CN114164160A - Fermentation medium for efficiently producing plasmid DNA and fermentation production method - Google Patents

Fermentation medium for efficiently producing plasmid DNA and fermentation production method Download PDF

Info

Publication number
CN114164160A
CN114164160A CN202111511464.5A CN202111511464A CN114164160A CN 114164160 A CN114164160 A CN 114164160A CN 202111511464 A CN202111511464 A CN 202111511464A CN 114164160 A CN114164160 A CN 114164160A
Authority
CN
China
Prior art keywords
fermentation
medium
plasmid dna
proline
leucine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111511464.5A
Other languages
Chinese (zh)
Inventor
焦顺昌
张嵘
张凤娇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Dingcheng Taiyuan Biotechnology Co ltd
Original Assignee
Beijing Dingcheng Taiyuan Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Dingcheng Taiyuan Biotechnology Co ltd filed Critical Beijing Dingcheng Taiyuan Biotechnology Co ltd
Priority to CN202111511464.5A priority Critical patent/CN114164160A/en
Publication of CN114164160A publication Critical patent/CN114164160A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides a fermentation medium for efficiently producing plasmid DNA and a fermentation production method, and relates to the technical field of plasmid production. The fermentation culture medium comprises a fermentation basal culture medium and a feeding culture medium which are independently packaged, and the combination of the fermentation basal culture medium and the feeding culture medium can meet the requirement of high-density fermentation and improve the yield of plasmid DNA. When the fermentation medium is used for fermentation production of escherichia coli, the average plasmid yield can reach 181mg/L, the volume of a bioreactor can be reduced, the purity of downstream purified plasmid DNA can be improved, the production period can be shortened, the equipment investment can be reduced, the production cost can be reduced, and the purpose of improving the production efficiency can be achieved.

Description

Fermentation medium for efficiently producing plasmid DNA and fermentation production method
Technical Field
The invention belongs to the technical field of plasmid production, and particularly relates to a fermentation medium for efficiently producing plasmid DNA and a fermentation production method.
Background
With the further development of DNA vaccines, cell therapy and gene therapy, the demand of plasmid DNA increases, higher requirements are put forward for industrial production, plasmid DNA production becomes more and more important, and the large-scale preparation of clinical-grade plasmid DNA by using Escherichia coli is receiving more and more attention. Most of the efforts developed to date for plasmid DNA production processes have focused on downstream processing. However, the quality and yield of the product ultimately depends on the fermentation strategy. By optimizing the growth environment of the recombinant bacteria, the thallus yield, the plasmid yield and the plasmid quality can be improved. Therefore, in order to be able to provide a sufficient amount of plasmid DNA to meet the needs of clinical trials and future markets, an efficient plasmid DNA fermentation production process is required to provide a large amount of plasmid DNA.
The plasmid DNA production process is also dependent on culture conditions and medium composition, except for the influence of vector design, host bacteria, and purification methods. The composition of the culture medium obviously influences the plasmid yield, and further influences the production cost, so that the optimization of the culture medium is also one of important methods for improving the plasmid yield, but at present, an adaptive culture medium and a method which can meet the requirements of high-density cell fermentation and plasmid DNA production do not exist.
Disclosure of Invention
In view of the above, the present invention aims to provide a fermentation medium and a fermentation production method for efficiently producing plasmid DNA, wherein a basic culture medium and a supplemented culture medium are optimized to determine a new fermentation basic culture medium and a new supplemented culture medium, and finally, the plasmid yield is significantly improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a fermentation medium for efficiently producing plasmid DNA, which comprises a fermentation basal medium and a supplementary medium which are independently packaged;
every 4000mL of the fermentation basal medium comprises the following raw materials: 48g of yeast extract, 96g of soybean peptone, 32g of glycerol, 16g of sodium chloride, 2.5g of magnesium sulfate heptahydrate, 2g of calcium chloride, 2.5g of ammonium sulfate, 2g of thiamine, 5g of leucine, 3g of proline, 10mL of trace element liquid and 200mL of phosphate solution;
every 500mL of the feed medium comprises the following raw materials: 94g of glycerol, 56g of yeast extract, 32g of soybean peptone, 20g of ammonium chloride, 8g of leucine, 8g of proline and 2g of magnesium sulfate heptahydrate.
Preferably, the trace element liquid comprises the following raw materials in concentration: 20g/L of ferrous sulfate heptahydrate, 2g/L of zinc sulfate heptahydrate, 2g/L of cobalt chloride hexahydrate, 2g/L of sodium molybdate dihydrate, 0.76g/L of anhydrous calcium chloride, 1.27g/L of anhydrous copper sulfate, 1.6g/L of manganese sulfate monohydrate, 0.5g/L of boric acid and 3mL/L of hydrochloric acid with the volume percentage content of 36-38%.
The invention also provides a preparation method of the fermentation culture medium, wherein the preparation method of every 4000mL of the fermentation basal culture medium comprises the following steps: mixing the yeast extract, soybean peptone, glycerol, sodium chloride, magnesium sulfate heptahydrate, calcium chloride, ammonium sulfate and water to 3700mL for sterilization, and mixing with 10mL of trace element liquid, 200mL of phosphate solution and 90mL of premixed solution to obtain a fermentation basal medium; the solutes of the premix solution comprise thiamine, leucine, and proline;
the preparation method of every 500mL of the feed medium comprises the steps of mixing glycerol, yeast extract, soybean peptone, ammonium chloride, magnesium sulfate heptahydrate and water to 450mL in proportion, sterilizing, and mixing with 50mL of a mixed solution of leucine and proline filtered by 0.22 mu m to obtain the feed medium.
Preferably, the preparation method of the premix solution comprises the following steps: and (3) dissolving thiamine, leucine and proline independently or in a mixed manner, filtering by using a 0.22-micrometer filter membrane, and collecting filtrate to obtain the premixed solution.
The invention also provides the application of the fermentation medium in the efficient fermentation production of plasmid DNA.
Preferably, the plasmid DNA is derived from recombinant Escherichia coli.
The invention also provides a method for producing plasmid DNA by high-efficiency fermentation, which comprises the following steps: (1) inoculating the secondary seed liquid of the escherichia coli to a fermentation basic culture medium, carrying out aerobic fermentation, and controlling the pH value to be 6.9-7.0 in the fermentation process;
(2) fermenting to OD600When the number is 11-13, adding a supplementary culture medium;
(3) fermenting to OD600When the culture temperature is 15-20 ℃, increasing the culture temperature toCulturing at 42 deg.C until fermentation is finished.
Preferably, in the aerobic fermentation in the step (1), the volume content of dissolved oxygen is controlled to be 30-40%.
Preferably, the feeding speed of the feed medium in the step (2) is 2-2.8 mL/min.
Preferably, the fermentation in step (3) further comprises collecting the thallus and extracting the plasmid.
Has the advantages that: the invention provides a fermentation medium for efficiently producing plasmid DNA, which can meet the requirement of high-density fermentation and improve the yield of the plasmid DNA. When the fermentation medium is used for fermentation production of escherichia coli, the average plasmid yield can reach 181mg/L, the volume of a bioreactor can be reduced, the purity of downstream purified plasmid DNA can be improved, the production period can be shortened, the equipment investment can be reduced, the production cost can be reduced, and the purpose of improving the production efficiency can be achieved.
Drawings
FIG. 1 is a graph showing the growth of strains and the growth of plasmids in the basal medium formulation 1;
FIG. 2 is a graph showing the growth of strains and the growth of plasmids in basal medium formulation 2;
FIG. 3 is a graph showing the growth of the strain and the growth of the plasmid in the feed medium formulation A;
FIG. 4 is a graph showing the growth of the strain and the growth of the plasmid in the feed medium formulation B.
Detailed Description
The invention provides a fermentation medium for efficiently producing plasmid DNA, which comprises a fermentation basal medium and a supplementary medium which are independently packaged;
every 4000mL of the fermentation basal medium comprises the following raw materials: 48g of yeast extract, 96g of soybean peptone, 32g of glycerol, 16g of sodium chloride, 2.5g of magnesium sulfate heptahydrate, 2g of calcium chloride, 2.5g of ammonium sulfate, 2g of thiamine, 5g of leucine, 3g of proline, 10mL of trace element liquid and 200mL of phosphate solution;
every 500mL of the feed medium comprises the following raw materials: 94g of glycerol, 56g of yeast extract, 32g of soybean peptone, 20g of ammonium chloride, 8g of leucine, 8g of proline and 2g of magnesium sulfate heptahydrate.
The fermentation medium optimizes the dosage and components of reagents in the medium by improving the formulas of a basic medium and a supplemented medium, so that the thalli can be subjected to high-density fermentation.
The trace element liquid preferably comprises the following raw materials in concentration: 20g/L of ferrous sulfate heptahydrate, 2g/L of zinc sulfate heptahydrate, 2g/L of cobalt chloride hexahydrate, 2g/L of sodium molybdate dihydrate, 0.76g/L of anhydrous calcium chloride, 1.27g/L of anhydrous copper sulfate, 1.6g/L of manganese sulfate monohydrate, 0.5g/L of boric acid and 3mL/L of 36-38% (v/v) of hydrochloric acid.
The invention also provides a preparation method of the fermentation culture medium, wherein the preparation method of every 4000mL of the fermentation basal culture medium comprises the following steps: mixing the yeast extract, soybean peptone, glycerol, sodium chloride, magnesium sulfate heptahydrate, calcium chloride, ammonium sulfate, thiamine, leucine and proline with water to 3700mL for sterilization, and mixing with 10mL of trace element liquid, 200mL of phosphate solution and 90mL of premixed solution to obtain a fermentation basal medium; the solutes of the premix solution comprise thiamine, leucine, and proline;
the preparation method of every 500mL of the feed medium comprises the steps of mixing glycerol, yeast extract, soybean peptone, ammonium chloride, magnesium sulfate heptahydrate and water to 450mL in proportion, sterilizing, and mixing with 50mL of a mixed solution of leucine and proline filtered by 0.22 mu m to obtain the feed medium.
The preparation method of the premix solution preferably comprises the following steps: and (3) dissolving thiamine, leucine and proline independently or in a mixed manner, filtering by using a 0.22-micrometer filter membrane, and collecting filtrate to obtain the premixed solution. The sterilization of the invention is preferably carried out by autoclaving at 121 ℃, and the sterilization time is preferably 30 min.
In the preparation of the feed medium of the present invention, the sterilization is preferably the same as described above, and will not be described herein.
The invention also provides the application of the fermentation medium in the efficient fermentation production of plasmid DNA.
The plasmid DNA is preferably derived from Escherichia coli, including wild strains, variant strains or recombinant strains of Escherichia coli.
The invention also provides a method for producing plasmid DNA by high-efficiency fermentation, which comprises the following steps:
(1) inoculating the secondary seed liquid of the escherichia coli to a fermentation basic culture medium, carrying out aerobic fermentation, and controlling the pH value to be 6.9-7.0 in the fermentation process;
(2) fermenting to OD600When the number is 11-13, adding a supplementary culture medium;
(3) fermenting to OD600And when the temperature is 15-20 ℃, increasing the culture temperature to 42 ℃, and culturing until the fermentation is finished.
The secondary seed liquid of the escherichia coli is inoculated on a fermentation basic culture medium for aerobic fermentation, and the pH value in the fermentation process is controlled to be 6.9-7.0. The preparation method of the secondary seed solution preferably comprises the steps of activating the cryopreserved bacterial solution, inoculating the activated bacterial solution into an LB liquid culture medium containing ampicillin to obtain a primary seed solution, and inoculating the primary seed solution into the LB liquid culture medium containing ampicillin to obtain a secondary seed solution.
The bacteria liquid activation of the invention preferably comprises streaking the frozen bacteria liquid on LB culture medium containing ampicillin, placing the streaked bacteria liquid upside down in an incubator, and culturing overnight at 37 ℃. The method preferably picks a monoclonal on an LB solid plate, inoculates the monoclonal on 5mL LB liquid culture medium with ampicillin resistance, and performs shaking culture at 37 ℃ and 220rpm for 6-10 h to obtain a first-stage seed solution. After the primary seed solution is obtained, 400 mu L of the primary seed solution is preferably inoculated in 400mL of LB liquid culture medium with ampicillin resistance, and is subjected to shake culture at 37 ℃ and 220rpm for 14-18 h to obtain the secondary seed solution. In the present invention, the concentration of ampicillin in the LB medium is preferably 50. mu.g/mL.
The secondary seed liquid is inoculated to a fermentation basal medium for aerobic fermentation, and the inoculation amount (v/v) of the inoculation is preferably 5-10%. In the aerobic fermentation of the present invention, the volume content of dissolved oxygen is preferably controlled to be 30-40%, and as in the embodiment of the present invention, the aerobic fermentation is performed in a fermentation tank, and the following fermentation initial parameters are set: the pressure in the tank is as follows: not more than 0.02 Mpa; temperature setting: 37.0 +/-1 ℃, and the pH control range is as follows: 6.90-7.10; rotating speed: 300-900 rpm; air ventilation volume: 1-8L/min. In the aerobic fermentation process, when the rotating speed and the ventilation quantity are not enough to maintain the dissolved oxygen at 40 percent, oxygen can be introduced.
After the aerobic fermentation is carried out, the fermentation is carried out until the fermentation reaches OD600When the concentration is 11-13, a feed medium is added. The addition speed of the fed-batch culture medium is preferably 2-2.8 mL/min until the fed-batch culture medium is exhausted. In the present invention, when the fermentation basal medium is 4000mL, the volume of the corresponding feed medium is preferably 500 mL.
Culturing to 0D600When the temperature is 15-20 ℃, the culture temperature is increased to 42 ℃, and the culture is carried out until the fermentation is finished. The present invention preferably further comprises collecting the cells and extracting plasmids after the fermentation is completed, and the method for collecting the cells and extracting the plasmids is not particularly limited, and may be performed by a conventional method in the art.
The following examples are provided to describe the fermentation medium and the fermentation method for efficiently producing plasmid DNA according to the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Activating strains: mu.L of the cryopreserved stbl3 recombinant strain liquid (the bacteria used in the experiment are recombinant bacteria, and the objective plasmid is transformed into stbl3 competent cells) is taken to streak an ampicillin resistant LB solid plate, and the plate is placed upside down in an incubator and cultured overnight at 37 ℃.
2. Preparing a first-level seed solution: a single colony on an LB solid plate was picked, inoculated into 5mL of LB liquid medium having ampicillin resistance, and cultured with shaking at 37 ℃ and 220rpm for 7.5 hours.
3. Preparing a secondary seed liquid: 400 μ L of the primary seed liquid was inoculated into 400mL of LB liquid medium having ampicillin resistance, and subjected to shaking culture at 37 ℃ and 220rpm for 16 hours for subsequent fermentation.
4. Fermentation pH control solution
Diluting the phosphoric acid solution to 15% (wt); the aqueous ammonia solution was diluted to a concentration of 5% (v/v) and sterilized by 0.22 μm filtration. Used for regulating and controlling the pH value in the fermentation process.
5. Solution of trace elements:
20g/L of ferrous sulfate heptahydrate, 2g/L of zinc sulfate heptahydrate, 2g/L of cobalt chloride hexahydrate, 2g/L of sodium molybdate dihydrate, 0.76g/L of anhydrous calcium chloride, 1.27g/L of anhydrous copper sulfate, 1.6g/L of manganese sulfate monohydrate, 0.5g/L of boric acid, 3mL/L of 36-38% (v/v) of hydrochloric acid, and 0.22 mu m of filtration sterilization.
6. Fermentation culture
(1) Adjusting pH of the fermentation medium to 7.0 with ammonia water or phosphoric acid, inoculating the secondary seed solution into a fermentation tank according to an inoculation ratio of 5%, and starting fermentation culture.
(2) Setting initial fermentation parameters: the pressure in the tank is as follows: not more than 0.02 Mpa; temperature setting: 37.0 ℃, pH control: 7.0; rotating speed: 300 rpm; air ventilation volume: 3L/min; and monitoring the temperature, pH, rotating speed and dissolved oxygen in the fermentation process by taking the completion of inoculation as the culture starting time.
7. Comparison of different fermentation basal media
(1) Phosphate solution:
28g of dipotassium phosphate and 8g of monopotassium phosphate are dissolved by using a proper amount of purified water, the volume is determined to be 200mL, and autoclaving is carried out at 121 ℃ for 30 min.
(2) The fermentation basal medium formula comprises:
basal medium formula 1: 48g of yeast extract, 72g of soybean peptone, 16g of glycerol, 8g of sodium chloride, 2.5g of magnesium sulfate heptahydrate, 2g of calcium chloride, 2.5g of ammonium sulfate and 2g of thiamine. Dissolving the above components except thiamine with purified water, diluting to 3700mL, sterilizing at 121 deg.C for 30min under high pressure; thiamine was dissolved in 90mL of water, filtered through a 0.22 μm filter, and added sequentially with 200mL of phosphate and 10mL of trace elements.
Basal medium formula 2: 48g of yeast extract, 96g of soybean peptone, 32g of glycerol, 16g of sodium chloride, 2.5g of magnesium sulfate heptahydrate, 2g of calcium chloride, 2.5g of ammonium sulfate, 2g of thiamine, 5g of leucine and 3g of proline. Dissolving all the components except for the methionine, the leucine and the proline by using purified water, fixing the volume to 3700mL, and carrying out autoclaving at 121 ℃ for 30 min;
thiamine, leucine and proline are prepared into 90mL solution, filtered by a 0.22 mu m filter membrane, and added with 200mL phosphate and 10mL trace elements in sequence.
(3) Fermentation process control
The dissolved oxygen is controlled to be about 40 percent in the fermentation process; in the fermentation process, diluted phosphoric acid and ammonia water are used for adjusting the pH value to 6.9-7.0; when fermenting OD600When the temperature is 9.2 ℃, the culture temperature is adjusted from 37 ℃ to 42 ℃, and the culture is carried out until the fermentation is finished.
The strain is collected by centrifugation at 10000rpm for 30min at low temperature, and the plasmid is extracted by using a Tiangen plasmid miniprep kit, the strain growth and plasmid growth curve graphs are shown in figures 1-2 and table 1, and higher plasmid yield can be obtained in the same time by using a basic culture medium of a formula 2.
TABLE 1 comparison of fermentation results of different basic media for recombinant bacteria
/ Basal Medium 1 experiment Basal Medium 2 experiment
OD600Value of 16.9 20.7
Harvest bacterial size (wet weight) 155g 201g
Total plasmid yield 173mg 241mg
Average plasmid yield 43.3mg/L 60.3mg/L
8. Comparison of different feed media
(1) Fermentation basal medium: basal medium formula 2.
(2) A supplemented medium:
feed medium a: 94g of glycerol, 56g of yeast extract, 32g of soybean peptone, 20g of ammonium chloride and 2g of magnesium sulfate heptahydrate, metering the volume to 450mL, and autoclaving at 121 ℃ for 30 min;
8g of leucine and 8g of proline are dissolved in 50mL of water, filtered through a 0.22 mu m filter membrane and added into a sterilized fermentation basal medium.
Supplemented medium B: 94g/L of glycerol, 56g of yeast extract and 64g of soybean peptone are added, the volume is adjusted to 500mL, and autoclaving is carried out at 121 ℃ for 30 min.
(4) Fermentation process control
Controlling the dissolved oxygen to be about 40% in the fermentation process, and introducing oxygen when the rotating speed and the ventilation quantity are not enough to maintain the dissolved oxygen to be about 40%; in the fermentation process, diluted phosphoric acid and ammonia water are used for adjusting the pH value to 6.9-7.0; when OD is reached600Starting to feed the feed culture medium at the speed of 2.8mL/min from 11-13 until 500mL of the feed culture medium is used up; when fermenting OD600And when the temperature is 15-20 ℃, adjusting the culture temperature from 37 ℃ to 42 ℃, and culturing until the fermentation is finished.
The same method as in the comparison with the fermentation basal medium was used to collect the cells and extract the plasmids. As shown in FIGS. 3 to 4 and Table 2, the feed medium A significantly increased the amount of bacteria and the amount of extracted plasmid.
TABLE 2 comparison of fermentation results of different supplemented media for recombinant bacteria
Figure BDA0003393969020000071
Figure BDA0003393969020000081
Example 2
1. Fermentation culture
(1) Basic culture medium: 48g of yeast extract, 96g of soybean peptone, 32g of glycerol, 16g of sodium chloride, 2.5g of magnesium sulfate heptahydrate, 2g of calcium chloride, 2.5g of ammonium sulfate, 2g of thiamine, 5g of leucine and 3g of proline. Dissolving the above components with purified water, diluting to 3700mL, autoclaving at 121 deg.C for 30 min;
thiamine, leucine and proline are prepared into 90mL of solution, filtered by a 0.22 mu m filter membrane, and then sequentially added with 200mL of phosphate solution and 10mL of trace element solution.
(2) A supplemented medium: 94g of glycerol, 56g of yeast extract, 32g of soybean peptone, 20g of ammonium chloride, 8g of leucine, 8g of proline and 2g of magnesium sulfate heptahydrate, wherein the volume is fixed to 450mL, and autoclaving is carried out at 121 ℃ for 30 min;
8g of leucine and 8g of proline are dissolved in 50mL of water, filtered through a 0.22 mu m filter membrane and added into a sterilized fermentation basal medium.
(3) Fermentation process control
The conditions of strain activation, first-stage seed liquid preparation, second-stage seed liquid preparation and fermentation were the same as in example 1. Controlling the dissolved oxygen to be about 40% in the fermentation process, and introducing oxygen when the rotating speed and the ventilation quantity are not enough to maintain the dissolved oxygen to be about 40%; in the fermentation process, diluted phosphoric acid and ammonia water are used for adjusting the pH value to 6.9-7.0; when OD is reached600Starting to feed the feed medium at 11.9 at a rate of 2.8mL/min until 500mL of feed medium is exhausted; when fermenting OD600When the temperature is 15.6 ℃, the culture temperature is adjusted from 37 ℃ to 42 ℃, and the culture is carried out until the fermentation is finished.
(4) As a result: after the fermentation was completed, 429g of the cells (wet weight) were harvested, 813mg of the total plasmid yield and 181mg/L of the average plasmid yield.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A fermentation culture medium for efficiently producing plasmid DNA is characterized by comprising a fermentation basal culture medium and a supplementary culture medium which are independently packaged;
every 4000mL of the fermentation basal medium comprises the following raw materials: 48g of yeast extract, 96g of soybean peptone, 32g of glycerol, 16g of sodium chloride, 2.5g of magnesium sulfate heptahydrate, 2g of calcium chloride, 2.5g of ammonium sulfate, 2g of thiamine, 5g of leucine, 3g of proline, 10mL of trace element liquid and 200mL of phosphate solution;
every 500mL of the feed medium comprises the following raw materials: 94g of glycerol, 56g of yeast extract, 32g of soybean peptone, 20g of ammonium chloride, 8g of leucine, 8g of proline and 2g of magnesium sulfate heptahydrate.
2. The fermentation medium of claim 1, wherein the trace element liquid comprises the following concentrations of raw materials: 20g/L of ferrous sulfate heptahydrate, 2g/L of zinc sulfate heptahydrate, 2g/L of cobalt chloride hexahydrate, 2g/L of sodium molybdate dihydrate, 0.76g/L of anhydrous calcium chloride, 1.27g/L of anhydrous copper sulfate, 1.6g/L of manganese sulfate monohydrate, 0.5g/L of boric acid and 3mL/L of hydrochloric acid with the volume percentage content of 36-38%.
3. The method of preparing the fermentation medium of claim 1 or 2, wherein the method of preparing the fermentation basal medium per 4000mL comprises: mixing the yeast extract, soybean peptone, glycerol, sodium chloride, magnesium sulfate heptahydrate, calcium chloride, ammonium sulfate and water to 3700mL for sterilization, and mixing with 10mL of trace element liquid, 200mL of phosphate solution and 90mL of premixed solution to obtain a fermentation basal medium; the solutes of the premix solution comprise thiamine, leucine, and proline;
the preparation method of every 500mL of the feed medium comprises the steps of mixing glycerol, yeast extract, soybean peptone, ammonium chloride, magnesium sulfate heptahydrate and water to 450mL in proportion, sterilizing, and mixing with 50mL of a mixed solution of leucine and proline filtered by 0.22 mu m to obtain the feed medium.
4. The method of claim 3, wherein the premix solution is prepared by a method comprising: and (3) dissolving thiamine, leucine and proline independently or in a mixed manner, filtering by using a 0.22-micrometer filter membrane, and collecting filtrate to obtain the premixed solution.
5. Use of the fermentation medium according to claim 1 or 2 for the efficient fermentative production of plasmid DNA.
6. The use of claim 5, wherein the plasmid DNA is derived from recombinant E.coli.
7. A method for producing plasmid DNA by high-efficiency fermentation is characterized by comprising the following steps:
(1) inoculating the secondary seed liquid of the escherichia coli to the fermentation basal medium in the claim 1 or 2, and carrying out aerobic fermentation, wherein the pH value in the fermentation process is controlled to be 6.9-7.0;
(2) fermenting to OD600When 11-13, adding the feed medium of claim 1 or 2;
(3) fermenting to OD600And when the temperature is 15-20 ℃, increasing the culture temperature to 42 ℃, and culturing until the fermentation is finished.
8. The method according to claim 7, wherein the volume content of dissolved oxygen is controlled to be 30-40% in the aerobic fermentation in the step (1).
9. The method according to claim 7, wherein the feeding rate of the feed medium in the step (2) is 2-2.8 mL/min.
10. The method of claim 7, further comprising collecting the biomass and extracting the plasmid after the fermentation in step (3) is completed.
CN202111511464.5A 2021-12-06 2021-12-06 Fermentation medium for efficiently producing plasmid DNA and fermentation production method Pending CN114164160A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111511464.5A CN114164160A (en) 2021-12-06 2021-12-06 Fermentation medium for efficiently producing plasmid DNA and fermentation production method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111511464.5A CN114164160A (en) 2021-12-06 2021-12-06 Fermentation medium for efficiently producing plasmid DNA and fermentation production method

Publications (1)

Publication Number Publication Date
CN114164160A true CN114164160A (en) 2022-03-11

Family

ID=80485625

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111511464.5A Pending CN114164160A (en) 2021-12-06 2021-12-06 Fermentation medium for efficiently producing plasmid DNA and fermentation production method

Country Status (1)

Country Link
CN (1) CN114164160A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116751731A (en) * 2023-08-11 2023-09-15 赛奥斯博生物科技(北京)有限公司 Fermentation process for large-scale plasmid production

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050233421A1 (en) * 2004-04-08 2005-10-20 Boehringer Ingelheim Austria Gmbh Fed-batch fermentation process and culture medium for the production of plasmid DNA in E. coli on a manufacturing scale
WO2005098002A1 (en) * 2004-04-08 2005-10-20 Boehringer Ingelheim Austria Gmbh Method for producing plasmid dna on a manufacturing scale by fermentation of the escherichia coli k-12 strain jm108
CN106566795A (en) * 2016-09-30 2017-04-19 广州白云山拜迪生物医药有限公司 Culture medium and culture method for efficiently expressing plasmid DNA through Escherichia coli engineering bacteria
CN112725223A (en) * 2020-12-22 2021-04-30 北京艺妙神州医药科技有限公司 Method for improving plasmid fermentation yield
CN112725231A (en) * 2020-12-31 2021-04-30 上海汉尼生物细胞技术有限公司 Fermentation method for large-scale efficient expression of supercoiled plasmid DNA by escherichia coli

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050233421A1 (en) * 2004-04-08 2005-10-20 Boehringer Ingelheim Austria Gmbh Fed-batch fermentation process and culture medium for the production of plasmid DNA in E. coli on a manufacturing scale
WO2005098002A1 (en) * 2004-04-08 2005-10-20 Boehringer Ingelheim Austria Gmbh Method for producing plasmid dna on a manufacturing scale by fermentation of the escherichia coli k-12 strain jm108
CN106566795A (en) * 2016-09-30 2017-04-19 广州白云山拜迪生物医药有限公司 Culture medium and culture method for efficiently expressing plasmid DNA through Escherichia coli engineering bacteria
CN112725223A (en) * 2020-12-22 2021-04-30 北京艺妙神州医药科技有限公司 Method for improving plasmid fermentation yield
CN112725231A (en) * 2020-12-31 2021-04-30 上海汉尼生物细胞技术有限公司 Fermentation method for large-scale efficient expression of supercoiled plasmid DNA by escherichia coli

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AARON E. CARNES等: "Inducible Escherichia coli fermentation for increased plasmid DNA production", 《BIOTECHNOL APPL BIOCHEM》, vol. 45, no. 3, pages 155 - 166, XP071712813, DOI: 10.1042/BA20050223 *
AARON E. CARNES等: "Plasmid Fermentation Process for DNA Immunization Applications", 《METHODS MOL BIOL.》, vol. 1143, pages 197 - 217 *
张泉: "重组质粒DNA的规模化生产工艺及质量鉴定研究", 《中国博士学位论文全文数据库基础科学辑》, no. 4, pages 006 - 35 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116751731A (en) * 2023-08-11 2023-09-15 赛奥斯博生物科技(北京)有限公司 Fermentation process for large-scale plasmid production
CN116751731B (en) * 2023-08-11 2023-11-24 赛奥斯博生物科技(北京)有限公司 Fermentation process for large-scale plasmid production

Similar Documents

Publication Publication Date Title
KR102132132B1 (en) Fermentation method to improve recombinant human collagen production level
CN108220175B (en) High-density culture method and pH regulation and control method for saccharomyces cerevisiae
KR102015829B1 (en) Coenzyme Q10 Fermentation Production Process Based on Integrated Control of Online Oxygen Consumption and Conductivity
JP2020534787A (en) Continuous culture of black aspergillus and citric acid production method using it
CN103397005B (en) Production method of glucose oxidase
CN105368766A (en) Genetically engineered bacterium for producing pentamethylene diamine and method for preparing pentamethylene diamine
CN104845896B (en) Produce the bacterial strain and method of Weilan gum
CN111733200A (en) Fermentation process for improving production level of recombinant collagen by adjusting addition mode of PTM1
CN111748480B (en) Candida virginiana and application thereof
CN114164160A (en) Fermentation medium for efficiently producing plasmid DNA and fermentation production method
CN101842491B (en) Ethanol plant by-products is used for the purposes of yeast growth
CN116179356B (en) Method for high-density heterotrophic culture of chlamydomonas reinhardtii and application thereof
CN112608963A (en) Method for culturing pichia pastoris engineering bacteria through semi-continuous fermentation
EP2697381A1 (en) High efficiency fermentation process
CN109266578B (en) Escherichia coli ACThr1032 and application thereof in fermentation production of L-threonine
CN109402203A (en) Culture medium for expressing O-type foot-and-mouth disease inclusion body engineering bacteria
FI71766C (en) FRAMSTAELLNING AV ETHANOL GENOM HOEGEFFEKTIV BAKTERIEJAESNING.
CN115261361A (en) Industrial fermentation method for generating hyaluronidase by recombinant pichia pastoris
CN113969257B (en) Culture medium for producing insulin glargine
CN107988293B (en) Fermentation process for improving production level of recombinant human-derived collagen by adjusting pressure
CN108048511B (en) Fermentation process for adjusting pH and improving production level of recombinant human collagen
CN114507611B (en) High-temperature-resistant yeast for producing alpha-ketoglutarate and fermentation method
CN111574390A (en) Efficient green production and extraction process of amino acid
CN118006716B (en) Method for preparing recombinant human albumin with high expression and low O-glycosylation level
CN117487722B (en) Feed medium for high-density fermentation of soluble protein and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20220311