production costs and the risk of contamination, is suitable for large-scale continuous fermentation of Pichia pastoris to produce exogenous products, and has good practicality.

[0142] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A continuous fermentation process suitable for Pichia pastoris, characterized in that: The continuous fermentation process comprises: (1) Shake flask seed culture: Inoculating the fermentation bacteria species Pichia pastoris into a shake flask seed culture medium, culturing the culture flask, and obtaining a seed solution after the culture is completed; (2) Primary seed tank culture: The seed liquid is inoculated into the first-level seed tank culture medium for cultivation. When the basic glycerol in the first-level seed tank culture medium is consumed and the DO rebounds quickly, the cultivation is terminated to obtain the first-level seed tank fermentation liquid; (3) Basic fermentation culture: Transfer the fermentation liquid from the first-stage seed tank to the fermentation tank, and when the basic glycerol in the fermentation tank is consumed and the DO rebounds quickly, the cultivation is terminated; (4) Growth and cultivation: Add feed medium to the fermenter, and then feed growth medium according to the DO positive regulation feed strategy. When the carbon source in the tank is consumed, DO rebounds quickly and the growth culture ends; (5) Induction culture: The DO positive control feeding strategy is adopted to feed the induction medium to the fermentation tank after the growth culture is completed, and the induction culture is carried out after the feeding is completed until the growth of Pichia pastoris and the expression of exogenous protein enter the plateau phase to obtain the fermentation liquid; (6) Continuous induction culture: Discharging part of the fermentation liquid in the fermentation tank, adding continuous culture medium into the fermentation tank, and continuing induction culture until bacterial growth and exogenous protein expression enter a plateau phase; (7) Repeat the continuous induction culture steps multiple times until the cell density and protein expression level show a significant decrease and the viability of the Pichia pastoris cells decreases, and the fermentation is terminated.

2. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (1), the shake flask seed culture medium is sterilized, and the shake flask seed culture medium is Pichia pastoris YPD shake flask seed culture medium.

3. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (1), the shaking flask culture conditions are: culturing at 30.0±1.0°C and 220±10rpm until the OD600 value of the fermentation bacteria Pichia pastoris in the seed liquid reaches 2-8.

4. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (2), before inoculating the seed solution, the rotation speed of the culture medium in the first-level seed tank is adjusted to 300 rpm, the ventilation volume is 12 L / min, and the temperature is 30° C., and the pH of the culture medium in the first-level seed tank is controlled to 5.0 with concentrated ammonia water.

5. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (2), the inoculation amount of the seed solution is 10%.

6. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (2), the composition of the primary seed tank culture medium is: 85wt% H3PO4 13.35mL / L, CaSO4·2H2O 0.465g / L, K2SO49.1g / L, MgSO4·7H2O 7.45g / L, KOH 2.065g / L, glycerol 20g / L, PTM1 solution 2mL / L, and purified water to volume.

7. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (3), the fermenter is added with a sterilized fermenter medium, and the components of the fermenter medium include: 85wt% H3PO4 17.8mL / L, CaSO4·2H2O 0.62g / L, K2SO4 12.13g / L, MgSO4·7H2O 9.33g / L, KOH 0.53g / L, glycerol 26.67g / L, PTM1 solution 2.67mL / L, purified water to volume; Before inoculation, the speed in the fermenter was adjusted to 200 rpm, the ventilation volume to 90 L / min, and the temperature to 30 °C.

8. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (4), before adding feed medium, the ventilation volume in the fermenter was controlled to be 120 L / min, the temperature to be 30° C., and the fermentation pH value to be 5.

0.

9. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (4), the requirement for adding the feed medium is: adding the feed medium until the components in the fermentation tank are restored to the same salt concentration as the inorganic salt in the conventional BSM medium; The ingredients of the feed medium are: 85wt% H3PO4 53.4mL / L, CaSO4·2H2O 1.86g / L, K2SO4 36.4g / L, MgSO4·7H2O 29.8g / L, KOH 1.59g / L, make up to volume with purified water.

10. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (4), the DO positive regulation feeding strategy is: when DO≥30%, feed addition of growth medium is started, and when DO<30%, feed addition of growth medium is stopped.

11. The continuous fermentation process suitable for Pichia pastoris according to claim 10, characterized in that: The composition of the growth medium is: 50% glycerol (mass to volume ratio), and 12 mL of PTM1 is added per liter of glycerol.

12. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (5), the DO positive regulation feeding strategy is: when DO ≥ 20%, feed addition of induction medium is started, and when DO < 20%, feed addition of induction medium is stopped; The components of the induction medium include: methanol, with 12 mL of PTM1 added per liter of methanol; The induction culture conditions are: temperature 30° C., pH 5.

0.

13. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (6), part of the fermentation liquid in the fermenter is released, and continuous culture medium is added to the fermenter, so that the OD600 of the fermentation liquid after the continuous culture medium is added is 120±10; and the total volume of the fermentation liquid after the continuous culture medium is added is 65-75% of the total volume of the fermenter; The components of the continuous culture medium are: 85% H3PO4 26.7mL / L, CaSO4·2H2O 0.93g / L, K2SO4 18.2g / L, MgSO4·7H2O 14.9g / L, KOH 4.13g / L, make up to volume with purified water.

14. The continuous fermentation process suitable for Pichia pastoris according to claim 1, characterized in that: In step (7), the continuous induction culture step is repeated multiple times until the cell density and protein expression level of the fermentation bacteria Pichia pastoris are 10% or more lower than the detection results during the induction fermentation in step (5). At this time, the activity of the Pichia pastoris cells decreases and the fermentation is terminated.

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