Fermentation medium and method for expressing recombinant bovine lactoferricin in pichia

Abstract

The present application relates to the Pichia pastoris fermentation medium and fermentation method for expressing recombinant bovine lactoferrin, and belongs to the technical field of biochemistry, which solves the technical problem of improving the expression amount of bovine lactoferrin in the process of industrialized and large-scale preparation of recombinant bovine lactoferrin. The components and their proportions in the Pichia pastoris fermentation medium aqueous solution for expressing recombinant bovine lactoferrin are as follows: 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 defoaming agent 1-2 ml / L. The high-density fermentation method of the present application is realized by controlling the technical parameters such as the dissolved oxygen amount, pH value, temperature and feeding medium rate in the fermentation process, so that the expression amount of bovine lactoferrin is improved to 4-5 g / L.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, and specifically relates to the fermentation culture medium and fermentation method of Pichia pastoris expressing recombinant bovine lactoferrin. Background Technology

[0002] Lactoferrin is a glycoprotein with a relative molecular mass of approximately 80 kDa, primarily found in human milk. It is a natural antibacterial glycoprotein, possessing strong bactericidal and bacteriostatic abilities against both Gram-positive and Gram-negative bacteria due to its specific iron-binding capacity. Studies have also found that lactoferrin has antiviral, antioxidant, and immunomodulatory effects. Therefore, lactoferrin is widely used in various fields such as milk powder, food, and pharmaceuticals.

[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—Standard for the Use of Food Fortifiers included lactoferrin in GB14880-2012, recognizing it as a food fortifier.

[0004] Experimental studies have found that bovine lactoferrin and human lactoferrin have extremely similar biological activities. Currently, bovine lactoferrin is mainly produced from cow's milk, which is difficult and costly to isolate. With the development of gene editing technology, bovine lactoferrin can now be expressed from a variety of genetically engineered bacteria.

[0005] After ligating the bovine lactoferrin gene and PAOX1 promoter to the pPIC9K vector and transforming it into Pichia pastoris strain GS115, industrial-scale production can be achieved using high-density cell fermentation technology. This yields large quantities of bovine lactoferrin with the same activity as human lactoferrin, showing broad application prospects in biomedicine, health foods, and cosmetics. Correspondingly, how to increase the expression level of recombinant bovine lactoferrin during Pichia pastoris fermentation, thereby improving yield and production efficiency, has become a technical problem that needs to be solved. Summary of the Invention

[0006] To address the above-mentioned technical problems, this invention provides a Pichia pastoris fermentation medium and fermentation method for expressing recombinant bovine lactoferrin, in order to solve the technical problem of increasing the expression level of bovine lactoferrin in the process of industrial and large-scale preparation of recombinant bovine lactoferrin.

[0007] The objective of this invention is mainly achieved through the following technical solutions:

[0008] First, this invention provides a fermentation medium for Pichia pastoris expressing recombinant bovine lactoferrin. The components and their proportions in the aqueous solution of the medium are as follows: yeast extract 10-30 g / L, phosphate 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, and PTM1 trace element solution 4-6 ml / L. The feed medium A is an aqueous solution containing 500-600 g / L glycerol and 10-20 ml / L PTM1 trace element solution; the feed medium B is a 100% methanol solution containing 10-20 ml / L PTM1 trace element solution; the feed medium C is an aqueous solution containing 20-60 g / L mannitol or sorbitol; the feed medium D is an aqueous solution containing 100-200 g / L yeast extract and 200-400 g / L peptone.

[0009] Furthermore, the phosphoric acid aqueous solution is of analytical grade and has an initial mass percentage content of ≥85%; the defoamer is an organosilicon or polyether defoamer.

[0010] Furthermore, the PTM1 trace element solution is an aqueous solution containing 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, cobalt chloride hexahydrate 0.45-0.55 g / L, biotin 0.45-0.55 g / L, sulfuric acid 5-7 ml / L, and ferrous sulfate 145-155 g / L.

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

[0012] Step S1, Seed culture preparation: The working seed of Pichia pastoris was spread on YPD solid medium plates and cultured. After culture, a single colony was picked and inoculated into a shake flask containing YPD liquid medium to obtain the seed culture.

[0013] Step S2, Batch Fermentation: The seed liquid is aseptically transferred into a fermenter containing Pichia pastoris fermentation medium. After the initial aeration ratio, initial stirring speed, and initial relative tank pressure stabilize, the dissolved oxygen (DO) is corrected to 100%. Then, the dissolved oxygen (DO) is controlled to 10-60% during the process by increasing the stirring speed and aeration ratio. The pH is controlled between 5.5 and 6.0, and the temperature is 30±1℃.

[0014] Step S3, Fed-feed fermentation: Observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen (DO) exceeds 80%, start feeding culture medium A. At the same time, take samples regularly to detect the OD of the fermentation broth. 600 Value, when OD 600 When the value exceeds 150, stop feeding culture medium A. Control the dissolved oxygen (DO) during the process by adjusting the stirring speed and aeration ratio to 10-60%, maintain the pH between 5.5 and 6.0, and keep the temperature at 30±1℃.

[0015] Step S4, Induction of Fermentation: Stop feeding fed medium A, allow the cells to starve for 30±1 min, then start feeding fed medium B, fed medium C and fed medium D to begin induction of expression. Control the dissolved oxygen (DO) during the process by adjusting the stirring speed and aeration ratio to 10-60%, the pH to 5.0-5.5, and the temperature to 22-25℃. Induction of expression for 30-40 h will end the fermentation.

[0016] Furthermore, in step S1, the working seed of Pichia pastoris is GS115 / pPIC9K-blf, and recombinant bovine lactoferrin is expressed intracellularly in the Pichia pastoris. 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 It is 5-10.

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

[0019] Furthermore, in step S3, the feeding rate of the feed medium A is 30 g / L / h to 50 g / L / h, based on the volume of the Pichia pastoris fermentation medium in the fermenter.

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

[0021] Furthermore, in steps S2-S4, ammonia water is used to control the pH value, the stirring speed is 200-1000 rpm, the air flow 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 fermentation medium of Pichia pastoris of the present invention contains a portion of yeast extract, ammonium chloride and defoamer. The yeast extract contains various nutrients required for the growth of Pichia pastoris and is easily utilized by the cells, and has a significant effect in the cell amplification stage. Ammonium chloride serves as an inorganic nitrogen source, supplementing and adapting the nitrogen source during the cell growth process. The defoamer prevents the fermentation liquid from foaming, ensuring oxygen transfer efficiency, substrate transfer and uniformity.

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

[0025] (3) The present invention increases the ferrous sulfate content in the PTM1 trace element solution from 65g / L in the conventional formula to about 150g / L. This is because bovine lactoferrin contains iron ions, which not only serve as a substance to nourish cells, but also provide a better precursor for the generation 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 rate during the fermentation process, so as to increase the expression level of bovine lactoferrin to 4-5 g / L.

[0027] (5) The Pichia pastoris fermentation medium and high-density fermentation method proposed in this invention use mostly inorganic salts as raw materials, which are inexpensive and provide a good example for the industrial and large-scale production of lactoferrin. Detailed Implementation

[0028] The following detailed description of the fermentation medium and fermentation method of Pichia pastoris expressing recombinant bovine lactoferrin, with reference to specific embodiments, is provided. These embodiments are for comparative and illustrative purposes only, and the present invention is not limited to these embodiments.

[0029] In existing technologies, Pichia pastoris is widely used in protein expression, mainly in the fields of medicine, industry, and biology. Through the Pichia pastoris expression system, it is possible to achieve efficient expression and large-scale production of a variety of important proteins. Specifically, one such method is to link the bovine lactoferrin gene and PAOX1 promoter with the vector pPIC9K and transform them into GS115 type Pichia pastoris, and then use high-density cell fermentation technology to achieve large-scale industrial production.

[0030] In view of the need for the industrial production of recombinant bovine lactoferrin using Pichia pastoris, this invention provides a Pichia pastoris fermentation medium, based on the BSM basic inorganic salt medium formula, with several materials optimized and added. The specific components and their proportions in the aqueous solution are as follows: yeast extract 10-30 g / L, phosphate 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. / L, PTM1 trace element solution 4-6ml / L, and defoamer 1-2ml / L; Feed medium A is an aqueous solution containing 500-600g / L glycerol and 10-20ml / L PTM1 trace element solution; Feed medium B is a 100% methanol solution containing 10-20ml / L PTM1 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 remaining substances are all analytical grade. The initial mass percentage of the phosphoric acid aqueous solution is ≥85%. The defoamer is an organosilicon or polyether defoamer, such as Antifoam 204.

[0032] Specifically, the PTM1 trace element solution is an aqueous solution containing 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, cobalt chloride hexahydrate 0.45-0.55 g / L, biotin 0.45-0.55 g / L, sulfuric acid 5-7 ml / L, and ferrous sulfate 145-155 g / 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: 85% phosphate aqueous solution 26.7 ml / L, calcium sulfate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate heptahydrate 14.9 g / L, potassium hydroxide 4.13 g / L, glycerol 40 g / L, PTM1 trace element solution 4.35 ml / L, of which PTM1 trace element solution contains: sodium iodide 0.08 g / L, copper sulfate pentahydrate 6 g / L, manganese sulfate monohydrate 3 g / L, sodium molybdate dihydrate 0.2 g / L, boric acid 0.02 g / L, zinc chloride 20 g / L, cobalt chloride hexahydrate 0.5 g / L, biotin 0.2 g / L, sulfuric acid 5 ml / L, and ferrous sulfate 65 g / L. Based on actual production needs, this invention modifies the content of the main components of the BSM basic inorganic salt culture medium and the component content of the PTM1 trace element solution from point values ​​to ranges. Simultaneously, it increases the content of boric acid, zinc chloride, biotin, and ferrous sulfate in the PTM1 trace element solution. Furthermore, it adds 10-30 g / L of yeast extract, 4-5 g / L of ammonium chloride, and 1-2 ml / L of antifoaming agent. The yeast extract contains various nutrients required for the growth of Pichia pastoris and is easily utilized directly by the cells, playing a significant role in the cell amplification stage. Ammonium chloride serves as an inorganic nitrogen source, supplementing and adapting the nitrogen source during cell growth. The antifoaming agent prevents foaming in the fermentation broth, ensuring oxygen transfer efficiency, substrate transfer, and homogeneity.

[0034] On the other hand, since bovine lactoferrin contains iron ions, which not only serve as a substance for nourishing cells but also provide a better precursor for the production of bovine lactoferrin, this invention increases the ferrous sulfate content in the PTM1 trace element solution added to the Pichia pastoris fermentation medium, fed medium A, and fed 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 non-inhibitory carbon sources, are beneficial for increasing bovine lactoferrin expression during the induction phase. Yeast extract and peptone in feed medium D are beneficial for increasing bovine lactoferrin expression during the induction phase and preventing the degradation of the target protein.

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

[0037] Step S1, Seed culture preparation: The working seed of Pichia pastoris was spread on YPD solid medium plates and cultured. After culture, a single colony was picked and inoculated into a shake flask containing YPD liquid medium to obtain the seed culture.

[0038] Step S2, Batch Fermentation: The seed liquid is aseptically transferred into a fermenter containing Pichia pastoris fermentation medium. After the initial aeration ratio, initial stirring speed, and initial relative tank pressure stabilize, the dissolved oxygen (DO) is corrected to 100%. Then, the dissolved oxygen (DO) is controlled to 10-60% during the process by increasing the stirring speed and aeration ratio. The pH is controlled between 5.5 and 6.0, and the temperature is 30±1℃.

[0039] Step S3, Fed-feed fermentation: Observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen (DO) exceeds 80%, start feeding culture medium A. At the same time, take samples regularly to detect the OD of the fermentation broth. 600 Value, when OD 600 When the value exceeds 150, stop feeding culture medium A. Control the dissolved oxygen (DO) during the process by adjusting the stirring speed and aeration ratio to 10-60%, maintain the pH between 5.5 and 6.0, and keep the temperature at 30±1℃.

[0040] Step S4, Induction of Fermentation: Stop feeding fed medium A, allow the cells to starve for 30±1 min, then start feeding fed medium B, fed medium C and fed medium D to begin induction of expression. Control the dissolved oxygen (DO) during the process by adjusting the stirring speed and aeration ratio to 10-60%, the pH to 5.0-5.5, and the temperature to 22-25℃. Induction of expression for 30-40 h will end the fermentation.

[0041] Specifically, in step S1, the GenbanK ID of the bovine lactoferrin gene is JX294418.1, and the working seed of Pichia pastoris is the GS115 type Pichia pastoris strain transformed by ligating the bovine lactoferrin gene, PAOX1 promoter, and vector pPIC9K; that is, the working seed of Pichia pastoris is GS115 / pPIC9K-blf. 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 medium consists of: 0.9-1.1 g / 100 ml yeast extract, 1.8-2.2 g / 100 ml peptone, 1.8-2.2 g / 100 ml glucose, 1.8-2.2 g / 100 ml agar, and 0.18-0.22 g / 100 ml genimycin G418. It is then sterilized at 120-122℃ for 18-22 minutes and cooled to approximately 58-62℃. Under aseptic conditions in a laminar flow hood, the solution is poured into 9 cm diameter petri dishes, each containing approximately 25 ml of YPD solid medium.

[0043] The aqueous solution of YPD liquid culture medium consists of: 0.9-1.1 g / 100 ml yeast extract, 1.8-2.2 g / 100 ml peptone, 1.8-2.2 g / 100 ml glucose, and 0.18-0.22 g / 100 ml genimycin G418. Prepare 400 ml of the solution. Each 500 ml shake flask contains approximately 200 ml of culture medium. Then, place the flask in an autoclave and autoclave it at 120-122°C for 18-22 minutes.

[0044] Specifically, the working seed culture of Pichia pastoris was spread onto YPD solid medium plates, with approximately 0.05-0.1 ml of the seed culture on each plate. The plates were then incubated at 30°C for 2-3 days. Next, single colonies from 1-5 plates were inoculated into 1L shake flasks containing 200 ml of YPD liquid medium. The flasks were placed in a shaker at 28-32°C and 210-230 rpm for 16-22 hours to obtain the OD (Oxygen Species) of Pichia pastoris. 600 Seed solution with an OD of 5-10 600 It is the absorbance of the seed liquid at a wavelength of 600nm.

[0045] Specifically, in step S2, the components and their proportions in the Pichia pastoris fermentation medium aqueous solution are as follows: yeast extract 10-30 g / L, phosphate 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 0.06-0.1 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, cobalt chloride hexahydrate 0.45-0.55 g / L, biotin 0.45-0.55 g / L, sulfuric acid 5-7 ml / L, and ferrous sulfate 145-155 g / L. When preparing the fermentation medium for Pichia pastoris, first prepare an aqueous solution of yeast extract, phosphate solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride. Sterilize this aqueous solution at 120-122℃ for 18-22 minutes, and then add PTM1 trace element solution after cooling to 28-32℃. The PTM1 trace element solution is prepared using ultrapure water and sterilized by filtration through a 0.22μm sieve.

[0046] In step S2, the fermenter volume is 5L or more, the volume of the Pichia pastoris fermentation medium in the fermenter accounts for 50-70% of the fermenter volume, and the seed liquid volume is 3-10% of the volume of the Pichia pastoris fermentation medium in the fermenter. Aeration is performed using air, with the initial aeration ratio controlled at (2.0, ε)VVM, the initial stirring speed at (200, ε)rpm, and the initial relative tank pressure at (0.05, ε)MPa, meaning the initial tank pressure is (0.05, ε)MPa higher than atmospheric pressure. Here, ε represents 10%, indicating the maximum deviation from the set value. For example, an initial aeration ratio of (2.0, ε)VVM means the initial aeration ratio is (2.0 ± 2.0 × 10%)VVM, i.e., 1.8-2.2VVM, and so on. The dissolved oxygen (DO) calibration at DO100% is to define the DO value at that time as DO100%, and then control the dissolved oxygen at DO10-60% during the process by increasing the stirring speed and the aeration ratio. Ammonia water is used to control the process pH between 5.5 and 6.0, the temperature at 30±1℃, the stirring speed at 200-1000 rpm, the aeration ratio at 2.0-4.0 VVM, and the relative tank pressure at 0.05±0.02 MPa.

[0047] Specifically, in step S3, the supplemental culture 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, and boric acid 0.036-0.044 g / L. The feed consisted of 27-33 g / L zinc chloride, 0.45-0.55 g / L cobalt chloride hexahydrate, 0.45-0.55 g / L biotin, 5-7 ml / L sulfuric acid, and 145-155 g / L ferrous sulfate. To prepare the feed, glycerol was sterilized at 120-122℃ for 18-22 min, then cooled to 28-32℃ before adding the PTM1 trace element solution. The PTM1 trace element solution was prepared using ultrapure water and filtered through a 0.22 μm sieve for sterilization. The feed rate of medium A was 30 g / L / h-50 g / L / h, based on the volume of the Pichia pastoris fermentation medium in the fermenter.

[0048] Regularly sample and test the OD of the fermentation broth 600 Values, such as sampling and testing once per hour; using ammonia to control the process pH between 5.5 and 6.0, temperature 30±1℃, stirring speed 200-1000rpm, air flow ratio 2.0-4.0VVM, relative tank pressure 0.05±0.02MPa, and dissolved oxygen content DO 10-60%.

[0049] Specifically, in step S4, the feed medium B is a 100% methanol solution containing 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 27-33 g / L, and chloride hexahydrate. The feed consisted of 0.45-0.55 g / L cobalt chloride, 0.45-0.55 g / L biotin, 5-7 ml / L sulfuric acid, and 145-155 g / L ferrous sulfate. The PTM1 trace element solution was prepared with ultrapure water and sterilized by filtration through a 0.22 μm sieve. Feed medium C was an aqueous solution containing 20-60 g / L mannitol or sorbitol; feed medium D was an aqueous solution containing 100-200 g / L yeast extract and 200-400 g / L peptone. Feed media B, C, and D were added simultaneously. Based on the volume of Pichia pastoris fermentation medium in the fermenter, the feed medium B was added at a rate of 1-3 g / L / h, the feed medium C at a rate of 2-5 g / L / h, and the feed medium D at a rate of 2-5 g / L / h. The process is controlled by using ammonia water to maintain the pH between 5.0 and 5.5, the temperature between 22 and 25°C, the stirring speed between 200 and 1000 rpm, the air flow ratio between 2.0 and 4.0 VVM, the relative tank pressure between 0.05 and 0.02 MPa, and the dissolved oxygen content between DO and 10% during the process.

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

[0051] The Pichia pastoris fermentation medium provided by this invention can promote the expression of bovine lactoferrin while meeting the growth and metabolic needs of the cells. Using the Pichia pastoris fermentation medium and fermentation method provided by this invention, the expression level of bovine lactoferrin can be increased to 4-5 g / L, representing a technological breakthrough compared to the 1-4 g / L protein expression level of existing fermentation processes. Furthermore, the raw materials used are mostly inorganic salts, resulting in low cost and bringing more high-quality products to the market while also bringing better economic benefits to enterprises.

[0052] Comparative Example

[0053] A fermentation method using Pichia pastoris includes the following steps:

[0054] Step S1, Seed culture preparation: The working seed of Pichia pastoris was spread on YPD solid medium plates and cultured. After culture, a single colony was picked and inoculated into a shake flask containing YPD liquid medium to obtain the seed culture.

[0055] The GenbanK ID of the bovine lactoferrin gene is JX294418.1, and the working seed of Pichia pastoris is GS115 / pPIC9K-blf. 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 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 genimycin G418. After sterilization at 121°C for 20 min, the solution is cooled to 60°C and then aseptically poured into 9 cm diameter petri dishes under a laminar flow hood. Each plate contains 25 ml of YPD solid medium.

[0057] The YPD liquid culture medium aqueous solution is prepared with the following composition: 1.0g / 100ml yeast extract, 2.0g / 100ml peptone, 2.0g / 100ml glucose, and 0.2g / 100ml genimycin G418. Prepare 400ml of the solution. Each 500ml shake flask contains 200ml of the culture medium. Then, place the flask in an autoclave and autoclave at 121℃ for 20min.

[0058] Specifically, the working seed culture of Pichia pastoris was spread onto YPD solid medium plates, with 0.75 ml of the seed culture on each plate. The plates were incubated at 30°C for 2.5 days. Then, single colonies from the three plates were inoculated into 1L shake flasks containing 200 ml of YPD liquid medium. The flasks were placed in a shaker and incubated at 30°C and 220 rpm for 19 hours to obtain the OD (October Result). 600 The seed solution was 7.5.

[0059] Step S2, Batch Fermentation: The seed liquid is aseptically transferred into a fermenter containing Pichia pastoris fermentation medium. After the initial aeration ratio, initial stirring speed, and initial relative tank pressure stabilize, the dissolved oxygen (DO) is corrected to 100%. Then, the dissolved oxygen (DO) is controlled to 35% by increasing the stirring speed and aeration ratio. The pH is controlled to 5.75 and the temperature to 30℃.

[0060] The specific components and their proportions in the aqueous fermentation medium of Pichia pastoris are as follows: 26.7 ml / L of 85% phosphoric acid aqueous solution, 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 fermentation medium for Pichia pastoris, first prepare an aqueous solution containing 85% phosphoric acid, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride. Sterilize this aqueous solution at 121℃ for 20 minutes, and then add the PTM1 trace element solution after cooling to 30℃. The PTM1 trace element solution is prepared using ultrapure water and sterilized by filtration through a 0.22μm sieve.

[0061] The fermenter has a volume of 30L. The volume of the Pichia pastoris fermentation medium in the fermenter accounts for 60% of the fermenter volume, and the volume of the seed liquid is 7% of the volume of the Pichia pastoris fermentation medium. The initial aeration ratio is controlled at 2.0 VVM, the initial stirring speed is 200 rpm, and the initial relative tank pressure is 0.05 MPa, i.e., the initial tank pressure is 0.05 MPa higher than atmospheric pressure. The dissolved oxygen (DO) is corrected to 100% by defining the DO value at that time as DO100%, and then controlling the dissolved oxygen to DO35% during the process by increasing the stirring speed and aeration ratio. Ammonia is used to control the process pH at 5.75, the temperature at 30℃, the stirring speed at 200-1000 rpm, the aeration ratio at 2.0-4.0 VVM, and the relative tank pressure at 0.05 ± 0.02 MPa.

[0062] Step S3, Fed-feed fermentation: Observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen (DO) exceeds 80%, start feeding the culture medium A′. At the same time, take samples regularly to detect the OD of the fermentation broth. 600 Value, when OD 600 When the value exceeds 150, stop feeding culture medium A′. Control the dissolved oxygen (DO) at 35%, pH at 5.75, and temperature at 30℃ during the process by adjusting the stirring speed and aeration ratio.

[0063] The fed-batch medium A′ was an aqueous solution containing 550 g / L glycerol and 15 ml / L PTM1 trace element solution. The PTM1 trace element solution contained the following components and their proportions: sodium iodide 0.08 g / L, copper sulfate pentahydrate 6 g / L, manganese sulfate monohydrate 3 g / L, sodium molybdate dihydrate 0.2 g / L, boric acid 0.02 g / L, zinc chloride 20 g / L, cobalt chloride hexahydrate 0.5 g / L, biotin 0.2 g / L, sulfuric acid 5 ml / L, and ferrous sulfate 65 g / L. During preparation, glycerol was sterilized at 121°C for 20 min, cooled to 30°C, and then the PTM1 trace element solution was added. The PTM1 trace element solution was prepared using ultrapure water and sterilized by filtration through a 0.22 μm sieve. The fed-batch medium A′ was added at a rate of 40 g / L / h, based on the volume of the Pichia pastoris fermentation medium in the fermenter.

[0064] Regularly sample and test the OD of the fermentation broth 600 The pH value is sampled and tested once per hour; ammonia water is used to control the pH between 5.5 and 6.0, the dissolved oxygen content is controlled at DO 35%, the temperature is 30℃, the stirring speed is 200-1000rpm, the air flow ratio is 2.0-4.0VVM, and the relative tank pressure is 0.05±0.02MPa.

[0065] Step S4, Induction of fermentation: Stop feeding the fed medium A′ and starve the cells for 30 minutes. Then start feeding the fed medium B′ to begin induction of expression. Control the dissolved oxygen (DO) to 35% and the pH to 5.75 and the temperature to 30℃ by adjusting the stirring speed and aeration ratio. Induce expression for 35 hours to end the fermentation.

[0066] The fed medium B′ is a 100% methanol solution containing 15 ml / L PTM1 trace element solution. The PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.08 g / L, copper sulfate pentahydrate 6 g / L, manganese sulfate monohydrate 3 g / L, sodium molybdate dihydrate 0.2 g / L, boric acid 0.02 g / L, zinc chloride 20 g / L, cobalt chloride hexahydrate 0.5 g / L, biotin 0.2 g / L, sulfuric acid 5 ml / L, and ferrous sulfate 65 g / L. The PTM1 trace element solution is prepared using ultrapure water and sterilized by filtration through a 0.22 μm sieve. The fed medium B′ is added at a rate of 2 g / L / h, based on the volume of Pichia pastoris fermentation medium in the fermenter. The process pH was controlled at 5.75 using ammonia water, the dissolved oxygen content was controlled at 35% DO, the temperature was 30℃, the stirring speed was 200-1000 rpm, the air flow ratio was 2.0-4.0 VVM, and the relative tank pressure was 0.05±0.02 MPa.

[0067] After fermentation, the expression level of bovine lactoferrin was determined to be 2.3 g / L by liquid chromatography.

[0068] Example 1

[0069] A fermentation method using Pichia pastoris includes the following steps:

[0070] Step S1, Seed culture preparation: The working seed of Pichia pastoris was spread on YPD solid medium plates and cultured. After culture, a single colony was picked and inoculated into a shake flask containing YPD liquid medium to obtain the seed culture.

[0071] The GenbanK ID of the bovine lactoferrin gene is JX294418.1, and the working seed of Pichia pastoris is GS115 / pPIC9K-blf. 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 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 genimycin G418. After sterilization at 121°C for 20 min, the solution is cooled to 60°C and then aseptically poured into 9 cm diameter petri dishes under a laminar flow hood. Each plate contains 25 ml of YPD solid medium.

[0073] The YPD liquid culture medium aqueous solution is prepared with the following composition: 1.0g / 100ml yeast extract, 2.0g / 100ml peptone, 2.0g / 100ml glucose, and 0.2g / 100ml genimycin G418. Prepare 400ml of the solution. Each 500ml shake flask contains 200ml of the culture medium. Then, place the flask in an autoclave and autoclave at 121℃ for 20min.

[0074] Specifically, the working seed culture of Pichia pastoris was spread onto YPD solid medium plates, with 0.75 ml of the seed culture on each plate. The plates were incubated at 30°C for 2.5 days. Then, single colonies from the three plates were inoculated into 1L shake flasks containing 200 ml of YPD liquid medium. The flasks were placed in a shaker and incubated at 30°C and 220 rpm for 19 hours to obtain the OD (October Result). 600 The seed solution was 7.5.

[0075] Step S2, Batch Fermentation: The seed liquid is aseptically transferred into a fermenter containing Pichia pastoris fermentation medium. After the initial aeration ratio, initial stirring speed, and initial relative tank pressure stabilize, the dissolved oxygen (DO) is corrected to 100%. Then, the dissolved oxygen (DO) is controlled to 35% by increasing the stirring speed and aeration ratio. The pH is controlled to 5.75 and the temperature is 30℃.

[0076] The specific components and their proportions in the Pichia pastoris fermentation medium aqueous solution are as follows: yeast extract 20 g / L, 85% phosphoric acid aqueous solution 30 g / L, calcium sulfate 2.0 g / L, potassium sulfate 20 g / L, potassium hydroxide 4.5 g / L, glycerol 40 g / L, magnesium sulfate heptahydrate 10 g / L, ammonium chloride 4.5 g / L, PTM1 trace element solution 5 ml / L, and Antifoam 204 defoamer 1.5 ml / L; the PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.08 g / L, copper sulfate pentahydrate 6 g / L, manganese sulfate monohydrate 3.0 g / L, sodium molybdate dihydrate 0.2 g / L, boric acid 0.04 g / L, zinc chloride 30 g / L, cobalt chloride hexahydrate 0.5 g / L, biotin 0.5 g / L, sulfuric acid 6 ml / L, and ferrous sulfate 150 g / L. When preparing the fermentation medium for Pichia pastoris, first prepare an aqueous solution of yeast extract, 85% phosphate solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride. Sterilize this aqueous solution at 121℃ for 20 minutes, and then add PTM1 trace element solution after cooling to 30℃. The PTM1 trace element solution is prepared using ultrapure water and sterilized by filtration through a 0.22μm sieve.

[0077] The fermenter has a volume of 30L. The volume of the Pichia pastoris fermentation medium in the fermenter accounts for 60% of the fermenter volume, and the volume of the seed liquid is 7% of the volume of the Pichia pastoris fermentation medium. The initial aeration ratio is controlled at 2.0 VVM, the initial stirring speed is 200 rpm, and the initial relative tank pressure is 0.05 MPa, i.e., the initial tank pressure is 0.05 MPa higher than atmospheric pressure. The dissolved oxygen (DO) is corrected to 100% by defining the DO value at that time as DO100%, and then controlling the dissolved oxygen to DO35% during the process by increasing the stirring speed and aeration ratio. Ammonia is used to control the process pH at 5.75, the temperature at 30℃, the stirring speed at 200-1000 rpm, the aeration ratio at 2.0-4.0 VVM, and the relative tank pressure at 0.05 ± 0.02 MPa.

[0078] Step S3, Fed-feed fermentation: Observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen (DO) exceeds 80%, start feeding culture medium A. At the same time, take samples regularly to detect the OD of the fermentation broth. 600 Value, when OD 600 When the value exceeds 150, stop feeding culture medium A. Control the dissolved oxygen (DO) at 35%, pH at 5.75, and temperature at 30℃ during the process by adjusting the stirring speed and aeration ratio.

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

[0080] Regularly sample and test the OD of the fermentation broth 600 The pH value is sampled and tested once per hour; ammonia water is used to control the process pH at 5.75, the dissolved oxygen content is controlled at DO 35%, the temperature is 30℃, the stirring speed is 200-1000rpm, the air flow ratio is 2.0-4.0VVM, and the relative tank pressure is 0.05±0.02MPa.

[0081] Step S4, Induction of fermentation: Stop feeding fed medium A, starve the cells for 30 minutes, then start feeding fed medium B, fed medium C and fed medium D to begin induction of expression. Control the dissolved oxygen (DO) to 35% and the pH to 5.25 and the temperature to 22℃ by adjusting the stirring speed and aeration ratio. Induce expression for 35 hours to end fermentation.

[0082] Feed medium B is a 100% methanol solution containing 15 ml / L PTM1 trace element solution. The PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.08 g / L, copper sulfate pentahydrate 6 g / L, manganese sulfate monohydrate 3.0 g / L, sodium molybdate dihydrate 0.2 g / L, boric acid 0.04 g / L, zinc chloride 30 g / L, cobalt chloride hexahydrate 0.5 g / L, biotin 0.5 g / L, sulfuric acid 6 ml / L, and ferrous sulfate 150 g / L. The PTM1 trace element solution is prepared using ultrapure water and sterilized by filtration through a 0.22 μm sieve. Feed medium C is an aqueous solution containing 40 g / L sorbitol. Feed medium D is an aqueous solution containing 150 g / L yeast extract and 300 g / L peptone. Feed media B, feed media C, and feed media D were added simultaneously. Based on the volume of Pichia pastoris fermentation medium in the fermenter, the feed media B was added at a rate of 2 g / L / h, feed media C at a rate of 3.5 g / L / h, and feed media D at a rate of 3.5 g / L / h. Ammonia was used to control the process pH at 5.25, dissolved oxygen (DO) at 35%, temperature at 22℃, stirring speed at 200-1000 rpm, aeration ratio at 2.0-4.0 VVM, and relative tank pressure at 0.05 ± 0.02 MPa.

[0083] After fermentation, the expression level of bovine lactoferrin was determined to be 4.3 g / L by liquid chromatography.

[0084] Example 2

[0085] A fermentation method using Pichia pastoris includes the following steps:

[0086] Step S1, Seed culture preparation: The working seed of Pichia pastoris was spread on YPD solid medium plates and cultured. After culture, a single colony was picked and inoculated into a shake flask containing YPD liquid medium to obtain the seed culture.

[0087] The GenbanK ID of the bovine lactoferrin gene is JX294418.1, and the working seed of Pichia pastoris is GS115 / pPIC9K-blf. 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 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 genimycin G418. After sterilization at 121°C for 20 min, the solution is cooled to 60°C and then aseptically poured into 9 cm diameter petri dishes under a laminar flow hood. Each plate contains 25 ml of YPD solid medium.

[0089] The YPD liquid culture medium aqueous solution is prepared with the following composition: 1.0g / 100ml yeast extract, 2.0g / 100ml peptone, 2.0g / 100ml glucose, and 0.2g / 100ml genimycin G418. Prepare 400ml of the solution. Each 500ml shake flask contains 200ml of the culture medium. Then, place the flask in an autoclave and autoclave at 121℃ for 20min.

[0090] Specifically, the working seed culture of Pichia pastoris was spread onto YPD solid medium plates, with 0.05 ml of the seed culture on each plate. The plates were incubated at 30°C for 2 days. Then, a single colony from each plate was inoculated into a 1L shake flask containing 200 ml of YPD liquid medium. The flask was placed in a shaker and incubated at 28°C and 210 rpm for 16 hours to obtain the OD (Oxygen Demand). 600 Seed solution with a concentration of 5.

[0091] Step S2, Batch Fermentation: The seed liquid is aseptically transferred into a fermenter containing Pichia pastoris fermentation medium. After the initial aeration ratio, initial stirring speed, and initial relative tank pressure stabilize, the dissolved oxygen (DO) is corrected to 100%. Then, the dissolved oxygen (DO) is controlled to 10% by increasing the stirring speed and aeration ratio. The pH is controlled to 5.5 and the temperature is 30℃.

[0092] The specific components and their proportions in the Pichia pastoris fermentation medium aqueous solution are as follows: yeast extract 10 g / L, 85% phosphate aqueous solution 25 g / L, calcium sulfate 1.0 g / L, potassium sulfate 18 g / L, potassium hydroxide 4.0 g / L, glycerol 30 g / L, magnesium sulfate heptahydrate 7 g / L, ammonium chloride 4 g / L, PTM1 trace element solution 4 ml / L, and Antifoam 204 defoamer 1 ml / L; the PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.06 g / L, copper sulfate pentahydrate 5 g / L, manganese sulfate monohydrate 2.5 g / L, sodium molybdate dihydrate 0.18 g / L, boric acid 0.036 g / L, zinc chloride 27 g / L, cobalt chloride hexahydrate 0.45 g / L, biotin 0.45 g / L, sulfuric acid 5 ml / L, and ferrous sulfate 145 g / L. When preparing the fermentation medium for Pichia pastoris, first prepare an aqueous solution of yeast extract, 85% phosphate solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride. Sterilize this aqueous solution at 120℃ for 18 minutes, and then add PTM1 trace element solution after cooling to 28℃. The PTM1 trace element solution is prepared using ultrapure water and sterilized by filtration through a 0.22μm sieve.

[0093] The fermenter has a volume of 5L. The volume of the Pichia pastoris fermentation medium in the fermenter occupies 50% of the fermenter volume, and the volume of the seed liquid is 3% of the volume of the Pichia pastoris fermentation medium. The initial aeration ratio is controlled at 2.0 VVM, the initial stirring speed is 200 rpm, and the initial relative tank pressure is 0.05 MPa, i.e., the initial tank pressure is 0.05 MPa higher than atmospheric pressure. The dissolved oxygen (DO) is corrected to 100% by defining the DO value at that time as DO100%, and then controlling the dissolved oxygen to DO10% during the process by increasing the stirring speed and aeration ratio. Ammonia is used to control the process pH at 5.5, the temperature at 30℃, the stirring speed at 200-1000 rpm, the aeration ratio at 2.0-4.0 VVM, and the relative tank pressure at 0.05 ± 0.02 MPa.

[0094] Step S3, Fed-feed fermentation: Observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen (DO) exceeds 80%, start feeding culture medium A. At the same time, take samples regularly to detect the OD of the fermentation broth. 600 Value, when OD 600 When the value exceeds 150, stop feeding culture medium A. Control the dissolved oxygen (DO) during the process by increasing the stirring speed and aeration ratio to 10%, control the pH to 5.5, and maintain the temperature at 30℃.

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

[0096] Regularly sample and test the OD of the fermentation broth 600 The pH value is sampled and tested once per hour; ammonia water is used to control the process pH 5.5, the dissolved oxygen content is controlled at DO 10%, the temperature is 30℃, the stirring speed is 200-1000rpm, the air flow ratio is 2.0-4.0VVM, and the relative tank pressure is 0.05±0.02MPa.

[0097] Step S4, Induction of fermentation: Stop feeding fed medium A, starve the cells for 30 minutes, then start feeding fed medium B, fed medium C and fed medium D to begin induction of expression. Control the dissolved oxygen (DO) to 10% by increasing the stirring speed and aeration ratio, control the pH to 5.0, and maintain the temperature at 25℃. Induction of expression for 30 hours will end the fermentation.

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

[0099] After fermentation, the expression level of bovine lactoferrin was determined to be 4 g / L by liquid chromatography.

[0100] Example 3

[0101] A fermentation method using Pichia pastoris includes the following steps:

[0102] Step S1, Seed culture preparation: The working seed of Pichia pastoris was spread on YPD solid medium plates and cultured. After culture, a single colony was picked and inoculated into a shake flask containing YPD liquid medium to obtain the seed culture.

[0103] The GenbanK ID of the bovine lactoferrin gene is JX294418.1, and the working seed of Pichia pastoris is GS115 / pPIC9K-blf. 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 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 genimycin G418. After sterilization at 121°C for 20 min, the solution is cooled to 60°C and then aseptically poured into 9 cm diameter petri dishes under a laminar flow hood. Each plate contains 25 ml of YPD solid medium.

[0105] The YPD liquid culture medium aqueous solution is prepared with the following composition: 1.0g / 100ml yeast extract, 2.0g / 100ml peptone, 2.0g / 100ml glucose, and 0.2g / 100ml genimycin G418. Prepare 400ml of the solution. Each 500ml shake flask contains 200ml of the culture medium. Then, place the flask in an autoclave and autoclave at 121℃ for 20min.

[0106] Specifically, the working seed culture of Pichia pastoris was spread onto YPD solid medium plates, with 0.1 ml of the seed culture on each plate. The plates were then incubated at 30°C for 3 days. Next, single colonies from each of the five plates were inoculated into a 1L shake flask containing 200 ml of YPD liquid medium. The flask was then placed in a shaker and incubated at 32°C and 230 rpm for 22 hours to obtain the OD (October Resultant). 600 The seed solution is 10.

[0107] Step S2, Batch Fermentation: The seed liquid is aseptically transferred into a fermenter containing Pichia pastoris fermentation medium. After the initial aeration ratio, initial stirring speed, and initial relative tank pressure stabilize, the dissolved oxygen (DO) is adjusted to 100%. Then, the dissolved oxygen (DO) is controlled to 60% by increasing the stirring speed and aeration ratio. The pH is controlled to 6.0 and the temperature is 30℃.

[0108] The specific components and their proportions in the Pichia pastoris fermentation medium aqueous solution are as follows: yeast extract 30 g / L, 85% phosphoric acid aqueous solution 35 g / L, calcium sulfate 3.0 g / L, potassium sulfate 22 g / L, potassium hydroxide 5.0 g / L, glycerol 50 g / L, magnesium sulfate heptahydrate 13 g / L, ammonium chloride 5 g / L, PTM1 trace element solution 6 ml / L, and Antifoam 204 defoamer 2 ml / L; the PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.1 g / L, copper sulfate pentahydrate 7 g / L, manganese sulfate monohydrate 3.5 g / L, sodium molybdate dihydrate 0.22 g / L, boric acid 0.044 g / L, zinc chloride 33 g / L, cobalt chloride hexahydrate 0.55 g / L, biotin 0.55 g / L, sulfuric acid 7 ml / L, and ferrous sulfate 155 g / L. When preparing the fermentation medium for Pichia pastoris, first prepare an aqueous solution of yeast extract, 85% phosphate solution, calcium sulfate, potassium sulfate, potassium hydroxide, glycerol, magnesium sulfate heptahydrate, and ammonium chloride. Sterilize this aqueous solution at 122℃ for 22 minutes, and then add PTM1 trace element solution after cooling to 32℃. The PTM1 trace element solution is prepared using ultrapure water and sterilized by filtration through a 0.22μm sieve.

[0109] The fermenter has a volume of 200L. The volume of the Pichia pastoris fermentation medium in the fermenter occupies 70% of the fermenter volume, and the volume of the seed liquid is 10% of the volume of the Pichia pastoris fermentation medium. The initial aeration ratio is controlled at 2.0 VVM, the initial stirring speed is 200 rpm, and the initial relative tank pressure is 0.05 MPa, i.e., the initial tank pressure is 0.05 MPa higher than atmospheric pressure. The dissolved oxygen (DO) is corrected to 100% by defining the DO value at that time as DO100%, and then controlling the dissolved oxygen to DO60% during the process by increasing the stirring speed and aeration ratio. Ammonia is used to control the process pH at 6.0, the temperature at 30℃, the stirring speed at 200-1000 rpm, the aeration ratio at 2.0-4.0 VVM, and the relative tank pressure at 0.05 ± 0.02 MPa.

[0110] Step S3, Fed-feed fermentation: Observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen (DO) exceeds 80%, start feeding culture medium A. At the same time, take samples regularly to detect the OD of the fermentation broth. 600 Value, when OD 600 When the value exceeds 150, stop feeding culture medium A. Control the dissolved oxygen (DO) to 60% by increasing the stirring speed and aeration ratio, control the pH to 6.0, and maintain the temperature at 30℃.

[0111] Feed medium A is an aqueous solution containing 600 g / L glycerol and 20 ml / L PTM1 trace element solution. The PTM1 trace element solution contains the following components and their proportions: sodium iodide 0.1 g / L, copper sulfate pentahydrate 7 g / L, manganese sulfate monohydrate 3.5 g / L, sodium molybdate dihydrate 0.22 g / L, boric acid 0.044 g / L, zinc chloride 33 g / L, cobalt chloride hexahydrate 0.55 g / L, biotin 0.55 g / L, sulfuric acid 7 ml / L, and ferrous sulfate 155 g / L. During preparation, glycerol is sterilized at 122°C for 22 min, then cooled to 32°C before adding the PTM1 trace element solution. The PTM1 trace element solution is prepared using ultrapure water and sterilized by filtration through a 0.22 μm sieve. The feed medium A is added at a rate of 50 g / L / h, based on the volume of the Pichia pastoris fermentation medium in the fermenter.

[0112] Regularly sample and test the OD of the fermentation broth 600 The pH value is sampled and tested once per hour; ammonia water is used to control the process pH 6.0, the dissolved oxygen content is controlled at DO 60%, the temperature is 30℃, the stirring speed is 200-1000rpm, the air flow ratio is 2.0-4.0VVM, and the relative tank pressure is 0.05±0.02MPa.

[0113] Step S4, Induction of fermentation: Stop feeding fed medium A, starve the cells for 30 minutes, then start feeding fed medium B, fed medium C and fed medium D to begin induction of expression. Control the dissolved oxygen (DO) to 60% by increasing the stirring speed and aeration ratio, control the pH to 5.5, and the temperature to 23.5℃. Induction of expression for 40 hours ends the fermentation.

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

[0115] After fermentation, the expression level of bovine lactoferrin was determined to be 5 g / L by liquid chromatography.

[0116] As can be seen from Examples 1-3 above, the Pichia pastoris fermentation medium and fermentation method of the present invention resulted in a bovine lactoferrin expression level of 4-5 g / L. The comparative example used a conventional BSM basal inorganic salt medium, and the fed medium used a conventional glycerol and methanol-based medium. The temperature of the induction fermentation stage in step S4 was 30°C and the pH was 5.75. The remaining process parameters were exactly the same as in Example 1. However, the bovine lactoferrin expression level of the comparative example was only 2.3 g / L, which is about 50% of that in Example 1. This indicates that the Pichia pastoris fermentation medium and fermentation method of the present invention can effectively improve the expression level of recombinant bovine lactoferrin in Pichia pastoris.

[0117] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermentation method for Pichia pastoris expressing recombinant bovine lactoferrin, characterized in that, Includes the following steps: Step S1, Seed culture preparation: The working seed of Pichia pastoris was spread on YPD solid medium plates and cultured. After culture, a single colony was picked and inoculated into a shake flask containing YPD liquid medium to obtain the seed culture. Step S2, Batch Fermentation: The seed culture is aseptically transferred into a fermenter containing Pichia pastoris fermentation medium. After the initial aeration ratio, initial stirring speed, and initial relative tank pressure stabilize, the dissolved oxygen (DO) is corrected to 100%. Then, the DO is controlled to 10-60% during the process by adjusting the stirring speed and aeration ratio. The pH is controlled between 5.5 and 6.0, and the temperature is 30±1℃. The components and their proportions in the Pichia pastoris fermentation medium are as follows: yeast extract 10-30 g / L, phosphate 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. Step S3, Fed-feed fermentation: Observe the fermentation process. When the pH value is higher than 6.5 and the dissolved oxygen (DO) exceeds 80%, start feeding culture medium A. At the same time, take samples regularly to detect the OD of the fermentation broth. 600 Value, when OD 600 When the value exceeds 150, stop feeding the culture medium A. Control the dissolved oxygen (DO) during the process by adjusting the stirring speed and aeration ratio to 10-60%, maintain the pH between 5.5 and 6.0, and keep the temperature at 30±1℃. The culture medium A is an aqueous solution containing 500-600 g / L glycerol and 10-20 ml / L TTM1 trace element solution. Step S4, Induction of Fermentation: Stop feeding fed medium A, allow the cells to starve for 30±1 min, then start feeding fed mediums B, C, and D to begin induction of expression. Control the dissolved oxygen (DO) to 10-60% and the pH to 5.0-5.5, and the temperature to 22-25℃ by adjusting the stirring speed and aeration ratio. Induce expression for 30-40 h to end fermentation. Feed medium B is a 100% methanol solution containing 10-20 ml / L of 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. The PTM1 trace element solution is an aqueous solution containing 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, cobalt chloride hexahydrate 0.45-0.55 g / L, biotin 0.45-0.55 g / L, sulfuric acid 5-7 ml / L, and ferrous sulfate 145-155 g / L.

2. The fermentation method of Pichia pastoris expressing recombinant bovine lactoferrin according to claim 1, characterized in that, The phosphoric acid aqueous solution is of analytical grade and has an initial mass percentage content of ≥85%; the defoamer is an organosilicon or polyether defoamer.

3. The fermentation method of Pichia pastoris expressing recombinant bovine lactoferrin according to claim 1, characterized in that, In 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 Pichia pastoris genome is 4-6.

4. The fermentation method of Pichia pastoris expressing recombinant bovine lactoferrin according to claim 1, characterized in that, In step S1, the OD of the seed solution 600 It is 5-10.

5. The fermentation method of Pichia pastoris expressing recombinant bovine lactoferrin according to claim 1, characterized in that, In step S2, the fermenter has a volume of 5L or more, the volume of the Pichia pastoris fermentation medium in the fermenter accounts for 50-70% of the fermenter volume, and the seed liquid volume is 3-10% of the volume of the Pichia pastoris fermentation medium in the fermenter; the initial aeration ratio is (2.0, ε) VVM, the initial stirring speed is (200, ε) rpm, and the initial relative tank pressure is (0.05, ε) MPa, where ε is 10%, representing the maximum deviation from the set value.

6. The fermentation method of Pichia pastoris expressing recombinant bovine lactoferrin according to claim 1, characterized in that, In step S3, the feeding rate of the fed-batch culture medium A is 30 g / L / h to 50 g / L / h, based on the volume of the Pichia pastoris fermentation medium in the fermenter.

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

8. The fermentation method of Pichia pastoris expressing recombinant bovine lactoferrin according to claim 1, characterized in that, In steps S2-S4, ammonia is used to control the pH value, the stirring speed is 200-1000 rpm, the air flow ratio is 2.0-4.0 VVM, and the relative tank pressure is 0.05±0.02 MPa.