A fermentation culture method for increasing the yield of CRM197 protein

Abstract

The present application relates to a fermentation method for increasing the yield of CRM197 protein, including seed tank culture: adding a bacterial seed solution and a liquid culture medium into a seed tank and mixing them into a bacterial seed culture solution, and culturing at a temperature of 36.5±0.5°C until an OD 600 When the value is greater than 10.0, the bacterial solution in the seed tank is harvested; the dissolved oxygen in the bacterial culture solution is controlled to be greater than 40% during the culture; and, the production tank culture: the bacterial solution in the seed tank and the liquid culture medium are put into the production tank and mixed into the bacterial fermentation liquid, and cultured to OD 600 When the value is >40.0, the bacterial liquid in the production tank is harvested; OD 600 When the value is less than 15.0, the culture temperature is controlled at 27.0-37.5℃, the dissolved oxygen is greater than 40%; OD 600 When the value is ≥15.0, the culture temperature is controlled to be 27.0-34.5°C and the dissolved oxygen is 10-40%. This method achieves the purpose of increasing the yield of CRM197 protein by controlling the temperature and dissolved oxygen content during the fermentation process.

Description

Technical Field

[0001] The present application relates to the technical field of microbial culture, and in particular to a fermentation culture method for increasing the yield of CRM197 protein. Background Art

[0002] Diphtheria toxin (DT) is a protein toxin synthesized and secreted by toxin-producing strains of Corynebacterium diphtheriae. CRM197 protein is a non-toxic form of diphtheria toxin, but it is immunologically indistinguishable from diphtheria toxin. It is produced by Corynebacterium diphtheriae (referred to as CRM197 bacteria in this article) infected with the non-toxigenic bacteriophage β197tox. Since the non-toxic CRM197 protein can still bind to the receptors of sensitive cells, that is, the CRM197 protein retains the immunogenicity of diphtheria toxin while losing its enzymatic activity and toxicity, it is an ideal polysaccharide-conjugated vaccine carrier protein.

[0003] The existing method for preparing CRM197 protein is mainly to purify the culture of CRM197 bacteria by fermentation. Therefore, whether CRM197 bacteria are natural strains or recombinant engineered strains, the cultivation method is an important link restricting the mass production of CRM197 protein. In order to increase the yield of CRM197 protein, the applicant optimized the culture medium used for fermentation. For details, please refer to the Chinese invention patent with application number 201910651534.3.

[0004] However, the yield of CRM197 protein is still at a low level when the strain is fermented according to the above-mentioned culture method. Therefore, it is urgent to develop a more excellent fermentation culture method so that CRM197 bacteria can be amplified on a large scale to obtain a fermentation liquid with high expression level. Summary of the invention

[0005] The present application provides a fermentation culture method for increasing the yield of CRM197 protein, which specifically achieves the purpose of increasing the yield of CRM197 protein by controlling the temperature and dissolved oxygen content during the fermentation process.

[0006] This application adopts the following technical solutions:

[0007] A fermentation method for increasing the yield of CRM197 protein comprises the following steps:

[0008] Seed pot culture:

[0009] The CRM197 bacterial culture liquid and liquid culture medium were added to the seed tank and mixed into the bacterial culture liquid, and cultured at 36.5±0.5℃ until the OD 600 When the value is >10.0, the bacterial liquid in the seed tank is harvested;

[0010] During the culture of the bacterial strain, the dissolved oxygen is controlled to be greater than 40%;

[0011] Production tank culture:

[0012] The bacterial solution in the seed tank and the liquid culture medium are put into the production tank to mix into bacterial fermentation liquid, and cultured to OD 600 When the value is >40.0, the bacterial liquid in the production tank is harvested;

[0013] When OD 600 When the value is less than 15.0, the culture temperature is controlled to be 27.0-37.5°C, and the dissolved oxygen in the fermentation liquid of the strain is greater than 40%;

[0014] When OD 600 When the value is ≥15.0, the culture temperature is controlled to be 27.0-34.5°C, and the dissolved oxygen in the bacterial fermentation liquid is 10-40%.

[0015] Different strains of bacteria will have large differences in dissolved oxygen and temperature during cultivation. The CRM197 bacteria tested in this application are easy to grow and need to be cultivated in a suitable temperature and aerobic environment. To this end, during fermentation culture, this application controls the culture temperature of the seed tank at 36.5°C and the dissolved oxygen at >40%. This is based on the fact that seed tank culture does not need to consider whether the CRM197 bacteria express secretory proteins. Within a certain temperature and oxygen capacity range, the higher the temperature and dissolved oxygen, the faster the fermentation efficiency of the strain, thereby enabling the strain to be rapidly expanded.

[0016] When the present application is cultured in a production tank, the production tank controls the culture temperature at 27.0-37.5°C and the dissolved oxygen at >40% in the early stage, and controls the culture temperature at 27.0-34.5°C and the dissolved oxygen at 10-40% in the later stage. This is because the strain needs to be expanded to a certain amount in the early stage, and then the strain is controlled to express and secrete CRM197 protein in large quantities in the later stage. The inventors have found through research and experiments that when controlling the strain to express secreted proteins, the dissolved oxygen is not the higher the better, nor the lower the better. It is necessary to leave it vacant in different ranges at different growth stages to express CRM197 protein at a higher level. If the dissolved oxygen is too low, it will affect the growth and reproduction rate of the CRM197 strain; if the dissolved oxygen is too high, the CRM197 strain will reproduce in large quantities to produce CRM197 protein, and it will also produce substances that degrade CRM197 protein, thereby reducing the harvest of CRM197 protein. In addition, lowering the culture temperature and controlling different dissolved oxygen at different growth stages can appropriately slow down the reproduction of the strain and promote the strain to grow and express secreted protein, thereby achieving the purpose of higher yield. Therefore, according to the temperature and dissolved oxygen control method of the present application, the CRM197 bacteria can be effectively promoted to reproduce and express and secrete CRM197 protein, thereby increasing the yield of CRM197 protein, and can be better applied to the production process of CRM197 protein.

[0017] Preferably, the method comprises the following steps:

[0018] Seed pot culture:

[0019] The CRM197 bacterial culture liquid and liquid culture medium were added to the seed tank and mixed into the bacterial culture liquid, and cultured at 36.5±0.5℃ until the OD 600 When the value is >10.0, the bacterial liquid in the seed tank is harvested;

[0020] When OD 600 When the value is less than 2.0, the dissolved oxygen in the bacterial culture solution is controlled to be 45-50%;

[0021] When OD 600 When the value is 2.0-10.0, the dissolved oxygen in the bacterial culture solution is controlled to be 55-60%;

[0022] Production tank culture:

[0023] The bacterial solution in the seed tank and the liquid culture medium are put into the production tank to mix into bacterial fermentation liquid, and cultured to OD 600 When the value is >40.0, the bacterial liquid in the production tank is harvested;

[0024] When OD 600 When the value is less than 15.0, the culture temperature is controlled to be 27.0-37.5°C, and the dissolved oxygen in the fermentation liquid of the strain is 55-60%;

[0025] When OD 600 When the value is 15.0-40.0, the culture temperature is controlled to be 27.0-34.5°C, and the dissolved oxygen in the bacterial fermentation liquid is 25-30%;

[0026] When OD 600 When the value is greater than 40.0, the culture temperature is controlled to be 27.0-33.0°C, and the dissolved oxygen in the fermentation liquid of the strain is 12-15%.

[0027] When this application is cultured in seed tanks, OD 600 When the value is less than 2.0, the amount of bacteria is relatively small, and the dissolved oxygen is maintained at 45-50% to meet the reproduction needs of the bacteria. 600 When the value reaches 2.0-10.0, the amount of bacteria begins to increase gradually. At this time, the dissolved oxygen is appropriately increased to 55-60%. This not only allows the bacteria to quickly expand to the set amount, but also reduces the energy waste caused by oxygen supply.

[0028] This application is used in production tank culture, OD 600 When the value is less than 15.0, it is mainly for bacterial amplification. 600The OD value is maintained at 15.0-40.0 mainly to promote the expression and secretion of CRM197 protein. At this time, the culture temperature and dissolved oxygen should not be too low or too high. Controlling the temperature at 27.0-34.5℃ and dissolved oxygen at 25-30% can ensure the stable growth of the strain while secreting a large amount of CRM197 protein. 600 When the value is greater than 40.0, the fermentation efficiency of the strain slows down and the protein production capacity decreases. The present application further reduces the temperature and dissolved oxygen to effectively slow down the metabolic rate of the strain, thereby reducing the amount of metabolites in the fermentation broth of the strain, so that the CRM197 protein is stably present in the fermentation broth, thereby being able to harvest a high yield of CRM197 protein.

[0029] Preferably, in the seed tank culture, the dissolved oxygen of the bacterial culture solution is controlled by the following steps:

[0030] When OD 600 When the value is less than 2.0, maintain the ventilation volume at 0.1-0.3vvm and control the stirring speed at 200-500rpm;

[0031] When OD 600 When the value is 2.0-10.0, maintain the ventilation volume at 0.1-0.3vvm, control the stirring speed at 200-500rpm, and when the stirring speed reaches 500rpm, control the ventilation volume at 0.2-2vvm.

[0032] Preferably, in the seed tank culture, the dissolved oxygen of the bacterial culture solution is controlled by the following steps:

[0033] When OD 600 When the value is less than 2.0, the ventilation volume is maintained at 0.2vvm and the stirring speed is controlled at 200-500rpm;

[0034] When OD 600 When the value is 2.0-10.0, maintain the ventilation volume at 0.2vvm, increase the stirring speed to 500rpm, and control the ventilation volume at 0.5-1.0vvm.

[0035] Preferably, in the production tank culture, the dissolved oxygen of the bacterial fermentation liquid is controlled by the following steps:

[0036] When OD 600 When the value is ≤40.0, maintain the ventilation volume at 0.05-0.2vvm, control the stirring speed at 50-300rpm, and when the stirring speed reaches 300rpm, control the ventilation volume at 0.2-2.0vvm;

[0037] When OD 600 When the value is greater than 40.0, maintain the speed at 300 rpm and control the ventilation volume at 0.2-2.0 vvm.

[0038] Preferably, in the production tank culture, the dissolved oxygen of the bacterial fermentation liquid is controlled by the following steps: when OD 600 When the value is ≤40.0, maintain the ventilation volume at 0.1vvm, control the stirring speed at 50-300rpm, and when the stirring speed reaches 300rpm, control the ventilation volume at 0.5-1.0vvm;

[0039] When OD 600 When the value is greater than 40.0, maintain the speed at 300 rpm and control the ventilation volume at 0.5-1.0 vvm.

[0040] In the actual fermentation and cultivation process, the ventilation volume and stirring speed will directly affect the contact effect between the strains and oxygen, and the ventilation volume and stirring speed requirements of different strains are different; for this reason, after a lot of research and experiments, the present application found that if the ventilation volume is too high and the stirring speed is slow, it is easy for the fermentation efficiency of strains in different regions to have a large deviation; if the ventilation volume is too low and the stirring speed is fast, it is easy for the strains to have insufficient contact time with oxygen and affect their growth and / or reproduction. The above ventilation volume and stirring speed are used to control different fermentation degrees (OD 600 value), can promote the bacteria to express CRM197 protein at high density, so this application takes it as a further preference.

[0041] When the strain is cultured in a seed tank, the ventilation volume of 0.1-0.3vvm can be specifically 0.1vvm, 0.15vvm, 0.2vvm, 0.25vvm, 3.0vvm, etc., and the ventilation volume of 0.2vvm is further preferred. The stirring speed is allowed to be adjusted between 200-500rpm; when the stirring speed reaches 500rpm, the present application adjusts the ventilation volume to 0.2-2vvm, which can be specifically 0.5vvm, 0.6vvm, 0.7vvm, 0.8vvm, 0.9vvm, 1.0vvm, etc., and the ventilation volume of 0.5-1.0vvm is further preferred, thereby facilitating the rapid adjustment of the ventilation volume in the seed tank.

[0042] Similarly, when the strain is cultured in a production tank, the ventilation volume of 0.05-0.2vvm can be specifically 0.05vvm, 0.1vvm, 0.15vvm, 0.2vvm, etc., and the ventilation volume of 0.1vvm is further preferred. The stirring speed is allowed to be adjusted between 50-300rpm; when the stirring speed reaches 300rpm, the present application adjusts the ventilation volume to 0.2-2vvm, which can be specifically 0.5vvm, 0.6vvm, 0.7vvm, 0.8vvm, 0.9vvm, 1.0vvm, etc., and the ventilation volume of 0.5-1.0vvm is further preferred, thereby facilitating the rapid adjustment of the ventilation volume in the production tank.

[0043] Preferably, during the seed tank culture process, the volume ratio of the bacterial liquid to the liquid culture medium is 1:(10-15); during the production tank culture process, the volume ratio of the bacterial liquid in the seed tank to the liquid culture medium is 1:(5-10).

[0044] When the strain of the present application is cultured in a seed tank, the strain reproduces in large quantities and consumes a large amount of nutrients. Therefore, the amount of liquid culture medium added needs to be appropriately excessive to ensure the healthy growth and reproduction of the strain. After the strain is transferred to the production tank, the amount of liquid culture medium added is reduced. On the one hand, this is because there are excess nutrients in the seed tank that can continue to participate in the fermentation. On the other hand, the strain amount in the production tank remains within a stable range after the amplification is completed. At this time, the nutrients are difficult to maintain the reproduction needs of the strain, which in turn prompts the strain to begin to express and secrete CRM197 protein in large quantities. Among them, the fermentation broth cultured according to the above-mentioned volume ratio in the present application has a high content of CRM197 protein, so it is further preferred.

[0045] Preferably, during the production tank cultivation process, when OD 600 When the value is >40.0, the OD 600 When the value increases to ≤2.0, the culture is stopped.

[0046] Preferably, during the production tank cultivation process, when OD 600 When the value is >40.0, the OD 600 When the value increases to 0.5-1.0, stop the cultivation.

[0047] The inventors have found that, during the harvesting stage, the concentration of various metabolites of CRM197 strains is relatively high, so a half-hour difference in harvesting will also result in a significant difference in yield. 600 When the value is greater than 40.0, the production rate of CRM197 protein slows down. 600 When the OD value increased ≤ 2.0, the increase in CRM197 protein production tended to be at a lower level. 600 When the OD value increased ≤ 1.0, the production of CRM197 protein hardly increased. 600 When the value increases by ≤0.5, the yield of CRM197 protein decreases instead of increases. 600 The growth value of the value is used as a reference to timely control the point at which the culture of the strain is stopped, thereby harvesting a higher content of CRM197 protein.

[0048] Therefore, according to the fermentation harvest control method of the present application, a suitable harvest endpoint is selected to capture the target CRM197 protein at the apex of the yield curve. Under the premise of controlling metabolic side reaction substances, the yield of CRM197 protein can be maximized, and through scale-up verification, it can be repeatedly implemented and applied to the production process of CRM197 protein.

[0049] In summary, this application has the following beneficial effects:

[0050] 1. The present application maintains a high dissolved oxygen content and a suitable temperature in the seed tank, maintains a certain growth and amplification level of the strain, and then maintains a moderate dissolved oxygen content and bottom temperature in the production tank, so as to control the growth rate of the strain within a suitable range for efficient expression and secretion of CRM197 protein, thereby achieving the purpose of high yield of CRM197 protein, and can be well applied to the production process of CRM197 protein;

[0051] 2. The present application preferably uses rotation speed and ventilation volume to control dissolved oxygen, thereby achieving high-density expression of CRM197 protein;

[0052] 3. This application preferably uses OD 600 The growth value of the value is used as a reference for timely control of the point at which the culture of the strain is stopped, which is helpful to harvest a higher content of CRM197 protein. DETAILED DESCRIPTION

[0053] Raw material preparation

[0054] 1. CRM197 strain

[0055] The CRM197 strain in this application is from the American Type Culture Collection (ATCC), and the strain number is ATCC39255. In addition, the CRM197 strain in this application can also be selected from other strains with strain numbers such as ATCC39526, ATCC11049, ATCC51926, and ATCC51280. Before using the above strains, use 1 mL of liquid culture medium to resuscitate and activate them.

[0056] 2. Liquid culture medium

[0057] Each liter is prepared with the following ingredients: 250 mL of basic nutrient solution, 7.2 mL of acetic acid, 30 g of maltose monohydrate, 2 mL of sodium lactate, 0.5 g of tyrosine, 1.96 g of potassium dihydrogen phosphate, 5.1 g of potassium dihydrogen phosphate, 6.13 mL of growth factor a, 8 mL of growth factor b, 8 mL of growth factor c and 1.3 g of calcium chloride, with the remainder being water.

[0058] Each liter of the basic nutrient solution is composed of the following ingredients: 2 g arginine, 2 g asparagine, 0.5 g aspartic acid, 0.5 g glutamic acid, 2 g L-glutamine, 0.5 g glycine, 1.5 g histidine, 2 g L-isoleucine, 2 g leucine, 2 g lysine hydrochloride, 0.6 g methionine, 1 g phenylalanine, 2 g proline, 2 g serine, 1 g threonine, 0.6 g tryptophan, 1.5 g valine and 0.5 g β-alanine, and the balance is water;

[0059] Each liter of growth factor a is composed of the following ingredients: 2 g L-cystine and 2 mL hydrochloric acid, the balance being water;

[0060] Each liter of growth factor b is composed of the following ingredients: 6.5 g nicotinic acid, 0.5 g anhydrous copper sulfate, 0.15 g pimelic acid, 30 mL hydrochloric acid, 200 g magnesium sulfate heptahydrate, 0.8 g zinc sulfate heptahydrate and 0.393 g manganese chloride tetrahydrate, the balance being water;

[0061] Each liter of growth factor C is composed of the following ingredients: 0.1g vitamin B1, 0.75g vitamin B5 and 0.075g vitamin B6, with the remainder being water.

[0062] The method for preparing the above liquid culture medium comprises the following steps:

[0063] ①, dissolve 0.5g of tyrosine in 20wt% sodium hydroxide at a ratio of 10% (W / V) to obtain a tyrosine solution;

[0064] ②, add an appropriate amount of water for injection at 55°C (fluctuation between 50°C and 60°C is allowed) into the container, accurately weigh or measure 7.2mL of acetic acid, 30g of maltose monohydrate, 2mL of sodium lactate, all of the tyrosine solution prepared in step ①, 1.96g of potassium dihydrogen phosphate and 5.1g of potassium dihydrogen phosphate, add to the water for injection and mix well to obtain a primary mixed solution;

[0065] ③. When the initial mixed solution cools to room temperature, prepare 25L of basic nutrient solution, 6.13mL of growth factor a, 8mL of growth factor b and 8mL of growth factor c and add them in turn. Mix thoroughly and make up to volume with injection water. Adjust the pH to 7.4 with 20wt% sodium hydroxide. Mix well to obtain a liquid to be layered.

[0066] ④. Add 1.3g calcium chloride to the liquid to be separated. Let it stand for 30 minutes until it is separated after it is fully dissolved. Siphon the supernatant with a silicone tube and obtain the liquid culture medium after sterilization and filtration.

[0067] The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0068] Example 1

[0069] A fermentation method for increasing the yield of CRM197 protein comprises the following steps:

[0070] (I) Shake flask culture

[0071] 1. First generation liquid shake bottle:

[0072] Open a tube of CRM197 and inoculate it into 50 mL of liquid culture medium. Add 2.5 mL of yeast extract (10 g / L, purchased from OXIOD) and shake the flask at 36.5 ± 0.5 °C and 200 rpm (allowed to fluctuate between 100 and 300 rpm) until the OD reaches 0. 600 When the concentration is above 1.0, the first generation of liquid shake flask culture solution is harvested.

[0073] 2. Second generation liquid shake bottle:

[0074] Inoculate 1.5 mL of the first-generation liquid shake flask culture medium into 500 mL of liquid culture medium, add 25 mL of yeast extract (10 g / L, purchased from OXIOD), this is the second-generation liquid shake flask culture medium, shake the flask at 36.5 ± 0.5 ° C, 200 rpm (allowed to fluctuate between 100-300 rpm) until OD 600 When the concentration is above 2.0, the second generation liquid shake flask culture solution is harvested.

[0075] (II) Bioreactor culture

[0076] 3. Seed tank cultivation:

[0077] Inoculate all the second-generation liquid shake flask bacterial solution into 7000 mL of liquid culture medium to mix as bacterial culture solution, and culture at 36.5°C (allowed to fluctuate within ±0.5°C) until OD 600 When the value is >10.0, the bacterial liquid in the seed tank is harvested;

[0078] When OD 600 When the value is less than 2.0, the ventilation volume is maintained at 0.2vvm, and the stirring speed is adjusted within the range of 200-500rpm to control the dissolved oxygen of the bacterial culture solution to 50%;

[0079] When OD 600 When the value is 2.0-10.0, maintain the ventilation volume at 0.2vvm, accelerate the stirring speed to 500rpm and maintain the speed, adjust the ventilation volume within the range of 0.5-1.0vvm to control the dissolved oxygen in the bacterial culture solution to 56%.

[0080] 4. Production tank culture:

[0081] Inoculate all the bacterial liquid in the seed tank into 60000mL liquid culture medium to mix as bacterial fermentation liquid and culture until OD 600Value>40.0 and OD within 30min of culture 600 When the value increases to 1.0, the culture is stopped and the bacterial liquid in the production tank is harvested, and the fermentation culture of the CRM197 strain is completed;

[0082] When OD 600 When the value is less than 15.0, the culture temperature is controlled at 36.5°C, the ventilation volume is maintained at 0.1vvm, and the stirring speed is adjusted within the range of 50-300rpm to control the dissolved oxygen in the fermentation liquid of the strain to 55%;

[0083] When OD 600 When the value is 15.0-40.0, control the culture temperature to 34.5°C, maintain the ventilation volume at 0.1vvm, adjust the stirring speed within the range of 50-300rpm, maintain the stirring speed after reaching 300rpm, and adjust the ventilation volume within the range of 0.5-1.0vvm to control the dissolved oxygen in the fermentation liquid of the strain to 28%;

[0084] When OD 600 When the value is greater than 40.0, the culture temperature is controlled at 33.0°C, the stirring speed is maintained at 300 rpm, and the ventilation volume is controlled at 0.5-1.0 vvm to control the dissolved oxygen in the fermentation liquid of the strain to 12%.

[0085] Example 2

[0086] In Example 2-5, based on the method of Example 1, the dissolved oxygen content was adjusted. The adjustment details are shown in Table 1 below.

[0087] Table 1 Adjustment parameters of dissolved oxygen content in Examples 1-5 (unit: %)

[0088]

[0089] Comparative Example 1

[0090] In this comparative example, based on the method of Example 1, the dissolved oxygen content of the seed tank culture was adjusted, specifically: OD 600 When the value is ≤10.0, the dissolved oxygen is controlled at 25%.

[0091] Comparative Example 2

[0092] In this comparative example, based on the method of Example 1, the dissolved oxygen content of the production tank culture was adjusted to: OD 600 When the value is ≤40.0, the dissolved oxygen is controlled at 25%.

[0093] Performance testing

[0094] According to the methods of Examples 1-7 and Comparative Examples 1-2, 9 batches of CRM197 strains were fermented, and the batch numbers were recorded as A202204001-A202204007 and D202204001-D202204002 respectively. The CRM197 protein yield of the harvested production tank culture liquid was determined according to the flocculent unit (Lf) determination method recorded in the 2015 edition of the "Chinese Pharmacopoeia".

[0095] Among them, the flocculent unit is a direct indicator for evaluating the protein expression level, indicating the concentration level of a specific protein contained in the fermentation culture medium, and the higher the flocculent protein level, the higher the protein expression. The measurement results are shown in Table 2 below.

[0096] Table 2 Detection results of CRM197 protein yield in the production tank culture liquid of Examples 1-7

[0097]

[0098]

[0099] Combined with the test results in Table 2, it can be seen that the present application controls the dissolved oxygen in the seed tank culture to be >40%, and the dissolved oxygen in the production tank culture to be 600 When the value is less than 15.0, it is controlled at more than 40%, and at OD 600 When the value is ≥15.0 and is controlled at 10-40%, the flocculent unit (Lf) of the harvested production tank culture liquid (A202204001-A202204007) is significantly higher than the flocculent unit (Lf) of the harvested production tank culture liquid (D202204001-D202204002) in the control example. It can be seen that the dissolved oxygen control of the present application can effectively increase the CRM197 protein yield.

[0100] Moreover, according to the test results of fermentation batches A202204001-A202204007, if the dissolved oxygen in the seed tank culture is at OD 600 When the value is less than 2.0, it should be controlled at 45-50%. 600 The value is 2.0-10.0 and is controlled at 55-60%. At the same time, the dissolved oxygen in the production tank is controlled at OD 600 When the value is less than 15.0, it is controlled at 55-60%. 600 When the value is 15.0-40.0, control it at 25-30%, then OD 600 When the value is greater than 40.0, it is controlled at 12-15%, and the flocculent unit (Lf) of the harvested production tank culture liquid (A202204001-A202204003) is relatively higher, that is, the CRM197 protein yield is higher, and the production tank culture liquid of A202204001 is further preferred.

[0101] Examples 8-10

[0102] Examples 8-10 take Example 1 as a preferred example. Based on the method of Example 1, the culture temperature in the production tank is adjusted. The adjustment details are shown in Table 3 below.

[0103] Table 3 Adjustment parameters of culture temperature in Example 1 and Examples 8-10 (unit: °C)

[0104]

[0105] Comparative Example 3

[0106] In this comparative example, based on the method of Example 1, the culture temperature in the production tank was maintained at 36.5°C (fluctuation within ±0.5°C was allowed).

[0107] Performance testing

[0108] Four batches of CRM197 strains were fermented according to the methods of Examples 8-10 and Comparative Example 3, and the batch numbers were recorded as A202204008-A2022040010 and D202204003 respectively. The CRM197 protein yield of the harvested production tank culture liquid was determined by the flocculent unit (Lf) determination method recorded in the 2015 edition of the "Chinese Pharmacopoeia". The test results are shown in Table 4 below.

[0109] Table 4 Detection results of CRM197 protein yield in Examples 1, 8-10 and Comparative Example 3

[0110] Fermentation batch <![CDATA[Final OD 600 > Flocculent unit Lf / mL A202204001 41.5 195 A202204008 42.2 187 A202204009 41.3 184 A2022040010 42.0 190 D202204003 41.8 155

[0111] Combined with the test results in Table 4, it can be seen that when the strains of the present application are cultured, the culture temperature of the production tank has a certain influence on the CRM197 protein yield. 600 When the value is less than 15.0, it should be controlled at 27.0-37.5℃. 600 When the value is 15.0-40.0, it is controlled at 27.0-34.5℃, and then OD 600 When the value is greater than 40.0 and is controlled within the range of 27.0-33.0", the yield of CRM197 protein harvested is higher, which can be further optimized.

[0112] From the test results in Tables 2 and 4, it can be seen that the present application maintains a high dissolved oxygen content and a suitable temperature in the seed tank to maintain a certain growth and amplification level of the strain, and then maintains a moderate dissolved oxygen content and bottom temperature in the production tank to control the growth rate of the strain within a suitable range for efficient expression and secretion of CRM197 protein, thereby achieving the purpose of high yield of CRM197 protein.

[0113] Examples 11-16

[0114] Based on the method of Example 1, Examples 11-16 make corresponding adjustments to the ventilation volume. For specific adjustments, please see Table 5 below.

[0115] Table 5 Ventilation adjustment parameter table of Examples 11-16 (unit: vvm)

[0116]

[0117] Performance testing

[0118] According to the method of Examples 11-16, 6 batches of CRM197 strains were fermented, and the batch numbers were recorded as A2022040011-A2022040016. The CRM197 protein yield of the harvested production tank culture liquid was determined by the flocculent unit (Lf) determination method recorded in the 2015 edition of the "Chinese Pharmacopoeia". The test results are shown in Table 6 below.

[0119] Table 6 Detection results of CRM197 protein yield in Example 1 and Examples 11-16

[0120]

[0121]

[0122] Combined with the test results in Table 6, it can be obtained that during the fermentation and cultivation process of the strains of the present application, the ventilation volume and stirring speed will directly affect the contact effect between the strains and oxygen. If the ventilation volume is too high and the stirring speed is slow, there will be a large deviation in the fermentation efficiency of the strains in different regions; if the ventilation volume is too low and the stirring speed is too fast, the strains will easily have insufficient contact time with oxygen, which will affect their growth and reproduction.

[0123] To this end, the present application further prefers to use a variable-speed ventilation volume of 0.2vvm (the ventilation volume when the stirring speed is allowed to be adjusted between 200-500rpm) and a uniform-speed ventilation volume of 0.5-1.0vvm (the ventilation volume when the stirring speed reaches 500rpm) during seed tank cultivation, and a variable-speed ventilation volume of 0.1vvm (the ventilation volume when the stirring speed is allowed to be adjusted between 50-300rpm) and a uniform-speed ventilation volume of 0.5-1.0vvm (the ventilation volume when the stirring speed reaches 300rpm) during production tank cultivation.

[0124] Examples 17-20

[0125] In Examples 17-20, based on the method of Example 1, the volume ratio of the second-generation liquid shake flask bacterial solution to the liquid culture medium and the volume ratio of the seed tank bacterial solution to the liquid culture medium were adjusted. The specific adjustments are shown in Table 7 below.

[0126] Table 7 Volume ratio of bacterial liquid to liquid culture medium in Example 1 and Examples 17-20

[0127]

[0128] Performance testing

[0129] Four batches of CRM197 strains were fermented according to the methods of Examples 17-20, and the batch numbers were recorded as A2022040017-A2022040020. The CRM197 protein yield of the harvested production tank culture liquid was determined by the flocculent unit (Lf) determination method recorded in the 2015 edition of the "Chinese Pharmacopoeia". The test results are shown in Table 8 below.

[0130] Table 8 Detection results of CRM197 protein yield in Example 1 and Examples 17-20

[0131] Fermentation batch <![CDATA[Final OD 600 > Flocculent unit Lf / mL A202204001 41.5 195 A2022040017 41.2 186 A2022040018 42.0 190 A2022040019 41.8 180 A2022040020 42.5 182

[0132] Combined with the test results in Table 8, it can be obtained that in the seed tank culture process, the volume ratio of the second-generation liquid shake flask culture medium is limited to 1: (10-15), and in the production tank culture process, the volume ratio of the seed tank culture medium is limited to 1: (5-10), which can harvest fermentation culture medium with a higher CRM197 protein content, so it is further preferred.

[0133] Harvest endpoint performance testing

[0134] The CRM197 strain was fermented according to the method of Example 1. During the fermentation process of the production tank culture, the CRM197 protein yield was measured by the flocculent unit (Lf) determination method recorded in the 2015 edition of the "Chinese Pharmacopoeia" every 30 minutes. The test results are shown in Table 9 below.

[0135] Table 9 Detection results of CRM197 protein production of the strains of Example 1 at different culture times

[0136] Cultivation time / min 30 60 90 120 150 180 210 240 270 <![CDATA[OD 600 Value increase]]> 18.0 12.0 6.0 4.0 2.0 1.0 0.7 0.5 0.1 Flocculent unit Lf / mL 31 76 121 162 188 195 196 190 181

[0137] As can be seen from Table 9, the CRM197 strain has a high concentration of various metabolites during the harvesting stage, so a half-hour difference in harvesting will also result in a significant difference in yield. 600 After the value was >40.0 (150 min of culture), the production rate of CRM197 protein slowed down, and the OD 600 When the OD value increased ≤ 2.0, the increase in CRM197 protein production tended to be at a lower level. 600When the OD value increased ≤ 1.0, the production of CRM197 protein hardly increased. 600 When the value increased by ≤0.5, the production of CRM197 protein decreased instead of increased.

[0138] Based on this, this application is based on OD 600 The growth value of the value is used as a reference, and the node at which the strain stops cultivation is controlled in time, thereby harvesting a higher content of CRM197 protein. Among them, the OD value within 30 minutes is preferred 600 When the value increases to ≤2.0 (cultivation for 150 min), the culture is stopped, and the OD value is preferably within 30 min. 600 When the value increases by 0.5-1.0 (180-240 min of culture), the culture is stopped, and the content of CRM197 protein obtained is relatively high.

[0139] In summary, the present invention controls dissolved oxygen by rotating speed and ventilation volume at a set culture temperature to achieve high-density expression of CRM197 protein, and finally uses OD 600 The growth value of the value is used as a reference to timely control the node when the culture of the strain is stopped, and the growth rate of the strain is controlled within the appropriate range for efficient expression and secretion of CRM197 protein, thereby achieving the purpose of high yield of CRM197 protein, which can be better applied to the production process of CRM197 protein.

[0140] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A fermentation method for increasing the yield of CRM197 protein, characterized in that: The following steps are involved: Seed pot culture: The CRM197 bacterial culture liquid and liquid culture medium were added to the seed tank and mixed into the bacterial culture liquid, and cultured at 36.5±0.5℃ until the OD 600 When the value is greater than 10.0, the bacterial liquid in the seed tank is harvested; the CRM197 bacterial liquid is from the American Type Culture Collection (ATCC), and the strain number is ATCC39255; When OD 600 When the value is less than 2.0, the dissolved oxygen in the bacterial culture solution is controlled to be 45%-50%; When OD 600 When the value is 2.0-10.0, the dissolved oxygen in the bacterial culture solution is controlled to be 55%-60%; Production tank culture: The bacterial solution in the seed tank and the liquid culture medium are put into the production tank to mix into bacterial fermentation liquid, and cultured to OD 600 When the value is >40.0, the bacterial liquid in the production tank is harvested; When OD 600 When the value is less than 15.0, the culture temperature is controlled to be 27.0-37.5°C, and the dissolved oxygen in the fermentation liquid of the strain is 55%-60%; When OD 600 When the value is 15.0-40.0, the culture temperature is controlled to be 27.0-34.5°C, and the dissolved oxygen in the fermentation liquid of the strain is 25%-30%; When OD 600 When the value is greater than 40.0, the culture temperature is controlled to be 27.0-33.0°C, and the dissolved oxygen in the fermentation liquid of the strain is 12%-15%.

2. The fermentation culture method according to claim 1, characterized in that: In the seed tank culture, the dissolved oxygen of the bacterial culture solution is controlled by the following steps: When OD 600 When the value is less than 2.0, maintain the ventilation volume at 0.1-0.3vvm and control the stirring speed at 200-500rpm; When OD 600 When the value is 2.0-10.0, maintain the ventilation volume at 0.1-0.3vvm, control the stirring speed at 200-500rpm, and when the stirring speed reaches 500rpm, control the ventilation volume at 0.2-2vvm.

3. The fermentation culture method according to claim 2, characterized in that: In the seed tank culture, the dissolved oxygen of the bacterial culture solution is controlled by the following steps: When OD 600 When the value is less than 2.0, the ventilation volume is maintained at 0.2vvm and the stirring speed is controlled at 200-500rpm; When OD 600 When the value is 2.0-10.0, maintain the ventilation volume at 0.2vvm, increase the stirring speed to 500rpm, and control the ventilation volume at 0.5-1.0vvm.

4. The fermentation culture method according to claim 1, characterized in that: In the production tank culture, the dissolved oxygen of the bacterial fermentation liquid is controlled by the following steps: When OD 600 When the value is ≤40.0, maintain the ventilation volume at 0.05-0.2vvm, control the stirring speed at 50-300rpm, and when the stirring speed reaches 300rpm, control the ventilation volume at 0.2-2.0vvm; When OD 600 When the value is greater than 40.0, the stirring speed is maintained at 300 rpm and the ventilation volume is controlled at 0.2-2.0 vvm.

5. The fermentation culture method according to claim 4, characterized in that: In the production tank culture, the dissolved oxygen of the bacterial fermentation liquid is controlled by the following steps: When OD 600 When the value is ≤40.0, maintain the ventilation volume at 0.1vvm, control the stirring speed at 50-300rpm, and when the stirring speed reaches 300rpm, control the ventilation volume at 0.5-1.0vvm; When OD 600 When the value is greater than 40.0, the stirring speed is maintained at 300 rpm and the ventilation volume is controlled at 0.5-1.0 vvm.

6. The fermentation culture method according to claim 1, characterized in that: During the seed tank culture process, the volume ratio of the bacterial liquid to the liquid culture medium is 1: (10-15); during the production tank culture process, the volume ratio of the bacterial liquid in the seed tank to the liquid culture medium is 1: (5-10).

7. The fermentation culture method according to claim 1, characterized in that: During the production tank cultivation process, when OD 600 When the value is >40.0, the OD 600 When the value increases to ≤2.0, the culture is stopped.

8. The fermentation culture method according to claim 7, characterized in that: During the production tank cultivation process, when OD 600 When the value is >40.0, the OD 600 When the value increases to 0.5-1.0, stop the cultivation.

Images