Pichia pastoris fermentation medium for expressing recombinant bovine lactoferrin and fermentation method

Abstract

The invention relates to a pichia pastoris fermentation medium for expressing recombinant bovine lactoferrin and a fermentation method, belongs to the technical field of biochemistry, and solves the technical problem of increasing the expression quantity of the bovine lactoferrin in the process of industrially preparing the recombinant bovine lactoferrin on a large scale. The aqueous solution of the pichia pastoris fermentation culture medium for expressing the recombinant bovine lactoferrin comprises the following components in proportion: 10-30 g / L of yeast extract, 25-35 g / L of phosphoric acid aqueous solution, 1.0-3.0 g / L of calcium sulfate, 18-22 g / L of potassium sulfate, 4.0-5.0 g / L of potassium hydroxide, 30-50 g / L of glycerol, 7-13 g / L of magnesium sulfate heptahydrate, 4-5 g / L of ammonium chloride, 4-6 ml / L of PTM1 trace element solution and 1-2 ml / L of defoaming agent. The high-density fermentation method disclosed by the invention is realized by controlling the technical parameters such as dissolved oxygen, pH value, temperature and fed-batch culture medium rate in the fermentation process, so that the expression quantity of the bovine lactoferrin is increased to 4-5g / L.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and particularly relates to a Pichia yeast fermentation medium and a fermentation method for expressing recombinant bovine lactoferrin. Background Art

[0002] Lactoferrin is a glycoprotein with a relative molecular mass of about 80KD, which is mainly found in human milk. Lactoferrin is a natural antibacterial glycoprotein. Due to its specific iron ion binding ability, it has strong bactericidal and antibacterial abilities against both Gram-positive and Gram-negative bacteria. Studies have also found that lactoferrin also has antiviral, antioxidant and immunomodulatory effects. Therefore, lactoferrin is widely used in many fields such as milk powder, food and medicine.

[0003] The U.S. Food and Drug Administration, Japan, South Korea and other countries currently allow lactoferrin to be used as a food additive in sports and functional foods; on March 30, 2012, China's "National Food Safety Standard - Food Nutrient Fortifier Usage Standard" included lactoferrin in GB14880-2012, and recognized it as a food nutrient fortifier.

[0004] Experimental studies have found that bovine lactoferrin and human lactoferrin have extremely similar biological activities. At present, bovine lactoferrin is mainly produced from cow milk at home and abroad, which is difficult to separate and has high costs. With the development of gene editing technology, bovine lactoferrin can now be expressed from a variety of genetically engineered bacteria.

[0005] The bovine lactoferrin gene, PAOX1 promoter and vector pPIC9K were connected and transformed into GS115 Pichia pastoris. High-density cell fermentation technology was used to achieve industrial production, and a large amount of bovine lactoferrin with the same activity as human lactoferrin was obtained, which has broad application prospects in the fields of biomedicine, health food, cosmetics, etc. Accordingly, how to increase the expression of recombinant bovine lactoferrin in the fermentation process of Pichia pastoris, improve the yield and production efficiency, has become a technical problem that needs to be solved. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a Pichia yeast fermentation medium and a fermentation method for expressing recombinant bovine lactoferrin, so as to solve the technical problem of increasing the expression amount of bovine lactoferrin in the process of industrial and large-scale preparation of recombinant bovine lactoferrin.

[0007] The purpose of the present invention is mainly achieved through the following technical solutions:

[0008] First, the present invention provides a Pichia yeast fermentation medium for expressing recombinant bovine lactoferrin, wherein the components and their proportions in the aqueous medium solution are: 10-30 g / L yeast extract, 25-35 g / L phosphoric acid aqueous solution, 1.0-3.0 g / L calcium sulfate, 18-22 g / L potassium sulfate, 4.0-5.0 g / L potassium hydroxide, 30-50 g / L glycerol, 7-13 g / L magnesium sulfate heptahydrate, 4-5 g / L ammonium chloride, and 4-6 ml / L PTM1 trace element solution. , and 1-2 ml / L of defoaming agent; feed medium A is an aqueous solution containing 500-600 g / L glycerol and 10-20 ml / L PTM1 trace element solution; feed medium B is a 100% methanol solution containing 10-20 ml / L PTM1 trace element solution; feed medium C is an aqueous solution containing 20-60 g / L mannitol or sorbitol; feed medium D is an aqueous solution containing 100-200 g / L yeast extract and 200-400 g / L peptone.

[0009] Furthermore, the specification of the phosphoric acid aqueous solution is analytically pure, and the initial mass percentage is ≥85%; the defoaming agent is an organosilicon or polyether defoaming agent.

[0010] Furthermore, the PTM1 trace element solution is an aqueous solution, comprising the following components and their proportions: sodium iodide 0.06-0.1g / L, copper sulfate pentahydrate 5-7g / L, manganese sulfate monohydrate 2.5-3.5g / L, sodium molybdate dihydrate 0.18-0.22g / L, boric acid 0.036-0.044g / L, zinc chloride 27-33g / L, cobalt chloride hexahydrate 0.45-0.55g / L, biotin 0.45-0.55g / L, sulfuric acid 5-7ml / L, and ferrous sulfate 145-155g / L.

[0011] Secondly, the present invention also provides a Pichia yeast fermentation method for expressing recombinant bovine lactoferrin, comprising the following steps:

[0012] Step S1, seed solution preparation: Pichia pastoris working seeds are spread on YPD solid culture medium plates for cultivation, and after cultivation, single colonies are picked and inoculated into a shake flask containing YPD liquid culture medium to obtain seed solution.

[0013] Step S2, batch fermentation: aseptically introducing the seed liquid into a fermentation tank containing a Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, correcting the dissolved oxygen content DO100%, and then controlling the dissolved oxygen content DO10-60% during the process by increasing the stirring speed and ventilation ratio, controlling the process pH between 5.5-6.0, and the temperature at 30±1°C;

[0014] Step S3, fed-batch fermentation: observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen content (DO) exceeds 80%, start feeding the feed medium A and take samples regularly to detect the OD of the fermentation liquid. 600 Value, when OD 600 When the value exceeds 150, stop feeding feed medium A, control the dissolved oxygen DO10-60% by stirring speed and ventilation ratio, control the pH between 5.5-6.0, and the temperature at 30±1℃;

[0015] Step S4, inducing fermentation: stop feeding feed medium A, starve the bacteria for 30±1 min, start feeding feed medium B, feed medium C and feed medium D, start inducing expression, control the dissolved oxygen content DO10-60% during the process by stirring speed and ventilation ratio, control the process pH between 5.0-5.5, and the temperature at 22-25°C. Induce expression for 30-40 hours to end the fermentation.

[0016] Furthermore, in step S1, the working seed of Pichia pastoris is GS115 / pPIC9K-blf, the recombinant bovine lactoferrin is expressed intracellularly in the Pichia pastoris, and the copy number of the bovine lactoferrin gene in the Pichia pastoris genome is 4-6.

[0017] Furthermore, in step S1, the OD of the seed solution 600 Is 5-10.

[0018] Further, in step S2, the volume of the fermentation tank is more than 5L, the volume of the Pichia fermentation medium in the fermentation tank accounts for 50-70% of the volume of the fermentation tank, and the volume of the seed liquid is 3-10% of the volume of the Pichia fermentation medium in the fermentation tank; the volume of the seed liquid is 3-10% of the volume of the Pichia fermentation medium in the fermentation tank; the initial ventilation ratio is (2.0, ε) VVM, the initial stirring speed is (200, ε) rpm, the initial relative tank pressure is (0.05, ε) MPa, ε is 10%, indicating the maximum deviation from the fixed value.

[0019] Further, in step S3, based on the volume of the Pichia yeast fermentation medium in the fermenter, the rate of adding feed medium A is 30 g / L / h-50 g / L / h.

[0020] Further, in step S4, based on the volume of the Pichia yeast fermentation medium in the fermenter, the rate of feeding the feed medium B is 1-3 g / L / h, the rate of feeding the feed medium C is 2-5 g / L / h, and the rate of feeding the feed medium D is 2-5 g / L / h.

[0021] Furthermore, in step S2 to step S4, ammonia water is used to control the pH value, the stirring speed is 200-1000 rpm, the ventilation ratio is 2.0-4.0 VVM, and the relative tank pressure is 0.05±0.02 MPa.

[0022] Compared with the prior art, the present invention can achieve at least one of the following technical effects:

[0023] (1) The Pichia yeast fermentation medium of the present invention is added with a portion of yeast extract, ammonium chloride and a defoaming agent. The yeast extract contains various nutrients required for the growth of Pichia yeast and is easily directly utilized by the bacteria, and has an obvious effect in the bacterial proliferation stage; the ammonium chloride is used as an inorganic nitrogen source to supplement and adapt the nitrogen source during the bacterial growth process; the defoaming agent prevents the fermentation liquid from foaming, thereby ensuring the oxygen transfer efficiency, the transfer and uniformity of the matrix.

[0024] (2) The present invention adds mannitol or sorbitol to the feed medium as a non-inhibitory carbon source, which is beneficial to increasing the expression level of bovine lactoferrin during the induction stage; the present invention adds yeast extract and peptone to the feed medium, which is beneficial to increasing the expression level of bovine lactoferrin during the induction stage and preventing the decomposition of the target protein.

[0025] (3) The present invention increases the ferrous sulfate content in the PTM1 trace element solution from 65 g / L in the conventional formula to about 150 g / L. This is because bovine lactoferrin contains iron ions, which not only serve as a substance for nutrient cells, but also provide a better precursor for the production of bovine lactoferrin.

[0026] (4) The high-density fermentation method of the present invention is achieved by controlling technical parameters such as dissolved oxygen, pH value, temperature and feed medium rate during the fermentation process, so that the expression level of bovine lactoferrin is increased to 4-5 g / L.

[0027] (5) The Pichia yeast fermentation medium and high-density fermentation method proposed in the present invention use mostly inorganic salts as raw materials, which are low in cost and provide a good example for the industrial and large-scale production of lactoferrin. DETAILED DESCRIPTION

[0028] The following is a further detailed description of the Pichia yeast fermentation medium and fermentation method for expressing recombinant bovine lactoferrin in conjunction with specific examples. These examples are only for comparison and explanation purposes, and the present invention is not limited to these examples.

[0029] In the prior art, Pichia pastoris is widely used in protein expression, mainly in the fields of medicine, industry, and biology. Through the Pichia pastoris expression system, efficient expression and large-scale production of a variety of important proteins can be achieved. Specifically, the bovine lactoferrin gene, PAOX1 promoter and vector pPIC9K are connected and then transformed into GS115 type Pichia pastoris, and high-density cell fermentation technology is used to achieve large-scale industrial production, which is one of them.

[0030] In view of the need to use Pichia pastoris to industrially produce recombinant bovine lactoferrin, the present invention provides a Pichia pastoris fermentation medium, which is based on the BSM basic inorganic salt medium formula, and several materials are optimized and added. The specific components and their proportions in the aqueous solution are: yeast extract 10-30g / L, phosphoric acid aqueous solution 25-35g / L, calcium sulfate 1.0-3.0g / L, potassium sulfate 18-22g / L, potassium hydroxide 4.0-5.0g / L, glycerol 30-50g / L, magnesium sulfate heptahydrate 7-13g / L, ammonium chloride 4-5g / L. / L, 4-6ml / L PTM1 trace element solution, and 1-2ml / L defoaming agent; feed medium A is an aqueous solution containing 500-600g / L glycerol and 10-20ml / LPTM1 trace element solution; feed medium B is a 100% methanol solution containing 10-20ml / LPTM1 trace element solution; feed medium C is an aqueous solution containing 20-60g / L mannitol or sorbitol; feed medium D is an aqueous solution containing 100-200g / L yeast extract and 200-400g / L peptone.

[0031] Specifically, the yeast extract, defoamer and peptone in the above formula are biochemical reagents, and the specifications of the other substances are analytically pure. The initial mass percentage of phosphoric acid aqueous solution is ≥85%. The defoamer is a silicone or polyether defoamer, such as Antifoam 204.

[0032] Specifically, the PTM1 trace element solution is an aqueous solution, which contains the following components and their proportions: sodium iodide 0.06-0.1g / L, copper sulfate pentahydrate 5-7g / L, manganese sulfate monohydrate 2.5-3.5g / L, sodium molybdate dihydrate 0.18-0.22g / L, boric acid 0.036-0.044g / L, zinc chloride 27-33g / L, cobalt chloride hexahydrate 0.45-0.55g / L, biotin 0.45-0.55g / L, sulfuric acid 5-7ml / L, ferrous sulfate 145-155g / L.

[0033] It should be noted that the main components and contents of the conventional BSM basic inorganic salt culture medium aqueous solution are as follows: 26.7 ml / L of 85% phosphoric acid aqueous solution by mass, 0.93 g / L of calcium sulfate, 18.2 g / L of potassium sulfate, 14.9 g / L of magnesium sulfate heptahydrate, 4.13 g / L of potassium hydroxide, 40 g / L of glycerol, and 4.35 ml / L of PTM1 trace element solution, wherein the PTM1 trace element solution contains: 0.08 g / L of sodium iodide, 6 g / L of copper sulfate pentahydrate, 3 g / L of manganese sulfate monohydrate, 0.2 g / L of sodium molybdate dihydrate, 0.02 g / L of boric acid, 20 g / L of zinc chloride, 0.5 g / L of cobalt chloride hexahydrate, 0.2 g / L of biotin, 5 ml / L of sulfuric acid, and 65 g / L of ferrous sulfate. According to the present invention, the content of the main components of the BSM basic inorganic salt culture medium and the content of the components in the PTM1 trace element solution are modified from point values ​​to ranges based on actual production needs, and the contents of boric acid, zinc chloride, biotin and ferrous sulfate in the PTM1 trace element solution are increased; in addition, 10-30 g / L of yeast extract, 4-5 g / L of ammonium chloride and 1-2 ml / L of defoamer are added, wherein the yeast extract contains various nutrients required for the growth of Pichia pastoris, is easily directly utilized by the bacteria, and has an obvious effect in the bacteria amplification stage; the ammonium chloride is used as an inorganic nitrogen source to supplement and adapt the nitrogen source in the bacteria growth process; the defoamer prevents the fermentation liquid from bubbling, ensures the oxygen transfer efficiency, and the transfer and uniformity of the matrix.

[0034] On the other hand, since bovine lactoferrin contains iron ions, iron ions not only serve as a substance for nutrient cells, but also can better provide precursors for the production of bovine lactoferrin. Therefore, the present invention increases the ferrous sulfate content in the PTM1 trace element solution added to the Pichia yeast fermentation medium, feed medium A and feed medium B from 65 g / L in the conventional formula to about 150 g / L.

[0035] It should be noted that mannitol or sorbitol in feed medium C, as a non-inhibitory carbon source, is conducive to increasing the expression of bovine lactoferrin in the induction stage. Yeast extract and peptone in feed medium D are conducive to increasing the expression of bovine lactoferrin in the induction stage and preventing the decomposition of the target protein.

[0036] Furthermore, the present invention also provides a fermentation method of Pichia pastoris, comprising the following steps:

[0037] Step S1, seed solution preparation: Pichia pastoris working seeds are spread on YPD solid culture medium plates for cultivation, and after cultivation, single colonies are picked and inoculated into a shake flask containing YPD liquid culture medium to obtain seed solution.

[0038] Step S2, batch fermentation: aseptically introducing the seed liquid into a fermentation tank containing a Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, correcting the dissolved oxygen content DO100%, and then controlling the dissolved oxygen content DO10-60% during the process by increasing the stirring speed and ventilation ratio, controlling the process pH between 5.5-6.0, and the temperature at 30±1°C;

[0039] Step S3, fed-batch fermentation: observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen content (DO) exceeds 80%, start feeding the feed medium A and take samples regularly to detect the OD of the fermentation liquid. 600 Value, when OD 600 When the value exceeds 150, stop feeding feed medium A, control the dissolved oxygen DO10-60% by stirring speed and ventilation ratio, control the pH between 5.5-6.0, and the temperature at 30±1℃;

[0040] Step S4, inducing fermentation: stop feeding feed medium A, starve the bacteria for 30±1 min, start feeding feed medium B, feed medium C and feed medium D, start inducing expression, control the dissolved oxygen content DO10-60% during the process by stirring speed and ventilation ratio, control the process pH between 5.0-5.5, and the temperature at 22-25°C. Induce expression for 30-40 hours to end the fermentation.

[0041] Specifically, in step S1, the GenbanK ID of the bovine lactoferrin gene is JX294418.1, and the Pichia pastoris working seed is the GS115 type Pichia pastoris into which the bovine lactoferrin gene, the PAOX1 promoter and the vector pPIC9K are connected and transformed, that is, the Pichia pastoris working seed is GS115 / pPIC9K-blf. The recombinant bovine lactoferrin is expressed intracellularly in Pichia pastoris, and the copy number of the bovine lactoferrin gene in the Pichia pastoris genome is 4-6, such as 5.88.

[0042] The aqueous solution of YPD solid culture medium is prepared as follows: 0.9-1.1g / 100ml yeast extract, 1.8-2.2g / 100ml peptone, 1.8-2.2g / 100ml glucose, 1.8-2.2g / 100ml agar, 0.18-0.22g / 100ml geneticin G418, and then sterilized at 120-122℃ for 18-22min. When cooled to about 58-62℃, aseptically operated in a clean bench and poured into a 9cm diameter plate. Each plate contains about 25ml of YPD solid culture medium.

[0043] The aqueous solution of YPD liquid culture medium is prepared with the following composition: 0.9-1.1g / 100ml yeast extract, 1.8-2.2g / 100ml peptone, 1.8-2.2g / 100ml glucose, 0.18-0.22g / 100ml Geneticin G418. 400ml is prepared, and each 500ml shake flask contains about 200ml of culture medium. Then, it is placed in a high-pressure steam sterilizer and sterilized at 120-122℃ for 18-22min.

[0044] Specifically, the Pichia pastoris working seeds were spread on YPD solid culture medium plates, with each plate being spread with about 0.05-0.1 ml of the Pichia pastoris working seeds, and placed in a 30°C incubator for 2-3 days. Then, a single colony of the Pichia pastoris on 1-5 plates was inoculated into a 1L shake flask containing 200 ml of YPD liquid culture medium, and placed in a shaker, and the temperature was controlled at 28-32°C, the rotation speed was 210-230 rpm, and the culture was carried out for 16-22 hours to obtain the OD 600 The seed solution is 5-10, OD 600 It is the absorbance of the seed solution at a wavelength of 600nm.

[0045] Specifically, in step S2, the specific components and their proportions in the aqueous solution of Pichia yeast fermentation medium are: yeast extract 10-30 g / L, phosphoric acid aqueous solution 25-35 g / L, calcium sulfate 1.0-3.0 g / L, potassium sulfate 18-22 g / L, potassium hydroxide 4.0-5.0 g / L, glycerol 30-50 g / L, magnesium sulfate heptahydrate 7-13 g / L, ammonium chloride 4-5 g / L, PTM1 trace element solution 4-6 ml / L, and defoamer 1-2 ml / L; PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.06-0.1g / L, copper sulfate pentahydrate 5-7g / L, manganese sulfate monohydrate 2.5-3.5g / L, sodium molybdate dihydrate 0.18-0.22g / L, boric acid 0.036-0.044g / L, zinc chloride 27-33g / L, cobalt chloride hexahydrate 0.45-0.55g / L, biotin 0.45-0.55g / L, sulfuric acid 5-7ml / L, ferrous sulfate 145-155g / L. When preparing the Pichia yeast fermentation medium, first prepare an aqueous solution of yeast extract, phosphoric acid solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride, sterilize the aqueous solution at 120-122° C. for 18-22 minutes, and add the PTM1 trace element solution after the temperature drops to 28-32° C.; the PTM1 trace element solution is prepared using ultrapure water and sterilized by filtering with a 0.22 μm pore size sieve.

[0046] In step S2, the volume of the fermentation tank is more than 5L, the volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 50-70% of the volume of the fermentation tank, and the volume of the seed liquid is 3-10% of the volume of the Pichia yeast fermentation medium in the fermentation tank. Ventilation is air ventilation, and the initial ventilation ratio is controlled to be (2.0, ε) VVM, the initial stirring speed is (200, ε) rpm, and the initial relative tank pressure is (0.05, ε) MPa, that is, the initial tank pressure is higher than the atmospheric pressure by (0.05, ε) MPa, wherein ε is 10%, indicating the maximum deviation from the fixed value, such as the initial ventilation ratio (2.0, ε) VVM, indicating that the initial ventilation ratio is (2.0±2.0×10%) VVM, i.e. 1.8-2.2 VVM, and others are similar. To correct the dissolved oxygen content DO100%, the DO value at that time is defined as DO100%, and then the dissolved oxygen content in the process is controlled to be DO10-60% by increasing the stirring speed and ventilation ratio; ammonia water is used to control the process pH between 5.5-6.0, the temperature is 30±1℃, the stirring speed is 200-1000rpm, the ventilation ratio is 2.0-4.0VVM, and the relative tank pressure is 0.05±0.02MPa.

[0047] Specifically, in step S3, feed medium A is an aqueous solution containing 500-600 g / L glycerol and 10-20 ml / L PTM1 trace element solution; the PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.06-0.1 g / L, copper sulfate pentahydrate 5-7 g / L, manganese sulfate monohydrate 2.5-3.5 g / L, sodium molybdate dihydrate 0.18-0.22 g / L, boric acid 0.036-0.044 g / L , zinc chloride 27-33g / L, cobalt chloride hexahydrate 0.45-0.55g / L, biotin 0.45-0.55g / L, sulfuric acid 5-7ml / L, ferrous sulfate 145-155g / L; during preparation, sterilize glycerol at 120-122℃ for 18-22min, add PTM1 trace element solution after cooling to 28-32℃; PTM1 trace element solution is prepared with ultrapure water and sterilized by filtration with a 0.22μm pore size sieve. Based on the volume of Pichia yeast fermentation medium in the fermenter, the rate of feeding medium A is 30g / L / h-50g / L / h.

[0048] Take samples regularly to detect the OD of the fermentation liquid 600 value, such as sampling and testing once every hour; use ammonia water to control the process pH between 5.5-6.0, temperature 30±1℃, stirring speed 200-1000rpm, ventilation ratio 2.0-4.0VVM, relative tank pressure 0.05±0.02MPa, and control the dissolved oxygen content in the process to DO10-60%.

[0049] Specifically, in step S4, feed medium B is a 100% methanol solution containing 10-20 ml / L PTM1 trace element solution, and the PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.06-0.1 g / L, copper sulfate pentahydrate 5-7 g / L, manganese sulfate monohydrate 2.5-3.5 g / L, sodium molybdate dihydrate 0.18-0.22 g / L, boric acid 0.036-0.044 g / L, zinc chloride 27-33 g / L, chlorine hexahydrate Cobalt oxide 0.45-0.55g / L, biotin 0.45-0.55g / L, sulfuric acid 5-7ml / L, ferrous sulfate 145-155g / L, wherein the PTM1 trace element solution is prepared with ultrapure water and sterilized by filtration with a 0.22μm pore size sieve; feed medium C is an aqueous solution containing 20-60g / L mannitol or sorbitol; feed medium D is an aqueous solution containing 100-200g / L yeast extract and 200-400g / L peptone. Feed medium B, feed medium C and feed medium D are fed at the same time, and based on the volume of the Pichia yeast fermentation medium in the fermenter, the rate of feeding feed medium B is 1-3g / L / h, the rate of feeding feed medium C is 2-5g / L / h, and the rate of feeding feed medium D is 2-5g / L / h. Ammonia water is used to control the process pH between 5.0-5.5, the temperature is 22-25°C, the stirring speed is 200-1000rpm, the ventilation ratio is 2.0-4.0VVM, the relative tank pressure is 0.05±0.02MPa, and the dissolved oxygen content in the process is controlled to be DO10-60%.

[0050] It should be noted that, during conventional induced fermentation, the pH is between 5.6-6.0 and the temperature is 26-30°C. In the present invention, the pH value during the induced fermentation is set to between 5.0-5.5 and the temperature is set to 22-25°C. Practice has shown that such pH and temperature are more conducive to induced fermentation.

[0051] The Pichia yeast fermentation medium provided by the present invention can promote the expression of bovine lactoferrin by the bacteria while meeting the growth and metabolism requirements of the bacteria. The Pichia yeast fermentation medium and the fermentation method provided by the present invention can increase the expression of bovine lactoferrin to 4-5 g / L, which is a technical breakthrough compared to the protein expression of 1-4 g / L in the existing fermentation process. The raw materials used are mostly inorganic salts, which are low in cost and bring more high-quality products to the market while also bringing better economic benefits to the enterprise.

[0052] Comparative Example

[0053] A fermentation method of Pichia pastoris comprises the following steps:

[0054] Step S1, seed solution preparation: Pichia pastoris working seeds are spread on YPD solid culture medium plates for cultivation, and after cultivation, single colonies are picked and inoculated into a shake flask containing YPD liquid culture medium to obtain seed solution.

[0055] The Genban ID of the bovine lactoferrin gene is JX294418.1, and the Pichia pastoris working seed is GS115 / pPIC9K-blf. The recombinant bovine lactoferrin is expressed intracellularly in Pichia pastoris, and the copy number of the bovine lactoferrin gene in the Pichia pastoris genome is 5.88.

[0056] The aqueous solution of YPD solid culture medium is prepared as follows: 1.0 g / 100 ml yeast extract, 2.0 g / 100 ml peptone, 2.0 g / 100 ml glucose, 2.0 g / 100 ml agar, and 0.2 g / 100 ml Geneticin G418. It is then sterilized at 121°C for 20 min, cooled to 60°C, and aseptically operated on a clean bench. The solution is poured into a 9 cm diameter plate, with each plate containing 25 ml of YPD solid culture medium.

[0057] The composition of the YPD liquid culture medium aqueous solution is as follows: 1.0g / 100ml yeast extract, 2.0g / 100ml peptone, 2.0g / 100ml glucose, 0.2g / 100ml Geneticin G418. Prepare 400ml, each 500ml shake flask contains 200ml of culture medium, and then put it into a high-pressure steam sterilizer and sterilize it at 121℃ for 20min.

[0058] Specifically, the Pichia pastoris working seeds were spread on YPD solid culture medium plates, 0.75 ml of Pichia pastoris working seeds were spread on each plate, and the plates were placed in a 30°C incubator for 2.5 days. Then, a single colony of Pichia pastoris on three plates was inoculated into a 1L shake flask containing 200 ml of YPD liquid culture medium, and the shaker was placed in a shaker. The temperature was controlled at 30°C and the rotation speed was 220 rpm. The OD was obtained. 600 It is 7.5 seed liquid.

[0059] Step S2, batch fermentation: aseptically introducing the seed liquid into a fermentation tank containing a Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, correcting the dissolved oxygen content DO100%, and then controlling the dissolved oxygen content DO35% during the process by increasing the stirring speed and ventilation ratio, and controlling the process pH to 5.75 and the temperature to 30°C;

[0060] The specific components and their proportions in the aqueous solution of the Pichia yeast fermentation medium are as follows: 26.7 ml / L of 85% phosphoric acid aqueous solution by mass percentage, 0.93 g / L of calcium sulfate, 18.2 g / L of potassium sulfate, 14.9 g / L of magnesium sulfate heptahydrate, 4.13 g / L of potassium hydroxide, 40 g / L of glycerol, and 4.35 ml / L of PTM1 trace element solution, wherein the PTM1 trace element solution contains: 0.08 g / L of sodium iodide, 6 g / L of copper sulfate pentahydrate, 3 g / L of manganese sulfate monohydrate, 0.2 g / L of sodium molybdate dihydrate, 0.02 g / L of boric acid, 20 g / L of zinc chloride, 0.5 g / L of cobalt chloride hexahydrate, 0.2 g / L of biotin, 5 ml / L of sulfuric acid, and 65 g / L of ferrous sulfate. When preparing the Pichia yeast fermentation medium, first prepare an aqueous solution of 85% by weight of phosphoric acid aqueous solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride, sterilize the aqueous solution at 121° C. for 20 minutes, and add the PTM1 trace element solution after the temperature drops to 30° C.; the PTM1 trace element solution is prepared using ultrapure water and sterilized by filtering with a 0.22 μm pore size sieve.

[0061] The volume of the fermentation tank is 30L, the volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 60% of the volume of the fermentation tank, and the volume of the seed liquid is 7% of the volume of the Pichia yeast fermentation medium in the fermentation tank. The initial ventilation ratio is controlled to be 2.0VVM, the initial stirring speed is 200rpm, and the initial relative tank pressure is 0.05MPa, that is, the initial tank pressure is 0.05MPa higher than the atmospheric pressure; the correction of the dissolved oxygen content DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen content in the process to be DO35% by increasing the stirring speed and ventilation ratio; ammonia water is used to control the process pH to 5.75, the temperature to 30°C, the stirring speed to 200-1000rpm, the ventilation ratio to 2.0-4.0VVM, and the relative tank pressure to 0.05±0.02MPa.

[0062] Step S3, fed-batch fermentation: observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen content (DO) exceeds 80%, start feeding the feed medium A' and take samples regularly to detect the OD of the fermentation liquid. 600 Value, when OD 600 When the value exceeds 150, stop feeding the feed medium A′, and control the dissolved oxygen DO35% and pH5.75 and temperature30°C in the process by stirring speed and ventilation ratio;

[0063] Feed medium A' is an aqueous solution containing 550g / L glycerol and 15ml / L PTM1 trace element solution; the PTM1 trace element solution contains the following components and their proportions: 0.08g / L sodium iodide, 6g / L copper sulfate pentahydrate, 3g / L manganese sulfate monohydrate, 0.2g / L sodium molybdate dihydrate, 0.02g / L boric acid, 20g / L zinc chloride, 0.5g / L cobalt chloride hexahydrate, 0.2g / L biotin, 5ml / L sulfuric acid, 65g / L ferrous sulfate. During preparation, glycerol was sterilized at 121°C for 20min, and after cooling to 30°C, the PTM1 trace element solution was added; the PTM1 trace element solution was prepared with ultrapure water and sterilized by filtering with a 0.22μm pore size sieve. Based on the volume of the Pichia yeast fermentation medium in the fermenter, the rate of feeding feed medium A' was 40g / L / h.

[0064] Take samples regularly to detect the OD of the fermentation liquid 600 value, sampling and testing once every hour; use ammonia water to control the process pH between 5.5-6.0, control the dissolved oxygen content in the process to DO35%, temperature to 30℃, stirring speed to 200-1000rpm, ventilation ratio to 2.0-4.0VVM, relative tank pressure to 0.05±0.02MPa.

[0065] Step S4, inducing fermentation: stop feeding the feed medium A', let the bacteria starve for 30 minutes, start feeding the feed medium B', start inducing expression, control the dissolved oxygen content DO35% during the process by stirring speed and ventilation ratio, control the process pH5.75, the temperature is 30°C, and induce expression for 35 hours to end the fermentation.

[0066] The feed medium B′ is a 100% methanol solution containing 15 ml / L PTM1 trace element solution, and the PTM1 trace element solution contains the following components and their proportions: 0.08 g / L sodium iodide, 6 g / L copper sulfate pentahydrate, 3 g / L manganese sulfate monohydrate, 0.2 g / L sodium molybdate dihydrate, 0.02 g / L boric acid, 20 g / L zinc chloride, 0.5 g / L cobalt chloride hexahydrate, 0.2 g / L biotin, 5 ml / L sulfuric acid, and 65 g / L ferrous sulfate, wherein the PTM1 trace element solution is prepared with ultrapure water and sterilized by filtration with a 0.22 μm pore size sieve; based on the volume of the Pichia yeast fermentation medium in the fermenter, the rate of feeding the feed medium B′ is 2 g / L / h. Ammonia water was used to control the pH value of the process to 5.75, the dissolved oxygen content during the process was controlled to be DO35%, the temperature was 30°C, the stirring speed was 200-1000rpm, the ventilation ratio was 2.0-4.0VVM, and the relative tank pressure was 0.05±0.02MPa.

[0067] After the fermentation was completed, the expression level of bovine lactoferrin was measured by liquid chromatography quantitative analysis and was 2.3 g / L.

[0068] Example 1

[0069] A fermentation method of Pichia pastoris comprises the following steps:

[0070] Step S1, seed solution preparation: Pichia pastoris working seeds are spread on YPD solid culture medium plates for cultivation, and after cultivation, single colonies are picked and inoculated into a shake flask containing YPD liquid culture medium to obtain seed solution.

[0071] The Genban ID of the bovine lactoferrin gene is JX294418.1, and the Pichia pastoris working seed is GS115 / pPIC9K-blf. The recombinant bovine lactoferrin is expressed intracellularly in Pichia pastoris, and the copy number of the bovine lactoferrin gene in the Pichia pastoris genome is 5.88.

[0072] The aqueous solution of YPD solid culture medium is prepared as follows: 1.0 g / 100 ml yeast extract, 2.0 g / 100 ml peptone, 2.0 g / 100 ml glucose, 2.0 g / 100 ml agar, and 0.2 g / 100 ml Geneticin G418. It is then sterilized at 121°C for 20 min, cooled to 60°C, and aseptically operated on a clean bench. The solution is poured into a 9 cm diameter plate, with each plate containing 25 ml of YPD solid culture medium.

[0073] The composition of the YPD liquid culture medium aqueous solution is as follows: 1.0g / 100ml yeast extract, 2.0g / 100ml peptone, 2.0g / 100ml glucose, 0.2g / 100ml Geneticin G418. Prepare 400ml, each 500ml shake flask contains 200ml of culture medium, and then put it into a high-pressure steam sterilizer and sterilize it at 121℃ for 20min.

[0074] Specifically, the Pichia pastoris working seeds were spread on YPD solid culture medium plates, 0.75 ml of Pichia pastoris working seeds were spread on each plate, and the plates were placed in a 30°C incubator for 2.5 days. Then, a single colony of Pichia pastoris on three plates was inoculated into a 1L shake flask containing 200 ml of YPD liquid culture medium, and the shaker was placed in a shaker. The temperature was controlled at 30°C and the rotation speed was 220 rpm. The OD was obtained. 600 It is 7.5 seed liquid.

[0075] Step S2, batch fermentation: aseptically introducing the seed liquid into a fermentation tank containing a Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, correcting the dissolved oxygen content DO100%, and then controlling the dissolved oxygen content DO35% during the process by increasing the stirring speed and ventilation ratio, controlling the process pH to 5.75, and the temperature to 30°C;

[0076] The specific components and their proportions in the aqueous solution of the Pichia yeast fermentation medium are: 20 g / L yeast extract, 30 g / L 85% phosphoric acid aqueous solution by mass percentage, 2.0 g / L calcium sulfate, 20 g / L potassium sulfate, 4.5 g / L potassium hydroxide, 40 g / L glycerol, 10 g / L magnesium sulfate heptahydrate, 4.5 g / L ammonium chloride, 5 ml / L PTM1 trace element solution, and 1.5 ml / L Antifoam 204 defoaming agent; the PTM1 trace element solution contains the following components and their proportions: 0.08 g / L sodium iodide, 6 g / L copper sulfate pentahydrate, 3.0 g / L manganese sulfate monohydrate, 0.2 g / L sodium molybdate dihydrate, 0.04 g / L boric acid, 30 g / L zinc chloride, 0.5 g / L cobalt chloride hexahydrate, 0.5 g / L biotin, 6 ml / L sulfuric acid, and 150 g / L ferrous sulfate. When preparing the Pichia yeast fermentation medium, first prepare yeast extract, 85% by weight phosphoric acid aqueous solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride aqueous solution, sterilize the aqueous solution at 121° C. for 20 minutes, and add the PTM1 trace element solution after the temperature drops to 30° C.; the PTM1 trace element solution is prepared using ultrapure water and sterilized by filtering with a 0.22 μm pore size sieve.

[0077] The volume of the fermentation tank is 30L, the volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 60% of the volume of the fermentation tank, and the volume of the seed liquid is 7% of the volume of the Pichia yeast fermentation medium in the fermentation tank. The initial ventilation ratio is controlled to be 2.0VVM, the initial stirring speed is 200rpm, and the initial relative tank pressure is 0.05MPa, that is, the initial tank pressure is 0.05MPa higher than the atmospheric pressure; the correction of the dissolved oxygen content DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen content in the process to be DO35% by increasing the stirring speed and ventilation ratio; ammonia water is used to control the process pH to 5.75, the temperature to 30°C, the stirring speed to 200-1000rpm, the ventilation ratio to 2.0-4.0VVM, and the relative tank pressure to 0.05±0.02MPa.

[0078] Step S3, fed-batch fermentation: observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen content (DO) exceeds 80%, start feeding the feed medium A and take samples regularly to detect the OD of the fermentation liquid. 600 Value, when OD 600 When the value exceeds 150, stop feeding the feed medium A, and control the dissolved oxygen DO35% and pH5.75 and temperature30℃ in the process by stirring speed and ventilation ratio;

[0079] Feed medium A is an aqueous solution containing 550g / L glycerol and 15ml / LPTM1 trace element solution; the PTM1 trace element solution contains the following components and their proportions: 0.08g / L sodium iodide, 6g / L copper sulfate pentahydrate, 3.0g / L manganese sulfate monohydrate, 0.2g / L sodium molybdate dihydrate, 0.04g / L boric acid, 30g / L zinc chloride, 0.5g / L cobalt chloride hexahydrate, 0.5g / L biotin, 6ml / L sulfuric acid, and 150g / L ferrous sulfate. During preparation, glycerol was sterilized at 121°C for 20min, and after cooling to 30°C, the PTM1 trace element solution was added; the PTM1 trace element solution was prepared with ultrapure water and sterilized by filtering with a 0.22μm pore size sieve. Based on the volume of the Pichia yeast fermentation medium in the fermenter, the rate of feeding feed medium A was 40g / L / h.

[0080] Take samples regularly to detect the OD of the fermentation liquid 600 value, sampling and testing once every hour; use ammonia water to control the process pH to 5.75, the dissolved oxygen content in the process is controlled to be DO35%, the temperature is 30℃, the stirring speed is 200-1000rpm, the ventilation ratio is 2.0-4.0VVM, and the relative tank pressure is 0.05±0.02MPa.

[0081] Step S4, inducing fermentation: stop feeding feed medium A, let the bacteria starve for 30 minutes, start feeding feed medium B, feed medium C and feed medium D, start inducing expression, control the dissolved oxygen content DO35% during the process by stirring speed and ventilation ratio, control the process pH to 5.25, the temperature to 22°C, and induce expression for 35 hours to end the fermentation.

[0082] Feed medium B is a 100% methanol solution containing 15ml / L PTM1 trace element solution, and the PTM1 trace element solution contains the following components and their proportions: 0.08g / L sodium iodide, 6g / L copper sulfate pentahydrate, 3.0g / L manganese sulfate monohydrate, 0.2g / L sodium molybdate dihydrate, 0.04g / L boric acid, 30g / L zinc chloride, 0.5g / L cobalt chloride hexahydrate, 0.5g / L biotin, 6ml / L sulfuric acid, 150g / L ferrous sulfate, wherein the PTM1 trace element solution is prepared with ultrapure water and sterilized by filtration with a 0.22μm pore size sieve; Feed medium C is an aqueous solution containing 40g / L sorbitol; Feed medium D is an aqueous solution containing 150g / L yeast extract and 300g / L peptone. Feed medium B, feed medium C and feed medium D were added simultaneously, and based on the volume of the Pichia fermentation medium in the fermenter, the rate of feed medium B was 2 g / L / h, the rate of feed medium C was 3.5 g / L / h, and the rate of feed medium D was 3.5 g / L / h. Ammonia water was used to control the pH of the process to 5.25, the dissolved oxygen content in the process was controlled to DO35%, the temperature was 22°C, the stirring speed was 200-1000 rpm, the ventilation ratio was 2.0-4.0 VVM, and the relative tank pressure was 0.05±0.02 MPa.

[0083] After the fermentation was completed, the expression level of bovine lactoferrin was measured by liquid chromatography quantitative analysis and was 4.3 g / L.

[0084] Example 2

[0085] A fermentation method of Pichia pastoris comprises the following steps:

[0086] Step S1, seed solution preparation: Pichia pastoris working seeds are spread on YPD solid culture medium plates for cultivation, and after cultivation, single colonies are picked and inoculated into a shake flask containing YPD liquid culture medium to obtain seed solution.

[0087] The Genban ID of the bovine lactoferrin gene is JX294418.1, and the Pichia pastoris working seed is GS115 / pPIC9K-blf. The recombinant bovine lactoferrin is expressed intracellularly in Pichia pastoris, and the copy number of the bovine lactoferrin gene in the Pichia pastoris genome is 5.88.

[0088] The aqueous solution of YPD solid culture medium is prepared as follows: 1.0 g / 100 ml yeast extract, 2.0 g / 100 ml peptone, 2.0 g / 100 ml glucose, 2.0 g / 100 ml agar, and 0.2 g / 100 ml Geneticin G418. It is then sterilized at 121°C for 20 min, cooled to 60°C, and aseptically operated on a clean bench. The solution is poured into a 9 cm diameter plate, with each plate containing 25 ml of YPD solid culture medium.

[0089] The composition of the YPD liquid culture medium aqueous solution is as follows: 1.0g / 100ml yeast extract, 2.0g / 100ml peptone, 2.0g / 100ml glucose, 0.2g / 100ml Geneticin G418. Prepare 400ml, each 500ml shake flask contains 200ml of culture medium, and then put it into a high-pressure steam sterilizer and sterilize it at 121℃ for 20min.

[0090] Specifically, the Pichia pastoris working seeds were spread on YPD solid culture medium plates, 0.05 ml of Pichia pastoris working seeds were spread on each plate, and the plates were placed in a 30°C incubator for 2 days. Then, a single colony of Pichia pastoris on one plate was inoculated into a 1L shake flask containing 200 ml of YPD liquid culture medium, placed in a shaker, and the temperature was controlled at 28°C and the rotation speed was 210 rpm. The OD was obtained. 600 5% seed liquid.

[0091] Step S2, batch fermentation: aseptically introducing the seed liquid into a fermentation tank containing Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, calibrating the dissolved oxygen content DO100%, and then controlling the dissolved oxygen content DO10% during the process by increasing the stirring speed and ventilation ratio, controlling the process pH to 5.5, and the temperature to 30°C;

[0092] The specific components and their proportions in the aqueous solution of the Pichia yeast fermentation medium are: 10 g / L yeast extract, 25 g / L 85% phosphoric acid aqueous solution by mass percentage, 1.0 g / L calcium sulfate, 18 g / L potassium sulfate, 4.0 g / L potassium hydroxide, 30 g / L glycerol, 7 g / L magnesium sulfate heptahydrate, 4 g / L ammonium chloride, 4 ml / L PTM1 trace element solution, and 1 ml / L Antifoam 204 defoaming agent; the PTM1 trace element solution contains the following components and their proportions: 0.06 g / L sodium iodide, 5 g / L copper sulfate pentahydrate, 2.5 g / L manganese sulfate monohydrate, 0.18 g / L sodium molybdate dihydrate, 0.036 g / L boric acid, 27 g / L zinc chloride, 0.45 g / L cobalt chloride hexahydrate, 0.45 g / L biotin, 5 ml / L sulfuric acid, and 145 g / L ferrous sulfate. When preparing the Pichia yeast fermentation medium, first prepare yeast extract, 85% by weight phosphoric acid aqueous solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride aqueous solution, sterilize the aqueous solution at 120° C. for 18 minutes, and add the PTM1 trace element solution after the temperature drops to 28° C.; the PTM1 trace element solution is prepared using ultrapure water and sterilized by filtering with a 0.22 μm pore size sieve.

[0093] The volume of the fermentation tank is 5L, the volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 50% of the volume of the fermentation tank, and the volume of the seed liquid is 3% of the volume of the Pichia yeast fermentation medium in the fermentation tank. The initial ventilation ratio is controlled to be 2.0VVM, the initial stirring speed is 200rpm, and the initial relative tank pressure is 0.05MPa, that is, the initial tank pressure is 0.05MPa higher than the atmospheric pressure; the correction of the dissolved oxygen content DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen content in the process to be DO10% by increasing the stirring speed and ventilation ratio; ammonia water is used to control the process pH to 5.5, the temperature to 30°C, the stirring speed to 200-1000rpm, the ventilation ratio to 2.0-4.0VVM, and the relative tank pressure to 0.05±0.02MPa.

[0094] Step S3, fed-batch fermentation: observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen content (DO) exceeds 80%, start feeding the feed medium A and take samples regularly to detect the OD of the fermentation liquid. 600 Value, when OD 600 When the value exceeds 150, stop feeding the feed medium A, and control the dissolved oxygen DO10% by increasing the stirring speed and the aeration ratio, and control the pH of the process to 5.5 and the temperature to 30°C;

[0095] Feed medium A is an aqueous solution containing 500g / L glycerol and 10ml / L PTM1 trace element solution; the PTM1 trace element solution contains the following components and their proportions: 0.06g / L sodium iodide, 5g / L copper sulfate pentahydrate, 2.5g / L manganese sulfate monohydrate, 0.18g / L sodium molybdate dihydrate, 0.036g / L boric acid, 27g / L zinc chloride, 0.45g / L cobalt chloride hexahydrate, 0.45g / L biotin, 5ml / L sulfuric acid, 145g / L ferrous sulfate; during preparation, glycerol is sterilized at 120°C for 18min, and after cooling to 28°C, the PTM1 trace element solution is added; the PTM1 trace element solution is prepared with ultrapure water and sterilized by filtering with a 0.22μm pore size sieve. Based on the volume of the Pichia yeast fermentation medium in the fermenter, the rate of feeding feed medium A is 30g / L / h.

[0096] Take samples regularly to detect the OD of the fermentation liquid 600 value, sampling and testing once every hour; use ammonia water to control the process pH to 5.5, the dissolved oxygen content in the process is controlled to be DO10%, the temperature is 30℃, the stirring speed is 200-1000rpm, the ventilation ratio is 2.0-4.0VVM, and the relative tank pressure is 0.05±0.02MPa.

[0097] Step S4, inducing fermentation: stop feeding feed medium A, starve the bacteria for 30 minutes, start feeding feed medium B, feed medium C and feed medium D, start inducing expression, control the dissolved oxygen content DO10% during the process by increasing the stirring speed and ventilation ratio, control the process pH to 5.0, and the temperature to 25°C. Induce expression for 30 hours to end the fermentation.

[0098] Feed medium B is a 100% methanol solution containing 10ml / L PTM1 trace element solution, and the PTM1 trace element solution contains the following components and their proportions: 0.06g / L sodium iodide, 5g / L copper sulfate pentahydrate, 2.5g / L manganese sulfate monohydrate, 0.18g / L sodium molybdate dihydrate, 0.036g / L boric acid, 27g / L zinc chloride, 0.45g / L cobalt chloride hexahydrate, 0.45g / L biotin, 5ml / L sulfuric acid, 145g / L ferrous sulfate, wherein the PTM1 trace element solution is prepared with ultrapure water and sterilized by filtration with a 0.22μm pore size sieve; Feed medium C is an aqueous solution containing 20g / L mannitol; Feed medium D is an aqueous solution containing 100g / L yeast extract and 200g / L peptone. Feed medium B, feed medium C and feed medium D were added simultaneously, and based on the volume of the Pichia fermentation medium in the fermenter, the rate of feed medium B was 1 g / L / h, the rate of feed medium C was 2 g / L / h, and the rate of feed medium D was 2 g / L / h. Ammonia water was used to control the pH of the process to 5.0, the dissolved oxygen content during the control process was DO10%, the temperature was 25°C, the stirring speed was 200-1000 rpm, the ventilation ratio was 2.0-4.0 VVM, and the relative tank pressure was 0.05±0.02 MPa.

[0099] After the fermentation was completed, the expression level of bovine lactoferrin was measured by liquid chromatography quantitative analysis and was 4 g / L.

[0100] Example 3

[0101] A fermentation method of Pichia pastoris comprises the following steps:

[0102] Step S1, seed solution preparation: Pichia pastoris working seeds are spread on YPD solid culture medium plates for cultivation, and after cultivation, single colonies are picked and inoculated into a shake flask containing YPD liquid culture medium to obtain seed solution.

[0103] The Genban ID of the bovine lactoferrin gene is JX294418.1, and the Pichia pastoris working seed is GS115 / pPIC9K-blf. The recombinant bovine lactoferrin is expressed intracellularly in Pichia pastoris, and the copy number of the bovine lactoferrin gene in the Pichia pastoris genome is 5.88.

[0104] The aqueous solution of YPD solid culture medium is prepared as follows: 1.0 g / 100 ml yeast extract, 2.0 g / 100 ml peptone, 2.0 g / 100 ml glucose, 2.0 g / 100 ml agar, and 0.2 g / 100 ml Geneticin G418. It is then sterilized at 121°C for 20 min, cooled to 60°C, and aseptically operated on a clean bench. The solution is poured into a 9 cm diameter plate, with each plate containing 25 ml of YPD solid culture medium.

[0105] The composition of the YPD liquid culture medium aqueous solution is as follows: 1.0g / 100ml yeast extract, 2.0g / 100ml peptone, 2.0g / 100ml glucose, 0.2g / 100ml Geneticin G418. Prepare 400ml, each 500ml shake flask contains 200ml of culture medium, and then put it into a high-pressure steam sterilizer and sterilize it at 121℃ for 20min.

[0106] Specifically, the Pichia pastoris working seeds were spread on YPD solid culture medium plates, 0.1 ml of Pichia pastoris working seeds were spread on each plate, and the plates were placed in a 30°C incubator for 3 days. Then, a single colony of Pichia pastoris on 5 plates was inoculated into a 1L shake flask containing 200 ml of YPD liquid culture medium, placed in a shaker, and the temperature was controlled at 32°C and the rotation speed was 230 rpm. The OD was obtained. 600 For 10% seed liquid.

[0107] Step S2, batch fermentation: aseptically introducing the seed liquid into a fermentation tank containing a Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, correcting the dissolved oxygen content DO100%, and then controlling the dissolved oxygen content DO60% during the process by increasing the stirring speed and ventilation ratio, controlling the process pH to 6.0, and the temperature to 30°C;

[0108] The specific components and their proportions in the aqueous solution of the Pichia yeast fermentation medium are: 30 g / L yeast extract, 35 g / L 85% phosphoric acid aqueous solution by mass percentage, 3.0 g / L calcium sulfate, 22 g / L potassium sulfate, 5.0 g / L potassium hydroxide, 50 g / L glycerol, 13 g / L magnesium sulfate heptahydrate, 5 g / L ammonium chloride, 6 ml / L PTM1 trace element solution, and 2 ml / L Antifoam 204 defoaming agent; the PTM1 trace element solution contains the following components and their proportions: 0.1 g / L sodium iodide, 7 g / L copper sulfate pentahydrate, 3.5 g / L manganese sulfate monohydrate, 0.22 g / L sodium molybdate dihydrate, 0.044 g / L boric acid, 33 g / L zinc chloride, 0.55 g / L cobalt chloride hexahydrate, 0.55 g / L biotin, 7 ml / L sulfuric acid, and 155 g / L ferrous sulfate. When preparing the Pichia yeast fermentation medium, first prepare yeast extract, 85% by weight phosphoric acid aqueous solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride aqueous solution, sterilize the aqueous solution at 122° C. for 22 minutes, and add the PTM1 trace element solution after the temperature drops to 32° C.; the PTM1 trace element solution is prepared using ultrapure water and sterilized by filtering with a 0.22 μm pore size sieve.

[0109] The volume of the fermentation tank is 200L, the volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 70% of the volume of the fermentation tank, and the volume of the seed liquid is 10% of the volume of the Pichia yeast fermentation medium in the fermentation tank. The initial ventilation ratio is controlled to be 2.0VVM, the initial stirring speed is 200rpm, and the initial relative tank pressure is 0.05MPa, that is, the initial tank pressure is 0.05MPa higher than the atmospheric pressure; the correction of the dissolved oxygen content DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen content in the process to be DO60% by increasing the stirring speed and ventilation ratio; ammonia water is used to control the process pH to 6.0, the temperature to 30°C, the stirring speed to 200-1000rpm, the ventilation ratio to 2.0-4.0VVM, and the relative tank pressure to 0.05±0.02MPa.

[0110] Step S3, fed-batch fermentation: observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen content (DO) exceeds 80%, start feeding the feed medium A and take samples regularly to detect the OD of the fermentation liquid. 600 Value, when OD 600 When the value exceeds 150, stop feeding the feed medium A, and control the dissolved oxygen DO60% by increasing the stirring speed and the ventilation ratio, and control the process pH to 6.0 and the temperature to 30°C;

[0111] Feed medium A is an aqueous solution containing 600g / L glycerol and 20ml / L PTM1 trace element solution; the PTM1 trace element solution contains the following components and their proportions: 0.1g / L sodium iodide, 7g / L copper sulfate pentahydrate, 3.5g / L manganese sulfate monohydrate, 0.22g / L sodium molybdate dihydrate, 0.044g / L boric acid, 33g / L zinc chloride, 0.55g / L cobalt chloride hexahydrate, 0.55g / L biotin, 7ml / L sulfuric acid, 155g / L ferrous sulfate; during preparation, glycerol is sterilized at 122°C for 22min, and after cooling to 32°C, the PTM1 trace element solution is added; the PTM1 trace element solution is prepared with ultrapure water and sterilized by filtering with a 0.22μm pore size sieve. Based on the volume of the Pichia yeast fermentation medium in the fermenter, the rate of feeding feed medium A is 50g / L / h.

[0112] Take samples regularly to detect the OD of the fermentation liquid 600 value, sampling and testing once every hour; use ammonia water to control the process pH to 6.0, the dissolved oxygen content in the process is controlled to be DO60%, the temperature is 30°C, the stirring speed is 200-1000rpm, the ventilation ratio is 2.0-4.0VVM, and the relative tank pressure is 0.05±0.02MPa.

[0113] Step S4, inducing fermentation: stop feeding feed medium A, starve the bacteria for 30 minutes, start feeding feed medium B, feed medium C and feed medium D, start inducing expression, control the dissolved oxygen content DO60% during the process by increasing the stirring speed and ventilation ratio, control the process pH to 5.5, and the temperature to 23.5°C. Induce expression for 40 hours to end the fermentation.

[0114] Feed medium B is a 100% methanol solution containing 20ml / L PTM1 trace element solution, and the PTM1 trace element solution contains the following components and their proportions: 0.1g / L sodium iodide, 7g / L copper sulfate pentahydrate, 3.5g / L manganese sulfate monohydrate, 0.22g / L sodium molybdate dihydrate, 0.044g / L boric acid, 33g / L zinc chloride, 0.55g / L cobalt chloride hexahydrate, 0.55g / L biotin, 7ml / L sulfuric acid, and 155g / L ferrous sulfate, wherein the PTM1 trace element solution is prepared with ultrapure water and sterilized by filtration with a 0.22μm pore size sieve; Feed medium C is an aqueous solution containing 60g / L mannitol; Feed medium D is an aqueous solution containing 200g / L yeast extract and 400g / L peptone. Feed medium B, feed medium C and feed medium D were added simultaneously, and based on the volume of the Pichia fermentation medium in the fermenter, the rate of feed medium B was 3 g / L / h, the rate of feed medium C was 5 g / L / h, and the rate of feed medium D was 5 g / L / h. Ammonia water was used to control the pH of the process to 5.5, the dissolved oxygen content during the control process was DO60%, the temperature was 23.5°C, the stirring speed was 200-1000 rpm, the ventilation ratio was 2.0-4.0 VVM, and the relative tank pressure was 0.05±0.02 MPa.

[0115] After the fermentation was completed, the expression level of bovine lactoferrin was measured by liquid chromatography quantitative analysis and was 5 g / L.

[0116] It can be seen from the above Examples 1-3 that the Pichia yeast fermentation medium and the fermentation method of the present invention make the expression amount of bovine lactoferrin 4-5 g / L; the comparative example uses a conventional BSM basic inorganic salt medium, the feed medium uses a conventional glycerol and methanol medium, the temperature of the induction fermentation stage in step S4 is 30° C., the pH is 5.75, and the other process parameters are exactly the same as those in Example 1, but the expression amount of bovine lactoferrin in the comparative example is only 2.3 g / L, which is about 50% of that in Example 1, indicating that the Pichia yeast fermentation medium and the fermentation method of the present invention can effectively increase the expression amount of recombinant bovine lactoferrin in Pichia yeast.

[0117] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A Pichia yeast fermentation medium for expressing recombinant bovine lactoferrin, characterized in that: The components and their proportions in the aqueous solution of the culture medium are: 10-30 g / L yeast extract, 25-35 g / L phosphoric acid aqueous solution, 1.0-3.0 g / L calcium sulfate, 18-22 g / L potassium sulfate, 4.0-5.0 g / L potassium hydroxide, 30-50 g / L glycerol, 7-13 g / L magnesium sulfate heptahydrate, 4-5 g / L ammonium chloride, 4-6 ml / L PTM1 trace element solution, and 1-2 ml / L defoamer; feed Culture medium A is an aqueous solution containing 500-600 g / L glycerol and 10-20 ml / L PTM1 trace element solution; feed culture medium B is a 100% methanol solution containing 10-20 ml / L PTM1 trace element solution; feed culture medium C is an aqueous solution containing 20-60 g / L mannitol or sorbitol; feed culture medium D is an aqueous solution containing 100-200 g / L yeast extract and 200-400 g / L peptone.

2. The culture medium according to claim 1, characterized in that The specification of the phosphoric acid aqueous solution is analytically pure, and the initial mass percentage is ≥85%; the defoaming agent is an organosilicon or polyether defoaming agent.

3. The culture medium according to claim 1, characterized in that The PTM1 trace element solution is an aqueous solution, comprising the following components and their proportions: sodium iodide 0.06-0.1g / L, copper sulfate pentahydrate 5-7g / L, manganese sulfate monohydrate 2.5-3.5g / L, sodium molybdate dihydrate 0.18-0.22g / L, boric acid 0.036-0.044g / L, zinc chloride 27-33g / L, cobalt chloride hexahydrate 0.45-0.55g / L, biotin 0.45-0.55g / L, sulfuric acid 5-7ml / L, and ferrous sulfate 145-155g / L.

4. A Pichia yeast fermentation method for expressing recombinant bovine lactoferrin, characterized in that: The method uses the culture medium according to any one of claims 1 to 3, and the method comprises the following steps: Step S1, seed solution preparation: spreading Pichia pastoris working seeds on a YPD solid culture medium plate for cultivation, picking a single colony after cultivation and inoculating it into a shake flask containing YPD liquid culture medium, and cultivating to obtain a seed solution; Step S2, batch fermentation: aseptically introducing the seed liquid into a fermentation tank containing a Pichia yeast fermentation medium, and after the initial ventilation ratio, initial stirring speed, and initial relative tank pressure are stable, correcting the dissolved oxygen content DO100%, and then controlling the dissolved oxygen content DO10-60% during the process by adjusting the stirring speed and ventilation ratio, controlling the process pH between 5.5-6.0, and the temperature at 30±1°C; Step S3, fed-batch fermentation: observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen content (DO) exceeds 80%, start feeding the feed medium A and take samples regularly to detect the OD of the fermentation liquid. 600 Value, when OD 600 When the value exceeds 150, stop feeding feed medium A, control the dissolved oxygen DO10-60% by stirring speed and ventilation ratio, control the pH between 5.5-6.0, and the temperature at 30±1℃; Step S4, inducing fermentation: stop feeding feed medium A, starve the bacteria for 30±1 min, start feeding feed medium B, feed medium C and feed medium D, start inducing expression, control the dissolved oxygen content DO10-60% during the process by stirring speed and ventilation ratio, control the process pH between 5.0-5.5, and the temperature at 22-25°C. Induce expression for 30-40 hours to end the fermentation.

5. The method according to claim 4, characterized in that In the step S1, the working seed of Pichia pastoris is GS115 / pPIC9K-blf, the recombinant bovine lactoferrin is expressed intracellularly in Pichia pastoris, and the copy number of the bovine lactoferrin gene in the genome of Pichia pastoris is 4-6.

6. The method according to claim 4, characterized in that In step S1, the OD of the seed solution 600 Is 5-10.

7. The method according to claim 4, characterized in that In step S2, the volume of the fermentation tank is more than 5L, the volume of the Pichia yeast fermentation medium in the fermentation tank accounts for 50-70% of the volume of the fermentation tank, and the volume of the seed liquid is 3-10% of the volume of the Pichia yeast fermentation medium in the fermentation tank; the initial ventilation ratio is (2.0, ε) VVM, the initial stirring speed is (200, ε) rpm, the initial relative tank pressure is (0.05, ε) MPa, ε is 10%, indicating the maximum deviation from the fixed value.

8. The method according to claim 4, characterized in that In the step S3, the rate of adding feed medium A is 30 g / L / h-50 g / L / h, based on the volume of the Pichia yeast fermentation medium in the fermenter.

9. The method according to claim 4, characterized in that In the step S4, based on the volume of the Pichia fermentation medium in the fermenter, the rate of the feed medium B is 1-3 g / L / h, the rate of the feed medium C is 2-5 g / L / h, and the rate of the feed medium D is 2-5 g / L / h.

10. The method according to claim 4, characterized in that In the steps S2 to S4, the pH value is controlled by using aqueous ammonia, the stirring speed is 200-1000 rpm, the ventilation ratio is 2.0-4.0 VVM, and the relative tank pressure is 0.05±0.02 MPa.