Culture medium of methylotrophic pichia and fermentation method thereof

Abstract

The application belongs to the field of microbial fermentation, and particularly relates to a culture medium of methylotrophic Pichia pastoris and a fermentation method thereof. The culture medium comprises a seed solid culture medium, a seed liquid culture medium and a fermentation culture medium. Through specific formula design, the Pichia pastoris can be efficiently cultured and fermented with methanol as the only carbon source, and on this basis, a fermentation liquor with high biomass and high protein content is obtained.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, and in particular relates to a culture medium for methyl-nutritive Pichia pastoris and its fermentation method. Background Technology

[0002] Pichia pastoris ( Komagataella phaffii Pichia pastoris is a methyltrophic yeast known for its highly efficient methanol-inducible promoter (AOX1), strong protein secretion capacity, and eukaryotic post-translational modification system. As a highly efficient eukaryotic expression system, it can correctly fold, post-translationally modify, and secrete recombinant proteins, offering advantages such as high growth density, low culture cost, and relatively simple genetic manipulation. Developing fermentation processes for Pichia pastoris can significantly improve the yield and quality of recombinant proteins (such as insulin, antibody fragments, vaccine antigens, and industrial enzymes), meeting the large-scale production needs of biopharmaceuticals, food processing, and bioenergy, while simultaneously reducing downstream purification costs, providing a reliable technological platform for the green manufacturing of high-value-added bioproducts.

[0003] Pichia pastoris, with its complete methyl-trophic metabolic system, can oxidize methanol to formaldehyde and isolate toxic intermediates through peroxisomal compartmentalization. Simultaneously, it utilizes a two-branched pathway consisting of dihydroxyacetone synthase and formaldehyde / formate dehydrogenase to direct formaldehyde towards assimilation and dissimilation, achieving efficient methanol utilization and detoxification balance. However, in actual fermentation, high concentrations of methanol can easily trigger reactive oxygen species bursts, cellular oxidative stress, and metabolic toxicity, inhibiting cell growth. Furthermore, methanol metabolism has extremely high oxygen consumption, often leading to dissolved oxygen transfer bottlenecks in high-density cultures. Additionally, the AOX promoter requires high cell density to be effectively induced, making it difficult to rapidly accumulate biomass using methanol as a single carbon source. Therefore, existing processes typically require the addition of auxiliary carbon sources such as glycerol or sorbitol to construct a mixed nutrient model, balancing biomass and efficient expression of the target protein. Although patent CN116716193A discloses a fermentation method using methanol as the sole carbon source, it is only applicable to specific strains and only uses methanol as the sole carbon source during the fermentation stage, limiting its applicability. Therefore, there is an urgent need to develop a culture medium and corresponding process suitable for general Pichia pastoris that can use methanol as the sole carbon source throughout the entire culture and fermentation process, so as to achieve high expression of both biomass and protein. Summary of the Invention

[0004] To address the above problems, the present invention provides a culture medium for methyl-trophic Pichia pastoris, the culture medium comprising a seed solid culture medium, a seed liquid culture medium, and a fermentation culture medium; The seed solid culture medium is a medium based on YPM solid culture medium, with methanol added to a final concentration of 2~3 g / L. The seed liquid culture medium is a medium based on YPM liquid culture medium, with methanol added to a final concentration of 4~5 g / L. The fermentation medium comprises (NH4)2SO4 at a final concentration of 1-5 g / L, KH2PO4 at 10-15 g / L, MgSO4·7H2O at 0.1-1 g / L, vitamin solution at 0.5-1.5 mL / L, trace element solution at 1-3 mL / L, and methanol at 7-13 g / L.

[0005] Furthermore, the seed solid culture medium is a medium based on YPM solid culture medium, with methanol added to a final concentration of 3 g / L. The YPM solid culture medium comprises yeast extract powder at a final concentration of 1-10 g / L, peptone at 10-20 g / L, and agar at 10-20 g / L.

[0006] Furthermore, the seed liquid culture medium is a medium based on YPM liquid culture medium with added methanol at a final concentration of 5 g / L. The YPM liquid culture medium comprises yeast extract powder at a final concentration of 1-10 g / L and peptone at a final concentration of 10-20 g / L.

[0007] Furthermore, the fermentation medium comprises (NH4)2SO4 at a final concentration of 2.5 g / L, KH2PO4 at 14.4 g / L, MgSO4·7H2O at 0.5 g / L, vitamin solution at 1 mL / L, trace element solution at 2 mL / L, and methanol at 10 g / L; the pH value is 5.6.

[0008] Further, the vitamin solution comprises D-biotin at a final concentration of 0.05 g / L, D-calcium pantothenate at 1.00 g / L, thiamine hydrochloride at 1.00 g / L, pyridoxine hydrochloride at 1.00 g / L, nicotinic acid at 0.2 g / L, aminobenzoic acid at 25 g / L, and inositol at a pH of 6.5.

[0009] The trace element solution comprises 3 g / L FeSO4•7H2O, 4.5 g / L ZnSO4•7H2O, 4.5 g / L CaCl2•2H2O, 1.00 g / L MnCl2•4H2O, 0.3 g / L CoCl2•6H2O, 0.3 g / L CuSO4•5H2O, 0.4 g / L Na2MoO4•2H2O, 1.0 g / L H3BO3, 0.1 g / L KI, and 19.0 g / L Na2EDTA•2H2O; the pH value is 6.0.

[0010] This invention also provides a fermentation method for methyl-trophic Pichia pastoris, comprising the following steps: 1) Take a Pichia pastoris strain, streak it onto a seed solid medium, and culture it to obtain a single colony of Pichia pastoris; 2) Take a single colony of Pichia pastoris, inoculate it into seed liquid culture medium, and culture it to obtain seed liquid; 3) Take the seed culture and inoculate it into the fermentation medium for culture; The seed solid culture medium is obtained by dissolving YPM solid culture medium in water, sterilizing it, adding methanol to a concentration of 2-3 g / L before solidification, mixing and solidifying. The seed liquid culture medium is YPM liquid culture medium, which is sterilized, cooled, and then methanol is added to a concentration of 4-5 g / L, and the medium is mixed well to obtain the culture medium. The fermentation medium is prepared by weighing the raw materials according to the following ratio, dissolving the inorganic salts in water, adjusting the pH value, sterilizing, cooling, and then adding vitamin solution, trace element solution and methanol and mixing well. 1~5 g / L (NH4)2SO4, 10~15 g / L KH2PO4, 0.1~1 g / L MgSO4·7H2O, 0.5~1.5 mL / L vitamin solution, 1~3 mL / L trace element solution, 7~13 g / L methanol, and the remainder is water.

[0011] Furthermore, the methanol concentration in the seed solid culture medium is 3 g / L; The methanol concentration in the seed liquid culture medium was 5 g / L; The ratio of raw materials in the fermentation medium is: 2.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 1 mL / L vitamin solution, 2 mL / L trace element solution, and 10 g / L methanol. The vitamin solution comprises 0.05 g / L of D-biotin, 1.00 g / L of D-calcium pantothenate, 1.00 g / L of thiamine hydrochloride, 1.00 g / L of pyridoxine hydrochloride, 1.00 g / L of nicotinic acid, 0.2 g / L of aminobenzoic acid and 25 g / L of inositol; the pH is 6.5.

[0012] The trace element solution comprises 3 g / L FeSO4•7H2O, 4.5 g / L ZnSO4•7H2O, 4.5 g / L CaCl2•2H2O, 1.00 g / L MnCl2•4H2O, 0.3 g / L CoCl2•6H2O, 0.3 g / L CuSO4•5H2O, 0.4 g / L Na2MoO4•2H2O, 1.0 g / L H3BO3, 0.1 g / L KI, and 19.0 g / L Na2EDTA•2H2O; the pH value is 6.0.

[0013] Furthermore, the cultivation conditions described in step 1) are a temperature of 30°C and a time of 48 hours.

[0014] Further, the cultivation conditions in step 2) are a rotation speed of 220 r / min, a temperature of 30℃, and a time of 20–24 h.

[0015] Further, in step 3), the initial OD value of the fermentation medium after inoculation with the seed liquid is 0.2 to 0.5; the culture conditions are 30°C, 220 r / min, and 60 to 65 h.

[0016] This invention provides a culture medium suitable for methyl-trophic Pichia pastoris, comprising a seed culture medium and a fermentation culture medium. Through a specific formulation design, this culture medium enables Pichia pastoris to be efficiently cultured and fermented using methanol as the sole carbon source, resulting in a fermentation broth with high biomass and high protein content. Compared to traditional processes that require the addition of auxiliary carbon sources such as glucose and glycerol, this invention significantly reduces sugar consumption and production costs during fermentation. Furthermore, compared to processes applicable only to specific strains and only using methanol as the sole carbon source during the fermentation stage, this invention's culture medium has a wider range of applications and higher practical application value.

[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0019] Figure 1 Results of OD values ​​and protein content for different fermentation systems. Detailed Implementation

[0020] The raw materials, reagents, and equipment used in the specific embodiments of this invention were obtained through commercial purchase. Pichia pastoris CN-SCP 00 (accession number: CCTCC NO: M2025657), deposited at the China Center for Type Culture Collection, was provided by Shanghai Ruikang Biotechnology R&D Co., Ltd., and its related technical information has been publicly disclosed through Chinese Patent CN120737986 A. YPD solid culture medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; YPD liquid culture medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; YPM solid culture medium and YPM liquid culture medium were both self-made, with the specific formulas as follows: Example 1: Pichia pastoris fermentation method of the present invention (a) Preparation of culture medium Seed solid culture medium: Prepare YPM solid culture medium, sterilize it, add methanol to a concentration of 3 g / L before solidification, mix well, dispense into petri dishes, and solidify to obtain the seed solid culture medium. Seed liquid culture medium: Prepare YPM liquid culture medium, sterilize, cool, add methanol to a concentration of 5 g / L, and mix well; Fermentation medium: Weigh the raw materials according to the following formula, dissolve the inorganic salts in water, adjust the pH to 5.6, sterilize at 115 ℃ for 30 min, cool, add vitamin solution, trace element solution and methanol to dissolve, and mix well to obtain the culture medium; Note: The vitamin solution is prepared by adding the following vitamins to water in the following order: 0.05 g D-biotin, 1.00 g D-calcium pantothenate, 1.00 g thiamine hydrochloride, 1.00 g pyridoxine hydrochloride, 1.00 g niacin, 0.2 g aminobenzoic acid, and 25 g inositol per 1 L of water; after adding D-biotin, adjust the pH to 6.5 with 1 M HCl or H2SO4; after adding aminobenzoic acid, adjust the pH to 6.5 with 1 M NaOH.

[0021] The method for preparing trace element solutions is as follows: Add the following components to water in sequence, with 3 g FeSO4•7H2O, 4.5 g ZnSO4•7H2O, 4.5 g CaCl2•2H2O, 1.00 g MnCl2•4H2O, 0.3 g CoCl2•6H2O, 0.3 g CuSO4•5H2O, 0.4 g Na2MoO4•2H2O, 1.0 g H3BO3, 0.1 g KI, and 19.0 g Na2EDTA•2H2O added sequentially per 1 L of water. After each addition of a trace element, the pH should be adjusted to 6.0 using NaOH solution.

[0022] (ii) Pichia pastoris fermentation 1) Take Pichia pastoris strain, streak it onto seed solid medium, and culture it at 30℃ for 48h to obtain single colonies of Pichia pastoris; 2) Take a single colony of Pichia pastoris, inoculate it into seed liquid culture medium, and culture it at 30℃ and 220r / min for 20-24 h to obtain seed liquid; 3) Take the seed liquid and inoculate it into the fermentation medium. The initial OD value of the fermentation medium after inoculation should be 0.2 to 0.5. Then, culture it at 30℃ and 220r / min for 60 to 65 hours to obtain the final product.

[0023] The following experimental examples further illustrate the beneficial effects of the present invention: Effects of different culture systems on Pichia pastoris fermentation in experimental cases I. Methods Experimental Group 1: Glucose System Culture and Fermentation (Pichia pastoris-1) S1 Stranding Isolation of Single Clones: Culture Medium: YPD Solid Plates; Procedure: Stranding isolation of single clones; S2 Seed Culture: Culture medium: YPD liquid medium (5 mL); Culture conditions: 30℃, 220 r / min; Culture time: 18 h; Final seed culture OD: 9.005 Preparation of S3 fermentation medium (50 mL per bottle) ① Prepare Delft basic salt medium (50 mL / 250 mL shake flask) (NH4)2SO4: 2.5 g / L, KH2PO4: 14.4 g / L, MgSO4·7H2O: 0.5 g / L; pH: 5.6; Sterilization: 115℃, 30 min; ②Add other ingredients After sterilization, Delft basal salt medium is supplemented with vitamin solution: 1 mL / L, trace element solution: 2 mL / L, and methanol 10 g / L.

[0024] S4 Inoculation and Culture The seed culture obtained from S2 was inoculated into the fermentation medium obtained from S3. The initial OD was 0.2-0.5. The culture conditions were 30℃ and 220r / min. The culture time was about 60 h. Experimental Group 2: Cultivation and fermentation of Pichia pastoris using a methanol-substituted glucose system (referred to as Pichia pastoris-2). S1 Stranding Isolation of Single Clones: Culture medium: YPM solid plates, with methanol concentration: 3 g / L; Procedure: Stranding isolation of single clones; S2 Seed Culture (Methanol System): Culture medium: YPM liquid medium (5 mL), with methanol concentration: 5 g / L; Culture conditions: 30℃, 220 r / min; Culture time: 23 h; Final seed culture OD: 6.9 Preparation of S3 fermentation medium (50 mL per bottle) ① Prepare Delft basic salt medium (50 mL / 250 mL shake flask) (NH4)2SO4: 2.5 g / L, KH2PO4: 14.4 g / L, MgSO4·7H2O: 0.5 g / L; pH: 5.6; Sterilization: 115℃, 30 min; ②Add other ingredients After sterilization, Delft basal salt medium is supplemented with vitamin solution: 1 mL / L, trace element solution: 2 mL / L, and methanol: 10 g / L; S4 Inoculation and Culture The seed culture obtained from S2 was inoculated into the fermentation medium obtained from S3. The initial OD was 0.2–0.5. The culture conditions were 30℃ and 220 r / min. The culture time was 64 h. Endpoint: Protein content measured upon bacterial collection. The formulas for the trace element solution and vitamin solution used in the above experimental group are as follows: II. Testing During step S4 of experimental groups 1 and 2, the OD value of the fermentation broth was monitored by spectrophotometry at the following time points: 16 h, 23 h, and 64 h; and at the endpoint, the protein content was determined by Kjeldahl method after collecting the cells.

[0025] III. Results The results of biomass and protein content detection for different fermentation systems are shown in Table 1 and 2. Figure 1 Table 1. Detection results of different fermentation systems The results show that although the initial growth rate of the strain was slightly lower than that of the glucose system under methanol as the sole carbon source, the final biomass (OD value) was essentially the same as that of the glucose system as the fermentation process progressed. This indicates that methanol, as a carbon source, can effectively support cell growth and metabolism. Simultaneously, the protein expression level remained stable, comparable to that of the glucose system. This demonstrates that under optimized process conditions, methanol can replace glucose as a carbon source and achieve a fermentation process without sacrificing product quality.

[0026] In summary, the culture medium of this invention, through its specific formulation, enables Pichia pastoris to be efficiently cultured and fermented using methanol as the sole carbon source, thereby obtaining a fermentation broth with high biomass and high protein content. Compared with traditional processes that require the addition of auxiliary carbon sources such as glucose and glycerol, this invention significantly reduces sugar consumption and production costs during fermentation. Furthermore, compared to processes that are only applicable to specific strains and can only use methanol as the sole carbon source during the fermentation stage, the culture medium of this invention has a wider range of applications and higher practical application value.

Claims

1. A culture medium for methyl-trophic Pichia pastoris, characterized in that: The culture medium includes seed solid culture medium, seed liquid culture medium, and fermentation culture medium; The seed solid culture medium is a medium based on YPM solid culture medium, with methanol added to a final concentration of 2~3 g / L. The seed liquid culture medium is a medium based on YPM liquid culture medium, with methanol added to a final concentration of 4~5 g / L. The fermentation medium comprises (NH4)2SO4 at a final concentration of 1-5 g / L, KH2PO4 at 10-15 g / L, MgSO4·7H2O at 0.1-1 g / L, vitamin solution at 0.5-1.5 mL / L, trace element solution at 1-3 mL / L, and methanol at 7-13 g / L.

2. The culture medium according to claim 1, characterized in that: The seed solid culture medium is a medium based on YPM solid culture medium, with methanol added to a final concentration of 3 g / L. The YPM solid culture medium comprises yeast extract powder at a final concentration of 1-10 g / L, peptone at 10-20 g / L, and agar at 10-20 g / L.

3. The culture medium according to claim 1, characterized in that: The seed liquid culture medium is a medium based on YPM liquid culture medium with added methanol at a final concentration of 5 g / L. The YPM liquid culture medium comprises yeast extract powder at a final concentration of 1-10 g / L and peptone at a final concentration of 10-20 g / L.

4. The culture medium according to claim 1, characterized in that: The fermentation medium comprises (NH4)2SO4 at a final concentration of 2.5 g / L, KH2PO4 at 14.4 g / L, MgSO4·7H2O at 0.5 g / L, vitamin solution at 1 mL / L, trace element solution at 2 mL / L, and methanol at 10 g / L; pH value is 5.

6.

5. The culture medium according to claim 1, characterized in that: The vitamin solution comprises D-biotin at a final concentration of 0.05 g / L, D-calcium pantothenate at 1.00 g / L, thiamine hydrochloride at 1.00 g / L, pyridoxine hydrochloride at 1.00 g / L, nicotinic acid at 0.2 g / L, aminobenzoic acid at 25 g / L, and inositol at 6.5; pH 6.

5. The trace element solution comprises 3 g / L FeSO4•7H2O, 4.5 g / L ZnSO4•7H2O, 4.5 g / L CaCl2•2H2O, 1.00 g / L MnCl2•4H2O, 0.3 g / L CoCl2•6H2O, 0.3 g / L CuSO4•5H2O, 0.4 g / L Na2MoO4•2H2O, 1.0 g / L H3BO3, 0.1 g / L KI, and 19.0 g / L Na2EDTA•2H2O; pH 6.

0.

6. A fermentation method for methyl-trophic Pichia pastoris, characterized in that: Includes the following steps: 1) Take a Pichia pastoris strain, streak it onto a seed solid medium, and culture it to obtain a single colony of Pichia pastoris; 2) Take a single colony of Pichia pastoris, inoculate it into seed liquid culture medium, and culture it to obtain seed liquid; 3) Take the seed culture and inoculate it into the fermentation medium for culture; The seed solid culture medium is YPM solid culture medium, which is sterilized and then mixed with methanol to a concentration of 2-3 g / L before solidification to obtain the culture medium. The seed liquid culture medium is YPM liquid culture medium, which is sterilized, cooled, and then methanol is added to a concentration of 4-5 g / L, and the medium is mixed well to obtain the culture medium. The fermentation medium is prepared by weighing the raw materials according to the following ratio, dissolving the inorganic salts in water, adjusting the pH value, sterilizing, cooling, and then adding vitamin solution, trace element solution and methanol and mixing well. 1~5 g / L (NH4)2SO4, 10~15 g / L KH2PO4, 0.1~1 g / L MgSO4·7H2O, 0.5~1.5 mL / L vitamin solution, 1~3 mL / L trace element solution, 7~13 g / L methanol, and the remainder is water.

7. The fermentation method according to claim 6, characterized in that: The methanol concentration in the seed solid culture medium is 3 g / L; The methanol concentration in the seed liquid culture medium was 5 g / L; The ratio of raw materials in the fermentation medium is: 2.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 1 mL / L vitamin solution, 2 mL / L trace element solution, and 10 g / L methanol. The vitamin solution comprises 0.05 g / L of D-biotin, 1.00 g / L of D-calcium pantothenate, 1.00 g / L of thiamine hydrochloride, 1.00 g / L of pyridoxine hydrochloride, 1.00 g / L of nicotinic acid, 0.2 g / L of aminobenzoic acid, and 25 g / L of inositol; the pH is 6.

5. The trace element solution comprises 3 g / L FeSO4•7H2O, 4.5 g / L ZnSO4•7H2O, 4.5 g / L CaCl2•2H2O, 1.00 g / L MnCl2•4H2O, 0.3 g / L CoCl2•6H2O, 0.3 g / L CuSO4•5H2O, 0.4 g / L Na2MoO4•2H2O, 1.0 g / L H3BO3, 0.1 g / L KI, and 19.0 g / L Na2EDTA•2H2O; the pH value is 6.

0.

8. The fermentation method according to claim 6, characterized in that: The culture conditions described in step 1) are a temperature of 30°C and a time of 48 hours.

9. The fermentation method according to claim 6, characterized in that: Step 2) The culture conditions are 220 r / min rotation speed, 30℃ temperature, and 20-24 h time.

10. The fermentation method according to claim 6, characterized in that: Step 3) The initial OD value of the fermentation medium after inoculation with the seed liquid is 0.2 to 0.5; the culture conditions are 30℃, 220r / min, and 60 to 65 h.

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