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: inoculating a pichia pastoris fermentation seed solution into a fermentation culture medium, performing fermentation culture to a glycerol culture stage, and entering a methanol-induced culture stage after glycerol feeding is finished and pH is rebounded. According to the methanol-induced fermentation process for expressing the III-type recombinant human collagen, provided by the invention, after the glycerol feeding is finished, the pH rebound is taken as an index, and methanol induction is started after the pH rebound, so that thallus growth is obviously accelerated, and the protein expression quantity is improved, and therefore, the III-type recombinant human collagen can be industrially produced; and greater economic values can be created for enterprises.

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 a family of proteins widely distributed in animal connective tissues, accounting for about one-third of the total protein in the human body. It plays an important role in maintaining the normal physiological functions of cells, tissues, and organs and repairing injuries. Collagen has a wide range of applications in medicine, tissue engineering, food, cosmetics, and other fields due to its good physical properties and biological characteristics.

[0003] At present, collagen used in various fields is mainly extracted from animal skins, connective tissues and other tissues by acid or alkaline hydrolysis or enzymatic hydrolysis. However, it is difficult to separate the collagen produced by these methods into pure products, and it is easy to cause pathogenic contamination and rejection reactions in clinical practice, which greatly limits its medical application. In response to the above problems, the use of genetic engineering technology to recombinantly express human type III recombinant human collagen is a new type of collagen material with higher quality and safety, and the molecular weight is determined, and the production process conditions are mild, which is conducive to maintaining its biological activity.

[0004] However, there are also some problems in expressing natural human collagen using genetic recombination technology. For example, when obtaining secretory protein through Pichia pastoris fermentation, there is still the problem of low expression level during the fermentation process. Summary of the invention

[0005] In order to solve the problem of low expression of secretory proteins obtained by Pichia pastoris fermentation in the prior art, the present invention provides a methanol-induced fermentation process for expressing type III recombinant human collagen. The fermentation process uses pH rebound and no rebound as indicators. After fermentation rebound, bacterial growth is significantly accelerated and protein expression is higher, which solves the problem of low expression of secretory proteins obtained by Pichia pastoris fermentation in the prior art.

[0006] 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: inoculating Pichia yeast fermentation seed liquid into a fermentation medium, fermenting and culturing to a glycerol culture stage, and after the glycerol addition is completed, waiting for pH rebound and entering a methanol-induced culture stage.

[0007] Optionally, after the addition of glycerol is completed, the pH rebounds to above 5.04 and then enters the methanol induction culture stage.

[0008] Optionally, the fermentation culture reaches the glycerol culture stage, and the glycerol addition is terminated when the bacterial cell OD600 ranges from 100-150, 150-180, 180-200 or 200-240.

[0009] Optionally, after the fermentation culture reaches the glycerol culture stage, the glycerol addition is terminated when the bacterial OD600 ranges from 180 to 200.

[0010] Optionally, the Pichia pastoris is a Pichia pastoris expressing type III recombinant human collagen, and the deposit number is CCTCC M 20242213.

[0011] Optionally, the OD600 of the Pichia fermentation seed liquid is in the range of 15-25.

[0012] Optionally, the fermentation 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.

[0013] Optionally, the fermentation culture temperature is 30°C.

[0014] Optionally, the dissolved oxygen during the methanol induction culture stage is 20%-30%.

[0015] 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 takes pH rebound as an indicator after the glycerol addition is completed, and methanol induction is started again after the pH rebound, so that the bacterial growth is significantly accelerated and the protein expression amount is improved, thereby enabling the industrial production of type III recombinant human collagen, which helps to create greater economic value for enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The electrophoresis diagram of collagen produced by fermentation under pH rebound and pH non-rebound conditions and at different OD values ​​before induction; Figure 2 The electrophoresis diagram of collagen produced by methanol-induced fermentation at different times. DETAILED DESCRIPTION

[0017] 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.

[0018] In order to solve the problem of low expression of secretory protein obtained by Pichia pastoris fermentation in the prior art, the present invention provides a methanol-induced fermentation process for expressing type III recombinant human collagen, and the methanol-induced fermentation process comprises the following steps: inoculating Pichia pastoris fermentation seed liquid into a fermentation medium, fermenting and culturing to a glycerol culture stage, and after the glycerol addition is completed, waiting for pH rebound, entering a methanol-induced culture stage.

[0019] The methanol-induced fermentation process for expressing type III recombinant human collagen provided by the present invention takes pH rebound as an indicator after the glycerol addition is completed, and methanol induction is started again after the pH rebound, so that the bacterial growth is significantly accelerated and the protein expression amount is improved, thereby enabling the industrial production of type III recombinant human collagen, which helps to create greater economic value for enterprises.

[0020] Furthermore, the present invention preferably waits for pH rebound after the addition of glycerol is completed. Specifically, wait for a certain period of time, about 3-4 hours, detect the pH value, and enter the methanol induction culture stage after the pH value rebounds to above 5.04.

[0021] Specifically, in the methanol-induced fermentation process for expressing type III recombinant human collagen provided by the present invention, the fermentation culture reaches the glycerol culture stage. After the fermentation grows for 8-11 hours, 50% (W / V) glycerol is added to different OD value ranges. Preferably, the glycerol addition is terminated when the bacterial OD600 range is 100-150, 150-180, 180-200 or 200-240.

[0022] In order to further increase the protein expression amount, the present invention preferably ends the glycerol addition after the fermentation culture reaches the glycerol culture stage and the OD600 of the bacteria is in the range of 180-200.

[0023] The present invention preferably uses Pichia pastoris to express type III recombinant human collagen, and the Pichia pastoris strain is Pichia pastoris RX-2, which is constructed by our laboratory and deposited in the China Center for Type Culture Collection on October 15, 2024, with a deposit number of CCTCC M 20242213; and the OD600 range of the Pichia pastoris fermentation seed liquid is preferably 15-25.

[0024] Specifically, the preferred Pichia yeast fermentation seed liquid of the present invention is prepared according to the following method: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0025] 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.

[0026] Seed culture medium YPD formula: yeast powder 10g / L; peptone 20g / L; glucose 10g / L.

[0027] The preferred fermentation medium of the present invention is Pichia yeast fermentation medium, and its preferred 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; the preferred fermentation culture temperature is 30°C; and the preferred dissolved oxygen in the methanol induction culture stage is 20%-30%.

[0028] The present invention increases the expression amount of type III recombinant human collagen by adjusting the timing of methanol induction (bacterial age and bacterial quantity), pH rebound, induction time, etc., thereby further improving the production level of recombinant human collagen.

[0029] 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 with reference to the accompanying drawings.

[0030] Example 1 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0031] 100L tank was filled with 50L BSM medium. Before inoculation, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH = 5.0. First, 200mL PTM1 was inoculated, and then the prepared seed solution with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 120, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 60 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 2.13 g / L.

[0032] Example 2 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0033] 100L tank was filled with 50L BSM medium, and the speed was adjusted to 300rpm before inoculation, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed solution with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 100-150 and the wet weight was 180-220g / L, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 60 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 2.12-2.47 g / L.

[0034] According to the method provided in this example, multiple fermentations were performed, and therefore, the OD value and content were range values ​​obtained through multiple fermentations.

[0035] Example 3 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0036] 100L tank was filled with 50L BSM medium. Before inoculation, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed solution with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 150-180 and the wet weight was 210-240g / L, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 60 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 2.49-3.01 g / L.

[0037] According to the method provided in this example, multiple fermentations were performed, and therefore, the OD value and content were range values ​​obtained through multiple fermentations.

[0038] Example 4 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0039] 100L tank was filled with 50L BSM medium. Before inoculation, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed solution with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 180-200 and the wet weight was 260-280g / L, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 60 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 4.8-7.0 g / L.

[0040] According to the method provided in this example, multiple fermentations were performed, and therefore, the OD value and content were range values ​​obtained through multiple fermentations.

[0041] Example 5 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0042] 100L tank was filled with 50L BSM medium, and the speed was adjusted to 300rpm before inoculation, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed liquid with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 200-240 and the wet weight was 275-290g / L, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 60 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 0.84-1.0 g / L.

[0043] According to the method provided in this example, multiple fermentations were performed, and therefore, the OD value and content were range values ​​obtained through multiple fermentations.

[0044] Example 6 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0045] 100L tank was filled with 50L BSM medium, and the speed was adjusted to 300rpm before inoculation, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed liquid with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 120 and the wet weight was 180-220g / L, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 84 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 2.26 g / L.

[0046] Example 7 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0047] 100L tank was filled with 50L BSM medium. Before inoculation, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed solution with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 120 and the wet weight was 180-220g / L, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 96 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 2.53 g / L.

[0048] Example 8 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0049] 100L tank was filled with 50L BSM medium. Before inoculation, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed solution with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 120 and the wet weight was 180-220g / LL, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 108 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 2.87 g / L.

[0050] Example 9 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0051] 100L tank was filled with 50L BSM medium, and the speed was adjusted to 300rpm before inoculation, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed liquid with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 120 and the wet weight was 180-220g / L, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 120 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 3.12 g / L.

[0052] Example 10 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0053] 100L tank was filled with 50L BSM medium. Before inoculation, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH=5.0. First, 200mL PTM1 was inoculated, and then the prepared seed solution with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 120 and the wet weight was 180-220g / L, and the addition of 50% glycerol was stopped. 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 per flow, and 12mL of PTM1 was added), 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 130 hours of induction, the collagen content of the fermentation supernatant was determined by the BCA method to be 2.98 g / L.

[0054] Comparative Example 1 Seed solution preparation: Primary seed solution: Take out with a sterile pipette tip, add 1 mL of engineered bacteria (Pichia pastoris RX-2, deposit number CCTCCM20242213) into a 500 mL shake flask containing 120 mL of YPD medium, culture at 220 rpm, 30°C for 18-30 h, until OD600 = 8-12; Secondary seed solution: Divide 120 mL of the cultured primary seed solution 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 hours, until OD600 = 15-25.

[0055] 100L tank was filled with 50L BSM medium. Before inoculation, the speed was adjusted to 300rpm, the ventilation volume was 4L / min (2vvm), the fermentation temperature was set to 30°C, and the pH was adjusted with concentrated ammonia to control the fermentation pH = 5.0. First, 200mL PTM1 was inoculated, and then the prepared seed solution with an OD value of 15-25 was inoculated into the tank at 5%. When the carbon source was exhausted, the dissolved oxygen suddenly rose to 80%, and 50% (W / V) glycerol was added (1L 50% glycerol was added per flow, and 12mL of PTM1 was added) until the bacterial OD value was 120, and the addition of 50% glycerol was stopped. After the glycerol was exhausted, the dissolved oxygen suddenly rose to 80%, and the pH did not need to rebound, and then methanol was added (1L methanol was added per flow, and 12mL of PTM1 was added), 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 measure the OD value, wet weight and fermentation supernatant protein content. After testing, the collagen content in the fermentation supernatant was determined to be 0.23g / L by the BCA method after 60h of induction.

[0056] A 10% gel running experiment was performed on the supernatant samples of the above-mentioned embodiments and comparative examples. The specific method was as follows: 40 μL of the supernatant was taken, 10 μL of 5×Loading Buffer was added, and the mixture was boiled in a metal bath for 10 min.

[0057] See also Figure 1 As shown, 1 is Takara Premixed Marker: load 5 μL; 2 is OD600 of 100-150, fermentation supernatant sample for 60 hours, that is, the supernatant sample of Example 2, load 5 μL; 3 is OD600 of 150-180, fermentation supernatant sample for 60 hours, that is, the supernatant sample of Example 3, load 5 μL; 4 is OD600 of 180-200, fermentation supernatant sample for 60 hours, that is, the supernatant sample of Example 4, load 5 μL; 5 is OD600 of 200-240, fermentation supernatant sample for 60 hours, that is, the supernatant sample of Example 5, load 5 μL; 6 is blank; 7 is Takara Premixed Marker: 5 μL of sample was loaded; 8 is the fermentation supernatant sample of 60 h fermentation with no pH rebound after glycerol supplementation during the fermentation process, i.e., the supernatant sample of Comparative Example 1, 5 μL of sample was loaded; 9 is the fermentation supernatant sample of 60 h fermentation with pH rebound after glycerol supplementation during the fermentation process, i.e., the supernatant sample of Example 1, 5 μL of sample was loaded.

[0058] See also Figure 2As shown, 1 is Takara Premixed Marker: load 5 μL; 2 is the fermentation supernatant sample after fermentation for 60 hours, that is, the supernatant sample of Example 1, load 5 μL; 3 is the fermentation supernatant sample after fermentation for 84 hours, that is, the supernatant sample of Example 6, load 5 μL; 4 is the fermentation supernatant sample after fermentation for 96 hours, that is, the supernatant sample of Example 7, load 5 μL; 5 is the fermentation supernatant sample after fermentation for 108 hours, that is, the supernatant sample of Example 8, load 5 μL; 6 is the fermentation supernatant sample after fermentation for 120 hours, that is, the supernatant sample of Example 9, load 5 μL; 7 is the fermentation supernatant sample after fermentation for 130 hours, that is, the supernatant sample of Example 10, load 5 μL.

[0059] The yields of fermentation production under pH rebound and pH non-rebound conditions are shown in Table 1: Table 1 Example 1 Comparative Example 1 Does pH rebound? pH rebound pH does not rebound Fermentation tank time 60h 60h Induction timing OD600=120 OD600=120 Fermentation tank supernatant yield 2.13g / L 0.23g / L From Table 1 and Figure 1 It can be seen that the conventional methanol induction method is used, the dissolved oxygen is controlled at 20%-30% during induction, and before induction, pH rebound or no rebound is used as an indicator. After the fermentation pH rebounds, methanol induction is performed, and the bacterial growth is significantly accelerated and the protein expression is high. However, when the fermentation pH does not rebound and methanol induction is performed, the bacterial growth is slow and the protein is basically not expressed.

[0060] The yield of fermentation production at different OD values ​​before induction is shown in Table 2: Table 2 Example 2 Example 3 Example 4 Example 5 Does pH rebound? pH rebound pH rebound pH rebound pH rebound Fermentation tank time 60h 60h 60h 60h Induction timing OD600=100-150 OD600=150-180 OD600=180-200 OD600=200-240 Fermentation tank supernatant yield 2.12-2.47g / L 2.49-3.01g / L 4.80-7.00g / L 0.84-1.00g / L Table 2 and Figure 1 The data show that during induction, the dissolved oxygen was controlled at 20%-30%, and before induction, the pH rebounded to above 5.04, and 50% (W / V) glycerol was added to different OD values: 100-150; 150-180; 180-200; 200-240, and the addition of glycerol was terminated. Preferably, when the OD value of 50% (W / V) glycerol was added to 180-200, the expression amount of the target protein was better.

[0061] The yield of methanol-induced fermentation production at different times is shown in Table 3: Table 3 Example 1 Example 6 Example 7 Example 8 Example 9 Example 10 Does pH rebound? pH rebound pH rebound pH rebound pH rebound pH rebound pH rebound Fermentation tank time 60h 84h 96h 108h 120h 130h Induction timing OD600=120 OD600=120 OD600=120 OD600=120 OD600=120 OD600=120 Fermentation tank supernatant yield 2.13g / L 2.26g / L 2.53g / L 2.87g / L 3.12g / L 2.98g / L From Table 3 and Figure 2 It can be seen that when the methanol induction stage is entered, samples are taken at different induction time periods. The longer the time, the higher the expression of the target protein. When the fermentation time reaches more than 120 hours, the expression of the target protein does not show obvious degradation. More preferably, during the methanol induction process at different time periods, when the fermentation time reaches 120 hours, the effect is better and the expression of the target protein is the highest. As time goes by, although there are more bacteria, the increase in protein production does not change significantly. Considering the principle of production cost saving, the fermentation is no longer delayed.

[0062] 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: inoculating Pichia yeast fermentation seed liquid into fermentation medium, fermenting and culturing to a glycerol culture stage, and after the glycerol addition is completed, waiting for pH rebound and entering a methanol induction culture stage.

2. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 1, characterized in that: After the addition of glycerol was completed, the pH rebounded to above 5.04 and entered the methanol induction culture stage.

3. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 1, characterized in that: The fermentation culture reaches the glycerol culture stage, and the glycerol addition is terminated when the bacterial OD600 ranges from 100 to 150, 150 to 180, 180 to 200, or 200 to 240.

4. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 3, characterized in that: After the fermentation culture reaches the glycerol culture stage, the glycerol addition is terminated when the bacterial OD600 ranges from 180 to 200.

5. The methanol-induced fermentation process for expressing type III recombinant human collagen according to any one of claims 1 to 4, characterized in that: The Pichia pastoris is a Pichia pastoris expressing type III recombinant human collagen, and the deposit number is CCTCC M20242213.

6. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 5, characterized in that: The OD600 of the Pichia yeast fermentation seed liquid is in the range of 15-25.

7. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 5, characterized in that: The fermentation 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 5, characterized in that: The fermentation temperature was 30°C.

9. The methanol-induced fermentation process for expressing type III recombinant human collagen according to claim 5, characterized in that: The dissolved oxygen in the methanol induction culture stage is 20%-30%.

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