Fermentation method of recombinant A-type humanized III-type collagen strain

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation method of a recombinant A-type humanized III-type collagen strain. According to the fermentation method, degradation of collagen can be reduced, and meanwhile the fermentation yield can be increased. According to the method, ammonium citrate and bovine bone peptone are added at one time in the fermentation process, and then methanol fed-batch induced expression is carried out, so that the purpose of reducing collagen degradation is achieved. Results of the embodiment of the invention show that the fermentation method provided by the invention can improve the biosynthesis rate of the recombinant human collagen, increase the expression quantity of the recombinant human collagen and reduce the degradation rate of the collagen, and the fermentation yield level is improved by 10-20% or above compared with that of a common fermentation mode.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a fermentation method of a recombinant type A humanized type III collagen strain. Background Art

[0002] Collagen is the main component of animal connective tissue, and its common types are type I, type II, type III, type V and type XI. The collagen in human skin is mainly type I and type III. Natural collagen is generally composed of three single chains, and the molecular weight of each single chain is generally around 120kDa. Its amino acid composition is Gly-XY (X and Y represent amino acids, where Y is mostly proline), and there is also a certain proportion of hydroxyproline. Hydroxyproline plays an important role in maintaining the triple helix structure and biological properties of collagen.

[0003] At present, there are two main ways to obtain collagen. One is to extract collagen from animal tissues. The collagen obtained by this method is generally a mixture, unstable, and generally has the risk of cross-contamination. It has strong immunogenicity, which affects the application of collagen in medicine. The other is to obtain it through fermentation of recombinant strains.

[0004] With the emergence and rapid development of synthetic biology, gene recombination expression methods have become increasingly popular, and various cell hosts have been used to express foreign proteins. Chinese Patent 202410768271.5 discloses a recombinant type A humanized type III collagen and its expression engineered bacteria, but does not involve a fermentation method. Chinese Patent 201110327865.5 uses Pichia pastoris genetically engineered bacteria to express collagen, and obtains recombinant human collagen through fermentation culture, but at the end of fermentation, collagen is severely degraded, resulting in a low fermentation level, which is not conducive to industrial production. Therefore, at the end of fermentation, it is crucial to stabilize the product from being degraded by proteases. Chinese Patent 201810049618.5, after 84 hours of methanol induction, 0.2-0.5M L-lysine or L-arginine is added to the fermenter at one time to improve the stability of recombinant human collagen. The Pichia pastoris fermentation manual mentions that adding hydrolyzed peptone can effectively reduce the degradation of collagen, but hydrolyzed peptone is expensive, which will lead to higher production costs.

[0005] Therefore, there is an urgent need in the art for a recombinant bacterial fermentation method for reducing collagen degradation. Summary of the invention

[0006] The object of the present invention is to provide a fermentation method for a recombinant collagen strain. The method provided by the present invention can not only enable the recombinant strain to express a large amount of collagen, but also reduce the degradation of collagen during the fermentation process.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a fermentation method for a recombinant collagen strain, the method comprising:

[0009] The strain is inoculated into a fermentation medium for cultivation, glycerol is added after glycerol is consumed, and the addition of glycerol is stopped when the wet weight of the bacteria is 180-220 g / L, and then ammonium citrate and bovine bone peptone are added, and then methanol is added until the fermentation is completed; the amount of the glycerol added is such that the glycerol concentration in the culture medium is controlled to be 1.8%-2%; the amount of the methanol added is such that the methanol concentration in the culture medium is controlled to be 0.8%-1%.

[0010] Preferably, the strain is Pichia pastoris.

[0011] Preferably, the formula of the fermentation medium is: every 30L of fermentation medium includes 8.3g calcium sulfate, 182g potassium sulfate, 72.8g magnesium sulfate heptahydrate, 41.3g potassium hydroxide, 400g glycerol, 267ml concentrated phosphoric acid, and 10ml defoaming agent.

[0012] Preferably, the ammonium citrate and bovine bone peptone are added at once, with the total amount of ammonium citrate being 10 g / L and the total amount of bovine bone peptone being 20 g / L.

[0013] Preferably, the strain is inoculated into the fermentation medium at an inoculation rate of 5 to 12%.

[0014] Preferably, in the fermentation method, the fermentation temperature during the culture process is 25-30°C.

[0015] Preferably, in the fermentation method, the pH during the culture process is 4.5 to 5.4 and is adjusted with aqueous ammonia.

[0016] Preferably, in the fermentation method, the dissolved oxygen in the culture medium is not less than 20% during the culture process.

[0017] Preferably, the methanol is added for 90 to 120 hours.

[0018] Beneficial effects:

[0019] The fermentation method of the present invention can effectively prevent the target product from being degraded by proteases. In the fermentation process, after glycerol flow addition, ammonium citrate and bovine bone peptone are added once, and then methanol flow addition induction expression stage is carried out until the end of fermentation, so that the recombinant human collagen protein of the large tank fermentation product is stably retained by more than 95% and the expression amount is increased by about 20%, which significantly reduces the production cost, reduces the loss of human and material resources of the enterprise, stabilizes the supply, and generates huge economic value for the enterprise. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 It is the SDS-PAGE gel result figure of embodiment and comparative example. DETAILED DESCRIPTION

[0022] The invention provides a fermentation method of a recombinant collagen strain.

[0023] The Pichia yeast culture liquid is inoculated into the sterilized fermentation medium, and glycerol is fed. When the wet weight of the bacteria reaches 180-220 g / L, the feeding of glycerol is stopped. After adding ammonium citrate and bovine bone peptone at one time, methanol is fed and induced expression is carried out until the fermentation is completed.

[0024] The purpose of the present invention of adding glycerol is to supplement the carbon source, and the concentration is not limited, and 50% glycerol is preferred.

[0025] The amount of glycerol added in the present invention is to control the glycerol concentration in the culture medium to be 1.8%-2%, preferably 2%; the amount of methanol added is to control the methanol concentration in the culture medium to be 0.8%-1%, preferably 0.8%.

[0026] The formula of the Pichia pastoris 10L fermentation medium is 8.3g calcium sulfate, 182g potassium sulfate, 72.8g magnesium sulfate heptahydrate, 41.3g potassium hydroxide, 400g glycerol, 267ml concentrated phosphoric acid, and 10ml defoamer added into a 30L fermentation tank.

[0027] In the fermentation method of the present invention, the inoculation amount of the Pichia yeast liquid is 5-12%, the fermentation temperature is 25-30°C, the pH value is adjusted to 4.5-5.4 by ammonia water, the dissolved oxygen is not less than 20%, and the methanol induction time is 90-120 hours.

[0028] The adjustment method of dissolved oxygen (DO)>20% in the present invention includes: adjusting the rotation speed, tank pressure, air flow rate and methanol addition speed.

[0029] The timing of the end of fermentation in the present invention is when the fermentation product level no longer increases.

[0030] The fermentation method of the present invention is suitable for Pichia pastoris expressing collagen, preferably, an engineered bacterium prepared by the method described in Chinese Patent No. 202410768271.5.

[0031] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0032] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0033] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Referring to the method described in Chinese Patent No. 202410768271.5, an engineered Pichia pastoris expressing a recombinant type A humanized type III collagen gene was prepared and the following examples were performed.

[0035] Example 1

[0036] Fermentation medium (10 L): 8.3 g calcium sulfate, 182 g potassium sulfate, 72.8 g magnesium sulfate heptahydrate, 41.3 g potassium hydroxide, 400 g glycerol, 267 ml concentrated phosphoric acid, and 10 ml defoamer were added to a 30 L fermenter.

[0037] The seed liquid is added to a 30L fermentation tank containing 10L fermentation medium according to a 10% inoculum amount, the initial stirring speed is 200rpm, the tank pressure is 0.05MPa, the air flow and the speed are adjusted to make the dissolved oxygen (DO)>30%, and the pH is controlled to be 5.4 during the fermentation process. When the carbon source is exhausted, the dissolved oxygen rises suddenly, and 50% glycerol is added to supplement the carbon source, and the glycerol concentration in the culture medium is controlled to be 1.8%-2%, until the wet weight of the bacterial cell is 180-220g / L, and the addition of glycerol is stopped. At this time, the fermentation culture time is about 20 hours. After the glycerol is exhausted, ammonium citrate and bovine bone meal hydrolyzed peptone are added at one time, and the total amount of ammonium citrate is 10g / L and the total amount of bovine bone peptone is 20g / L. Methanol was started to be added, and the methanol concentration in the culture medium was controlled to be 0.8%-1%, and the methanol induction culture stage was entered. The dissolved oxygen (DO) was greater than 20% by adjusting the rotation speed, tank pressure, air flow rate and methanol addition rate. At the end of the fermentation, samples were taken for detection every 6 hours. After the fermentation was induced until the product level no longer increased, the fermentation was terminated, the fermentation broth was collected, and the supernatant of the fermentation broth was obtained by centrifugation. The collagen expression amount was calculated by running SDS-PAGE gel and scanning the grayscale.

[0038] Comparative Example 1

[0039] Fermentation medium (10 L): 8.3 g calcium sulfate, 182 g potassium sulfate, 72.8 g magnesium sulfate heptahydrate, 41.3 g potassium hydroxide, 400 g glycerol, 267 ml concentrated phosphoric acid, and 10 ml defoamer were added to a 30 L fermenter.

[0040] The seed liquid was added to a 30L fermentation tank containing 10L fermentation medium according to an inoculum of 10%, with an initial stirring speed of 200rpm and a tank pressure of 0.05MPa. The air flow rate and the speed were adjusted to make the dissolved oxygen (DO)>30%, and the pH was controlled to be 5.4 during the fermentation process. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerol was added to supplement the carbon source, and the glycerol concentration in the culture medium was controlled to be 1.8%-2%, until the wet weight of the bacterial cell was 180-220g / L, and the addition of glycerol was stopped. At this time, the fermentation culture time was about 20 hours. After the glycerol was exhausted, methanol was added once, and the methanol concentration in the culture medium was controlled to be 0.8%-1%, and the methanol induction culture stage was entered. The dissolved oxygen (DO)>20% was made by adjusting the speed, tank pressure, air flow and the speed of adding methanol. At the end of the fermentation, sampling and detection were performed every 6h. After the fermentation was induced to the product level, the fermentation was terminated, the fermentation liquid was collected, and the fermentation liquid supernatant was obtained by centrifugation. The collagen expression was calculated by running SDS-PAGE gel and scanning grayscale.

[0041] Comparative Example 2

[0042] Fermentation medium (10 L): 8.3 g calcium sulfate, 182 g potassium sulfate, 72.8 g magnesium sulfate heptahydrate, 41.3 g potassium hydroxide, 400 g glycerol, 267 ml concentrated phosphoric acid, and 10 ml defoamer were added to a 30 L fermenter.

[0043] The seed liquid was added to a 30L fermentation tank containing 10L fermentation medium at an inoculation rate of 10%, with an initial stirring speed of 200rpm and a tank pressure of 0.05MPa. The air flow rate and the speed were adjusted to make the dissolved oxygen (DO)>30%, and the pH was controlled to be 5.4 during the fermentation process. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerol was added to supplement the carbon source. The glycerol concentration in the culture medium was controlled to be 1.8%-2%, and the addition of glycerol was stopped until the wet weight of the bacteria reached 180-220g / L. At this time, the fermentation culture time was about 20 hours. After the glycerol is exhausted, ammonium citrate is added all at once, and the total amount of ammonium citrate is 10 g / L. Methanol is then added, and the methanol concentration in the culture medium is controlled to be 0.8%-1%. The methanol induction culture stage is entered, and the dissolved oxygen (DO) is greater than 20% by adjusting the rotation speed, tank pressure, air flow rate and methanol addition rate. At the end of the fermentation, sampling is performed every 6 hours. After the fermentation is induced until the product level no longer increases, the fermentation is terminated, the fermentation broth is collected, and the supernatant of the fermentation broth is obtained by centrifugation. The collagen expression amount is calculated by running SDS-PAGE gel and scanning grayscale.

[0044] Comparative Example 3

[0045] Fermentation medium (10 L): 8.3 g calcium sulfate, 182 g potassium sulfate, 72.8 g magnesium sulfate heptahydrate, 41.3 g potassium hydroxide, 400 g glycerol, 267 ml concentrated phosphoric acid, and 10 ml defoamer were added to a 30 L fermenter.

[0046] The seed liquid was added to a 30L fermentation tank containing 10L fermentation medium at an inoculation rate of 10%, with an initial stirring speed of 200rpm and a tank pressure of 0.05MPa. The air flow rate and the speed were adjusted to make the dissolved oxygen (DO)>30%, and the pH was controlled to be 5.4 during the fermentation process. When the carbon source was exhausted, the dissolved oxygen rose sharply, and 50% glycerol was added to supplement the carbon source. The glycerol concentration in the culture medium was controlled to be 1.8%-2%, and the addition of glycerol was stopped until the wet weight of the bacteria reached 180-220g / L. At this time, the fermentation culture time was about 20 hours. After the glycerol is exhausted, bovine bone peptone is added at one time, and the total amount of bovine bone peptone is 20 g / L. Methanol is then added in a flow manner, and the methanol concentration in the culture medium is controlled to be 0.8%-1%. The methanol induction culture stage is entered, and the dissolved oxygen (DO) is greater than 20% by adjusting the rotation speed, tank pressure, air flow rate and methanol addition rate. At the end of the fermentation, sampling is performed every 6 hours. After the fermentation is induced until the product level no longer increases, the fermentation is terminated, the fermentation broth is collected, and the fermentation broth supernatant is obtained by centrifugation. The collagen expression amount is calculated by running SDS-PAGE gel and scanning the grayscale.

[0047] The SDS-PAGE gel results of the above embodiments and comparative examples are as follows: Figure 1 As shown, after calculation: the overall expression of comparative example 1 is 12.8 g / L, but the degradation is serious, the main band ( Figure 1 The uppermost band in the sample has a molecular weight of about 45 kda) and only 2 g / L. The overall expression level of comparative example 2 is 13.8 g / L, but the degradation is serious, and the main band is only 3 g / L. The overall expression level of comparative example 3 is 14.8 g / L, but the degradation is serious, and the main band is only 3.5 g / L. The overall expression level of embodiment 1 is 15.4 g / L, and the main band is 5 g / L. Therefore, the fermentation method provided by the present invention can improve the biosynthesis rate of recombinant human collagen, increase the expression level of recombinant human collagen and reduce the degradation rate of collagen. The fermentation yield level is increased by more than 10-20% compared with the general fermentation method.

[0048] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fermentation method for a recombinant collagen strain, characterized in that: The method comprises: inoculating the strain into a fermentation medium for culturing, adding glycerol after glycerol is consumed, stopping adding glycerol when the wet weight of the bacterial body is 180-220 g / L, adding ammonium citrate and bovine bone peptone, and then adding methanol until the fermentation is completed; the amount of the glycerol added is such that the glycerol concentration in the culture medium is controlled to be 1.8%-2%; the amount of the methanol added is such that the methanol concentration in the culture medium is controlled to be 0.8%-1%.

2. The method according to claim 1, characterized in that The strain is Pichia pastoris.

3. The method according to claim 1, characterized in that The formula of the fermentation medium is as follows: every 30L of the fermentation medium includes 8.3g of calcium sulfate, 182g of potassium sulfate, 72.8g of magnesium sulfate heptahydrate, 41.3g of potassium hydroxide, 400g of glycerol, 267ml of concentrated phosphoric acid, and 10ml of a defoaming agent.

4. The method according to claim 1, characterized in that: The ammonium citrate and bovine bone peptone are added in a one-time manner, and the added amounts are: the total amount of ammonium citrate is 10 g / L, and the total amount of bovine bone peptone is 20 g / L.

5. The method according to claim 1, characterized in that The inoculation amount of the strain inoculated into the fermentation medium is 5-12%.

6. The method according to claim 1, characterized in that In the fermentation method, the fermentation temperature during the culturing process is 25 to 30°C.

7. The method according to claim 1, characterized in that In the fermentation method, the pH during the culture process is 4.5 to 5.4 and is adjusted with aqueous ammonia.

8. The method according to claim 1, characterized in that In the fermentation method, the dissolved oxygen in the culture medium is not less than 20% during the culture process.

9. The method according to claim 1, characterized in that: The time for adding methanol is 90 to 120 hours.

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