Pichia pastoris fermentation medium as well as fermentation method and application thereof

Abstract

The invention discloses a pichia pastoris fermentation medium as well as a fermentation method and application thereof, and belongs to the technical field of fermentation. The pichia pastoris fermentation medium comprises the following components: 10-20 g / L of a yeast extract, 30-35 g / L of a phosphoric acid aqueous solution, 55-80 g / L of a macroelement inorganic salt, 4.5-5.5 g / L of a pH regulator, 10-20 g / L of a carbon source and 4-6 mL / L of a trace element solution. When the pichia pastoris fermentation culture medium is used for fermenting recombinant pichia pastoris, the recombinant pichia pastoris can be promoted to express bovine lactoferrin, and the expression quantity of the bovine lactoferrin is increased.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, and in particular to a Pichia yeast fermentation medium and a fermentation method and application thereof. Background Art

[0002] Lactoferrin is a glycoprotein with a relative molecular mass of about 78KD. It mainly exists in human milk and is a natural antibacterial glycoprotein. Due to its specific iron ion binding ability, it has strong bactericidal and antibacterial abilities against Gram-positive and Gram-negative bacteria. Lactoferrin also has antiviral, antioxidant and immunomodulatory effects. Therefore, lactoferrin is widely used in many fields such as milk powder, food and medicine.

[0003] Bovine lactoferrin has extremely similar biological activities to human lactoferrin. At present, bovine lactoferrin is mainly produced from cow's 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. For example, the bovine lactoferrin gene, PAOX1 promoter and vector pPIC9K are connected and transformed into GS115 type Pichia pastoris, and bovine lactoferrin is prepared using high-density cell fermentation technology. However, in the existing fermentation process, the expression level of bovine lactoferrin is still relatively low, and there is an urgent need for a fermentation medium and fermentation process that can enable recombinant Pichia pastoris to express bovine lactoferrin in large quantities. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a Pichia fermentation medium and a fermentation method and application thereof, so as to overcome the problem of low expression of bovine lactoferrin in the existing Pichia fermentation process.

[0005] In a first aspect, the present invention provides a Pichia pastoris fermentation medium comprising the following components: 10-20 g / L yeast extract, 30-35 g / L phosphoric acid aqueous solution, 55-80 g / L macro-element inorganic salts, 4.5-5.5 g / L pH regulator, 10-20 g / L carbon source and 4-6 mL / L trace element solution.

[0006] Compared with the prior art, 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, and improve the expression amount of bovine lactoferrin. The present invention optimizes the selection and proportion of the components in the fermentation medium, especially strictly limits the concentration of each component, and cooperates with each component in a specific concentration range, ensuring the supply of carbon source, nitrogen source and trace elements in the bacteria growth process, and promoting the growth and division of cells. A large number of inorganic salts and trace element solutions provide sufficient trace elements for the growth and reproduction of Pichia yeast, and yeast extract can not only provide necessary nitrogen source and other trace elements in the fermentation process of Pichia yeast, promote the metabolic activity and proliferation of cells, but also enhance cell tolerance and stabilize the fermentation process by optimizing the cell growth environment, and reduce the influence of cell growth inhibitory factors. On the one hand, the phosphoric acid aqueous solution provides a suitable acid-base environment for the fermentation of Pichia yeast together with the pH regulator, and on the other hand, the elements contained therein can also nourish cells and promote the growth and reproduction of Pichia yeast.

[0007] Further, the inorganic salt of a large number of elements includes at least one of calcium salt, potassium salt, magnesium salt, ferrous salt and ammonium salt; and / or,

[0008] The calcium salt comprises at least one of calcium sulfate or calcium chloride; and / or,

[0009] The potassium salt includes at least one of potassium sulfate, potassium chloride and potassium dihydrogen phosphate; and / or,

[0010] The magnesium salt includes at least one of magnesium sulfate heptahydrate and magnesium chloride; and / or,

[0011] The ferrous salt is ferrous sulfate heptahydrate; and / or,

[0012] The ammonium salt includes at least one of ammonium chloride and ammonium sulfate.

[0013] Compared with the prior art, the above technical solution defines the inorganic salt of a large amount of elements in the Pichia fermentation medium as at least one of calcium salt, potassium salt, magnesium salt, ferrous salt and ammonium salt, and provides specific selection of each salt type. The above salt types can cooperate with other components in the Pichia fermentation medium, so that the Pichia can obtain sufficient nutrition during fermentation in the fermentation medium and can grow and reproduce better.

[0014] Furthermore, the inorganic salts of macroelements include calcium sulfate, potassium sulfate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate and ammonium chloride; the concentration of calcium sulfate in the culture medium is 2.0-3.0 g / L, the concentration of potassium sulfate is 40-50 g / L, the concentration of magnesium sulfate heptahydrate is 10-20 g / L, the concentration of ferrous sulfate heptahydrate is 0.08-0.1 g / L, and the concentration of ammonium chloride is 4-5 g / L.

[0015] Compared with the prior art, the above technical solution further defines the components and concentrations of the macro-element inorganic salts. The above specific types and concentrations of inorganic salts have an optimal concentration ratio. Within the above specific concentration range, the synergistic effect between the elements is optimal, which can further promote the growth and division of Pichia pastoris cells.

[0016] Furthermore, based on one liter of trace element solution, the trace element solution includes the following components: 0.06-0.08 g of sodium iodide, 4-6 g of copper sulfate pentahydrate, 3-4 g of manganese sulfate monohydrate, 0.2-0.3 g of sodium molybdate dihydrate, 0.03-0.05 g of boric acid, 20-40 g of zinc chloride, 0.3-0.5 g of cobalt chloride hexahydrate, 10-12 g of sulfuric acid and 0.4-0.6 g of biotin.

[0017] Compared with the prior art, the above technical scheme limits the selection and content ratio of each component in the trace element solution, so that the trace element solution can ensure the supply of cell trace elements at all stages of Pichia pastoris growth and reproduction. The trace elements play a variety of important functions in the fungus, such as supporting enzyme activity, maintaining cell structure, regulating metabolic pathways, and antioxidant defense. Therefore, the above trace element solution can effectively promote the growth of Pichia pastoris cells.

[0018] Furthermore, the volume concentration of the phosphoric acid aqueous solution is ≥85%; and / or the pH regulator is potassium hydroxide; and / or the carbon source includes at least one of glycerol or glucose.

[0019] Compared with the prior art, the volume concentration of the phosphoric acid aqueous solution in the above technical solution is ≥85%, and the pH regulator is potassium hydroxide. The two can cooperate with each other to not only ensure the acidity and alkalinity of the Pichia yeast fermentation medium, but also provide phosphorus and potassium elements for the cells to nourish the cells. The carbon source includes at least one of glycerol or glucose, both of which can provide sufficient carbon source for Pichia yeast fermentation.

[0020] In a second aspect, the present invention provides a fermentation method of Pichia pastoris, comprising the following steps:

[0021] inoculating the Pichia pastoris seed liquid into the Pichia pastoris fermentation medium for fermentation;

[0022] After culturing for 3 to 5 hours, when the DO of the fermentation liquid rises back to above 100%, feed medium for the growth phase is added;

[0023] When OD 600 When the value exceeds 80, stop feeding the feed medium;

[0024] After the Pichia pastoris was starved for 30 minutes, the temperature was controlled at 20-25°C, and the induction stage feed medium was fed to induce Pichia pastoris to express the target protein.

[0025] Compared with the prior art, in the above technical solution, during the fermentation process, the control of DO ensures the breathing needs of Pichia pastoris during the growth stage, which is beneficial to the respiratory metabolism of cells, promotes rapid cell proliferation, and achieves high-density fermentation, thereby improving the expression efficiency of the target protein. At the same time, controlling the temperature at 20-25°C is beneficial to maintaining the activity of the enzyme system in the bacteria, thereby promoting the growth and metabolism of the bacteria.

[0026] Furthermore, during the fermentation process, the pH of the fermentation liquid is controlled to be 5.5-6 with aqueous ammonia.

[0027] Compared with the prior art, the above technical solution uses ammonia water to control the pH of the fermentation liquid to 5.5-6 during the fermentation process. During the growth and reproduction of Pichia pastoris, its internal metabolic activities will produce acidic substances, resulting in a decrease in the pH of the fermentation liquid. Ammonia water can neutralize the produced acidic substances, keep the pH of the fermentation liquid at 5.5-6, ensure the activity of related enzymes in the bacteria, and promote the growth of Pichia pastoris and the expression of target proteins.

[0028] Further, the feed medium in the growth phase includes the following components: 500-700 g / L of glucose, 200-400 g / L of yeast extract, and 55-80 g / L of inorganic salts;

[0029] The flow rate of glucose is 30-40 g / L / h, the flow rate of yeast extract is 30-60 g / L / h, and the flow rate of inorganic salt is 3-6 g / L / h; and / or,

[0030] The inorganic salt includes magnesium salt, ammonium salt and ferrous salt; the magnesium salt is at least one of magnesium sulfate heptahydrate and magnesium chloride, the ammonium salt is at least one of ammonium chloride and ammonium sulfate, and the ferrous salt is ferrous sulfate heptahydrate; and / or,

[0031] The feed culture medium during the growth phase contains 10-20 g / L of magnesium sulfate heptahydrate, 4-5 g / L of ammonium chloride and 0.08-0.1 g / L of ferrous sulfate heptahydrate.

[0032] Compared with the prior art, the above technical solution defines the composition ratio of the feed medium and the flow rate of each component in the growth stage. Glucose, yeast extract and inorganic salts can replenish the nutrients consumed during the fermentation process, maintain the balance of various nutrient elements in the medium, maintain an ideal fermentation environment, promote the rapid proliferation of bacteria and the effective expression of exogenous proteins.

[0033] Further, the feed medium in the induction stage comprises the following components: 500-600 mL / L of methanol, 200-300 g / L of peptone and 20-22 mL / L of trace element solution; and / or,

[0034] The addition scheme of methanol in the feed medium during the induction phase was as follows: the flow rate of methanol was 0.5 ml / L / h from 0 to 2 h, and the flow rate of methanol was 1.5 to 2.0 ml / L / h after 2 h;

[0035] The feeding scheme of peptone in the feed medium during the induction phase can be: starting to feed peptone after 2 hours, with a flow rate of 4-5 ml / L / h;

[0036] The addition scheme of the trace element solution in the feed culture medium during the induction phase is as follows: the addition of the trace element solution begins after 2 hours, with a flow rate of 0.02-0.04 ml / L / h.

[0037] Compared with the prior art, the above technical solution defines the composition ratio of the feed medium in the induction stage and the flow rate of each component. Within a specific flow rate range, methanol can provide a carbon source for the growth and development of Pichia pastoris cells, and at the same time can start the expression system of Pichia pastoris cells, and start the Pichia pastoris cells to produce the target protein. In addition, the peptone and trace element solution contained in the feed medium in the induction stage can provide a nitrogen source and trace elements, which can not only provide the growth of Pichia pastoris cells, but also provide precursors for the production of the target protein.

[0038] In a third aspect, the present application provides an application of a Pichia pastoris fermentation medium, wherein the fermentation medium is used to culture recombinant Pichia pastoris to express bovine lactoferrin.

[0039] Compared with the prior art, the use of the Pichia fermentation medium of the present invention for culturing recombinant Pichia to express bovine lactoferrin can meet the growth and metabolic requirements of the Pichia cells while promoting the Pichia cells to express bovine lactoferrin. DETAILED DESCRIPTION

[0040] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0041] In a first aspect, an embodiment of the present invention provides a Pichia pastoris fermentation medium, comprising the following components: 10-20 g / L yeast extract, 30-35 g / L phosphoric acid aqueous solution, 55-80 g / L macro-element inorganic salts, 4.5-5.5 g / L pH adjuster, 10-20 g / L carbon source and 4-6 mL / L trace element solution.

[0042] For example, the concentration of the yeast extract in the Pichia fermentation medium can be 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L or 20 g / L, preferably 12-18 g / L.

[0043] For example, the above-mentioned phosphoric acid aqueous solution can be selected as a phosphoric acid aqueous solution with a volume concentration of ≥85%, and the concentration of the phosphoric acid aqueous solution in the Pichia fermentation medium can be 30g / L, 31g / L, 32g / L, 33g / L, 34g / L or 35g / L, preferably 32-34g / L.

[0044] For example, the concentration of the above-mentioned inorganic salts of macroelements in the Pichia fermentation medium can be 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L or 80 g / L, preferably 60-75 g / L.

[0045] For example, the pH regulator can be potassium hydroxide, and the concentration of the pH regulator in the Pichia fermentation medium can be 4.5 g / L, 4.7 g / L, 5 g / L, 5.3 g / L or 5.5 g / L, preferably 4.7-5.3 g / L.

[0046] For example, the carbon source can be selected as glycerol or glucose, and the concentration of the carbon source in the Pichia fermentation medium can be 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L or 20 g / L, preferably 12-18 g / L.

[0047] For example, the concentration of the trace element solution in the Pichia fermentation medium may be 4 mL / L, 4.5 mL / L, 5 mL / L, 5.5 mL / L or 6 mL / L, preferably 4.5 to 5.5 mL / L.

[0048] In the above technical scheme, the Pichia fermentation medium contains 10-20 g / L of yeast extract, 30-35 g / L of phosphoric acid aqueous solution, 55-80 g / L of inorganic salts of macroelements, 4.5-5.5 g / L of pH regulator, 10-20 g / L of carbon source and 4-6 mL / L of trace element solution. The above components have a good synergistic effect within their corresponding concentration ranges, which is beneficial to promote the growth and reproduction of Pichia, and also to promote the expression of target protein by recombinant Pichia. Yeast extract, inorganic salts of macroelements, carbon source and trace element solution ensure the supply of carbon source, nitrogen source, macroelements and trace elements during the growth of bacteria. The phosphoric acid aqueous solution and potassium hydroxide cooperate with each other to adjust the pH value in the Pichia fermentation medium, and also provide phosphorus and potassium elements for Pichia.

[0049] Further, based on the above embodiments, the inorganic salt of a large number of elements in the embodiments of the present invention includes at least one of calcium salt, potassium salt, magnesium salt, ferrous salt and ammonium salt.

[0050] In some embodiments, the calcium salt may be at least one of calcium sulfate or calcium chloride.

[0051] In some embodiments, the potassium salt can be at least one of potassium sulfate, potassium chloride, or potassium dihydrogen phosphate.

[0052] In some embodiments, the magnesium salt may be at least one of magnesium sulfate heptahydrate and magnesium chloride.

[0053] In some embodiments, the ferrous salt can be ferrous sulfate heptahydrate.

[0054] In some embodiments, the ammonium salt may be at least one of ammonium chloride and ammonium sulfate.

[0055] Preferably, the inorganic salt of a large number of elements includes calcium sulfate, potassium sulfate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate and ammonium chloride. Among them:

[0056] The concentration of calcium sulfate in the culture medium can be 2.0 to 3.0 g / L. For example, it can be 2.0 g / L, 2.2 g / L, 2.4 g / L, 2.6 g / L, 2.8 g / L or 3.0 g / L, preferably 2.4 to 2.6 g / L.

[0057] The concentration of potassium sulfate in the culture medium can be 40-50 g / L. For example, it can be 40 g / L, 42 g / L, 44 g / L, 46 g / L, 48 g / L or 50 g / L, preferably 44-46 g / L.

[0058] The concentration of magnesium sulfate heptahydrate in the culture medium can be 10 to 20 g / L. For example, it can be 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L or 20 g / L, preferably 14 to 16 g / L.

[0059] The concentration of ferrous sulfate heptahydrate in the culture medium may be 0.08 to 0.1 g / L, for example, it may be 0.08 g / L, 0.09 g / L or 0.1 g / L.

[0060] The concentration of ammonium chloride in the culture medium can be 4 to 5 g / L. For example, it can be 4, 4.2 g / L, 4.4 g / L, 4.6 g / L, 4.8 g / L or 5 g / L, preferably 4.4 to 4.6 g / L.

[0061] In the above technical scheme, calcium salts, potassium salts, magnesium salts, ferrous salts and ammonium salts can provide the calcium ions, potassium ions, magnesium ions, ferrous ions and inorganic nitrogen sources required for the growth, reproduction and metabolism of Pichia pastoris. The metabolic flow of Pichia pastoris cells can be regulated by the reasonable selection and ratio of these nutrients, thereby improving the synthesis efficiency of the target protein.

[0062] Further, on the basis of the above embodiments, the embodiment of the present invention is based on one liter of trace element solution, and the trace element solution includes the following components: 0.06-0.08 g of sodium iodide, 4-6 g of copper sulfate pentahydrate, 3-4 g of manganese sulfate monohydrate, 0.2-0.3 g of sodium molybdate dihydrate, 0.03-0.05 g of boric acid, 20-40 g of zinc chloride, 0.3-0.5 g of cobalt chloride hexahydrate, 10-12 g of sulfuric acid and 0.4-0.6 g of biotin.

[0063] Wherein, the content of sodium iodide can be 0.06g, 0.07g or 0.08g;

[0064] The content of copper sulfate pentahydrate can be 4g, 4.5g, 5g, 5.5g or 6g, preferably 5-6g;

[0065] The content of manganese sulfate monohydrate can be 3g, 3.2g, 3.4g, 3.6g, 3.8g or 4g, preferably 3.6-4g;

[0066] The content of sodium molybdate dihydrate can be 0.2g, 0.22g, 0.24g, 0.26g, 0.28g or 0.3g, preferably 0.2 to 0.24g;

[0067] The content of boric acid can be 0.03g, 0.04g or 0.05g;

[0068] The content of zinc chloride can be 20g, 25g, 30g, 35g or 40g, preferably 25-35g;

[0069] The content of cobalt chloride hexahydrate can be 0.3g, 0.35g, 0.4g, 0.45g or 0.5g, preferably 0.4 to 0.5g;

[0070] The content of sulfuric acid can be 10g, 10.5g, 11g, 11.5g or 12g, preferably 10-11g;

[0071] The content of biotin may be 0.4 g, 0.45 g, 0.5 g, 0.55 g or 0.6 g, preferably 0.45 to 0.55 g.

[0072] The above technical solution defines the content and ratio of each component in the trace element solution. Within the above specific components and ratio ranges, the activity of the enzyme system in Pichia pastoris can be better maintained, thereby promoting the growth and metabolism of Pichia pastoris.

[0073] In a second aspect, an embodiment of the present invention provides a fermentation method of Pichia pastoris, comprising the following steps:

[0074] S1. Inoculate the Pichia pastoris seed liquid into the Pichia pastoris fermentation medium for fermentation.

[0075] The Pichia pastoris can be selected as a recombinant Pichia pastoris. The preparation method of the recombinant Pichia pastoris can be referred to as follows: a bovine lactoferrin gene is combined with a protein containing P AOX1 The obtained recombinant expression vector was transformed into GS115 type Pichia pastoris to prepare recombinant Pichia pastoris.

[0076] The preparation method of the recombinant Pichia yeast seed solution in the above steps can be referred to as follows:

[0077] S11. Prepare YPD solid culture medium: 1-3% yeast extract, 2-5% peptone, 2-4% glucose, 1-3% agar, 1000-2000 μg / mL geneticin (G418), sterilize at 120-122°C for 20-30 min, cool to 40-60°C, perform aseptic operation in a clean bench, and pour into plates, each containing about 25 mL of culture medium.

[0078] For example, the YPD solid medium may include the following components: 1% yeast extract, 2% peptone, 2% glucose, 2% agar, and 2000 μg / mL Geneticin (G418).

[0079] S12. Prepare YPD liquid culture medium: 1-3% yeast extract, 2-5% peptone, 2-4% glucose, 1000-2000 μg / mL geneticin (G418), prepare 400 ml, each 500 ml shake flask contains 200 ml culture medium, put it in a high pressure steam sterilizer, and sterilize it at 120-122°C for 20-30 min.

[0080] For example, the YPD liquid medium may include the following components: 1% yeast extract, 2% peptone, 2% glucose, and 2000 μg / mL Geneticin (G418).

[0081] S13, prepare Pichia pastoris seed solution: spread the Pichia pastoris recombinant bacteria working seeds on a YPD solid culture medium plate, place it in an incubator at 30±1°C for 2-3 days, pick out larger colonies and inoculate them into a shaking bottle containing 200±20mL YPD liquid culture medium, place it in a shaking incubator, control the temperature at 30±1°C, the rotation speed at 200-220rpm, and culture for 16-22h to obtain the OD 600 Shake flask seed solution with a value of 7 to 9.

[0082] S2. Cultivate for 3 to 5 hours. When the DO of the fermentation liquid rises back to above 100%, feed medium for the growth stage is added and the fermentation is continued for 8 to 10 hours.

[0083] The specific operation process of this step can be referred to as follows: the cultured seed shake flask seed liquid is aseptically transferred into the sterilized and pH-adjusted fermentation tank, the initial ventilation ratio is controlled to be 1-2VVM, the initial stirring speed is 100-200rpm, the tank pressure is 0.03-0.06MPa, the DO is corrected to 100%, and the DO is controlled to be 20-60% by increasing the stirring speed and the ventilation ratio. Ammonia water is used to control the pH at 5.5-6. After culturing for about 3-5 hours, the pH and DO rebound. When the DO rebounds to above 100%, feeding is started, that is, feeding the culture medium during the fed growth stage.

[0084] During the above fermentation process, the control of DO ensures the respiration needs of Pichia pastoris during the growth stage, while the control of temperature and pH ensures the growth rate of Pichia pastoris during the cell growth and reproduction stage, the generation and activity maintenance of the enzyme system in the bacteria. The enzyme system plays many important roles in the growth and metabolism of the bacteria, such as controlling metabolic pathways and decomposing nutrients for the bacteria to use.

[0085] Further, the feed medium in the growth stage includes the following components: 500-700 g / L of glucose, 200-400 g / L of yeast extract and 55-80 g / L of inorganic salts. For example, glucose can be specifically selected as 500 g / L, 550 g / L, 600 g / L, 650 g / L or 700 g / L, preferably 550-650 g / L; yeast extract can be specifically selected as 200 g / L, 250 g / L, 300 g / L, 350 g / L or 400 g / L, preferably 250-350 g / L; inorganic salt can be specifically selected as 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L or 80 g / L, preferably 60-75 g / L.

[0086] Furthermore, the inorganic salt in the growth stage feed medium may be a magnesium salt, an ammonium salt and a ferrous salt, wherein the magnesium salt may be at least one of magnesium sulfate heptahydrate and magnesium chloride; the ammonium salt may be at least one of ammonium chloride and ammonium sulfate; and the ferrous salt may be ferrous sulfate heptahydrate. For example, the inorganic salt may be magnesium sulfate heptahydrate, ammonium chloride and ferrous sulfate heptahydrate.

[0087] The feed medium in the growth stage contains 10-20 g / L of magnesium sulfate heptahydrate, 4-5 g / L of ammonium chloride and 0.08-0.1 g / L of ferrous sulfate heptahydrate. For example, the concentration of magnesium sulfate heptahydrate can be 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L or 20 g / L, preferably 12-18 g / L; the concentration of ammonium chloride can be 4 g / L, 4.2 g / L, 4.4 g / L, 4.6 g / L, 4.8 g / L or 5 g / L, preferably 4.2-4.6 g / L; the concentration of ferrous sulfate heptahydrate can be 0.08 g / L, 0.09 g / L or 0.1 g / L.

[0088] In the above steps, when feeding the feed medium in the growth phase, the flow rate of glucose is 30-40 g / L / h, the flow rate of yeast extract is 30-60 g / L / h, and the flow rate of inorganic salt is 3-6 g / L / h. It should be understood that the above flow rates are all determined according to the volume of the fermentation liquid. For example, the flow rate of glucose is 30-40 g / L / h, which means that 30-40 g of glucose is fed per liter of fermentation liquid per hour.

[0089] For example, the flow rate of glucose can be specifically selected as 30g / L / h, 32g / L / h, 34g / L / h, 36g / L / h, 38g / L / h or 40g / L / h, preferably 32-36g / L / h; the flow rate of yeast extract can be specifically selected as 30g / L / h, 40g / L / h, 50g / L / h or 60g / L / h, preferably 40-50g / L / h; the flow rate of inorganic salt can be specifically selected as 3g / L / h, 4g / L / h, 5g / L / h or 6g / L / h, preferably 4-5g / L / h.

[0090] S3, when OD 600 When the pH value exceeded 80, the feeding of the feed medium was stopped.

[0091] S4. After starving the Pichia pastoris for 30 minutes, the temperature was controlled at 20-25°C, and the feed medium was added during the induction phase to induce the Pichia pastoris to express the target protein. The fermentation was terminated after 40-50 hours of induction.

[0092] Wherein, the feed medium in the induction stage may include the following components: 500-600 mL / L of methanol, 200-300 g / L of peptone, and 20-22 mL / L of trace element solution. For example, the concentration of methanol may be 500 mL / L, 520 mL / L, 540 mL / L, 560 mL / L, 580 mL / L or 600 mL / L, preferably 540-580 mL / L; the concentration of peptone may be 200 g / L, 220 g / L, 240 g / L, 260 g / L, 280 g / L or 300 g / L, preferably 240-280; the concentration of trace element solution may be 20 mL / L, 21 mL / L or 22 mL / L.

[0093] Furthermore, the methanol addition scheme in the induction stage can be: the methanol flow rate is 0.5 ml / L / h in the first 0 to 2 hours, and the methanol flow rate is 1.5 to 2.0 ml / L / h after 2 hours.

[0094] The peptone addition scheme in the induction stage can be: start adding peptone after 2 hours, with a flow rate of 4-5 ml / L / h.

[0095] The addition scheme of the trace element solution in the induction stage can be: start adding the trace element solution after 2 hours, and the flow rate is 0.02-0.04 ml / L / h.

[0096] It should be understood that the above flow rates are determined according to the volume of the fermentation liquid. For example, the flow rate of methanol from 0 to 2 hours is 0.5 ml / L / h, which means that 0.5 ml of methanol is added per liter of fermentation liquid per hour.

[0097] In the above induction stage, methanol can not only provide a carbon source for the growth and metabolism of Pichia pastoris cells, but also start the expression system to allow the cells to express and produce the target protein. Peptone, as a nitrogen source, together with the trace element solution, provides the nutrients required for bacterial growth and also provides precursors for the production of the target protein. The synergistic effect of the three promotes the expression yield of the target protein.

[0098] In a third aspect, an embodiment of the present invention provides an application of a Pichia pastoris fermentation medium, wherein the Pichia pastoris fermentation medium is used to culture recombinant Pichia pastoris to express bovine lactoferrin.

[0099] Compared with the prior art, the Pichia pastoris fermentation medium of the present invention can promote the expression of bovine lactoferrin by the Pichia pastoris while meeting the growth and metabolic requirements of the Pichia pastoris, thereby increasing the expression level of bovine lactoferrin.

[0100] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0101] Example 1

[0102] The present embodiment provides a Pichia fermentation medium, comprising the following components: 20 g / L yeast extract, 35 g / L phosphoric acid aqueous solution with a volume concentration of 85%, 3.0 g / L calcium sulfate, 50 g / L potassium sulfate, 20 g / L magnesium sulfate heptahydrate, 0.1 g / L ferrous sulfate heptahydrate, 5 g / L ammonium chloride, 5.5 g / L potassium hydroxide, 20 g / L glycerol, and 6 mL / L trace element solution. Each liter of the trace element solution comprises the following components: 0.08 g sodium iodide, 6 g copper sulfate pentahydrate, 4 g manganese sulfate monohydrate, 0.3 g sodium molybdate dihydrate, 0.05 g boric acid, 40 g zinc chloride, 0.5 g cobalt chloride hexahydrate, 12 g sulfuric acid, and 0.6 g biotin.

[0103] A method for fermenting Pichia pastoris using the Pichia pastoris fermentation medium comprises the following steps:

[0104] S1. Preparation of recombinant Pichia pastoris: bovine lactoferrin gene and P AOX1 The recombinant expression vector was prepared by connecting the vector pPIC9K with the promoter to transform the obtained recombinant expression vector into GS115 type Pichia pastoris to prepare recombinant Pichia pastoris. The Genban ID of the bovine lactoferrin gene is JX294418.1.

[0105] S2. Spread the recombinant Pichia pastoris working seeds on a YPD solid medium plate, place it in a 30°C incubator and culture it for 3 days. Pick the colonies and inoculate them into a 200 mL shake bottle containing YPD liquid medium. Place it in a shaker and control the temperature at 30°C and the speed at 220 rpm for 20 h. Obtain the OD 600 It is 7 to 9 shake flask seed liquid.

[0106] The YPD solid culture medium includes the following components: 1% yeast extract, 2% peptone, 2% glucose, 2% agar, and 2000 μg / mL Geneticin (G418).

[0107] YPD liquid medium includes the following components: 1% yeast extract, 2% peptone, 2% glucose, and 2000 μg / mL Geneticin (G418).

[0108] S3. Aseptically transfer the cultured seed shake flask liquid into the sterilized and pH-adjusted fermentation tank, control the initial aeration ratio to 2VVM, the initial stirring speed to 200rpm, the tank pressure to 0.05MPa, calibrate DO to 100%, control DO to 20-60% by increasing the stirring speed and aeration ratio, and use ammonia water to control pH to 5.5-6. After about 3-5 hours of cultivation, pH and DO rebounded. When they rebounded to above 100%, feed medium for the growth stage was added and culture continued for 10 hours.

[0109] The feed medium in the growth stage includes the following components: 700 g / L glucose, 400 g / L yeast extract, 20 g / L magnesium sulfate heptahydrate, 5 g / L ammonium chloride and 0.1 g / L ferrous sulfate heptahydrate.

[0110] The flow rate of glucose was 40 g / L / h, the flow rate of yeast extract was 60 g / L / h, and the flow rate of inorganic salt was 6 g / L / h.

[0111] S4, fermentation broth OD 600 When the temperature can reach above 80, the growth stage feed medium is stopped, the bacteria are starved for 30 minutes, the temperature is controlled at 20°C, and the induction stage feed medium is prepared. The fermentation ends after 40 hours of induction. The induction stage feed medium includes the following components: 600 mL / L methanol, 300 g / L peptone and 22 mL / L trace element solution.

[0112] When adding feed medium in the induction stage, the flow rate of methanol was 0.5 ml / L / h from 0 to 2 hours, and the flow rate of methanol was 2.0 ml / L / h after 2 hours. Peptone and trace element solution were added after 2 hours, with the flow rate of peptone being 5 mL / L / h and the flow rate of trace element solution being 0.04 mL / L / h.

[0113] Example 2

[0114] The present embodiment provides a Pichia fermentation medium, comprising the following components: 10 g / L yeast extract, 30 g / L phosphoric acid aqueous solution with a volume concentration of 85%, 2.0 g / L calcium sulfate, 40 g / L potassium sulfate, 10 g / L magnesium sulfate heptahydrate, 0.08 g / L ferrous sulfate heptahydrate, 4 g / L ammonium chloride, 4.5 g / L potassium hydroxide, 10 g / L glucose and 4 mL / L trace element solution. Each liter of the trace element solution comprises the following components: 0.06 g sodium iodide, 4 g copper sulfate pentahydrate, 3 g manganese sulfate monohydrate, 0.2 g sodium molybdate dihydrate, 0.03 g boric acid, 20 g zinc chloride, 0.3 g cobalt chloride hexahydrate, 10 g sulfuric acid and 0.4 g biotin.

[0115] The method for fermenting Pichia pastoris using the culture medium is different from that in Example 1 in that:

[0116] The feed medium in the growth phase included the following components: 500 g / L glucose, 200 g / L yeast extract, 10 g / L magnesium sulfate heptahydrate, 4 g / L ammonium chloride, and 0.08.1 g / L ferrous sulfate heptahydrate. The flow rate of glucose was 30 g / L / h, the flow rate of yeast extract was 30 g / L / h, and the flow rate of inorganic salts was 3 g / L / h.

[0117] After starving the bacteria for 30 minutes, the temperature was controlled at 25°C and the feed medium for the induction phase was prepared.

[0118] The feed medium in the induction phase included the following components: 500 mL / L methanol, 200 g / L peptone and 20 mL / L trace element solution.

[0119] When adding feed culture medium in the induction stage, the flow rate of methanol was 0.5 ml / L / h in the first 0-2 h, and the flow rate of methanol was 1.5 mL / L / h after 2 h. Peptone and trace element solution were added after 2 h, with the flow rate of peptone being 4 mL / L / h and the flow rate of trace element solution being 0.02 mL / L / h.

[0120] Example 3

[0121] The present embodiment provides a Pichia fermentation medium, comprising the following components: 20 g / L yeast extract, 35 g / L phosphoric acid aqueous solution with a volume concentration of 85%, 3.0 g / L calcium sulfate, 45 g / L potassium chloride, 15 g / L magnesium chloride, 0.1 g / L ferrous sulfate heptahydrate, 4.5 g / L ammonium sulfate, 5 g / L potassium hydroxide, 15 g / L glycerol, and 5 mL / L trace element solution. Each liter of the trace element solution comprises the following components: 0.08 g sodium iodide, 6 g copper sulfate pentahydrate, 4 g manganese sulfate monohydrate, 0.3 g sodium molybdate dihydrate, 0.05 g boric acid, 40 g zinc chloride, 0.5 g cobalt chloride hexahydrate, 12 g sulfuric acid, and 0.6 g biotin.

[0122] The method for fermenting Pichia pastoris using the culture medium is different from that in Example 1 in that:

[0123] The feed medium in the growth phase included the following components: 600 g / L glucose, 300 g / L yeast extract, 15 g / L magnesium chloride, 4.5 g / L ammonium sulfate, and 0.1 g / L ferrous sulfate heptahydrate. The flow rate of glucose was 35 g / L / h, the flow rate of yeast extract was 40 g / L / h, and the flow rate of inorganic salts was 4 g / L / h.

[0124] After starving the bacteria for 30 minutes, the temperature was controlled at 23°C and the feed medium for the induction phase was prepared.

[0125] The feed medium in the induction phase included the following components: 550 mL / L methanol, 250 g / L peptone and 20 mL / L trace element solution.

[0126] When adding feed culture medium in the induction stage, the flow rate of methanol was 0.5 ml / L / h in the first 0-2 h, and the flow rate of methanol was 1.7 mL / L / h after 2 h. Peptone and trace element solution were added after 2 h, with the flow rate of peptone being 4 mL / L / h and the flow rate of trace element solution being 0.03 mL / L / h.

[0127] Example 4

[0128] The present embodiment provides a Pichia fermentation medium, comprising the following components: 20 g / L yeast extract, 32 g / L phosphoric acid aqueous solution with a volume concentration of 85%, 3.0 g / L calcium chloride, 45 g / L potassium dihydrogen phosphate, 15 g / L magnesium sulfate heptahydrate, 0.1 g / L ferrous sulfate heptahydrate, 4.5 g / L ammonium sulfate, 5 g / L potassium hydroxide, 15 g / L glucose and 5 mL / L trace element solution. Each liter of the trace element solution comprises the following components: 0.07 g sodium iodide, 5 g copper sulfate pentahydrate, 3.5 g manganese sulfate monohydrate, 0.3 g sodium molybdate dihydrate, 0.04 g boric acid, 30 g zinc chloride, 0.4 g cobalt chloride hexahydrate, 11 g sulfuric acid and 0.5 g biotin.

[0129] The method for fermenting Pichia pastoris using the culture medium is different from that in Example 1 in that:

[0130] The feed medium in the growth phase included the following components: 600 g / L glucose, 300 g / L yeast extract, 15 g / L magnesium sulfate heptahydrate, 4.5 g / L ammonium sulfate, and 0.1 g / L ferrous sulfate heptahydrate. The flow rate of glucose was 35 g / L / h, the flow rate of yeast extract was 40 g / L / h, and the flow rate of inorganic salts was 4 g / L / h.

[0131] After starving the bacteria for 30 minutes, the temperature was controlled at 25°C and the feed medium for the induction phase was prepared.

[0132] The feed medium in the induction phase included the following components: 550 mL / L methanol, 250 g / L peptone and 20 mL / L trace element solution.

[0133] When adding feed culture medium in the induction stage, the flow rate of methanol was 0.5 ml / L / h in the first 0-2 h, and the flow rate of methanol was 1.7 mL / L / h after 2 h. Peptone and trace element solution were added after 2 h, with the flow rate of peptone being 4 mL / L / h and the flow rate of trace element solution being 0.03 mL / L / h.

[0134] Comparative Example 1

[0135] The recombinant Pichia pastoris was fermented according to the fermentation method of Example 1, with the only difference being that the distribution ratios of the various groups in the Pichia pastoris fermentation medium were different. The Pichia pastoris fermentation medium of Comparative Example 1 included the following components: 25 g / L yeast extract, 40 g / L 85% aqueous phosphoric acid solution by volume, 3.5 g / L calcium sulfate, 55 g / L potassium sulfate, 6 g / L potassium hydroxide, 25 g / L glycerol, 25 g / L magnesium sulfate heptahydrate, 0.15 g / L ferrous sulfate heptahydrate, 5.5 g / L ammonium chloride, and 8 mL / L trace element solution.

[0136] Comparative Example 2

[0137] The recombinant Pichia pastoris was fermented according to the fermentation method of Example 1, with the only difference being that the distribution ratios of the various groups in the Pichia pastoris fermentation medium were different. The Pichia pastoris fermentation medium of Comparative Example 1 included the following components: 8 g / L yeast extract, 25 g / L 85% aqueous phosphoric acid solution, 1.5 g / L calcium sulfate, 35 g / L potassium sulfate, 4 g / L potassium hydroxide, 5 g / L glycerol, 5 g / L magnesium sulfate heptahydrate, 0.05 g / L ferrous sulfate heptahydrate, 3.5 g / L ammonium chloride, and 3 mL / L trace element solution.

[0138] Comparative Example 3

[0139] The recombinant Pichia pastoris was fermented according to the fermentation method of Example 1, except that the feed medium during the growth phase was 500 g / L glycerol and the flow rate was 35 g / L / h.

[0140] Comparative Example 4

[0141] The recombinant Pichia pastoris was fermented according to the fermentation method of Example 1, except that the feed medium in the induction stage contained only methanol.

[0142] Test Case

[0143] The expression level of bovine lactoferrin in the fermentation broth after the fermentation was completed was detected, and the results are shown in Table 1.

[0144] Table 1 Expression of bovine lactoferrin in fermentation broth

[0145] Test Case Expression of bovine lactoferrin Example 1 3.6g / L Example 2 3.4g / L Example 3 3.5g / L Example 4 3.6g / L Comparative Example 1 2.5g / L Comparative Example 2 2.6g / L Comparative Example 3 2.4g / L Comparative Example 4 2.3g / L

[0146] The concentration of Pichia pastoris in the fermentation broth after fermentation was completed was detected, and the results are shown in Table 2.

[0147] Table 2 Pichia yeast cell concentration in fermentation broth

[0148]

[0149]

[0150] The above test results show that the expression level of bovine lactoferrin in the fermentation broth obtained by fermentation in Examples 1 to 4 of the present invention is 3.4 g / L or above, and the OD 600 The value can reach 600-700, while the distribution ratio of each group in the Pichia yeast fermentation medium in comparative examples 1-2 exceeds the established range. After the composition of the feed medium is changed in comparative examples 3-4, the expression amount and OD value of bovine lactoferrin in the fermentation broth are 600 It can be seen that the application of the Pichia yeast fermentation medium and fermentation method provided by the present invention can significantly increase the density of Pichia yeast cells in the fermentation broth and the yield of bovine lactoferrin.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Pichia yeast fermentation medium, characterized in that: The invention comprises the following components: 10-20 g / L yeast extract, 30-35 g / L phosphoric acid aqueous solution, 55-80 g / L inorganic salt of macroelements, 4.5-5.5 g / L pH regulator, 10-20 g / L carbon source and 4-6 mL / L trace element solution.

2. The fermentation medium according to claim 1, characterized in that The inorganic salt of a large number of elements includes at least one of calcium salt, potassium salt, magnesium salt, ferrous salt and ammonium salt; and / or, The calcium salt comprises at least one of calcium sulfate or calcium chloride; and / or, The potassium salt includes at least one of potassium sulfate, potassium chloride and potassium dihydrogen phosphate; and / or, The magnesium salt includes at least one of magnesium sulfate heptahydrate and magnesium chloride; and / or, The ferrous salt is ferrous sulfate heptahydrate; and / or, The ammonium salt includes at least one of ammonium chloride and ammonium sulfate.

3. The fermentation medium according to claim 2, characterized in that The inorganic salts of macroelements include calcium sulfate, potassium sulfate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate and ammonium chloride; the concentration of calcium sulfate in the culture medium is 2.0-3.0 g / L, the concentration of potassium sulfate is 40-50 g / L, the concentration of magnesium sulfate heptahydrate is 10-20 g / L, the concentration of ferrous sulfate heptahydrate is 0.08-0.1 g / L, and the concentration of ammonium chloride is 4-5 g / L.

4. The fermentation medium according to any one of claims 1 to 3, characterized in that Based on one liter of trace element solution, the trace element solution includes the following components: 0.06-0.08 g of sodium iodide, 4-6 g of copper sulfate pentahydrate, 3-4 g of manganese sulfate monohydrate, 0.2-0.3 g of sodium molybdate dihydrate, 0.03-0.05 g of boric acid, 20-40 g of zinc chloride, 0.3-0.5 g of cobalt chloride hexahydrate, 10-12 g of sulfuric acid and 0.4-0.6 g of biotin.

5. The fermentation medium according to claim 1, characterized in that The volume concentration of the phosphoric acid aqueous solution is ≥85%; and / or, The pH regulator is potassium hydroxide; and / or, The carbon source includes at least one of glycerol or glucose.

6. A fermentation method of Pichia pastoris, characterized in that: The following steps are involved: Inoculating Pichia pastoris seed liquid into the fermentation medium according to any one of claims 1 to 5 for fermentation; After culturing for 3 to 5 hours, when the DO of the fermentation liquid rises back to above 100%, feed medium for the growth phase is added; When OD 600 When the value exceeds 80, stop feeding the feed medium; After the Pichia pastoris was starved for 30 minutes, the temperature was controlled at 20-25°C, and the induction stage feed medium was fed to induce Pichia pastoris to express the target protein.

7. The fermentation method according to claim 6, characterized in that During the fermentation process, ammonia water is used to control the pH of the fermentation liquid to 5.5-6.

8. The fermentation method according to claim 6, characterized in that The growth stage feed medium includes the following components: 500-700 g / L glucose, 200-400 g / L yeast extract and 55-80 g / L inorganic salts; The glucose has a flow rate of 30 to 40 g / L / h, the yeast extract has a flow rate of 30 to 60 g / L / h, and the inorganic salt has a flow rate of 3 to 6 g / L / h; and / or, The inorganic salt includes magnesium salt, ammonium salt and ferrous salt; the magnesium salt is at least one of magnesium sulfate heptahydrate and magnesium chloride, the ammonium salt is at least one of ammonium chloride and ammonium sulfate, and the ferrous salt is ferrous sulfate heptahydrate; and / or, The growth stage supplement culture medium contains 10-20 g / L of magnesium sulfate heptahydrate, 4-5 g / L of ammonium chloride and 0.08-0.1 g / L of ferrous sulfate heptahydrate.

9. The fermentation method according to claim 6, characterized in that: The feed medium in the induction stage comprises the following components: 500-600 mL / L methanol, 200-300 g / L peptone and 20-22 mL / L trace element solution; and / or, The addition scheme of methanol in the feed medium during the induction phase was as follows: the flow rate of methanol was 0.5 ml / L / h from 0 to 2 h, and the flow rate of methanol was 1.5 to 2.0 ml / L / h after 2 h; The feeding scheme of peptone in the feed medium during the induction phase can be: starting to feed peptone after 2 hours, with a flow rate of 4-5 ml / L / h; The addition scheme of the trace element solution in the feed culture medium during the induction phase is as follows: the addition of the trace element solution begins after 2 hours, with a flow rate of 0.02-0.04 ml / L / h.

10. An application of a Pichia yeast fermentation medium, characterized in that: The fermentation medium according to any one of claims 1 to 5 is used to culture recombinant Pichia pastoris to express bovine lactoferrin.

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