CN116751731A - Fermentation process for large-scale plasmid production - Google Patents
Fermentation process for large-scale plasmid production Download PDFInfo
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Abstract
The application discloses a fermentation process for large-scale plasmid production, and relates to the technical field of microbial fermentation. The fermentation process is carried out by adopting a fermentation medium, wherein the fermentation medium comprises a basic medium and a feed medium; the basic culture medium comprises tryptone, yeast powder, sodium chloride, glycerol, monopotassium phosphate, dipotassium phosphate, magnesium sulfate heptahydrate and defoamer; the feed medium comprises tryptone, yeast powder, glycerol, magnesium sulfate heptahydrate, proline, leucine and defoamer. The application develops a new fermentation medium based on the existing fermentation medium, and the culture medium is used for carrying out the fermentation culture of the plasmid strain, so that the requirement of high-density fermentation can be met, the yield of plasmids is improved, the productivity of plasmid production is further improved, and the production cost is reduced.
Description
Technical Field
The application relates to the technical field of microbial fermentation, in particular to a fermentation process for large-scale plasmid production.
Background
In recent years, with the continued development of biologicals, DNA plasmid products, including DNA vaccines, viral vectors, and the like, are continually being used in new gene therapies. The fermentation of E.coli to obtain DNA plasmids is a common technique. Thus, the technology for producing high purity and high quality DNA plasmids by large scale fermentation is becoming increasingly important. However, plasmid mutation or loss occurs during the culture process, thereby affecting plasmid yield and increasing production cost. Plasmid mutation refers to the mutation of DNA plasmid during the process of culturing recombinant bacteria, and the original phenotypic characteristics are lost. The main reason for the loss of plasmid is that when the cells divide, the plasmid is not evenly distributed in the daughter cells due to the fast growth of microorganisms, and part of daughter cells do not contain the plasmid. Plasmid mutation and loss often occur in cases where the plasmid is mutated and lost for one or several passages; 2. during the long-time fermentation culture process. The recombinant genetically engineered bacteria ferment DNA plasmids on a large scale and are easily influenced by culture temperature, pH, rotating speed, DO, feeding speed, culture medium composition and the like. The existing fermentation process does not carry out multiparameter process development aiming at plasmids for lentivirus VSVG/RD114/REV/PMDLG, and as a result, the yield of plasmids is not high. The production by the existing DNA plasmid fermentation process still faces the problem of insufficient DNA plasmid yield. The current process method cannot meet the requirements of economy and scale, so that development and optimization of a new process method are necessary.
Disclosure of Invention
The application aims to provide a fermentation process for large-scale plasmid production, which solves the problems in the prior art, and the fermentation medium provided by the application is used for carrying out the fermentation culture of plasmid strains, so that the requirement of high-density fermentation can be met, and the yield of plasmids is improved; the fermentation medium is combined with an optimized fermentation process, so that the yield of plasmids can be further improved, the productivity of plasmid production can be effectively improved, and the production cost is reduced.
In order to achieve the above object, the present application provides the following solutions:
the application provides a fermentation medium for large-scale plasmid production, which comprises a basic medium and a feed medium;
the basal medium comprises the following components in parts by weight: 8.0-12.0g/L of tryptone, 4.0-6.0g/L of yeast powder, 9.0-11.0g/L of sodium chloride, 12.0-13.0g/L of glycerin, 1.2-2.0g/L of monopotassium phosphate, 2.0-2.5g/L of dipotassium phosphate, 0.2-0.3g/L of magnesium sulfate heptahydrate and 1.5-2.5mL/L of defoamer;
the feed medium comprises the following components in parts by weight: 70.0-72.0g/L of tryptone, 70.0-72.0g/L of yeast powder, 168.0-172.0g/L of glycerol, 1.5-2.5g/L of magnesium sulfate heptahydrate, 0.6-1.0g/L of proline, 0.6-1.0g/L of leucine and 0.8-1.2mL/L of defoamer.
Preferably, the basal medium comprises 10.0g/L tryptone, 5.0g/L yeast powder, 10.0g/L sodium chloride, 12.6g/L glycerol, 1.5g/L potassium dihydrogen phosphate, 2.3g/L dipotassium hydrogen phosphate, 0.25g/L magnesium sulfate heptahydrate and 2.0mL/L defoamer;
the feed medium comprises tryptone 71.0g/L, yeast powder 71.0g/L, glycerin 170.0g/L, magnesium sulfate heptahydrate 1.99g/L, proline 0.83g/L, leucine 0.83g/L and defoamer 1.0mL/L.
Further, the defoamer is anti-foam 204.
The application also provides application of the fermentation medium in large-scale plasmid production.
Furthermore, the genetically engineered strain containing the target plasmid used for large-scale plasmid production is an escherichia coli recombinant strain, and the target plasmid is a recombinant plasmid containing VSVG, RD114, REV or PMDLG genes.
The application also provides a fermentation process for large-scale plasmid production, which comprises the step of fermenting and culturing the genetically engineered strain containing the target plasmid by utilizing the fermentation medium.
Further, the genetically engineered strain is an escherichia coli recombinant strain, and the target plasmid is a recombinant plasmid containing VSVG, RD114, REV or PMDLG genes.
Further, the fermentation culture comprises: inoculating the genetic engineering strain into a basic culture medium of the fermentation culture medium, culturing for 5 hours, and performing exponential feed fermentation according to a feed rate F shown in the following formula:
wherein μ: specific growth rate of bacteria, V 0 : the volume of the culture medium in the feed starting tank; ρ Co : the quality concentration of bacteria in the tank when the material supplementing is started;Y X/s : the yield of the thalli; ρ sF : total sugar mass concentration of the feed medium; ρ s : starting the total sugar mass concentration in the tank in the feeding period; t: index feed time.
Further, the temperature control conditions of the fermentation culture are as follows: 0 to 21 hours at 33 ℃,21 to 24 hours at 35 ℃,24 to 30 hours at 42 ℃.
Further, the pH of the fermentation culture is 7.0; after 17 hours of fermentation culture, dissolved oxygen is controlled at 30%.
The application discloses the following technical effects:
the application is based on the existing fermentation culture medium and fermentation process, optimizes the fermentation process of plasmid strains, and forms a new fermentation culture medium and a matched fermentation process thereof. The fermentation medium comprises a basic medium and a feed supplement medium, and the plasmid strain is fermented and cultured by using the fermentation medium, so that the requirement of high-density fermentation can be met, and the yield of plasmids is improved. Meanwhile, the application further optimizes the fermentation process, specifically, after the fermentation is performed for a certain time by utilizing a basic fermentation medium in the early stage, the exponential feed-supplementing fermentation is performed by utilizing an optimized feed-supplementing medium at a limited specific growth rate, and the fermentation temperature control parameter is optimized so as to further improve the yield of plasmids. The culture medium and the matched fermentation process thereof are used for carrying out the fermentation production of plasmid strains, and the plasmid yield can reach 400-716 mg/L, so that the productivity of plasmid production can be effectively improved, and the production cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the fermentation growth and plasmid growth curves of the RD114 recombinant strains 0-28h of example 1.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
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 application. 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 application. 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 application 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 application. 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 application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application 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.
Description of the terminology:
the large-scale plasmid production referred to in the present application means that fermentation culture is performed using a fermenter having a capacity of 1L or more in the plasmid production.
The four recombinant strains used in the following examples are constructed by entrusting Saiosburg biotechnology (Beijing) limited company, specifically, a target gene sequence (VSVG/RD 114/REV/PMDLG) is synthesized firstly, then a circular plasmid is constructed by adopting a cloning mode of plasmid complete sequence synthesis and cyclization (according to a skeleton sequence pLenti-EF1 a-backbond (NG) of an expression vector plasmid obtained on an adedge website, ID: # 27963), the constructed plasmid is introduced into a Stbl3 escherichia coli host strain by a chemical conversion mode, and finally recombinant genetic engineering bacteria meeting requirements are identified and screened. Wherein the VSVG, RD114, REV and PMDLG gene sequences are shown in SEQ ID NO.1-4 respectively.
Example 1
Recombinant strains of VSVG, RD114, REV and PMDLG were subjected to the following cultivation operations, respectively:
1. strain resuscitation
And taking out the working strains from the freezer at the temperature of minus 80 ℃ and putting the working strains into a water bath at the temperature of 37 ℃ for 30min to completely melt.
2. Seed liquid culture
1. First-stage seed culture
50 mu L of the thawed working bacteria are sucked under aseptic conditions, inoculated into 5mL of LB culture medium (containing antibiotics), and placed on a shaking table at 37 ℃/200rpm for shaking culture for 17 hours, so that primary seed liquid is obtained.
2. Two-stage seed culture
Inoculating 5mL of the first-stage seed bacterial liquid into 250mL of LB culture medium (containing antibiotics) under aseptic condition, shaking and culturing for 5 hours at 37 ℃/200rpm by a shaking table, and detecting OD by aseptic sampling 600 The culture was stopped at a value of 1.2, whichIs a secondary seed liquid.
3. Fermentation culture
1. The basal medium formulation is shown in table 1:
TABLE 1
2. The feed medium (designated B1) formulation is shown in table 2:
TABLE 2
3. Fermentation tank preparation
And connecting the DO/pH electrode with a fermentation control cabinet, and energizing for 2h. The pH electrode was calibrated with a standard solution of 4.01/7.00 and both electrodes were mounted to the fermenter. And simultaneously binding a feed supplement pipe connected with the fermentation tank and adding 6L of fermentation medium. High-pressure damp-heat sterilization is carried out at 121 ℃/20min, and the cooled product is connected with a fermentation cabinet.
4. Fermentation process
(1) Fermentation tank control conditions before inoculation:
temperature: 33 ℃, ph=7, do correction 100%, aeration 5L/min, stirring 200rpm.
(2) The secondary seed 250mL of the bacterial liquid was aseptically inoculated into 6L of fermentation medium to which kana antibiotic was added at a final concentration of 50. Mu.g/mL.
Automatic control conditions of the fermentation tank after inoculation:
temperature: 0-21 h/33 ℃, 21-24 h/35 ℃, 24-30 h/42 ℃;
the pH value is 0-30 h/7.0;
DO linkage rotation speed: setting 0-30 h/DO to be 30%, and setting the rotation speed linkage range to be: 200-750 rpm;
ventilation volume: setting the concentration to be 5L/min, and switching on oxygen when DO is less than 10% at 17 h; controlling dissolved oxygen to be 30%;
after 5h of culture, carrying out exponential feed fermentation according to the feed rate F until the fermentation is finished:
annotation:
f: feeding rate, L/h.
Mu: specific growth rate of bacteria, h -1 In this example, μ was set to 0.693.
V 0 : and (5) feeding the volume of the culture medium in the starting tank and L.
ρ Co : and the mass concentration of bacteria in the tank, g/L, is carried out when the material supplementing is started.
Y X/s : yield of thallus,%.
ρ sF : total sugar mass concentration of feed medium, g/L.
ρ s : total sugar mass concentration in tank, g/L, at the beginning of the feeding period.
t: index feed time, h.
(3) Sampling and detecting:
sampling every 2 hours from 17 hours of culture, and measuring OD 600 Values.
Sampling every 1 hour from 23 hours of culture, and measuring OD 600 Values were obtained and the plasmid amount was measured.
(4) And (3) fermentation is finished:
after fermentation culture for 30 hours, the fermentation is finished.
In this example, the fermentation growth and plasmid growth curves of RD114 recombinant strains 0-28h are shown in FIG. 1.
Example 2
The only difference from example 1 is that the formulations of the basal medium and the feed medium are shown in tables 3 and 4, respectively.
TABLE 3 Table 3
TABLE 4 Table 4
Example 3
The only difference from example 1 is that the formulations of the basal medium and the feed medium are shown in tables 5 and 6, respectively.
TABLE 5
TABLE 6
Comparative example 1
The only difference from example 1 is that the feed medium was replaced with medium a shown in table 7.
TABLE 7
Comparative example 2
The only difference from example 1 is that the feed medium is replaced with medium C shown in Table 8.
TABLE 8
Comparative example 3
The same as in example 1 was followed except that the feed medium was replaced with medium D shown in Table 9.
TABLE 9
Comparative example 4
The only difference from example 1 is that the feed medium was replaced with medium E shown in Table 10.
Table 10
Comparative example 5
The difference with comparative example 1 is that the temperature in the self-control condition of the fermentation tank after the inoculation of the secondary seeds in the fermentation process is controlled to be 0-24 h/37 ℃ and 24-30 h/42 ℃.
Comparative example 6
The same as in comparative example 1 was differing only in that the feed rate in the self-controlling conditions of the fermenter after the inoculation of the secondary seeds in the fermentation process was adjusted to feed at a constant rate of 5 mL/min.
Example 1 and comparative examples 1-6 after fermentation was completed, the cells were collected by tank centrifugation (4000 rpm/20min/4 ℃) and plasmids were extracted (refer to the description of plasmid Mini kit), and wet cell weight, total plasmid amount, plasmid volume ratio and plasmid cell weight ratio were measured, and the results are shown in Table 7.
The recombinant genetically engineered bacteria are used for large-scale fermentation production of DNA plasmids, are easily influenced by factors such as culture temperature, feed supplement speed, culture medium composition and the like, and the application carries out process adjustment and formula optimization from the angles of the fermentation temperature, the feed supplement speed and the culture medium, uses the culture medium B as the feed supplement culture medium, and combines the optimized fermentation process (comprising adjustment of the fermentation temperature and the feed supplement speed), thereby effectively improving the yield of plasmids: as can be seen from the experimental data of example 1 and comparative examples 1-4, medium B is most suitable as a feed medium; as can be seen from the combination of the example 1 and the comparative example 5, compared with the temperature control condition of 0-24 h constant temperature 37 ℃ in the prior art, the optimized temperature control condition of the application obviously improves the plasmid yield; as can be seen from a combination of example 1 and comparative example 6, the exponential feed process employed in the present application also provides a significant improvement in plasmid yield over the constant rate feed process of the prior art.
TABLE 7
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Claims (10)
1. A fermentation medium for large-scale plasmid production, which is characterized by comprising a basic medium and a feed medium;
the basal medium comprises the following components in parts by weight: 8.0-12.0g/L of tryptone, 4.0-6.0g/L of yeast powder, 9.0-11.0g/L of sodium chloride, 12.0-13.0g/L of glycerin, 1.2-2.0g/L of monopotassium phosphate, 2.0-2.5g/L of dipotassium phosphate, 0.2-0.3g/L of magnesium sulfate heptahydrate and 1.5-2.5mL/L of defoamer;
the feed medium comprises the following components in parts by weight: 70.0-72.0g/L of tryptone, 70.0-72.0g/L of yeast powder, 168.0-172.0g/L of glycerol, 1.5-2.5g/L of magnesium sulfate heptahydrate, 0.6-1.0g/L of proline, 0.6-1.0g/L of leucine and 0.8-1.2mL/L of defoamer.
2. The fermentation medium of claim 1, wherein the basal medium comprises 10.0g/L tryptone, 5.0g/L yeast powder, 10.0g/L sodium chloride, 12.6g/L glycerol, 1.5g/L potassium dihydrogen phosphate, 2.3g/L dipotassium hydrogen phosphate, 0.25g/L magnesium sulfate heptahydrate, and 2.0mL/L defoamer;
the feed medium comprises tryptone 71.0g/L, yeast powder 71.0g/L, glycerin 170.0g/L, magnesium sulfate heptahydrate 1.99g/L, proline 0.83g/L, leucine 0.83g/L and defoamer 1.0mL/L.
3. The fermentation medium of claim 1, wherein the antifoaming agent is anti-foam 204.
4. Use of a fermentation medium according to any one of claims 1-3 for large scale plasmid production.
5. The use according to claim 4, wherein the genetically engineered strain containing the plasmid of interest used in the large-scale plasmid production is a recombinant strain of E.coli and the plasmid of interest is a recombinant plasmid containing VSVG, RD114, REV or PMDLG genes.
6. A fermentation process for large-scale plasmid production, comprising the step of fermenting and culturing a genetically engineered strain containing a plasmid of interest using the fermentation medium of any one of claims 1 to 3.
7. The fermentation process of claim 6, wherein the genetically engineered strain is an E.coli recombinant strain and the plasmid of interest is a recombinant plasmid containing VSVG, RD114, REV or PMDLG genes.
8. The fermentation process of claim 7, wherein the fermentation culture comprises: inoculating the genetic engineering strain into a basic culture medium of the fermentation culture medium, culturing for 5 hours, and performing exponential feed fermentation according to a feed rate F shown in the following formula:
wherein μ: specific growth rate of bacteria, V 0 : the volume of the culture medium in the feed starting tank; ρ Co : the quality concentration of bacteria in the tank when the material supplementing is started;Y X/s : the yield of the thalli; ρ sF : total sugar mass concentration of the feed medium; ρ s : starting the total sugar mass concentration in the tank in the feeding period; t: index feed time.
9. The fermentation process of claim 8, wherein the temperature control conditions of the fermentation culture are: 0 to 21 hours at 33 ℃,21 to 24 hours at 35 ℃,24 to 30 hours at 42 ℃.
10. The fermentation process of claim 8, wherein the fermentation broth has a pH of 7.0; after 17 hours of fermentation culture, dissolved oxygen is controlled at 30%.
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