Process for improving the fermentation level of recombinant collagen by adjusting the concentration and adding mode of phosphoric acid

Abstract

The application discloses a process for improving the fermentation level of recombinant collagen by adjusting the concentration and adding mode of phosphoric acid. The process inoculates the Pichia pastoris expressing recombinant collagen into sterilized fermentation medium, and lowers the initial concentration of phosphoric acid in the seed liquid culture stage in the early fermentation stage, and adopts the quantitative continuous feeding mode of phosphoric acid in the methanol expression stage. The application adjusts the growth and metabolism of the Pichia pastoris by adjusting the concentration of phosphoric acid in the initial culture medium and the adding mode of phosphoric acid in the methanol expression stage, improves the synthesis rate of the recombinant collagen, and the fermentation level is increased by 29.929% at most, and the fermentation time can be shortened by 16-32 hours correspondingly.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology and relates to a process for improving the fermentation level of recombinant collagen by adjusting the phosphate concentration and the method of addition. Background Technology

[0002] Phosphorus is an important nutrient source in microbial fermentation media, significantly influencing cell growth and product synthesis. The main phosphorus-containing components in cells are DNA, RNA, and polyphosphoric acid (polyPi), primarily existing intracellularly as inorganic phosphorus forms such as orthophosphate (Pi), pyrophosphate (PPi), or polyphosphate (polyPi), and as organic phosphorus forms such as nucleic acids, phosphate sugars, phosphorylated proteins, and phospholipids. Microorganisms can utilize phosphorus sources to regulate cellular metabolism. For example, when there is sufficient Pi in the culture medium, microorganisms generate large amounts of polyphosphoric acid kinase (PPK) to produce polyPi. When Pi is insufficient, PPK enzymes can use polyPi as a substrate to reverse the reaction and generate high-energy phosphate-bonded substances such as ATP and GTP.

[0003] Phosphorus plays a central role in carbon and energy metabolism because many enzymatic reactions require phosphate substrates, and the activities of key enzymes involved in these reactions are regulated by phosphorus concentration, such as phosphofructokinase, 3-phosphoglycerate kinase, and pyruvate kinase. Simultaneously, the free energy gained from substrate phosphorylation is stored in ATP synthesized from ADP and Pi. Furthermore, many important cellular regulatory mechanisms rely on phosphorus, such as in the phosphotransferase reactions of two-component signal transduction systems and serine / threonine protein kinases. In summary, the phosphorus content in the fermentation broth affects the products during microbial fermentation. Summary of the Invention

[0004] The purpose of this invention is to provide a process for improving the fermentation level of recombinant collagen by adjusting the concentration and addition method of phosphate. This process regulates the growth and metabolism rate of Pichia pastoris by reducing the initial concentration of phosphate in the fermentation medium and by adopting a constant-rate feeding method of phosphate during the product expression stage, thereby increasing the expression level and production capacity of recombinant collagen.

[0005] The technical solution for implementing the present invention is as follows:

[0006] The process of improving the fermentation level of recombinant collagen by adjusting the phosphate concentration and the method of addition includes the following steps:

[0007] The Pichia pastoris strain expressing recombinant collagen was inoculated into a sterile fermentation medium with a low phosphate concentration. After basal culture for 12–16 hours, 50% glycerol was added until the target cell wet weight was reached. After the glycerol addition was completed, methanol was added to induce expression, while 85% phosphate solution was added at a rate of 12.396–27.892 ml / h. The composition of the low phosphate concentration fermentation medium was as follows: 85% H3PO4 8.9–12.816 ml / L, CaSO4·2H2O 0.392–1.175 g / L, K2SO4 6.11–18.2 g / L, MgSO4·7H2O 5.6–18.2 g / L, KOH 1.326–4.13 g / L, glycerol 13.5–40.0 g / L, and PTM1 solution 1.029–4.35 ml / L.

[0008] Preferably, the 85% phosphoric acid flow rate is 12.396–20.144 ml / h.

[0009] Preferably, the Pichia pastoris strain expressing recombinant collagen is Pichia pastoris JY0201, which was deposited on September 12, 2018 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 16461 and has been published in Chinese Patent 201811589560.X.

[0010] The PTM1 solution described in this invention uses an existing formula and may contain: CuSO4·5H2O 6.0 g / L, NaI 0.08 g / L, MnSO4·H2O 3.0 g / L, NaMoO4·2H2O 0.2 g / L, H3BO3 0.02 g / L, CoCl 0.5 g / L, ZnCl2 20.0 g / L, FeSO4·7H2O 65.0 g / L, biotin 0.2 g / L, and H2SO4 5 ml / L.

[0011] Preferably, the inoculum size of the Pichia pastoris expressing recombinant collagen is 8-10%, the fermentation temperature is 28-32℃, the pH is adjusted to 4.8-6.5 with ammonia, and the dissolved oxygen in the fermentation medium is not less than 30% during the basic culture stage.

[0012] Preferably, during the methanol induction stage, the dissolved oxygen in the fermentation medium is not less than 10%, and the induction time is 80–120 h, more preferably 80–112 h.

[0013] Preferably, the target cell wet weight during the 50% glycerol feeding stage is 190–220 g / L.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] This invention regulates the growth and metabolism of Pichia pastoris JY0201 by lowering the initial phosphate concentration during the early fermentation stage (basal culture) and simultaneously employing a continuous feed-on phosphate method during the methanol-induced expression stage, thereby increasing the recombinant collagen synthesis rate. The fermentation yield increased by up to 29.929%, the fermentation level improved by up to 35.556%, and the fermentation time was shortened by 8–16 hours. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments.

[0017] In the specific embodiments and comparative examples described in this invention, the strain is Pichia pastoris JY0201, which expresses recombinant collagen. It was deposited at the China General Microbiological Culture Collection Center on September 12, 2018, with accession number CGMCC No. 16461, and has been published in Chinese Patent 201811589560.X.

[0018] The PTM1 solution described in this invention is formulated according to the Pichia pastoris operation manual of Invitrogen, and specifically includes: CuSO4·5H2O 6.0 g / L, NaI 0.08 g / L, MnSO4·H2O 3.0 g / L, NaMoO4·2H2O 0.2 g / L, H3BO3 0.02 g / L, CoCl 0.5 g / L, ZnCl2 20.0 g / L, FeSO4·7H2O 65.0 g / L, biotin 0.2 g / L, H2SO4 5 ml / L. After the solution is fully dissolved, it is filtered through a 0.22 μm filter membrane for sterilization.

[0019] Example 1

[0020] Fermentation medium: 85% H3PO4 8.9ml / L, CaSO4·2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4·7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L.

[0021] The fermentation process for recombinant collagen includes the following steps:

[0022] Pichia pastoris JY0201 was inoculated at a 9% inoculum in a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added to induce expression. Simultaneously, 85% phosphate was added at a flow rate of 20.144ml / h until fermentation terminated after 112h of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8h, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0023] Example 2

[0024] Fermentation medium: 85% H3PO4 11.169 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0025] The fermentation process for recombinant collagen includes the following steps:

[0026] Pichia pastoris JY0201 was inoculated at a 9% inoculum in a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added to induce expression. Simultaneously, 85% phosphate was added at a flow rate of 20.144ml / h until fermentation terminated after 112h of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8h, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0027] Example 3

[0028] Fermentation medium: 85% H3PO4 12.816 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0029] The fermentation process for recombinant collagen includes the following steps:

[0030] Pichia pastoris JY0201 was inoculated at a 9% inoculum in a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added to induce expression. Simultaneously, 85% phosphate was added at a flow rate of 20.144ml / h until fermentation terminated after 112h of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8h, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0031] Example 4

[0032] Fermentation medium: 85% H3PO4 9.345 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0033] The fermentation process for recombinant collagen includes the following steps:

[0034] Pichia pastoris JY0201 was inoculated at a 9% inoculum in a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added to induce expression. Simultaneously, 85% phosphate was added at a flow rate of 20.144ml / h until fermentation terminated after 112h of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8h, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0035] Example 5

[0036] Fermentation medium: 85% H3PO4 9.345 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0037] The recombinant collagen fermentation process includes the following steps:

[0038] Pichia pastoris JY0201 was inoculated at a 9% inoculum in a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added to induce expression. Simultaneously, 85% phosphate was added at a flow rate of 27.892ml / h until fermentation terminated after 112h of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8h, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0039] Example 6

[0040] Fermentation medium: 85% H3PO4 9.345 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0041] The fermentation process for recombinant collagen includes the following steps:

[0042] Pichia pastoris JY0201 was inoculated at a 9% inoculum in a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added for induction expression. Simultaneously, 85% phosphate was added at a flow rate of 12.396ml / h until methanol induction for 112h, at which point fermentation was terminated. During the methanol induction stage, the fermentation broth was collected every 8h, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0043] Comparative Example 1

[0044] Fermentation medium: 85% H3PO4 6.675 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0045] The fermentation process for recombinant collagen includes the following steps:

[0046] Pichia pastoris JY0201 was inoculated at a rate of 9% into a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added, causing a rapid increase in the wet weight of the cells within a short period. The maximum wet weight was 154.63g / L. Further addition of 50% glycerol failed to achieve sustained cell density expansion.

[0047] Comparative Example 2

[0048] Fermentation medium: 85% H3PO4 9.0 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0049] The fermentation process for recombinant collagen includes the following steps:

[0050] Pichia pastoris JY0201 was inoculated at a rate of 9% into a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to rapidly increase the wet weight of the cells within a short period. The maximum wet weight was 206.41g / L.

[0051] Comparative Example 3

[0052] Fermentation medium: 85% H3PO4 26.7ml / L, CaSO4·2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4·7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L.

[0053] The fermentation process for recombinant collagen includes the following steps:

[0054] Pichia pastoris JY0201 was inoculated at a rate of 9% into a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol feeding. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added to induce expression. Fermentation was terminated after 112 hours of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8 hours, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0055] Comparative Example 4

[0056] Fermentation medium: 85% H3PO4 20.025ml / L, CaSO4·2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4·7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L.

[0057] The fermentation process for recombinant collagen includes the following steps:

[0058] Pichia pastoris JY0201 was inoculated at a rate of 9% into a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol feeding. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added to induce expression. Fermentation was terminated after 112 hours of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8 hours, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0059] Comparative Example 5

[0060] Fermentation medium: 85% H3PO4 13.884 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0061] The fermentation process for recombinant collagen includes the following steps:

[0062] Pichia pastoris JY0201 was inoculated at a 9% inoculum in a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added to induce expression. Simultaneously, 85% phosphate was added at a flow rate of 20.144ml / h until fermentation terminated after 112h of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8h, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0063] Comparative Example 6

[0064] Fermentation medium: 85% H3PO4 9.345 ml / L, CaSO4·2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4·7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L.

[0065] The fermentation process for recombinant collagen includes the following steps:

[0066] Pichia pastoris JY0201 was inoculated at a 9% inoculum in a 200L fermenter with a culture medium volume of 130L. The culture temperature was 28.9℃, the pH was adjusted to 5.0 with ammonia, dissolved oxygen was maintained at no less than 30%, the fermenter pressure was maintained at 0.075MPa, and the initial stirring speed was 200rpm. When the dissolved oxygen (DO) rapidly recovered to 65-75%, 50% glycerol was added to achieve a cell wet weight of 206-208g / L and glycerol depletion. After stopping the glycerol addition, the recombinant protein expression stage was initiated with methanol. The pH was adjusted to 6.2, the fermenter pressure was 0.10MPa, dissolved oxygen was maintained at no less than 10%, and methanol medium was added for induction expression. Simultaneously, 85% phosphate was added at a flow rate of 4.649ml / h until fermentation terminated after 112h of methanol induction. During the methanol induction stage, the fermentation broth was collected every 8h, centrifuged at 14000g, and the supernatant was collected for HPLC analysis of the recombinant collagen concentration.

[0067] Table 1. Product concentrations and fermentation levels at different time points for each example and comparative example.

[0068]

[0069] Comparing Comparative Example 1 and Comparative Example 2, it was found that when the initial concentration of 85% phosphate in the initial culture medium was too low (6.675 ml / L), the expected bacterial density could not be achieved during the glycerol feeding stage; however, when the initial concentration of 85% phosphate was increased to 9 ml / L, the bacterial density could reach the target wet weight of 206–208 g / L. These results indicate that phosphorus is closely related to the carbon source metabolism of engineered bacteria, and bacterial density expansion stagnates when phosphorus is relatively scarce.

[0070] Comparing Examples 1-3 and Comparative Example 5, it can be seen that the initial 85% phosphate concentration of 8.9-12.816 ml / L is more suitable. When the initial 85% phosphate concentration is too high, the expression of recombinant protein will be inhibited.

[0071] The total amount of phosphate added in Example 4 was the same as that in Comparative Example 3, and the total amount of phosphate added in Example 6 was the same as that in Comparative Example 4. However, Examples 7 and 9 used different addition methods compared to Comparative Examples 3 and 4: Examples 4 and 6 used a method of first reducing the phosphate concentration in the initial culture medium, and then quantitatively and constant-rately adding 85% phosphate during the methanol induction phase for 112 hours; Comparative Examples 3 and 4 used a method of adding all phosphate to the initial culture medium at once. Comparison revealed that using the fermentation process of Example 4, by adjusting the phosphate addition method, the fermentation time of recombinant collagen could be shortened by approximately 16 hours compared to Comparative Example 3, and the fermentation level at 80 hours could be increased by approximately 17.368%, confirming the advantages of the fed-batch process. Comparison of Examples 7 and 9 with Comparative Examples 3 and 4 shows that appropriate phosphate concentration, addition amount, and fed-batch method can shorten the fermentation cycle and improve carbon source utilization. Specifically, Example 9 showed a recombinant collagen fermentation level approximately 29.929% higher than Comparative Example 4 after 112 hours of methanol induction, with the fermentation level increasing by a maximum of 29.929%, and the fermentation time was correspondingly shortened by 16–32 hours.

[0072] Examples 4-6 and Comparative Example 6 all employed a fed-batch phosphoric acid process, with the same initial phosphoric acid concentration in the culture medium. The difference lay in the constant-rate addition of different concentrations of phosphoric acid during the methanol induction phase. Comparing Examples 4 and 5, it was found that increasing the feed rate of 85% phosphoric acid to 27.892 ml / h significantly inhibited the fermentation of recombinant collagen, with almost no yield increase after 80 hours of methanol induction. Comparing Examples 4-6 and Comparative Example 6, it was found that decreasing the feed rate of 85% phosphoric acid to 4.649 ml / h resulted in insufficient phosphorus supply during the methanol induction phase, leading to stagnant fermentation yield.

[0073] The above description represents some preferred embodiments of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for improving the fermentation level of recombinant collagen by adjusting the phosphate concentration and the method of addition, characterized in that, Includes the following steps: The Pichia pastoris strain expressing recombinant collagen was inoculated into a sterile fermentation medium with a low phosphate concentration. After basal culture for 12-16 hours, 50% glycerol was added until the target cell wet weight was reached. After the glycerol addition was completed, methanol was added to induce expression, while 85% phosphate solution was added at a rate of 12.396 ml / h to 27.892 ml / h. The composition of the low phosphate concentration fermentation medium was as follows: 85% H3PO4 8.9-12.816 ml / L, CaSO4·2H2O 0.392-1.175 g / L, K2SO4 6.11-18.2 g / L, MgSO4·7H2O 5.6-18.2 g / L, KOH 1.326-4.13 g / L, glycerol 13.5-40.0 g / L, and PTM1 solution 1.029-4.35 ml / L.

2. The method according to claim 1, characterized in that, The flow rate of 85% phosphoric acid is 12.396~20.144 ml / h.

3. The method according to claim 1, characterized in that, The Pichia pastoris strain expressing recombinant collagen was Pichia pastoris JY0201, with accession number CGMCC No.16461.

4. The method according to claim 1, characterized in that, The formula of the PTM1 solution is 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 0.02 g / L, CoCl 0.5 g / L, ZnCl2 20.0 g / L, FeSO4·7H2O 65.0 g / L, biotin 0.2 g / L, and H2SO4 5 ml / L.

5. The method according to claim 1, characterized in that, The inoculum size of Pichia pastoris expressing recombinant collagen is 8-10%, the fermentation temperature is 28-32℃, the pH is adjusted to 4.8-6.5 with ammonia, and the dissolved oxygen in the fermentation medium is not less than 30% during the basic culture stage.

6. The method according to claim 1, characterized in that, During the methanol induction phase, the dissolved oxygen in the fermentation medium should not be lower than 10%, and the induction time should be 80–120 h.

7. The method according to claim 1, characterized in that, The induction time for the methanol induction phase is 80~112h.

8. The method according to claim 1, characterized in that, The target cell wet weight for the 50% glycerol fed-batch stage is 190~220 g / L.

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