Method for efficiently producing plasmids

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for efficiently producing plasmids. The method comprises the following steps: (1) preparing a fed-batch culture medium; (2) inoculating the strain into a fermentation culture medium, and carrying out fermentation culture for 4-6 hours; (3) using the fed culture medium for continuous feeding, controlling the first flow rate of the fed culture medium at 5-8 mL / h, continuously feeding for 10-14 h at the first flow rate, and then adjusting the flow rate of the fed culture medium to a second flow rate of 20-40 mL / h until fermentation is finished; wherein in the continuous feeding process, the specific growth rate of thalli is controlled to be 0.1-0.5 h <-1 >. According to the present invention, the high density fermentation requirement can be met and the plasmid yield can be improved by using the provided material supplementing culture medium to carry out the plasmid strain fermentation culture, and the fermentation process is further optimized, and the material supplementing culture medium is supplemented in the gradient manner by limiting the specific growth rate so as to further improve the plasmid yield.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for efficiently producing plasmids. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Plasmids are circular DNA elements that are free from the genome and are widely used in the field of gene and cell therapy. In addition, plasmids are also important carriers of DNA vaccines and play an important role in the field of medicine. As a gene carrier, plasmid DNA is often the main cost of gene drugs.

[0004] In the prior art, Fabiola Islas-Lugo et al. designed a single-factor experiment, designed to use ammonia water instead of sodium hydroxide solution to adjust the pH, increased the fermentation cell density, and increased the plasmid yield to 213 mg pDNA / L, 10 mgpDNA / g DCW, and the pDNA productivity increased by 60%. The production of acetic acid and lactic acid is caused by glycolysis and tricarboxylic acid cycle, which will lead to the loss of carbon source and thus reduce the plasmid yield. Gheorghe M Borja et al. knocked out the genes of metabolic byproducts such as acetic acid and lactic acid, so that the metabolic flow flowed more to plasmid synthesis, and the plasmid yield reached 186 mg / L pDNA. MAIRHOFER et al. further increased the plasmid yield to 1.1 g / L by transforming the plasmid into a non-resistance gene-dependent plasmid, and the total plasmid yield increased by 2 times.

[0005] However, the inventors found that although the prior art has optimized plasmid production, the current plasmid production efficiency is still low. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a method for efficiently producing plasmids to solve the problems existing in the above-mentioned prior art. The feed medium provided by the present invention is used for plasmid strain fermentation culture, which can meet the requirements of high-density fermentation and improve the yield of plasmids. The growth rate of the bacteria is controlled by controlling the feeding rate of the feed medium. The fed-batch fermentation medium in the present invention is combined with the feeding method to further improve the yield of plasmids, thereby effectively improving the production capacity of plasmid production and reducing production costs.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides a method for efficiently producing a plasmid, the method comprising:

[0009] (1) preparing a feed medium, wherein the feed medium comprises the following components: 0.001-0.005 g / L thiamine hydrochloride, 0.01-0.1 g / L CoCl2·6H2O, 0.01-0.2 g / L ZnSO4·6H2O, 0.01-0.5 g / L CaCl2·2H2O, 0.01-0.2 g / L FeSO4·7H2O, 5-20 g / L MgSO4, 100-500 g / L glucose, 20-50 g / L yeast extract, and 20-50 g / L tryptone;

[0010] (2) Inoculate the strain into the fermentation medium and ferment for 4-6 hours;

[0011] (3) Using the feed medium for continuous feeding, the first flow rate of the feed medium is 5-8 mL / h, the feeding is continuously performed for 10-14 h at the first flow rate, and then the flow rate of the feed medium is adjusted to a second flow rate of 20-40 mL / h until the bacterial cells stop growing and the fermentation is ended; wherein the bacterial cell specific growth rate is controlled at 0.1-0.5 h during the continuous feeding process. -1 .

[0012] The present invention provides a new feed medium, and the fermentation conditions are optimized with the feed medium, the bacterial growth is finely controlled, and the bacterial specific growth rate is controlled by controlling the feed rate, so that the plasmid production can be maximized. By optimizing the feed medium and the growth control strategy, the plasmid yield in the fermenter can reach 2.3g / L.

[0013] In a specific embodiment, the feed medium in step (1) comprises: 0.001 g / L thiamine hydrochloride, 0.07 g / L CoCl2·6H2O, 0.17 g / L ZnSO4·6H2O, 0.23 g / L CaCl2·2H2O, 0.17 g / L FeSO4·7H2O, 10 g / L MgSO4, 200 g / L glucose, 30 g / L yeast extract, and 50 g / L tryptone.

[0014] In a specific embodiment, the temperature control conditions of the fermentation culture in step (2) are 37° C., pH 7.0, and dissolved oxygen is controlled at 40%.

[0015] In a specific embodiment, 50 mg·mL of -1 kanamycin solution, 100×MgSO4·7H2O mother solution, 500 g / L glucose solution.

[0016] In a specific embodiment, the initial fermentation parameters in step (2) are set to 1 L of liquid volume, 400-800 rpm of linkage speed, 1-3 lpm of compressed air ventilation, and 0-1 lpm of oxygen.

[0017] In a specific embodiment, the strain in step (2) is DH5α.

[0018] In a specific embodiment, the composition of the fermentation medium in step (2) is: 24 g / L yeast powder, 12 g / L tryptone, 3.8 g / L potassium dihydrogen phosphate, 16.38 g / L potassium hydrogen phosphate, and 1.03 g / L MgSO4.7H2O.

[0019] In a specific embodiment, during the continuous feeding process in step (3), the specific growth rate of the bacteria is controlled at 0.12 h -1 .

[0020] In a specific embodiment, in step (3), the first flow rate is 7.3 mL / h, and the second flow rate is 31.9 mL / h.

[0021] In the second aspect of the present invention, there is provided an application of the method for efficiently producing a plasmid according to the first aspect in plasmid preparation.

[0022] The present invention has the following beneficial effects:

[0023] The present invention provides a novel fermentation feed medium, which can be used for plasmid strain fermentation culture, and can improve the plasmid yield while increasing the bacterial concentration. At the same time, the present invention further optimizes the fermentation process, and after a certain period of fermentation using the basic medium in the early stage, the feed medium is gradually added by limiting the specific growth rate to further improve the plasmid yield. The plasmid yield can reach 2.8 g / L by using the feed medium of the present invention and its supporting fermentation process for fermentation production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 This is a diagram of the plasmid yield in the fermentation tank in Example 1 of the present invention;

[0026] Figure 2 This is a diagram of the plasmid yield in the fermentation tank in Example 2 of the present invention;

[0027] Figure 3 This is a diagram of the plasmid yield in the fermentation tank in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, they are generally in accordance with conventional methods and conditions in the art, and such techniques and conditions are fully explained in the literature.

[0030] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Example 1

[0032] 1. Seed solution preparation

[0033] Transfer the DH5α glycerol bacteria to 100 mL of resistant LB liquid medium and culture at 37°C and 200 rpm for about 10 h until the bacterial OD600 reaches 3.5 to obtain seed solution;

[0034] 2. Culture medium preparation

[0035] The fermentation medium formula is as follows: yeast powder 24 g / L, tryptone 12 g / L, potassium dihydrogen phosphate 3.8 g / L, potassium hydrogen phosphate 16.38 g / L, MgSO4 .7H2O 1.03 g / L.

[0036] The feed medium formulation is as follows:

[0037] Glucose 200g / L.

[0038] 3. Fermentation tank inoculation

[0039] Before inoculation, add 500 μl of 50 mg / mL kanamycin solution, 10 mL of 100×MgSO4·7H2O mother solution, and 10 mL of 500 g / L glucose solution to the fermentation medium, and inoculate 100 mL of bacterial solution into the fermenter.

[0040] The initial fermentation parameters were set to 1 L of liquid volume, culture temperature of 37 °C, pH 7.0, dissolved oxygen control of 40%, linkage speed of 300-800 rpm, compressed air ventilation of 2 lpm, and oxygen of 0.5 lpm.

[0041] 4. Feeding method

[0042] When glucose was exhausted and dissolved oxygen suddenly increased, glucose was immediately replenished to 7.5 g / L.

[0043] 5. Sampling and data detection

[0044] Sampling was performed at intervals of 2.5 h using a sampler and a 20 mL disposable syringe equipped with the fermenter. The first sample was 9 mL, which was discarded. Then 5 mL was collected into a 15 mL centrifuge tube, and the sampling time and fermentation time were marked. 1 mL of bacterial solution was taken and diluted to an appropriate multiple to an absorbance of 0.5. The OD600 of the bacteria was determined using a visible-ultraviolet spectrophotometer. Each sample was run in triplicate. 1 mL of bacterial solution was centrifuged to collect the bacteria, the plasmid was extracted to determine the concentration, and the plasmid was eluted with 100 μL of sterile purified water. Each sample was run in triplicate. Figure 1 This is a graph of the plasmid yield in the fermentation tank in Example 1 of the present invention. As shown in the figure, the bacterial cell stopped growing after 11 hours of fermentation, and the final dry weight reached 22.0 g / L, and the plasmid yield was 2.2 g / L.

[0045] Example 2

[0046] 1. Seed solution preparation

[0047] Transfer the DH5α glycerol bacteria to 100 mL of resistant LB liquid medium and culture at 37°C and 200 rpm for about 10 h until the bacterial OD600 reaches 3.5 to obtain seed solution;

[0048] 2. Culture medium preparation

[0049] The fermentation medium formula is the same as that in Example 1;

[0050] The feed medium formula is as follows: thiamine hydrochloride 0.001 g / L, CoCl2·6H2O 0.07 g / L, ZnSO4·6H2O0.17 g / L, CaCl2·2H2O 0.23 g / L, FeSO4·7H2O 0.17 g / L, MgSO4 10 g / L, glucose 200 g / L, yeast extract 30 g / L, and tryptone 50 g / L.

[0051] 3. Fermentation tank inoculation

[0052] Before inoculation, add 500 μl of 50 mg mL-1 kanamycin solution, 10 mL 100×MgSO4·7H2O mother solution, 10 mL 500 g / L glucose solution, and inoculate 100 mL of bacterial solution into the fermenter.

[0053] The initial fermentation parameters were set to 1 L of liquid volume, culture temperature of 37 °C, pH 7.0, dissolved oxygen control of 40%, linkage speed of 600 rpm, compressed air ventilation of 2 lpm, and oxygen of 0.5 lpm.

[0054] 4. Feeding method

[0055] When the glucose was exhausted and the dissolved oxygen suddenly increased, 4 mL of the above feed medium was immediately added.

[0056] 5. Sampling and data detection

[0057] Sampling was performed at intervals of 2.5 h using a sampler and a 20 mL disposable syringe equipped with the fermenter. The first sample was 9 mL, which was discarded. Then 5 mL was collected into a 15 mL centrifuge tube, and the sampling time and fermentation time were marked. 1 mL of bacterial solution was taken and diluted to an appropriate multiple to an absorbance of 0.5. The OD600 of the bacteria was determined using a visible-ultraviolet spectrophotometer. Each sample was run in triplicate. 1 mL of bacterial solution was centrifuged to collect the bacteria, the plasmid was extracted to determine the concentration, and the plasmid was eluted with 100 μL of sterile purified water. Each sample was run in triplicate. Figure 2 This is a graph of the plasmid yield in the fermentation tank in Example 2 of the present invention. As shown in the figure, the bacterial cells stopped growing after 12 hours of fermentation, and the final dry weight reached 22.5 g / L, and the plasmid yield was 2.4 g / L.

[0058] Example 3

[0059] 1. Seed solution preparation

[0060] Transfer the DH5α glycerol bacteria to 100 mL of resistant LB liquid medium and culture at 37°C and 200 rpm for about 10 h until the bacterial OD600 reaches 3.5 to obtain seed solution;

[0061] 2. Culture medium preparation

[0062] The fermentation medium formula is the same as that in Example 1;

[0063] The feed medium formula is the same as that in Example 2

[0064] 3. Fermentation tank inoculation

[0065] Before inoculation, add 500 μL of 50 mg mL -1kanamycin solution, 10 mL 100×MgSO4·7H2O mother solution, 10 mL 500 g / L glucose solution, and 100 mL seed solution was inoculated into the fermenter.

[0066] The initial fermentation parameters were set to 1 L of liquid volume, culture temperature of 37 °C, pH 7.0, dissolved oxygen control of 40%, linkage speed of 600 rpm, compressed air ventilation of 2 lpm, and oxygen of 0.5 lpm.

[0067] 4. Feeding method:

[0068] After culturing in the fermentation medium for 5 h, the above feed medium was used for continuous feeding. First, the feed medium was fed at a first flow rate of 7.3 mL / h for 12 h, and the flow rate of the feed medium was adjusted to a second flow rate of 31.9 mL / h until the bacterial cells stopped growing and the fermentation was ended. During the continuous feeding process, the specific growth rate of the bacterial cells was controlled at 0.12 h -1 .

[0069] 5. Sampling and data detection:

[0070] Sampling was performed at intervals of 2.5 h using a sampler and a 20 mL disposable syringe equipped with the fermenter. The first sample was 9 mL, which was discarded. Then 5 mL was collected into a 15 mL centrifuge tube, and the sampling time and fermentation time were marked. 1 mL of bacterial solution was taken and diluted to an appropriate multiple to an absorbance of 0.5. The OD600 of the bacteria was determined using a visible-ultraviolet spectrophotometer. Each sample was run in triplicate. 1 mL of bacterial solution was centrifuged to collect the bacteria, the plasmid was extracted to determine the concentration, and the plasmid was eluted with 100 μL of sterile purified water. Each sample was run in triplicate. Figure 3 This is a graph of the plasmid yield in the fermentation tank in Example 3 of the present invention. As shown in the figure, the bacterial cell stopped growing after 29 hours of fermentation, and the final dry weight reached 43.3 g / L, and the plasmid yield was 2.8 g / L.

[0071] From the comparison of the results of Examples 1 to 3, it can be seen that the use of the feed medium provided in Example 3 of the present application and the gradient addition of the feed medium by limiting the specific growth rate can meet the requirements of high-density fermentation and increase the yield of the plasmid.

[0072] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for efficiently producing a plasmid, characterized in that: The method comprises: (1) preparing a feed medium, wherein the feed medium comprises the following components: 0.001-0.005 g / L thiamine hydrochloride, 0.01-0.1 g / L CoCl2·6H2O, 0.01-0.2 g / L ZnSO4·6H2O, 0.01-0.5 g / L CaCl2·2H2O, 0.01-0.2 g / L FeSO4·7H2O, 5-20 g / L MgSO4, 100-500 g / L glucose, 20-50 g / L yeast extract, and 20-50 g / L tryptone; (2) Inoculate the strain into the fermentation medium and ferment for 4 to 6 hours; (3) Using the feed medium for continuous feeding, the first flow rate of the feed medium is 5-8 mL / h, the first flow rate is continuously fed for 10-14 h, and then the flow rate of the feed medium is adjusted to a second flow rate of 20-40 mL / h until the fermentation ends; wherein the bacterial cell specific growth rate is controlled at 0.1-0.5 h during the continuous feeding process. -1 .

2. The method according to claim 1, characterized in that The feed medium comprises: 0.001 g / L thiamine hydrochloride, 0.07 g / L CoCl2·6H2O, 0.17 g / L ZnSO4·6H2O, 0.23 g / L CaCl2·2H2O, 0.17 g / L FeSO4·7H2O, 10 g / L MgSO4, 200 g / L glucose, 30 g / L yeast extract, and 50 g / L tryptone.

3. The method according to claim 1, characterized in that The temperature control conditions of the fermentation culture in step (2) are 37° C., pH 7.0, and dissolved oxygen is controlled at 40%.

4. The method according to claim 1, characterized in that The strain described in step (2) is DH5α.

5. The method according to claim 1, characterized in that The composition of the fermentation medium in step (2) is: 24 g / L yeast powder, 12 g / L tryptone, 3.8 g / L potassium dihydrogen phosphate, 16.38 g / L potassium hydrogen phosphate, and 1.07 g / L MgSO4.7H2O.

6. The method according to claim 1, characterized in that Before inoculation in step (2), 50 mg mL -1 kanamycin solution, 100×MgSO4·7H2O mother solution, 500 g / L glucose solution.

7. The method according to claim 1, characterized in that In step (2), the initial fermentation parameters are set to 1 L of liquid volume, 400-800 rpm of linkage speed, 1-3 lpm of compressed air ventilation, and 0-1 lpm of oxygen.

8. The method according to claim 1, characterized in that During the continuous feeding process in step (3), the specific growth rate of the bacteria was controlled at 0.12 h -1 .

9. The method according to claim 1, characterized in that In step (3), the first flow rate is 7.3 mL / h, and the second flow rate is 31.9 mL / h.

10. Use of the method for efficiently producing a plasmid according to any one of claims 1 to 9 in plasmid preparation.

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