Escherichia coli culture medium suitable for recombinant mussel mucin production and culture method

Abstract

The invention discloses an Escherichia coli fermentation culture medium suitable for recombinant mussel mucin production and a culture method. A common fermentation LB culture medium for escherichia coli is replaced, safer inorganic salt is adopted, the dosage of yeast powder and peptone is reduced, and ammonium salt with lower cost is used for replacing a traditional organic nitrogen source; by adopting the culture medium disclosed by the invention, the influence of the mussel mucin on the thallus activity is reduced and the thallus density and the expression quantity of the recombinant mussel mucin are improved by adding a nutrition promoter and a staged induction strategy in the fermentation process; on the other hand, by means of the culture method, the fermentation expression quantity of the recombinant mussel mucin is increased, so that the application requirements of the recombinant mussel mucin as an excellent bioactive raw material in the fields of functional skin care products, medical instruments, biomedicine and the like are met; the culture method disclosed by the invention has the characteristics of simple process and high yield, the expression quantity concentration of fermentation liquor can reach 2g / L or above, and the culture method is suitable for industrialization.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to an Escherichia coli culture medium suitable for the production of recombinant mussel adhesive protein and a culture method thereof. Background Art

[0002] Recombinant mussel adhesive protein has attracted much attention due to its wide potential in the fields of medicine and bioengineering. When producing such recombinant proteins in the Escherichia coli system, a significant problem is that when using LB medium for the soluble expression fermentation of recombinant mussel adhesive protein, cell growth is significantly inhibited and the yield drops substantially. Chinese Patent CN 116693692A discloses that after culturing co-rMfp151 protein in a 5-liter fermenter and going through the purification step, its yield only reaches a low level of 250 to 300 milligrams per liter. The Yuan Sheng team at Huazhong University of Science and Technology has also explored this. They successfully constructed various recombinant mussel adhesive proteins and carried out fermentation using LB medium. However, even for the four adhesive proteins M5, M151, 5LC, and 151LC, their crude protein yields are only approximately 170.55 milligrams per liter, 193.47 milligrams per liter, 203.10 milligrams per liter, and 200.80 milligrams per liter respectively, once again confirming the limitations of LB medium in increasing the yield of recombinant mussel adhesive protein and posing an obstacle to the commercialization and industrialization process of recombinant mussel adhesive protein.

[0003] In view of this, in order to accelerate the commercialization and industrialization of recombinant mussel adhesive protein, it is urgent to reduce the cell inhibitory effect during its production process and research and develop a new type of recombinant mussel adhesive protein culture medium and its preparation method, which will greatly increase the yield of recombinant protein and effectively reduce the production cost. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an Escherichia coli culture medium suitable for the production of recombinant mussel adhesive protein and a culture method thereof in view of the deficiencies of the above-mentioned prior art. The culture medium of the present invention contains specific nutritional promoters. Among them, glutathione, as a crucial metabolic regulator inside cells, can effectively reduce the stress on cells; while oleic acid and Tween 80, as highly efficient surfactant components, can significantly improve cell permeability; they act synergistically to not only relieve cell stress but also significantly enhance cell permeability.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is: An Escherichia coli culture medium suitable for the production of recombinant mussel adhesive protein, characterized in that it comprises a fermentation medium and a feeding medium; the concentrations of the components in the fermentation medium include: diammonium hydrogen phosphate 0.8 - 2.0 g / L, potassium dihydrogen phosphate 3.5 - 10.0 g / L, ammonium citrate 0.5 - 1.2 g / L, magnesium sulfate heptahydrate 0.5 - 2.0 g / L, glucose monohydrate 15 - 27 g / L, ferrous sulfate heptahydrate 40 - 90 mg / L, manganese sulfate pentahydrate 2 - 8 mg / L, zinc sulfate heptahydrate 10 - 25 mg / L, copper sulfate pentahydrate 5 - 20 mg / L, calcium chloride dihydrate 10 - 30 mg / L, borax 0.9 - 3 mg / L, ammonium molybdate 1.5 - 4 mg / L, organic silicon antifoaming agent 1 - 2 g / L, nutrient promoter 10 - 20 mL / L;

[0006] The nutrient promoter includes glutathione, oleic acid, Tween 80 and vitamin C ethyl ether; the concentrations of the components in the nutrient promoter are respectively: glutathione 6 - 16 g / L, oleic acid 2 - 5 g / L, Tween 80 2 - 5 g / L, vitamin C ethyl ether 10 - 20 g / L.

[0007] The above-mentioned Escherichia coli culture medium suitable for the production of recombinant mussel adhesive protein, characterized in that the feeding medium includes feeding medium A and feeding medium B, and the concentrations of the components in the feeding medium A include: magnesium sulfate heptahydrate 1.5 - 4.5 g / L, nutrient promoter 100 - 200 mL / L, ferrous sulfate heptahydrate 50 - 150 mg / L, manganese sulfate pentahydrate 5.0 - 15 mg / L, zinc sulfate heptahydrate 15 - 40 mg / L, copper sulfate pentahydrate 10 - 35 mg / L, calcium chloride dihydrate 20 - 50 mg / L, borax 3.0 - 8.0 mg / L, ammonium molybdate 3.0 - 7.0 mg / L; the feeding medium B is a glycerol solution.

[0008] The above-mentioned Escherichia coli culture medium suitable for the production of recombinant mussel adhesive protein, characterized in that the concentration of the glycerol solution is 400 - 700 g / L.

[0009] Furthermore, the present invention provides a method for culturing Escherichia coli for the production of recombinant mussel adhesive protein using the above-mentioned Escherichia coli culture medium, characterized in that it comprises the following steps:

[0010] Step 1: Inoculate Escherichia coli into a shake flask culture medium for activation;

[0011] Step 2: Inoculate the activated shake flask culture solution into a seed culture medium for seed culture;

[0012] Step 3: Inoculate the cultured seed liquid into the fermentation medium for fermentation culture and induction expression. During the fermentation culture and induction expression, a feeding medium is used for fed-batch feeding. Control the initial feeding rate of feeding medium A to be 30 - 40 mL / (L·h), and then increase it by 1 - 3 mL / (L·h) every 1 hour. Control the initial feeding rate of feeding medium B to be 15 - 25 mL / (L·h), and then increase it by 0.3 - 1 mL / (L·h) every 1 hour.

[0013] The method described above is characterized in that the formula of the shake flask medium in Step 1 includes: peptone 10 - 20 g / L, yeast extract powder 5 - 10 g / L, sodium chloride 5 - 20 g / L, glycerol 5 - 10 g / L; the activation culture conditions are: temperature is 34 - 39 °C, shaker speed is 150 - 250 rpm, culture time is 7 - 12 h, and culture until OD600 is 3 - 7.

[0014] The method described above is characterized in that the inoculation amount in Step 2 is 2 - 5% v / v; the formula of the seed medium includes: diammonium hydrogen phosphate 0.8 - 2.0 g / L, potassium dihydrogen phosphate 3.5 - 10.0 g / L, ammonium citrate 0.5 - 1.2 g / L, magnesium sulfate heptahydrate 0.5 - 2.0 g / L, glucose monohydrate 10 - 20 g / L, ferrous sulfate heptahydrate 30 - 80 mg / L, manganese sulfate pentahydrate 1.5 - 5.5 mg / L, zinc sulfate heptahydrate 8 - 20 mg / L, copper sulfate pentahydrate 4.5 - 15 mg / L, calcium chloride dihydrate 10 - 20 mg / L, borax 0.8 - 2 mg / L, ammonium molybdate 0.8 - 2.0 mg / L, and silicone antifoaming agent 0.5 - 1.0 g / L.

[0015] The method described above is characterized in that the seed culture conditions in Step 2 are: culture temperature is 34 - 39 °C, adjust the rotation speed and ventilation volume during the culture process to keep the dissolved oxygen level above 30%, adjust the pH to 6.5 - 7.0 with ammonia water, culture for 6 - 8 h, and culture until OD600 is 5 - 13.

[0016] The method described above is characterized in that the inoculation amount of the seed liquid in Step 3 is 3 - 6% v / v; the fermentation culture and induction expression conditions in Step 3 are: fermentation temperature 32 - 39 °C, adjust the pH to 6.5 - 7.0 with ammonia water, adjust the rotation speed and ventilation volume during the fermentation process to keep the dissolved oxygen level above 30%, and tank pressure 0.04 - 0.08 MPa.

[0017] The method described above is characterized in that an inducer is added in stages during the fermentation culture and induction expression process in Step 3, and the inducer is a 300 - 400 g / L lactose solution.

[0018] The above method is characterized in that the specific method of adding the inducer in stages is as follows: when the OD600 of the fermentation broth is 35-45, the inducer is added for the first time; when the OD600 of the fermentation broth is 65-75, the inducer is added for the second time; the addition amount of the inducer each time is 7-20 mL / L.

[0019] The present invention has the following advantages compared with the prior art:

[0020] 1. The fermentation medium and the feeding medium designed in the present invention contain specific nutritional promoters. Glutathione in the nutritional promoters, as a crucial metabolic regulator in cells, can effectively alleviate the oxidative stress response of cells; oleic acid and Tween 80, as highly efficient surfactant components, can significantly improve the cell permeability; vitamin C ethyl ether can maintain the redox balance in cells while promoting the growth and proliferation of cells. By controlling the contents of various substances in the nutritional promoters and coordinating with each other, it improves the cell permeability while alleviating the oxidative stress response of cells, and solves the problem that the cell membrane potential imbalance caused by the aggregation of lysine residues during the fermentation process of recombinant mussel adhesive protein, reducing the expression level of the target protein. Finally, the concentration of recombinant mussel adhesive protein in the obtained fermentation broth reaches more than 2 g / L.

[0021] 2. As the fermentation process continues, the present invention adopts a dynamic feeding strategy, timely adding the feeding medium, and through a staged and fine-tuned feeding strategy, timely supplementing the substrate trace elements and nutritional promoters consumed due to cell growth. On the one hand, it maintains the stability of the key nutrient concentration, so that the key nutrients can be transported in time to avoid the situation of energy metabolism imbalance, and can maintain the cell proliferation efficiency at a relatively high level; on the other hand, through the dynamic feeding strategy, it increases the cell membrane permeability and effectively solves the problem of cell inhibition of target protein expression caused by the molecular crowding effect generated during the fermentation process.

[0022] 3. Usually, isopropyl β-D-thiogalactopyranoside (IPTG) is used as an inducer in Escherichia coli fermentation to promote expression. However, IPTG is a synthetic compound, and its high cost is due to the complex production process; moreover, since IPTG is a non-physiological inducer, long-term or large-scale use may have an impact on cells; the present invention uses lactose as an inducer. Lactose is a natural sugar, with a wide source and relatively low price, and lactose can be decomposed into glucose and galactose by lactase in vivo, and then participate in the cell metabolism process, with little impact on the physiological functions of organisms and high biological safety.

[0023] 4. The present invention preferably adopts a staged induction strategy. By precisely controlling the addition timing and concentration of lactose, the induction pressure is effectively alleviated. Specifically, the first induction occurs in the early to middle logarithmic growth phase of the cells. During this stage, the cells gradually adapt to the inhibitory effect caused by the accumulation of recombinant mussel adhesive protein expression, laying a foundation for the subsequent induction process. Subsequently, the second induction is carried out in the middle and late logarithmic growth phase. This strategy ensures that the key enzymes in the synthesis pathway are fully expressed, thus significantly increasing the expression level of the target protein during the fermentation process.

[0024] 5. The cultivation method of the present invention has the characteristics of simple process and high yield. The fermentation expression level can reach above 2 g / L, which is suitable for industrialization. At the same time, the appearance color of the freeze-dried powder of the recombinant mussel adhesive protein prepared is white / whitish, and it can be used as an excellent bioactive raw material in the fields of functional skin care products, medical devices, biomedicine, etc.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0026] Figure 1 It is the appearance diagram of the freeze-dried powder of the recombinant mussel adhesive protein prepared in Example 1 and Comparative Example 1 of the present invention. Detailed Embodiments

[0027] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below with reference to the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] The present invention provides an Escherichia coli culture medium and a cultivation method suitable for the production of recombinant mussel adhesive protein. Among them, the amino acid sequence of the recombinant mussel adhesive protein is as follows (the recombinant mussel adhesive protein that can be produced by this method is not limited to the mussel adhesive protein with this sequence):

[0030] AKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKSSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSSAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYK

[0031] The Escherichia coli used in the present invention is the BL21(DE3) strain, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0032] Example 1

[0033] The culture media used in this example are as follows:

[0034] Flask culture medium: peptone 15 g / L, yeast extract powder 7.5 g / L, sodium chloride 7.5 g / L, glycerol 7.5 g / L, pH 6.8, sterilized at 121 °C and 0.1 MPa for 20 min;

[0035] Seed culture medium: diammonium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 5.5 g / L, ammonium citrate 0.8 g / L, magnesium sulfate heptahydrate 0.9 g / L, glucose monohydrate 15 g / L, ferrous sulfate heptahydrate 40 mg / L, manganese sulfate pentahydrate 2.0 mg / L, zinc sulfate heptahydrate 13 mg / L, copper sulfate pentahydrate 6.5 mg / L, calcium chloride dihydrate 13 mg / L, borax 1.3 mg / L, ammonium molybdate 1.0 mg / L, silicone antifoaming agent 0.8 g / L, pH 6.8, sterilized at 121 °C and 0.1 MPa for 20 min;

[0036] Fermentation culture medium: diammonium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 5.5 g / L, ammonium citrate 0.8 g / L, magnesium sulfate heptahydrate 0.9 g / L, glucose monohydrate 20 g / L, ferrous sulfate heptahydrate 60 mg / L, manganese sulfate pentahydrate 4 mg / L, zinc sulfate heptahydrate 15 mg / L, copper sulfate pentahydrate 10 mg / L, calcium chloride dihydrate 18 mg / L, borax 1.8 mg / L, ammonium molybdate 2 mg / L, silicone antifoaming agent 1.5 g / L, nutrient promoter 15 mL / L, pH 6.8, sterilized at 121 °C and 0.1 MPa for 20 min;

[0037] Feeding medium A: Magnesium sulfate heptahydrate 2 g / L, nutrient promoter 150 mL / L, Ferrous sulfate heptahydrate 70 mg / L, Manganese sulfate pentahydrate 8 mg / L, Zinc sulfate heptahydrate 20 mg / L, Copper sulfate pentahydrate 17 mg / L, Calcium chloride dihydrate 32 mg / L, Borax 5 mg / L, Ammonium molybdate 5 mg / L, pH 6.8, sterilized at 121 °C and 0.1 MPa for 20 min;

[0038] Feeding medium B is a 600 g / L glycerol solution, sterilized at 121 °C and 0.1 MPa for 20 min;

[0039] The nutrient promoter includes components with the following concentrations: Glutathione 10.0 g / L, Oleic acid 3.5 g / L, Tween 80 2.5 g / L, Vitamin C ethyl ether 15 g / L;

[0040] The method for producing the above recombinant mussel adhesive protein using the medium of this example includes the following steps:

[0041] Step 1: Inoculate Escherichia coli preserved with glycerol into a shake flask medium for activation; The activation culture conditions are: temperature 37 °C, shaker speed 200 rpm, and OD600 is 5.0 after culturing for 10 h;

[0042] Step 2: Inoculate the activated shake flask culture solution in Step 1 into a 30 L seed tank containing seed medium at an inoculation amount of 3.3% v / v for seed culture to obtain seed liquid; The culture conditions are: culture temperature 37 °C, adjust pH to 6.8 with ammonia water, tank pressure 0.07 MPa, adjust the rotation speed and ventilation volume during the culture process to keep the dissolved oxygen level above 30%, and OD600 is 6.0 when culturing for 7 h;

[0043] Step 3: Inoculate the cultured seed liquid into a 500 L fermenter containing 250 L fermentation medium at an inoculation amount of 3.3% v / v for culture, temperature 37 °C, adjust pH to 6.8 with ammonia water, tank pressure 0.05 MPa, adjust the rotation speed and ventilation volume during the fermentation process to keep the dissolved oxygen level above 30%;

[0044] When OD600 increases to 40, add 10 mL / L of a 350 g / L lactose solution, start the first induction, and at the same time start adding feeding medium A. The flow rate of feeding medium A is 35 mL / (L·h), and then increases by 2 mL / (L·h) every 1 hour until the fermentation ends; After the dissolved oxygen increases significantly (the dissolved oxygen increases by more than 30% in 1 minute), add feeding medium B. The flow rate of feeding medium B is 18 mL / (L·h), and then increases by 0.5 mL / (L·h) every 1 hour until the fermentation ends;

[0045] When the OD600 increases to 70, add 12 mL / L of a 350 g / L lactose solution all at once to start the secondary induction; control the fermentation temperature at 34 °C, adjust the pH to 6.8 with ammonia water, the tank pressure is 0.06 MPa, the dissolved oxygen is not less than 30%, induce for 6 h, when the OD600 of Escherichia coli cells no longer increases and the cells are senescent under microscopic examination, the entire fermentation process ends; after detection, the wet weight at the end of fermentation is 153.5 g / L, and the protein content in the fermentation broth is 2.25 g / L by high performance liquid chromatography detection.

[0046] Example 2

[0047] The culture media used in this example are as follows:

[0048] Shake flask culture medium: peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 5 g / L, glycerol 5 g / L, pH 6.8, sterilized at 121 °C and 0.1 MPa for 20 min;

[0049] Seed culture medium: diammonium hydrogen phosphate 0.8 g / L, potassium dihydrogen phosphate 3.5 g / L, ammonium citrate 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose monohydrate 10 g / L, ferrous sulfate heptahydrate 30 mg / L, manganese sulfate pentahydrate 1.5 mg / L, zinc sulfate heptahydrate 8.0 mg / L, copper sulfate pentahydrate 4.5 mg / L, calcium chloride dihydrate 10 mg / L, borax 0.8 mg / L, ammonium molybdate 0.8 mg / L, organosilicon antifoaming agent 0.5 g / L, pH 6.5, sterilized at 121 °C and 0.1 MPa for 20 min;

[0050] Fermentation culture medium: diammonium hydrogen phosphate 0.8 g / L, potassium dihydrogen phosphate 3.5 g / L, ammonium citrate 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose monohydrate 15 g / L, ferrous sulfate heptahydrate 40 mg / L, manganese sulfate pentahydrate 2.0 mg / L, zinc sulfate heptahydrate 10 mg / L, copper sulfate pentahydrate 5 mg / L, calcium chloride dihydrate 10 mg / L, borax 0.9 mg / L, ammonium molybdate 1.5 mg / L, organosilicon antifoaming agent 1.0 g / L, nutrient promoter 10 mL / L, pH 6.5, sterilized at 121 °C and 0.1 MPa for 20 min;

[0051] Feed culture medium A: magnesium sulfate heptahydrate 1.5 g / L, nutrient promoter 100 mL / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 5 mg / L, zinc sulfate heptahydrate 15 mg / L, copper sulfate pentahydrate 10 mg / L, calcium chloride dihydrate 20 mg / L, borax 3.0 mg / L, ammonium molybdate 3.0 mg / L, pH 6.5, sterilized at 121 °C and 0.1 MPa for 20 min;

[0052] Feed culture medium B is a 400 g / L glycerol solution, sterilized at 121 °C and 0.1 MPa for 20 min;

[0053] The nutrient promoter includes components at the following concentrations: glutathione 6.0 g / L, oleic acid 2 g / L, Tween 80 2 g / L, and vitamin C ethyl ether 10 g / L;

[0054] The method for producing the above-mentioned recombinant mussel adhesive protein using the culture medium of this example includes the following steps:

[0055] Step 1: Inoculate Escherichia coli preserved with glycerol into a shake flask culture medium for activation; the activation culture conditions are: temperature 34°C, shaker speed 150 rpm, and OD600 is 3.0 after culturing for 7 h;

[0056] Step 2: Inoculate the activated shake flask culture solution in Step 1 into a 30 L seed tank containing a seed culture medium at an inoculation amount of 2% v / v for seed culture to obtain a seed solution; the culture conditions are: culture temperature 34°C, adjust the pH to 6.5 with ammonia water, tank pressure 0.06 MPa, and adjust the rotation speed and ventilation volume during the culture process to keep the dissolved oxygen level above 30%. OD600 is 5 when culturing for 6 h;

[0057] Step 3: Inoculate the cultured seed solution into a 500 L fermenter containing 250 L of fermentation medium at an inoculation amount of 3.0% v / v for culture, temperature 32°C, adjust the pH to 6.5 with ammonia water, tank pressure 0.04 MPa, and adjust the rotation speed and ventilation volume during the fermentation process to keep the dissolved oxygen level above 30%;

[0058] When OD600 increases to 35, add 7 mL / L of a 300 g / L lactose solution to start the first induction, and at the same time start to supplement the feeding medium A. The feeding rate of the feeding medium A is 30 mL / (L·h), and then it increases by 1 mL / (L·h) every 1 hour until the end of fermentation; after a significant increase in dissolved oxygen (the dissolved oxygen increases by more than 30% in 1 minute), supplement the feeding medium B. The feeding rate of the feeding medium B is 15 mL / (L·h), and then it increases by 0.3 mL / (L·h) every 1 hour until the end of fermentation;

[0059] When OD600 increases to 65, add 7 mL / L of a 300 g / L lactose solution at one time to start the second induction; control the fermentation temperature at 32°C, adjust the pH to 6.5 with ammonia water, tank pressure 0.06 MPa, the dissolved oxygen is not less than 30%, induce for 6 h, and when the OD600 of Escherichia coli cells no longer increases and the cells are senescent under microscopic examination, the entire fermentation process ends; after detection, the wet weight at the end of fermentation is 135 g / L, and the protein content in the fermentation broth is 2.01 g / L by high performance liquid chromatography.

[0060] Example 3

[0061] The culture medium used in this example is as follows:

[0062] Flask medium: peptone 20 g / L, yeast extract powder 10 g / L, sodium chloride 20 g / L, glycerol 10 g / L, pH 6.8, sterilized at 121 °C and 0.1 MPa for 20 min;

[0063] Seed medium: diammonium hydrogen phosphate 2.0 g / L, potassium dihydrogen phosphate 10.0 g / L, ammonium citrate 1.2 g / L, magnesium sulfate heptahydrate 2.0 g / L, glucose monohydrate 20 g / L, ferrous sulfate heptahydrate 80 mg / L, manganese sulfate pentahydrate 5.5 mg / L, zinc sulfate heptahydrate 20 mg / L, copper sulfate pentahydrate 15 mg / L, calcium chloride dihydrate 20 mg / L, borax 2.0 mg / L, ammonium molybdate 2.0 mg / L, silicone antifoaming agent 1.0 g / L, pH 7.0, sterilized at 121 °C and 0.1 MPa for 20 min;

[0064] Fermentation medium: diammonium hydrogen phosphate 2.0 g / L, potassium dihydrogen phosphate 10.0 g / L, ammonium citrate 1.2 g / L, magnesium sulfate heptahydrate 2.0 g / L, glucose monohydrate 27 g / L, ferrous sulfate heptahydrate 90 mg / L, manganese sulfate pentahydrate 8.0 mg / L, zinc sulfate heptahydrate 25 mg / L, copper sulfate pentahydrate 20 mg / L, calcium chloride dihydrate 30 mg / L, borax 3.0 mg / L, ammonium molybdate 4.0 mg / L, silicone antifoaming agent 2.0 g / L, nutrient promoter 20 mL / L, pH 7.0, sterilized at 121 °C and 0.1 MPa for 20 min;

[0065] Feeding medium A: magnesium sulfate heptahydrate 4.5 g / L, nutrient promoter 200 mL / L, ferrous sulfate heptahydrate 150 mg / L, manganese sulfate pentahydrate 15.0 mg / L, zinc sulfate heptahydrate 40 mg / L, copper sulfate pentahydrate 35 mg / L, calcium chloride dihydrate 50 mg / L, borax 8 mg / L, ammonium molybdate 7.0 mg / L, pH 7.0, sterilized at 121 °C and 0.1 MPa for 20 min;

[0066] Feeding medium B is a 700 g / L glycerol solution, sterilized at 121 °C and 0.1 MPa for 20 min;

[0067] The nutrient promoter includes components with the following concentrations: glutathione 16.0 g / L, oleic acid 5.0 g / L, Tween 80 5.0 g / L, vitamin C ethyl ether 20 g / L;

[0068] The method for producing the above recombinant mussel adhesive protein using the medium of this example includes the following steps:

[0069] Step 1: Inoculate Escherichia coli preserved with glycerol into the flask medium for activation; the activation culture conditions are: temperature 39 °C, shaker speed 250 rpm, and OD600 is 7 after culturing for 12 h;

[0070] Step 2: Inoculate the activated shake flask culture medium in Step 1 into a 30 L seed tank containing seed medium at an inoculation amount of 5% v / v for seed culture to obtain a seed solution; the culture conditions are: culture temperature 39°C, adjust the pH to 7 with ammonia water, tank pressure 0.08 MPa, adjust the rotation speed and ventilation volume during the culture process to keep the dissolved oxygen level above 30%, and the OD600 is 13 at 8 h of culture;

[0071] Step 3: Inoculate the cultured seed solution into a 500 L fermenter containing 250 L of fermentation medium at an inoculation amount of 6.0% v / v for culture, temperature 39°C, adjust the pH to 7.0 with ammonia water, tank pressure 0.08 MPa, adjust the rotation speed and ventilation volume during the fermentation process to keep the dissolved oxygen level above 30%;

[0072] When OD600 increases to 45, add 20 mL / L of 400 g / L lactose solution to start the first induction, and at the same time start to supplement the feeding medium A. The feeding rate of the feeding medium A is 40 mL / (L·h), and then increase by 3.0 mL / (L·h) every 1 hour until the end of fermentation; after the dissolved oxygen rebounds significantly (the dissolved oxygen increases by more than 30% in 1 minute), supplement the feeding medium B. The feeding rate of the feeding medium B is 25 mL / (L·h), and then increase by 1 mL / (L·h) every 1 hour until the end of fermentation;

[0073] When OD600 increases to 75, add 20 mL / L of 400 g / L lactose solution at one time to start the second induction; control the fermentation temperature at 39°C, adjust the pH to 7.0 with ammonia water, tank pressure 0.08 MPa, the dissolved oxygen is not less than 30%, induce for 6 h, and when the OD600 of Escherichia coli cells no longer increases and the cells are examined microscopically and found to be senescent, the entire fermentation process ends; after detection, the wet weight at the end of fermentation is 148.5 g / L, and the protein content in the fermentation broth is 2.07 g / L by high performance liquid chromatography detection.

[0074] Comparative Example 1

[0075] The difference from Example 1 is that the fermentation medium in Comparative Example 1 is an existing Escherichia coli fermentation medium, specifically as follows:

[0076] The fermentation medium for the engineered Escherichia coli used in the fermenter comprises components at the following concentrations: 10.0 g / L of tryptone, 20.0 g / L of yeast extract, 30.0 g / L of glucose, 2.0 g / L of sodium chloride, 8.7 g / L of dipotassium hydrogen phosphate, 4.2 g / L of sodium dihydrogen phosphate, 5.6 g / L of ammonium sulfate, 10.0 g / L of gelatin (gelatin hydrolyzate), 2.5 g / L of magnesium sulfate heptahydrate, 1.0 g / L of EDTA, 100.0 mg / L of ferric chloride hexahydrate, 20.0 mg / L of manganese sulfate tetrahydrate, 8.0 mg / L of zinc sulfate, 2.5 mg / L of boric acid, 2.5 mg / L of sodium molybdate, 2.5 mg / L of cobalt chloride, and 2.5 mg / L of copper chloride.

[0077] At the end of fermentation, after detection, the wet weight of the broth discharged from the fermenter was 95.75 g / L, and the protein content in the fermentation broth detected by high performance liquid chromatography was 1.21 g / L.

[0078] Comparative Example 2

[0079] Based on Example 1, in this comparative example, only the ratio of the nutritional promoter is changed, and the specific settings are as follows:

[0080] The nutritional promoter comprises components at the following concentrations: 20.0 g / L of glutathione, 9.0 g / L of oleic acid, 9.0 g / L of Tween 80, 28.0 g / L of vitamin C ethyl ether, and other components remain unchanged.

[0081] At the end of fermentation, after detection, the wet weight of the broth discharged from the fermenter was 131.5 g / L, and the protein content in the fermentation broth detected by high performance liquid chromatography was 1.83 g / L.

[0082] Comparative Example 3

[0083] Based on Example 1, in this comparative example, only the concentration of the nutritional promoter in the fermentation medium and feeding medium A is changed, and the specific settings are as follows:

[0084] The concentration of the nutritional promoter in the fermentation medium is 30 mL / L; the concentration of the nutritional promoter in feeding medium A is 290 mL / L, and other components remain unchanged.

[0085] At the end of fermentation, after detection, the wet weight of the broth discharged from the fermenter was 130 g / L, and the protein content in the fermentation broth detected by high performance liquid chromatography was 1.85 g / L.

[0086] Comparative Example 4

[0087] Based on Example 1, in this comparative example, the flow rates of feeding medium A and feeding medium B are lower than the ranges specified in the present invention, and other components remain unchanged. The specific settings are as follows:

[0088] In step 3, the engineered Escherichia coli was inoculated into a 500 L fermenter containing 250 L of fermentation medium at an inoculation amount of 3.3% v / v, cultured at 37 °C, the pH was adjusted to 6.8 with ammonia water, the tank pressure was 0.05 MPa, and the dissolved oxygen was not less than 30%;

[0089] When OD600 increased to 40, 10 mL / L of a 350 g / L lactose solution was added to start the first induction. At the same time, feeding medium A was added, and the feeding rate of feeding medium A was 20 mL / (L·h), and then increased by 0.5 mL / (L·h) every 1 hour until the end of fermentation; when the dissolved oxygen increased significantly (the dissolved oxygen increased by more than 30% in 1 minute), feeding medium B was added, and the feeding rate of feeding medium B was 8 mL / (L·h), and then increased by 0.5 mL / (L·h) every 1 hour until the end of fermentation;

[0090] When OD600 increased to 70, 12 mL / L of a 350 g / L lactose solution was added at one time to start the second induction; the fermentation temperature was controlled at 34 °C, the pH was adjusted to 6.8 with ammonia water, the tank pressure was 0.06 MPa, the dissolved oxygen was not less than 30%, and the induction was carried out for 6 h. When the OD600 of Escherichia coli cells no longer increased and the cells were observed to be senescent under the microscope, the entire fermentation process ended.

[0091] After detection, the wet weight at the end of fermentation was 128.5 g / L, and the protein content in the fermentation broth was 1.76 g / L by high performance liquid chromatography.

[0092] Comparative Example 5

[0093] Based on Example 1, the feeding rates of feeding medium A and feeding medium B in this comparative example were higher than the ranges specified in the present invention, and other conditions remained unchanged. The specific settings were as follows:

[0094] Among them, in step S3, the following steps were included:

[0095] In step 3, the engineered Escherichia coli was inoculated into a 500 L fermenter containing 250 L of fermentation medium at an inoculation amount of 3.3% v / v, cultured at 37 °C, the pH was adjusted to 6.8 with ammonia water, the tank pressure was 0.05 MPa, and the dissolved oxygen was not less than 30%;

[0096] When OD600 increased to 40, 10 mL / L of a 350 g / L lactose solution was added to start the first induction. At the same time, feeding medium A was added, and the feeding rate of feeding medium A was 50 mL / (L·h), and then increased by 5 mL / (L·h) every 1 hour until the end of fermentation; when the dissolved oxygen increased significantly (the dissolved oxygen increased by more than 30% in 1 minute), feeding medium B was added, and the feeding rate of feeding medium B was 30 mL / (L·h), and then increased by 1 mL / (L·h) every 1 hour until the end of fermentation;

[0097] When the OD600 increases to 70, add 12 mL / L of 350 g / L lactose solution at one time to start the secondary induction; control the fermentation temperature at 34 °C, adjust the pH to 6.8 with ammonia water, the tank pressure is 0.06 MPa, the dissolved oxygen is not less than 30%, induce for 6 h, when the OD600 of Escherichia coli cells no longer increases and the cells are senescent under microscopic examination, the whole fermentation process ends.

[0098] After detection, the wet weight at the end of fermentation is 131.75 g / L, and the protein content in the fermentation broth is 1.81 g / L by high performance liquid chromatography.

[0099] Comparative Example 6

[0100] Based on Example 1, neither the fermentation medium nor the feeding medium A used in the fermenter contains a nutrient promoter, and other conditions remain unchanged.

[0101] After detection, the wet weight at the end of fermentation is 118.25 g / L, and the protein content in the fermentation broth is 1.63 g / L by high performance liquid chromatography.

[0102] Since no nutrient promoter is used in the fermentation medium and the feeding medium A in Comparative Example 6, the problem that the electrostatic interaction caused by the aggregation of lysine residues in recombinant mussel adhesive protein during fermentation leads to the imbalance of cell membrane potential and the decrease of membrane permeability cannot be improved. Therefore, the protein content in the recombinant mussel adhesive fermentation broth of Comparative Example 6 decreases.

[0103] Experimental Example

[0104] After the fermentation of Examples 1-3 and Comparative Examples 1-6 was completed, the fermentation broth was subjected to solid-liquid separation using a disc centrifuge; the cell precipitate was redissolved in purified water to 1.5 times the volume at the end of fermentation, the pH was adjusted to 5.0, 15 mM acetate (pH 5.0), 15 mM EDTA, and 2.0% Triton-100 were added, heated to 85 °C, air was introduced until the tank pressure reached 1.35 Mpa, and stirring was carried out at 50-150 rpm for 30 min. After cooling, the precipitate was collected by centrifugation, and acetic acid extraction was carried out overnight with acetic acid at 0.5 times the weight of the precipitate. The protein content of the acid extract was detected by high-performance liquid chromatography, and the results are shown in Table 1. The acid extract was subjected to salting-out impurity removal, modification oxidation, chromatography purification, and freeze-drying to obtain a freeze-dried powder of recombinant mussel adhesive protein, and the color of the freeze-dried powder is shown in Table 1. The liquid chromatography detection conditions are as follows: octadecylsilane-bonded silica gel was used as the filler (250 mm * 0.45 mm, 5 μm or other chromatographic columns with similar polarity); a mixture of 0.05% trifluoroacetic acid aqueous solution and 0.05% trifluoroacetic acid acetonitrile solution (95:5) was used as mobile phase A, and a mixture of 0.05% trifluoroacetic acid aqueous solution and 0.05% trifluoroacetic acid acetonitrile solution (20:80) was used as mobile phase B, and elution was carried out according to a gradient (0-5 min: 95% A: 5% B; 5-20 min: 85% A: 15% B; 20-40 min: 50% A: 50% B; 40-50 min: 95% A: 5% B); the column temperature was 25 °C, the sample tray temperature was 4 °C, the flow rate was 1.0 mL / min, and the detection wavelength was 280 nm.

[0105] Table 1 Concentration of recombinant mussel adhesive protein in the fermentation broth and the appearance color of the freeze-dried powder of recombinant mussel adhesive protein

[0106]

[0107] As can be seen from Table 1, compared with Examples 1-3, there were significant differences in the production of recombinant mussel adhesive protein in Comparative Examples 1-6. Examples 1-3 adopted a unique inorganic salt ion formula, nutritional promoter, and staged lactose induction strategy. This combination effectively alleviated the inhibitory effect of the expression and accumulation of recombinant mussel adhesive protein on the growth of the strain, and significantly promoted the growth of the strain and the expression of the target protein. Specifically, the average wet weight at the end of fermentation in Examples 1-3 was approximately around 145 g / L, and the concentration of recombinant mussel adhesive protein in the fermentation broth also reached more than 2.0 g / L.

[0108] In Comparative Example 1, a traditional peptone / yeast extract formulation was used and IPTG was used for induction. However, the wet weight at the end of fermentation and the concentration of recombinant mussel adhesive protein in Comparative Example 1 were much lower than those in Examples 1 to 3. Among them, the wet weight at the end of fermentation in Example 1 was increased by more than 50% compared with Comparative Example 1, and the concentration of recombinant mussel adhesive protein in Example 1 was increased by more than 80% compared with Comparative Example 1. From the appearance of the obtained recombinant mussel adhesive protein product, the recombinant mussel adhesive protein prepared in Examples 1 to 3 was white / off-white, while the product prepared in Comparative Example 1 was light yellow / light pink, which was not conducive to subsequent industrial applications.

[0109] In Comparative Examples 2 and 3, the concentration of the nutrient promoter was changed. The results showed that whether the concentration of the components in the nutrient promoter formulation increased, or the concentration of the nutrient promoter in the fermentation medium and the feeding medium increased, it would cause a significant decrease of about 15% in the wet weight at the end of fermentation and the concentration of recombinant mussel adhesive protein. This indicated that a high concentration of the nutrient promoter had an obvious inhibitory effect on cell growth and protein expression. This might be because excessive addition of glutathione, oleic acid, Tween 80 and vitamin C ethyl ether in the nutrient promoter would result in an over-reduction phenomenon, affecting the formation of disulfide bonds, leading to incorrect target protein structure, and the cell membrane permeability was too strong, which would instead damage the membrane structure and affect cell viability.

[0110] In Comparative Examples 4 and 5, the effects of too low and too high feeding medium flow rates on cell growth and protein expression were investigated respectively. The results showed that too low a flow rate would result in insufficient supplementation of trace elements, energy substances and nutrient promoters required for cell growth; while too high a flow rate would cause too high an osmotic pressure, resulting in cell metabolic imbalance and inhibiting cell growth. Both of these situations would have an adverse effect on cell growth and protein expression, resulting in a decrease of about 10% in the wet weight at the end of fermentation compared with the examples, and a decrease of about 17% in the expression level of recombinant mussel adhesive protein.

[0111] In Comparative Example 6, fermentation culture was carried out using a medium without a nutrient promoter. However, this method resulted in a decrease of about 20% in the wet weight of the harvested bacteria compared with the Examples, and the expression level of recombinant mussel adhesive protein also decreased by about 23%. This indicates that the nutrient promoter plays a crucial role in the fermentation process. Specifically, nutrient promoters such as glutathione, as an important regulatory metabolite in cells, can relieve cell stress; oleic acid and Tween 80, as surfactants, can increase cell permeability; vitamin C ethyl ether can maintain the redox balance in cells while promoting cell growth and proliferation. These nutrient promoters work together to improve cell permeability as a whole while relieving the cell oxidative stress response, and solve the problem of the decrease in the expression level of the target protein caused by the interference of the electrostatic interaction with the cell membrane potential balance during the fermentation of recombinant mussel adhesive protein, thereby increasing cell density and expression level; in addition, vitamin C ethyl ether and glutathione in the nutrient promoter play an antioxidant role and can also prevent the recombinant mussel adhesive protein expressed during the fermentation from being peroxidized. Therefore, the recombinant protein produced by the method of the present invention appears white or off-white. Compared with the common light yellow or light pink recombinant mussel adhesive protein products on the market, the recombinant mussel adhesive protein prepared by the present invention has a lighter color and a broader application prospect. In summary, the fermentation method provided by the present invention has achieved remarkable results in Examples 1 to 3. Compared with Comparative Examples 1 to 6, the obtained recombinant mussel adhesive protein not only has a high expression level but also has better appearance quality.

[0112] In summary, the present application provides a recombinant mussel adhesive protein Escherichia coli medium and its fermentation method. Based on the existing traditional Escherichia coli medium, by regulating the proportion of each nutrient component and adding a nutrient promoter to the fermentation medium and the feeding medium, the problem of low expression level of recombinant mussel adhesive protein during fermentation is solved; and by jointly using the dynamic feeding method, the staged induction strategy and lactose induction, it is further ensured that the key enzymes in the synthesis pathway are fully expressed, while increasing the expression level of the target protein, reducing the production cost and having good biocompatibility, which is convenient for large-scale production and popularization.

[0113] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. An Escherichia coli culture medium suitable for the production of recombinant mussel mucin, characterized in that: The method comprises a fermentation medium and a feed medium; the concentrations of the components in the fermentation medium include: 0.8-2.0 g / L diammonium hydrogen phosphate, 3.5-10.0 g / L potassium dihydrogen phosphate, 0.5-1.2 g / L ammonium citrate, 0.5-2.0 g / L magnesium sulfate heptahydrate, 15-27 g / L glucose monohydrate, 40-90 mg / L ferrous sulfate heptahydrate, 2-8 mg / L manganese sulfate pentahydrate, 10-25 mg / L zinc sulfate heptahydrate, 5-20 mg / L copper sulfate pentahydrate, 10-30 mg / L calcium chloride dihydrate, 0.9-3 mg / L borax, 1.5-4 mg / L ammonium molybdate, 1-2 g / L organosilicon defoamer, and 10-20 mL / L nutrition promoter; The nutrition promoter comprises glutathione, oleic acid, Tween 80 and vitamin C ethyl ether; the concentrations of the components in the nutrition promoter are respectively: glutathione 6-16 g / L, oleic acid 2-5 g / L, Tween 80 2-5 g / L, and vitamin C ethyl ether 10-20 g / L.

2. The Escherichia coli culture medium suitable for the production of recombinant mussel mucin according to claim 1, characterized in that: The feed medium comprises a feed medium A and a feed medium B, wherein the concentrations of the components in the feed medium A comprise: 1.5-4.5 g / L of magnesium sulfate heptahydrate, 100-200 mL / L of a nutrient promoter, 50-150 mg / L of ferrous sulfate heptahydrate, 5.0-15 mg / L of manganese sulfate pentahydrate, 15-40 mg / L of zinc sulfate heptahydrate, 10-35 mg / L of copper sulfate pentahydrate, 20-50 mg / L of calcium chloride dihydrate, 3.0-8.0 mg / L of borax, and 3.0-7.0 mg / L of ammonium molybdate; and the feed medium B is a glycerol solution.

3. The Escherichia coli culture medium suitable for the production of recombinant mussel mucin according to claim 2, characterized in that: The concentration of the glycerol solution is 400-700 g / L.

4. A method for culturing Escherichia coli for producing recombinant mussel mucin using the Escherichia coli culture medium as claimed in claim 2, characterized in that: The following steps are involved: Step 1, inoculating Escherichia coli into shake flask culture medium for activation; Step 2, inoculating the activated shake flask culture solution into the seed culture medium for seed culture; Step 3: inoculate the cultured seed liquid into the fermentation medium for fermentation culture and induced expression. During the fermentation culture and induced expression process, feed medium is used for flow feeding. The initial feed rate of feed medium A is controlled to be 30-40 mL / (L·h), and then increased by 1-3 mL / (L·h) every hour. The initial feed rate of feed medium B is controlled to be 15-25 mL / (L·h), and then increased by 0.3-1 mL / (L·h) every hour.

5. The method according to claim 4, characterized in that The formula of the shake flask culture medium in step 1 includes: 10-20 g / L peptone, 5-10 g / L yeast extract powder, 5-20 g / L sodium chloride, and 5-10 g / L glycerol; the activated culture conditions are: temperature of 34-39°C, shaker speed of 150-250 rpm, culture time of 7-12 h, and culture until OD600 is 3-7.

6. The method according to claim 4, characterized in that The inoculation amount of the inoculation in step 2 is 2-5% v / v; the formula of the seed culture medium includes: 0.8-2.0 g / L diammonium hydrogen phosphate, 3.5-10.0 g / L potassium dihydrogen phosphate, 0.5-1.2 g / L ammonium citrate, 0.5-2.0 g / L magnesium sulfate heptahydrate, 10-20 g / L glucose monohydrate, 30-80 mg / L ferrous sulfate heptahydrate, 1.5-5.5 mg / L manganese sulfate pentahydrate, 8-20 mg / L zinc sulfate heptahydrate, 4.5-15 mg / L copper sulfate pentahydrate, 10-20 mg / L calcium chloride dihydrate, 0.8-2 mg / L borax, 0.8-2.0 mg / L ammonium molybdate, and 0.5-1.0 g / L silicone defoamer.

7. The method according to claim 4, characterized in that The seed culture conditions in step 2 are: the culture temperature is 34-39° C., the rotation speed and ventilation volume are adjusted during the culture process to keep the dissolved oxygen level above 30%, the pH is adjusted to 6.5-7.0 with ammonia water, and the culture is carried out for 6-8 hours until the OD600 is 5-13.

8. The method according to claim 4, characterized in that The inoculation amount of the seed liquid in step three is 3-6% v / v; the conditions for fermentation culture and induced expression in step three are: fermentation temperature 32-39° C., ammonia water adjusting pH to 6.5-7.0, adjusting the rotation speed and ventilation volume during fermentation to keep the dissolved oxygen level above 30%, and tank pressure 0.04-0.08 MPa.

9. The method according to claim 4, characterized in that In step 3, during the fermentation culture and induced expression process, the inducer is added in stages, and the inducer is 300-400 g / L lactose solution.

10. The method according to claim 9, characterized in that The specific method of adding the inducer in stages is: when the OD600 of the fermentation liquid is 35-45, the inducer is added for the first time; when the OD600 of the fermentation liquid is 65-75, the inducer is added for the second time; and the amount of the inducer added each time is 7-20 mL / L.

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