Methanol-induced fermentation process for expressing III-type recombinant human collagen

Abstract

The invention relates to the technical field of biological fermentation, in particular to a methanol-induced fermentation process for expressing III-type recombinant human collagen, which comprises the following steps: adding YPD into a fermentation basal culture medium to obtain a compound fermentation culture medium; the method comprises the following steps: inoculating a pichia pastoris fermentation seed solution into a compound fermentation culture medium, performing fermentation culture to a glycerol culture stage, and after glycerol feeding is finished, entering a methanol induction culture stage. According to the methanol induced fermentation process for expressing the III-type recombinant human collagen, provided by the invention, a yeast extract powder peptone glucose culture medium YPD is added into a traditional fermentation basal culture medium to form a compound fermentation culture medium, so that a relatively high fermentation speed and a relatively high total fermentation yield can be achieved in a fermentation production process of the collagen; therefore, the expression quantity of the III-type recombinant human collagen can be improved, and the production level of the III-type recombinant human collagen is further improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation, and in particular to a methanol-induced fermentation process for expressing type III recombinant human collagen. Background Art

[0002] Collagen is the product of complete collagen hydrolysis, and plays an important role in maintaining the normal physiological functions of cells, tissues, and organs and repairing injuries. Collagen has good physical properties and biological characteristics, and is widely used in medicine, tissue engineering, food, cosmetics and other fields.

[0003] Currently, collagen used in various fields is mainly extracted from animal skins, connective tissues and other tissues by acid or alkali hydrolysis or enzymatic hydrolysis. However, the collagen produced by these methods is difficult to separate into pure products. The heterogeneity between animals and humans leads to the need to increase the removal of immunogenicity, and it is easy to cause pathogenic contamination and rejection reactions in clinical practice, affecting the application of collagen in medicine.

[0004] In order to promote its application in medicine, the recombinant expression of human type III recombinant human collagen using genetic engineering technology is a new type of collagen material with higher quality and greater safety, and its molecular weight is determined and the production process conditions are mild, which is conducive to maintaining its biological activity.

[0005] Existing recombinant collagen is generally obtained by fermenting Pichia pastoris, but the existing fermentation process for obtaining recombinant collagen by Pichia pastoris has the problem of low expression level. Summary of the invention

[0006] In order to solve the problem of low expression level in the fermentation process of obtaining recombinant collagen-like proteins through Pichia pastoris in the prior art, the present invention provides a methanol-induced fermentation process for expressing type III recombinant human collagen. By adding YPD to the fermentation basal medium, a faster fermentation speed and a higher total fermentation yield can be achieved in the fermentation production process of collagen, thereby increasing the expression level of type III recombinant human collagen, further improving the production level of type III recombinant human collagen, and solving the problem of low expression level in the fermentation process of obtaining recombinant collagen-like proteins through Pichia pastoris in the prior art.

[0007] The technical solution adopted by the present invention to solve its technical problem is: A methanol-induced fermentation process for expressing type III recombinant human collagen comprises the following steps: adding YPD to a fermentation basal medium to obtain a composite fermentation medium; inoculating Pichia yeast fermentation seed liquid into the composite fermentation medium, fermenting and culturing to a glycerol culture stage, and entering a methanol-induced culture stage after the glycerol addition is completed.

[0008] Optionally, the mass content of the YPD in the composite fermentation medium is 1-13%.

[0009] Optionally, vitamin C is added during the methanol induction stage.

[0010] Optionally, the concentration of vitamin C in the fermentation broth during the methanol induction culture stage is 3-15 mM.

[0011] Optionally, the methanol induction culture stage adopts a multi-stage methanol induction method for culture.

[0012] Optionally, the methanol induction culture stage adopts a secondary methanol induction method for culture.

[0013] Optionally, the fermentation basal medium is a Pichia fermentation medium, and its formula is: 85% H3PO4 26.7 mL / L; CaSO4·2H2O 0.93 g / L; K2SO4 18.2 g / L; MgSO4·7H2O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 solution 4 mL / L.

[0014] Optionally, the OD600 of the Pichia fermentation seed liquid is in the range of 15-25.

[0015] Optionally, after the glycerol addition is completed, the pH rebounds and the methanol induction culture stage is entered.

[0016] Optionally, after the addition of glycerol is completed, the pH rebounds to above 5.04 and then enters the methanol induction culture stage.

[0017] The beneficial effects of the present invention are: The methanol-induced fermentation process for expressing type III recombinant human collagen provided by the present invention forms a composite fermentation medium by adding yeast extract peptone glucose medium YPD to a traditional fermentation basal medium. During the fermentation production of collagen, a faster fermentation speed and a higher total fermentation yield can be achieved, thereby helping to increase the expression amount of type III recombinant human collagen and further improve the production level of type III recombinant human collagen. DETAILED DESCRIPTION

[0018] The present invention is now further described in detail. The embodiments described below are exemplary and intended to be used to explain the present invention, but cannot be understood as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0019] In order to solve the problem of low expression level in the fermentation process of obtaining recombinant collagen by Pichia pastoris in the prior art, the present invention provides a methanol-induced fermentation process for expressing type III recombinant human collagen, and the fermentation process comprises the following steps: adding YPD to a fermentation basal medium to obtain a composite fermentation medium; inoculating Pichia pastoris fermentation seed liquid into the composite fermentation medium, fermenting and culturing to a glycerol culture stage, and entering a methanol-induced culture stage after the glycerol addition is completed.

[0020] Among them, YPD medium has the advantages of rich nutrients, wide application range, and easy operation. These nutrients provide the carbon source, nitrogen source and vitamins required for the growth of yeast strains, and are suitable for the growth of yeast strains.

[0021] The methanol-induced fermentation process for expressing type III recombinant human collagen provided by the present invention forms a composite fermentation medium by adding yeast extract peptone dextrose medium YPD (Yeast Extract Peptone Dextrose Medium) to a traditional fermentation basal medium. During the fermentation production of collagen, a faster fermentation speed and a higher total fermentation yield can be achieved, thereby helping to increase the expression amount of type III recombinant human collagen and further improve the production level of type III recombinant human collagen.

[0022] In order to take into account both expression amount and economy, the mass content of YPD in the composite fermentation medium of the present invention is preferably 1-13%, and more preferably 1-10%.

[0023] In order to further increase the protein expression, the present invention preferably adds vitamin C during the methanol induction culture stage; by adding vitamin C, the synthesis of collagen can be promoted in an endogenous manner, thereby increasing protein expression.

[0024] In order to take into account both expression amount and economy, the present invention preferably has a vitamin C concentration of 3-15 mM in the fermentation broth during the methanol induction culture stage, and more preferably 10 mM.

[0025] In order to further increase the fermentation speed and shorten the time, the present invention preferably adopts a multi-stage methanol induction method for cultivation in the methanol induction culture stage, and further preferably adopts a two-stage methanol induction method for cultivation in the methanol induction culture stage.

[0026] To ensure the protein expression, the fermentation basal medium is Pichia yeast fermentation medium, and its formula is: 85% H3PO4 26.7 mL / L; CaSO4·2H2O 0.93 g / L; K2SO4 18.2 g / L; MgSO4·7H2O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 solution 4 mL / L.

[0027] The present invention preferably uses Pichia pastoris to express type III recombinant human collagen, and the Pichia pastoris is a species of Pichia pastoris ( Pichia pastoris ) RX-2, deposited in China Center for Type Culture Collection on October 15, 2024, with the deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number: CCTCC No.M 20242213; and the OD600 range of the Pichia yeast fermentation seed liquid is preferably 15-25.

[0028] In the preferred fermentation process of the present invention, after the addition of glycerol is completed, the pH rebounds and the methanol induction culture stage is entered; and further preferably, after the addition of glycerol is completed, a certain period of time, about 3-4 hours, is waited, the pH value is detected, and the pH rebounds to above 5.04, and the methanol induction culture stage is entered.

[0029] The present invention preferably performs fermentation culture to the glycerol culture stage, and after the fermentation grows for 8-11 hours, 50% (W / V) glycerol is added to different OD value ranges, and preferably the glycerol addition is terminated when the bacterial OD600 range is 100-150, 150-180, 180-200 or 200-240, and further preferably, after the fermentation culture reaches the glycerol culture stage, the glycerol addition is terminated when the bacterial OD600 range is 180-200.

[0030] The preferred fermentation culture temperature of the present invention is 30° C., and the dissolved oxygen in the methanol induction culture stage is 20%-30%.

[0031] Specifically, the methanol-induced fermentation process for expressing type III recombinant human collagen is preferably carried out as follows: 1) Primary seed liquid culture: Use a sterile pipette tip to take out 1 mL of the engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) and add it to a 500 mL shake flask containing 120 mL of YPD medium. Cultivate at 200 rpm and 30°C for 18-30 h until OD600 = 8-12. 2) Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 18-30 h until OD600 = 15-25; 3) Preparation of fermentation medium: Add YPD to the fermentation basal medium to form a composite fermentation medium, where YPD accounts for 1-13% of the mass of the fermentation medium and is sterilized for later use; 4) Fermentation process control: The seed liquid cultured in step 2) is inoculated into the composite fermentation medium sterilized in step 3) to start fermentation. 100L tank is filled with 50L BSM medium. Before inoculation, the rotation speed is adjusted to 300rpm, the ventilation volume is 4L / min (2vvm), the fermentation temperature is set to 30°C, and the pH is adjusted with concentrated ammonia water to control the fermentation pH=5.0. First, 200mL PTM1 is inoculated, and then the prepared seed liquid with an OD value of 15-25 is inoculated into the tank at 6%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and 50% (W / V) glycerol is added (1L 50% glycerol is added for each flow, and 12mL PTM1 is added) until the bacterial OD value is 180-200 and the wet weight is 260-280g / L, and the addition of 50% glycerol is stopped. After the glycerol is exhausted, the dissolved oxygen suddenly rises to 80%, and the pH rebounds to above 5.04, then methanol is added (12 mL of PTM1 is added for each 1 L of methanol added), and vitamin C is added, the amount of vitamin C added is 3-15 mM, and the methanol induction culture stage is entered. The rotation speed and compressed air flux are adjusted to make the dissolved oxygen greater than 20%, and the OD value is measured every 4 hours. After 60 hours of induction, the fermentation is terminated to obtain the fermentation liquid.

[0032] The formula of the seed culture medium in step 1) is, in percentage by mass, 1% yeast extract powder, 2% trypsin, 2% glucose, and the remainder water; the inoculation amount of the seed liquid in step 2) is 1-10%; YPD is used as a raw material component of the fermentation medium in the form of powder to participate in microbial fermentation.

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below.

[0034] In each embodiment and comparative example of the present invention, The PTM1 solution was prepared according to the Invitrogen manual. The specific formula was as follows: CuSO4·5H2O 6.0 g / L; NaI 0.08 g / L; MnSO4·H2O 3.0 g / L; NaMoO4·2H2O 0.2 g / L; H3BO3 0.02g / L; CoCl20.5g / L; ZnCl220.0g / L; FeSO4·7H2O 65.0g / L; Biotin0.2g / L; H2SO45.0mL / L, sterilized by filtration with a 0.22μm filter membrane, stored at 4°C. BSM medium formula: 85% H3PO426.7mL / L; CaSO4·2H2O 0.93g / L; K2SO418.2g / L; MgSO4·7H2O 14.9g / L; KOH 4.13g / L; Glycerol40.0g / L; PTM1 solution4mL / L.

[0035] Seed culture medium YPD formula: yeast powder 10g / L; peptone 20g / L; glucose 10g / L.

[0036] Example 1 The conventional methanol induction method was used to add 1% YPD to the fermentation basal medium for fermentation effect experiment: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0037] 100L tank with 50L liquid volume (99% BSM medium + 1% YPD medium), (1) 100L tank with 50L BSM medium, before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, add 200mL PTM1, and then add 5% of the prepared seed liquid with an OD value of 10-15 into the tank. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to add 50% (W / V) glycerol (add 12mL of PTM1 for each 1L 50% glycerol added) until the bacterial OD value is 180-200 and the wet weight is 260-280g / L, then stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added), and the methanol induction culture stage (methanol consumption was 10L) was entered. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to measure the OD value, wet weight and protein content of the fermentation supernatant. After 60 hours of induction, the collagen content of the fermentation supernatant was 7.0g / L by the BCA method.

[0038] Example 2 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 3% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0039] 100L tank liquid volume is 50L, (97% BSM medium + 3% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200 and the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added), and the methanol induction culture stage (methanol consumption was 10L) was entered. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to measure the OD value, wet weight and protein content of the fermentation supernatant. After 60 hours of induction, the collagen content of the fermentation supernatant was 8.0g / L by the BCA method.

[0040] Example 3 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0041] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added) (methanol consumption was 10L) to enter the methanol induction culture stage. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to measure the OD value, wet weight and protein content of the fermentation supernatant. After 60 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 9.0g / L.

[0042] Example 4 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 7% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0043] 100L tank liquid volume is 50L, (93% BSM medium + 7% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added) (methanol consumption was 10L) to enter the methanol induction culture stage. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to measure the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 8.5g / L.

[0044] Example 5 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 10% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0045] 100L tank liquid volume is 50L, (90% BSM medium + 10% YPD medium), before inoculation, the speed is adjusted to 300rpm, the ventilation volume is 4L / min (2vvm), the fermentation temperature is set to 30℃, and the pH is adjusted with concentrated ammonia water to control the fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and 50% (W / V) glycerol is added (12mL of PTM1 is added for each 1L of 50% glycerol) until the bacterial OD value is 180-200 and the wet weight is 260-280g / L, and then stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added) (methanol consumption was 10L) to enter the methanol induction culture stage. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to measure the OD value, wet weight and protein content of the fermentation supernatant. After 60 hours of induction, the collagen content of the fermentation supernatant was 6.5g / L by the BCA method.

[0046] Example 6 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 13% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0047] 100L tank liquid volume is 50L, (87% BSM medium + 13% YPD medium), before inoculation, the speed is adjusted to 300rpm, the ventilation volume is 4L / min (2vvm), the fermentation temperature is set to 30℃, and the pH is adjusted with concentrated ammonia water to control the fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and 50% (W / V) glycerol is added (12mL of PTM1 is added for each 1L of 50% glycerol) until the bacterial OD value is 180-200 and the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added) (methanol consumption was 10L) to enter the methanol induction culture stage. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to measure the OD value, wet weight and protein content of the fermentation supernatant. After 60 hours of induction, the collagen content of the fermentation supernatant was 5.0g / L by the BCA method.

[0048] Comparative Example 1: The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 0% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0049] 100L tank liquid volume 50L, (100% BSM medium + 0% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume 4L / min (2vvm), fermentation temperature set to 30℃, adjust pH with concentrated ammonia water, control fermentation pH = 5.0. First inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added) (methanol consumption was 10L) to enter the methanol induction culture stage. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to measure the OD value, wet weight and protein content of the fermentation supernatant. After 60 hours of induction, the collagen content of the fermentation supernatant was 6.0g / L by the BCA method.

[0050] By comparison, it can be seen that adding a certain amount of YPD to the fermentation basal medium can increase the yield from the original 6.0g / L to the current 9.0g / L, increasing the yield of type III recombinant human collagen by 30%.

[0051] The fermentation experiment results show that the expression of the target protein can be increased by adding different proportions of YPD to the fermentation basal medium. When more than 5% is added to the fermentation basal medium, the expression of the target protein decreases. Preferably, when 5% YPD is added to the fermentation basal medium, the expression of the target protein is better. The yield of the target protein by adding different proportions of YPD to the fermentation basal medium is shown in Table 1: Table 1 Example 7 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0052] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol is exhausted, the dissolved oxygen suddenly rises to 80%, and the pH rebounds to above 5.04, then methanol is added (12mL of PTM1 is added for each 1L of methanol added), (methanol consumption is 10L), and vitamin C is added at the same time. The concentration of vitamin C in the fermentation broth is 3mM, and the methanol induction culture stage is entered. The speed and compressed air flux are adjusted to make the dissolved oxygen greater than 20%, and samples are taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60h of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 9.5g / L.

[0053] Example 8 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0054] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol is exhausted, the dissolved oxygen suddenly rises to 80%, and the pH rebounds to above 5.04, then methanol is added (12mL of PTM1 is added for each 1L of methanol added), (methanol consumption is 10L), and vitamin C is added at the same time. The concentration of vitamin C in the fermentation broth is 10mM, and the methanol induction culture stage is entered. The speed and compressed air flux are adjusted to make the dissolved oxygen greater than 20%, and samples are taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60h of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 10g / L.

[0055] Example 9 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0056] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol is exhausted, the dissolved oxygen suddenly rises to 80%, and the pH rebounds to above 5.04, then methanol is added (12mL of PTM1 is added for each 1L of methanol added), (methanol consumption is 10L), and vitamin C is added at the same time. The concentration of vitamin C in the fermentation broth is 15mM, and the methanol induction culture stage is entered. The speed and compressed air flux are adjusted to make the dissolved oxygen greater than 20%, and samples are taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60h of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 8.5g / L.

[0057] The fermentation experiment results show that the expression of the target protein can be increased by adding different proportions of vitamin C during the methanol induction process, and the expression of the target protein is better when the vitamin C concentration is 10mM. The target protein yield at different vitamin C addition amounts is shown in Table 2: Table 2 BSM fermentation basal medium YPD complex medium Vitamin C concentration (mM) Collagen content in fermentation broth (g / L) Example 7 95% 5% 3 9.5 Example 8 95% 5% 10 10 Example 9 95% 5% 15 8.5 Example 10 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0058] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added), (methanol consumption was 8L), and vitamin C was added at the same time. The concentration of vitamin C in the fermentation broth was 10mM, and the methanol induction culture stage was entered. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60h of induction, the collagen content in the fermentation supernatant was 8.5g / L by the BCA method.

[0059] Embodiment 11 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0060] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol is exhausted, the dissolved oxygen suddenly rises to 80%, and the pH rebounds to above 5.04, then methanol is added (12mL of PTM1 is added for each 1L of methanol added), (methanol consumption is 10L), and vitamin C is added at the same time. The concentration of vitamin C in the fermentation broth is 10mM, and the methanol induction culture stage is entered. The speed and compressed air flux are adjusted to make the dissolved oxygen greater than 20%, and samples are taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60h of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 10g / L.

[0061] Example 12 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0062] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol is exhausted, the dissolved oxygen suddenly rises to 80%, and the pH rebounds to above 5.04, then methanol is added (12mL of PTM1 is added for each 1L of methanol added), (methanol consumption is 12L), and vitamin C is added at the same time. The concentration of vitamin C in the fermentation broth is 10mM, and the methanol induction culture stage is entered. The speed and compressed air flux are adjusted to make the dissolved oxygen greater than 20%, and samples are taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60h of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 12g / L.

[0063] Embodiment 13 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0064] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added), (methanol consumption was 14L), and vitamin C was added at the same time. The concentration of vitamin C in the fermentation broth was 10mM, and the methanol induction culture stage was entered. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60h of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 9.3g / L.

[0065] Embodiment 14 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0066] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (12mL of PTM1 was added for each 1L of methanol added), (methanol consumption was 16L), and vitamin C was added at the same time. The concentration of vitamin C in the fermentation broth was 10mM, and the methanol induction culture stage was entered. The speed and compressed air flux were adjusted to make the dissolved oxygen greater than 20%, and samples were taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 60h of induction, the collagen content in the fermentation supernatant was 8g / L by the BCA method.

[0067] The fermentation experiment results show that when the methanol consumption is 12L, the expression of the target protein is better. The target protein yield at different methanol consumption is shown in Table 3: Table 3 BSM fermentation basal medium YPD complex medium Vitamin C concentration (mM) Methanol consumption (L) Collagen content in fermentation broth (g / L) Example 10 95% 5% 10 8 8.5 Embodiment 11 95% 5% 10 10 10 Example 12 95% 5% 10 12 12 Embodiment 13 95% 5% 10 14 9.3 Embodiment 14 95% 5% 10 16 8 Embodiment 15 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0068] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (1L methanol was added for each flow, and 12mL of PTM1 was added). 180g methanol was added once for the first induction (1L methanol was added for each flow, and 12mL of PTM1 was added), and 200g methanol was added once for the second induction (1L methanol was added for each flow, and 12mL of PTM1 was added). (Total methanol consumption was 12L, and two methanol inductions were performed). Vitamin C was added at the same time. The concentration of vitamin C in the fermentation broth was 10mM, and the methanol induction culture stage was entered. The speed and compressed air flux were adjusted so that the dissolved oxygen was greater than 20%, and samples were taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 52h of induction, the collagen content in the fermentation supernatant was determined by the BCA method to be 14g / L.

[0069] Example 16 The conventional methanol induction method was used to perform the fermentation effect experiment under the condition of adding 5% YPD to the fermentation basal medium: Seed solution preparation: Primary seed solution culture: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris strain RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 200 rpm, 30°C for 24 h until OD600 = 8-12; Secondary seed liquid culture: Divide 120 mL of the cultured primary seed liquid into 4 portions, inoculate 30 mL of each portion into a 2000 mL shake flask containing about 720 mL of culture medium, culture at 220 rpm, 30°C for 24 h, until OD600 = 15-25.

[0070] 100L tank liquid volume is 50L, (95% BSM medium + 5% YPD medium), before inoculation, adjust the speed to 300rpm, ventilation volume to 4L / min (2vvm), fermentation temperature to 30℃, adjust pH with concentrated ammonia water, and control fermentation pH = 5.0. First, inoculate 200mL PTM1, and then inoculate the prepared seed liquid with an OD value of 10-15 into the tank at 5%. When the carbon source is exhausted, the dissolved oxygen suddenly rises to 80%, and start to flow 50% (W / V) glycerol (each flow of 1L 50% glycerol, add 12mL PTM1) until the bacterial OD value is 180-200, the wet weight is 260-280g / L, and stop adding 50% glycerol. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH rebounded to above 5.04, and then methanol was added (1L methanol was added for each flow, and 12mL of PTM1 was added). 180g methanol was added once for the first induction (1L methanol was added for each flow, and 12mL of PTM1 was added), 200g methanol was added once for the second induction (1L methanol was added for each flow, and 12mL of PTM1 was added), and 220g methanol was added once for the third induction (1L methanol was added for each flow, and 12mL of PTM1 was added), (the total methanol consumption was 12L, and three methanol inductions were performed), and vitamin C was added at the same time. The concentration of vitamin C in the fermentation broth was 10mM, and the methanol induction culture stage was entered. The speed and compressed air flux were adjusted so that the dissolved oxygen was greater than 20%, and samples were taken every 4 hours to determine the OD value, wet weight and protein content of the fermentation supernatant. After testing, after 58h of induction, the content of collagen in the fermentation supernatant was determined by the BCA method to be 12.9g / L.

[0071] The results of the fermentation experiment show that, when the total amount of methanol induction added is unchanged at 12L, adjusting the multi-stage induction (one methanol induction, two methanol induction, three methanol induction) helps to shorten the tank time, and the two methanol induction can take into account both the fermentation efficiency and the protein expression. The effect of multi-stage induction on the collagen production and the shortening of the time are shown in Table 4: Table 4 BSM fermentation basal medium YPD complex medium Vitamin C concentration (mM) Methanol consumption (L) Multi-level induction Collagen content in fermentation broth (g / L) Example 12 95% 5% 10 12 One-time induction 12 Embodiment 15 95% 5% 10 12 Secondary induction 14 Example 16 95% 5% 10 12 Three times induction 12.9 Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A methanol-induced fermentation process for expressing type III recombinant human collagen, characterized in that: The method comprises the following steps: adding YPD to a fermentation basal medium to obtain a composite fermentation medium; inoculating Pichia yeast fermentation seed liquid into the composite fermentation medium, fermenting and culturing to a glycerol culture stage, and entering a methanol induction culture stage after the glycerol addition is completed.

2. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 1, characterized in that: The mass content of the YPD in the composite fermentation medium is 1-13%.

3. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 2, characterized in that: Vitamin C was added during the methanol induction stage.

4. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 3, characterized in that: The concentration of vitamin C in the fermentation broth during the methanol induction culture stage is 3-15 mM.

5. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 1, characterized in that: The methanol induction culture stage adopts a multi-stage methanol induction method for culture.

6. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 5, characterized in that: The methanol induction culture stage adopts a secondary methanol induction method for culture.

7. The methanol-induced fermentation process for expressing type III recombinant human collagen according to any one of claims 1 to 6, characterized in that: The fermentation basal medium is a Pichia yeast fermentation medium, and its formula is: 85% H3PO4 26.7mL / L; CaSO4·2H2O 0.93g / L; K2SO4 18.2g / L; MgSO4·7H2O 14.9g / L; KOH 4.13g / L; glycerol 40.0g / L; PTM1 solution 4mL / L.

8. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 7, characterized in that: The OD600 of the Pichia yeast fermentation seed liquid is in the range of 15-25.

9. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 7, characterized in that: After the addition of glycerol is completed, wait for the pH to rebound and enter the methanol induction culture stage.

10. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 9, characterized in that: After the addition of glycerol was completed, the pH rebounded to above 5.04 and entered the methanol induction culture stage.

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