CN117701459A - Escherichia coli high-density fermentation medium and fermentation process - Google Patents
Escherichia coli high-density fermentation medium and fermentation process Download PDFInfo
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- CN117701459A CN117701459A CN202311785393.7A CN202311785393A CN117701459A CN 117701459 A CN117701459 A CN 117701459A CN 202311785393 A CN202311785393 A CN 202311785393A CN 117701459 A CN117701459 A CN 117701459A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 83
- 230000004151 fermentation Effects 0.000 title claims abstract description 83
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 30
- 239000012526 feed medium Substances 0.000 claims abstract description 29
- 239000002609 medium Substances 0.000 claims abstract description 16
- 239000007640 basal medium Substances 0.000 claims abstract description 11
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 claims abstract description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 10
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims abstract description 10
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 7
- 229960001031 glucose Drugs 0.000 claims abstract description 7
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 6
- 239000013530 defoamer Substances 0.000 claims abstract description 6
- 239000012138 yeast extract Substances 0.000 claims abstract description 6
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 claims abstract description 5
- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 claims abstract description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000004327 boric acid Substances 0.000 claims abstract description 5
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims abstract description 5
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims abstract description 5
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 claims abstract description 5
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims abstract description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 5
- 229960004642 ferric ammonium citrate Drugs 0.000 claims abstract description 5
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- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims abstract description 5
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims abstract description 5
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims abstract description 5
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 claims abstract description 5
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- 229960000999 sodium citrate dihydrate Drugs 0.000 claims abstract description 5
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims abstract description 5
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- 238000003756 stirring Methods 0.000 claims description 3
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a high-density fermentation medium for escherichia coli, which comprises a basic medium and a feed supplement medium; the basal medium comprises: 9.54g/L of disodium hydrogen phosphate, 6.80g/L of monopotassium phosphate, 1.9g/L of ammonium sulfate, 0.09g/L of ferric ammonium citrate and 0.1mL/L of defoamer; the feed medium comprises: 600g/L of glucose monohydrate, 29.58g/L of magnesium sulfate heptahydrate, 15g/L of yeast extract, 19.8g/L of ammonium sulfate, 2.3g/L of sodium citrate dihydrate, 0.88g/L of ferric chloride hexahydrate, 0.076g/L of cobalt chloride hexahydrate, 0.077g/L of sodium molybdate dihydrate, 0.08g/L of copper sulfate pentahydrate, 0.063g/L of manganese chloride tetrahydrate, 0.06g/L of zinc chloride, 0.08g/L of calcium chloride dihydrate, 0.03g/L of boric acid, 10.25g/L of vitamin B, 0.004g/L of riboflavin, 0.08g/L, D g/L of nicotinamide, 0.43g/L of calcium pantothenate, and 0.063g/L of pyrazolyl hydrochloride; the invention can solve the problems that the traditional feed medium contains animal source components, is easily influenced by exogenous pollution components such as endotoxin, exogenous DNA and bacteriophage, and the quality and the safety of the final product are influenced by the pollutants.
Description
Technical Field
The invention relates to the technical field of high-density fermentation culture of escherichia coli, in particular to a high-density fermentation culture medium and a fermentation process of escherichia coli.
Background
Coli is widely used as an engineering cell for the production of expressed recombinant proteins, virus-like particles, plasmid DNA, mRNA and various metabolites. In recent years, along with the development of various novel medicines, such as pDNA, gene therapy and mRNA vaccine, escherichia coli high-density fermentation is also promoted again.
High density fermentation of E.coli refers to the cultivation of E.coli cells at high cell concentrations (typically above 100 g/L) and at high specific growth rates (typically above 0.5-1 h). This can significantly improve productivity and yield of the target product and reduce consumption of raw materials and energy. The conventional escherichia coli high-density fermentation process is generally carried out in a bioreactor mainly because the high-density fermentation needs higher oxygen consumption, and the bioreactor can control various environmental parameters in the process of escherichia coli fermentation to be stable. At the same time, the high concentration of the feed medium and the feed-related control processes in conventional fed-batch fermentation are of vital importance, which directly affect the optimal growth environment and metabolic state of the cells.
The composition and quality of the feed medium can affect the growth and expression of E.coli. The feed medium should provide sufficient nutrients such as carbon, nitrogen, phosphorus, sulfur, trace elements and vitamins to support high density fermentation. However, the feed medium should also avoid excessive or undesired components, such as glucose, acetate, salts and antibiotics, which inhibit or interfere with the metabolism and protein synthesis of E.coli. In addition, the feed medium should be sterile, free of endotoxin, phage, foreign DNA and other contaminants, and the traditional feed medium contains animal-derived components, which are susceptible to exogenous contaminating components (endotoxin, exogenous DNA, phage) so that the contaminants affect the quality and safety of the final product.
Disclosure of Invention
The invention aims to solve the technical problems that the traditional feed medium contains animal source components and is easily influenced by exogenous pollution components, so that the quality and the safety of the final product are influenced by pollutants.
In a first aspect, the present invention provides a high-density fermentation medium for E.coli, the fermentation medium comprising a basal medium and a feed medium;
the basal medium comprises the following components in parts by weight: 8-12g/L of disodium hydrogen phosphate, 5-10g/L of monopotassium phosphate, 1-5g/L of ammonium sulfate, 0.05-0.1g/L of ferric ammonium citrate and 0.05-0.2mL/L of defoamer;
the feed medium comprises the following components in parts by weight: 400-800g/L of glucose monohydrate, 25-32g/L of magnesium sulfate heptahydrate, 10-20g/L of yeast extract, 15-25g/L of ammonium sulfate, 1-4g/L of sodium citrate dihydrate, 0.5-1.2g/L of ferric chloride hexahydrate, 0.05-0.1g/L of cobalt chloride hexahydrate, 0.05-0.1g/L of sodium molybdate dihydrate, 0.05-0.1g/L of copper sulfate pentahydrate, 0.05-0.1g/L of manganese chloride tetrahydrate, 0.05-0.1g/L of zinc chloride, 0.05-0.1g/L of calcium chloride dihydrate, 0.02-0.05g/L of boric acid and 1g/L of vitamin B
0.1-0.5g/L, 0.001-0.005g/L riboflavin, 0.05-0.1g/L, D g/L calcium pantothenate, 0.3-0.6g/L pyrazole alcohol hydrochloride, 0.05-0.1g/L.
In a preferred embodiment of the above escherichia coli high-density fermentation medium, the basal medium comprises: disodium hydrogen phosphate 9.54g/L, potassium dihydrogen phosphate 6.80g/L, ammonium sulfate 1.9g/L, ferric ammonium citrate 0.09g/L, and defoamer 0.1mL/L.
In the preferred technical scheme of the escherichia coli high-density fermentation medium, the feed medium comprises the following components: 600g/L of glucose monohydrate, 29.58g/L of magnesium sulfate heptahydrate, 15g/L of yeast extract, 19.8g/L of ammonium sulfate, 2.3g/L of sodium citrate dihydrate, 0.88g/L of ferric chloride hexahydrate, 0.076g/L of cobalt chloride hexahydrate, 0.077g/L of sodium molybdate dihydrate, 0.08g/L of copper sulfate pentahydrate, 0.063g/L of manganese chloride tetrahydrate, 0.06g/L of zinc chloride, 0.08g/L of calcium chloride dihydrate, 0.03g/L of boric acid, 10.25g/L of vitamin B, 0.004g/L of riboflavin, 0.08g/L, D g/L of nicotinamide, 0.43g/L of calcium pantothenate, and 0.063g/L of pyrazolyl hydrochloride.
In a second aspect, the invention also provides a high-density escherichia coli fermentation process, which comprises the fermentation medium.
In the preferred technical scheme of the escherichia coli high-density fermentation process, the fermentation process comprises the following steps of:
s101: emptying the fermentation tank for 30min, wherein the emptying temperature is 121 ℃, cooling, and adding the basic culture medium into the fermentation tank for actual digestion for 30min, wherein the actual digestion temperature is 121 ℃;
s102: aseptically adding a feed medium into the fermenter at 2.25% of the initial culture volume;
s103: inoculating fresh seed liquid cultured at 37 ℃ in a shake flask into the fermentation tank according to 1% of inoculation amount in a bottle-changing sterile operation, and setting the inoculation time of strain bacterial liquid as a fermentation zero point;
s104: setting a pH-related acid-base pump, wherein when the pH is lower than a set value, the pH pump controls 28% ammonia water to be added into the fermentation tank, and when the pH is higher than the set value, the pH pump controls 20% phosphoric acid to be added into the fermentation tank;
setting DO first-stage associated stirring and DO second-stage associated oxygen ventilation; when the dissolved oxygen is lower than a set value, the fermentation tank automatically adjusts the rotating speed, and when the rotating speed reaches 1000rpm and the dissolved oxygen is still lower than the set value, the fermentation tank automatically adjusts the oxygen ventilation;
s105: feeding associated DO, when the DO signal is higher than 45%, a feeding pump receiving signal starts a single feeding operation;
s106: when the thallus grows OD 600 And adding IPTG with the final concentration of 0.5mM when the concentration reaches 48-52 for induction, and ending fermentation after 8 hours of induction culture.
In the preferred technical scheme of the high-density escherichia coli fermentation process, in the step S102, the pH value in the fermentation tank is 7.00±0.10.
In the preferred embodiment of the high-density fermentation process of escherichia coli, in S105, when the DO is higher than 45%, the start of the single feeding is delayed for 6 seconds, the feeding is not performed if the DO is reduced to 45% or less in 6 seconds, and the single feeding is normally performed after 6 seconds but still higher than 45%.
The beneficial effects of the invention are as follows: the components of the feed medium are changed to realize that the feed medium does not contain animal source components, and most of the components are chemical components limited compounds, so that the feed medium is prevented from being influenced by exogenous pollution components such as endotoxin, exogenous DNA and bacteriophage, and the quality and the safety of the final product are further ensured.
Drawings
FIG. 1 is a graph showing the growth of fermenting bacteria in the control group at 0-26h in example 1;
FIG. 2 is a graph showing the comparison of the average bacterial sludge yield of the fermenting bacteria in the example 1 and the control group in 0-26 hours;
FIG. 3 is a graph showing the osmolarity comparison between 0-26h in example 1 and the control group;
FIG. 4 is a graph showing the comparison of glucose concentration and osmolarity at 0-24h in example 1.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The feed medium based on the background technology contains animal source components, is easily influenced by endotoxin, exogenous DNA and phage, and causes the problem that the quality and the safety of the final product are not up to standard. The invention provides a high-density fermentation medium for escherichia coli, which aims to ensure that the feed medium does not contain animal source components by changing the components of the feed medium, and most of the components are chemical components-limited compounds, so that the influence of exogenous pollution components (endotoxin, exogenous DNA and bacteriophage) is avoided, and the quality and the safety of a final product are ensured. Meanwhile, the invention provides a high-density fermentation process of escherichia coli, which aims to reduce residual sugar control in the fermentation process by correlating DO with feed, so as to reduce acetate accumulation phenomenon caused by excessively accumulated carbon sources, further reduce inhibition of acetate on bacterial growth, and further improve the growth density and bacterial yield of engineering strains to the greatest extent, thereby improving the expression quantity of target proteins.
Example 1:
1. fermentation preparation
Firstly, calibrating a pH electrode of a fermentation tank, and then respectively installing the pH electrode and an oxygen dissolving electrode at corresponding positions of a glass fermentation tank;
secondly, connecting an exhaust pipe, a condensate pipe, an acid liquor bottle, an alkali liquor bottle, a defoamer bottle, a feed supplementing bottle and a pipeline of a feed supplementing culture medium of the fermentation tank to corresponding positions, emptying the fermentation tank for 30min, wherein the emptying temperature is 121 ℃, adding a basic culture medium into the fermentation tank after cooling the fermentation tank, and performing actual disinfection for 30min, wherein the actual disinfection temperature is 121 ℃.
TABLE 1 basal medium component contents
2. Parameter setting
After the fermenter was connected to the control cabinet, the feed medium was added aseptically in a proportion of 2.25% of the initial culture volume.
Before inoculation, fermenter parameter setting:
tank pressure of fermentation tank: less than 0.1Mpa;
air relief valve pressure: 0.50bar;
large bubble air intake flow at bottom of fermenter: 1VVM;
fermentation tank rotation speed: 200rpm;
pH value: 7.00+/-0.10;
dissolved oxygen: 30+ -5%.
It should be noted that when the parameters are set, when the pH value in the fermentation tank exceeds or is lower than a preset value, the automatic control alkali supplementing bottle is started to conduct alkali supplementing operation so as to keep the pH value stable; when the dissolved oxygen is set for the fermentation tank, firstly, the dissolved oxygen electrode wire is pulled out, the dissolved oxygen is calibrated to be 0%, then the electrode wire is inserted, the rotating speed of the fermentation tank is adjusted to be 1000rpm, after the dissolved oxygen curve is stable, the dissolved oxygen is calibrated to be 100%, and after the calibration is completed, the dissolved oxygen is set to be 30+/-5%.
TABLE 2 feed Medium component contents
3. Inoculation of
Fresh seed liquid cultured at 37 ℃ in a shaking bottle is inoculated into a fermentation tank according to 1% of inoculation amount in a bottle replacement sterile operation, and the inoculation time of the set strain bacterial liquid is taken as a fermentation zero point, so that the starting moment of the fermentation process can be conveniently recorded and controlled, and the fermentation process can be accurately controlled.
4. PH and DO-related control
Setting pH and associating with an acid-base pump, after inoculation, when the pH is lower than a set value (7.00+/-0.10), starting the base pump to work, and automatically adding 28% ammonia water; when the pH is higher than the set value (7.00+/-0.10), the acid pump starts to work, and 20% of phosphoric acid is automatically added.
DO first-stage associated stirring (200-1000 rpm) is set, second-stage associated oxygen is aerated, when dissolved oxygen is lower than the set value, the rotating speed of the fermentation tank is automatically regulated, and when the rotating speed reaches 1000rpm and dissolved oxygen control cannot be met, the oxygen aeration is automatically regulated.
5. Feed supplement associated control
When dissolved oxygen begins to rise steeply, the batch culture phase ends and the fed-batch culture phase is entered.
In order to ensure that the excessive addition of nutrient substances in the culture process leads to accumulation of acetate byproducts which affect the growth of the strain and the expression of target proteins. The invention adopts the mode of associating the feeding with DO to control, when the carbon source is consumed, DO starts to rise, when DO is higher than 45%, the feeding pump receives a signal to start single feeding operation. After feeding, the cells begin to metabolize aerobically, and DO drops rapidly and remains around 30%. When the carbon source in the feed is depleted again, DO starts to rise for the second time, and once again, feeding starts after being higher than the set value. The cycle is repeated, so that the thallus fermentation is always at a lower fermentation nutrition limit. The single feeding amount is regulated according to the growth condition of the thalli, and is generally maintained at 0.5-2.5mL each time.
In order to further reduce misjudgment caused by D0 fluctuation, it is set that when DO signal is higher than 45%, single feeding is started after delaying for 6 seconds, if DO is reduced to below 45% in 6 seconds, feeding is not performed, and if DO is still higher than 45% after 6 seconds, single feeding is normally started.
6. Induction and harvesting
Along with the feeding, when the thallus grows OD 600 When the concentration reaches about 50 mM, IPTG with the final concentration of 0.5mM is added for induction, and after induction culture is carried out for 8 hours, fermentation is ended.
Example 2:
the only difference from example 1 is the composition of the basal medium. The basal medium in example 2 can be replaced by a simple PBS buffer solution, thereby meeting the production of escherichia coli bacterial cells and also reducing the possibility of pollution of the basal medium.
Comparative example:
the basic culture medium of the control group adopts a conventional TB culture medium with high nutrition abundance; the control group feed medium used high concentration glycerol.
TABLE 3 content of the components of TB Medium
TABLE 4 feed Medium component contents
The basal medium formulations in example 1 and example 2 were simple, compared to the comparative examples, and did not provide the carbon nitrogen source required for growth, only as a conventional buffer system. The simple basic salt culture medium is beneficial to large-scale preparation of culture medium configuration and facilitates subsequent solid powdering. Meanwhile, the simple basic salt culture medium does not contain complex organic matter components, so that the basic culture medium can have relatively long storage and quality guarantee period after being prepared; in addition, the simple inorganic salt component improves the sterilization simplicity of the culture medium to a certain extent, and the culture medium can be simply prepared into a high-concentration mother solution for sterilization treatment, and then the mother solution is diluted to normal concentration by sterile water according to the ratio of 1:1 for use, so that the energy consumption required by single-batch culture medium sterilization is reduced, and the culture medium is energy-saving, emission-reducing, environment-friendly and safe.
Compared with the comparative example, the nutrient components of the feed medium in the example 1 and the example 2 are sufficient, and the nutrient requirements of the fermentation culture process of microorganisms of different species can be met. The content of non-chemical component limiting components (hydrolysate) in the feed medium is less, most of the components are determined compounds, and the yeast powder (yeast extract) only plays an auxiliary role and has a small proportion of action on the whole thallus fermentation, so that the prepared components have good batch-to-batch difference controllability.
Referring to fig. 4, the feed control methods of examples 1 and 2 can maximally reduce the accumulation of acetate during the carbon source metabolism process, and reduce residual sugar control during fermentation process by correlating DO with feed, thereby reducing the accumulation of acetate caused by excessively accumulated carbon source, and further reducing the inhibition of acetate on the growth of bacterial cells, thereby maximally increasing the growth density and bacterial cell yield of the engineering strain, and thus increasing the expression level of the target protein, as compared with the comparative example.
The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and to implement the same, but are not intended to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (7)
1. The escherichia coli high-density fermentation medium is characterized by comprising a basic medium and a feed medium;
the basal medium comprises the following components in parts by weight: 8-12g/L of disodium hydrogen phosphate, 5-10g/L of monopotassium phosphate, 1-5g/L of ammonium sulfate, 0.05-0.1g/L of ferric ammonium citrate and 0.05-0.2mL/L of defoamer;
the feed medium comprises the following components in parts by weight: 400-800g/L of glucose monohydrate, 25-32g/L of magnesium sulfate heptahydrate, 10-20g/L of yeast extract, 15-25g/L of ammonium sulfate, 1-4g/L of sodium citrate dihydrate, 0.5-1.2g/L of ferric chloride hexahydrate, 0.05-0.1g/L of cobalt chloride hexahydrate, 0.05-0.1g/L of sodium molybdate dihydrate, 0.05-0.1g/L of copper sulfate pentahydrate, 0.05-0.1g/L of manganese chloride tetrahydrate, 0.05-0.1g/L of zinc chloride, 0.05-0.1g/L of calcium chloride dihydrate, 0.02-0.05g/L of boric acid, 10.1-0.5g/L of vitamin B, 0.001-0.005g/L of riboflavin, 0.05-0.1g/L, D g/L of calcium pantothenate and 0.3-0.6g/L of pyrazolyl alcohol hydrochloride.
2. The high-density escherichia coli fermentation medium as set forth in claim 1, wherein: the basal medium comprises: disodium hydrogen phosphate 9.54g/L, potassium dihydrogen phosphate 6.80g/L, ammonium sulfate 1.9g/L, ferric ammonium citrate 0.09g/L, and defoamer 0.1mL/L.
3. The high-density escherichia coli fermentation medium as set forth in claim 1, wherein: the feed medium comprises: glucose monohydrate 600g/L, magnesium sulfate heptahydrate 29.58g/L, yeast extract 15g/L, ammonium sulfate 19.8g/L, sodium citrate dihydrate 2.3g/L, ferric chloride hexahydrate 0.88g/L, cobalt chloride hexahydrate 0.076g/L, sodium molybdate dihydrate 0.077g/L, copper sulfate pentahydrate 0.08g/L, manganese chloride tetrahydrate 0.063g/L, zinc chloride 0.06g/L, calcium chloride dihydrate 0.08g/L, boric acid 0.03g/L, vitamin B1.25 g/L, riboflavin 0.004g/L, nicotinamide 0.08g/L, D calcium pantothenate 0.43g/L, and pyrazolyl hydrochloride 0.063g/L.
4. A high-density fermentation process of escherichia coli is characterized in that: comprising the step of subjecting E.coli-containing bacteria to high-density fermentation culture using the fermentation medium according to any one of claims 1 to 3.
5. The high-density fermentation process of Escherichia coli according to claim 4, wherein the fermentation process comprises the steps of:
s101: emptying the fermentation tank for 30min, wherein the emptying temperature is 121 ℃, cooling, and adding the basic culture medium into the fermentation tank for actual digestion for 30min, wherein the actual digestion temperature is 121 ℃;
s102: aseptically adding a feed medium into the fermenter at 2.25% of the initial culture volume;
s103: inoculating fresh seed liquid cultured at 37 ℃ in a shake flask into the fermentation tank according to 1% of inoculation amount in a bottle-changing sterile operation, and setting the inoculation time of strain bacterial liquid as a fermentation zero point;
s104: setting a pH-related acid-base pump, wherein when the pH is lower than a set value, the pH pump controls 28% ammonia water to be added into the fermentation tank, and when the pH is higher than the set value, the pH pump controls 20% phosphoric acid to be added into the fermentation tank;
setting DO first-stage associated stirring and DO second-stage associated oxygen ventilation; when the dissolved oxygen is lower than a set value, the fermentation tank automatically adjusts the rotating speed, and when the rotating speed reaches 1000rpm and the dissolved oxygen is still lower than the set value, the fermentation tank automatically adjusts the oxygen ventilation;
s105: feeding is related to the DO, and when the DO signal is higher than 45%, a feeding pump receives a signal to start a single feeding operation;
s106: when the thallus grows OD 600 When 48-52 is reached, IPTG with the final concentration of 0.5mM is added for induction, and after 8 hours of induction culture, the fermentation is ended.
6. The high-density fermentation process of Escherichia coli according to claim 5, wherein the fermentation process comprises the steps of: in S102, the pH value in the fermentation tank is 7.00+/-0.10.
7. The high-density fermentation process of Escherichia coli according to claim 5, wherein the fermentation process comprises the steps of: in S105, when the DO is higher than 45%, the single feeding is started after a delay of 6 seconds, and if the DO is lowered to 45% or lower in 6 seconds, the single feeding is not performed, and after 6 seconds, the DO is still higher than 45%, the single feeding is normally performed.
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