Recombinant pichia pastoris fermentation medium, preparation method thereof and application of recombinant pichia pastoris fermentation medium in production of recombinant A-type collagen

Abstract

The invention provides a recombinant pichia pastoris fermentation medium, a preparation method thereof and application of the recombinant pichia pastoris fermentation medium in production of recombinant A-type collagen, and relates to the technical field of biological fermentation. According to the recombinant pichia pastoris fermentation culture medium disclosed by the invention, by introducing a disodium hydrogen phosphate dihydrate-citric acid buffer system, it is accidentally found that the recombinant pichia pastoris fermentation culture medium can greatly reduce the dosage of ammonia water; by screening and optimizing a culture medium component formula and culture conditions, the fermentation expression level of the recombinant A-type collagen is obviously improved, the emission of ammonia nitrogen and total phosphorus in a fermentation system is obviously reduced, and the environmental pollution is reduced. And on the basis, the fermentation process is accurately controlled, so that high-efficiency expression of the recombinant A-type collagen is facilitated, and a foundation is laid for green and large-scale production of the recombinant A-type collagen.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a recombinant Pichia pastoris fermentation medium and its preparation method, and its application in the production of recombinant type A collagen. Background Technology

[0002] Collagen, as a major structural protein of the human extracellular matrix, plays a vital role in maintaining the structure and function of tissues such as skin, bone, and cartilage. Traditional animal-derived collagen has problems such as viral risks, immunogenicity risks, and batch-to-batch quality instability. With the development of genetic engineering and synthetic biology technologies, recombinant collagen has gradually become an alternative. It has advantages such as well-defined structure, no viral risks, high biocompatibility, and low immunogenicity, and has been widely used in medical repair, functional skin care, tissue engineering, and other fields.

[0003] Recombinant collagen expression systems mainly include bacteria, yeast, animal cells, and plant cells. Among them, Pichia pastoris has become an ideal host for large-scale production of recombinant collagen due to its advantages such as clear genetic background, simple operation, stable integration of exogenous genes, low culture cost, and no production of endotoxins or pathogenic factors.

[0004] Currently, high-density fermentation of Pichia pastoris commonly uses a culture medium composed of a basal salt medium (BSM) developed by Invitrogen and a PTM1 trace element solution. However, this culture medium system requires the use of large quantities of hazardous chemicals such as phosphate and potassium hydroxide, which not only pose a threat to the safety of operators but also corrode fermentation equipment. More importantly, the fermentation wastewater from this process has extremely high levels of ammonia nitrogen and total phosphorus. Direct discharge of such wastewater would cause serious pollution to aquatic environments, becoming one of the key bottlenecks restricting the greening and industrialization of this technology.

[0005] To address these issues, several attempts have been made in the existing technology field. For example, Chinese patent CN103923846A discloses a Pichia pastoris culture medium, and Chinese patent CN118853799A discloses a fermentation medium and fermentation method for the efficient production of type III collagen α1 chain mature peptides using recombinant Pichia pastoris. Although the technical solutions in these two patents replace strong acid and strong base hazardous chemicals such as potassium hydroxide and phosphoric acid in the culture medium, they do not solve the problem of residual ammonia nitrogen and total phosphorus in the process fermentation waste liquid. Another Chinese patent CN117535164A proposes to replace strong acids and strong bases with inorganic salts and potassium salts with sodium salts to reduce the irritation caused by potassium ion residues in the final product, but this still does not effectively control the emission levels of ammonia nitrogen and total phosphorus.

[0006] In summary, significantly reducing ammonia nitrogen and total phosphorus emissions during fermentation while ensuring efficient expression of recombinant collagen has become a pressing technical challenge in this field. Existing improvements are mostly limited to component replacement or localized adjustments, and a comprehensive solution that balances fermentation performance and environmental friendliness has not yet been developed. Therefore, developing a culture medium and supporting processes that can both support efficient Pichia pastoris fermentation and significantly reduce ammonia nitrogen and total phosphorus emissions has significant industrial value and environmental implications. Summary of the Invention

[0007] To address the environmental problems of high ammonia nitrogen and total phosphorus emissions and unfriendly practices in existing recombinant collagen fermentation processes, this invention provides a recombinant Pichia pastoris fermentation medium, its preparation method, and its application in the production of recombinant type A collagen. While ensuring efficient and stable production of recombinant type A collagen, it significantly reduces ammonia nitrogen and total phosphorus emissions in the fermentation system, achieving green and efficient production of the target protein.

[0008] To achieve the above technical objectives, firstly, this invention proposes a recombinant Pichia pastoris fermentation medium:

[0009] The culture medium, by total volume, comprises: 10.7–35.6 g / L disodium hydrogen phosphate dihydrate, 5.1–15.6 g / L citric acid, 3.96–15.84 g / L ammonium sulfate, 0.63–3.16 g / L calcium sulfate dihydrate, 10.4–22.6 g / L potassium sulfate, 3.3–17.1 g / L magnesium sulfate heptahydrate, 20–50 g / L glycerol, 0.3–1.2 g / L amino acids, 0.1–0.6 g / L vitamins, and 2.65–4.95 ml / L PTM1 solution; the pH of the culture medium is 4.6–6.9.

[0010] In existing technologies, the fermentation process of recombinant Pichia pastoris requires a large amount of ammonia water to provide the nitrogen source needed for cell growth and product synthesis, and to neutralize organic acids produced by metabolism and maintain a suitable pH environment. This directly leads to persistently high ammonia nitrogen content in the fermentation waste liquid. To address this issue, this invention achieves multiple synergistic and unexpected effects by systematically reconstructing the culture medium components. Specifically, the culture medium of this invention uses a buffer system composed of disodium hydrogen phosphate dihydrate and citric acid. Its initial purpose is to replace hazardous chemicals such as phosphate and potassium hydroxide in traditional culture media to stabilize the fermentation pH and improve operational safety. Surprisingly, the research team discovered during practice that this buffer system not only effectively maintains pH stability in the fermentation environment but also significantly reduces the amount of ammonia added during fermentation. The team hypothesizes that the disodium hydrogen phosphate dihydrate-citric acid buffer system in the culture medium optimizes the nitrogen source metabolism environment in the fermentation broth. This buffer system promotes more efficient utilization of ammonium sulfate as the basic nitrogen source, reducing acidic byproducts caused by incomplete nitrogen source metabolism, thereby reducing dependence on ammonia (used to neutralize metabolic acids) at the source. Ultimately, this significantly reduces the total input of ammonia nitrogen and its final discharge into the waste liquid. Furthermore, by controlling the concentration of added phosphate, the invention effectively controls the total phosphorus emission level while meeting the normal metabolic needs of the microorganisms. Simultaneously, by using sodium salt instead of potassium salt as the main component, the potential irritation caused by residual potassium ions in the final product is reduced, improving safety in use. In addition, the culture medium of the present invention is based on the synergistic design of the overall ratio, so that the components work together to construct a fermentation system that can support the efficient and stable expression of recombinant type A collagen, and can significantly reduce environmental emissions and improve product safety.

[0011] Examples of the invention demonstrate that it not only ensures the efficient and stable production of recombinant type A collagen, but also unexpectedly achieves a simultaneous improvement in environmental friendliness and product safety.

[0012] In a further example of the present invention, the amino acid includes at least two of glycine, proline, isoleucine, or arginine; optionally, when there are two amino acids, the amino acids are proline and arginine, or isoleucine and glycine; when there are three amino acids, the amino acids are glycine, proline, and arginine, or glycine, isoleucine, and arginine, or proline, isoleucine, and arginine, or glycine, proline, and isoleucine. And / or, the vitamin includes at least two of thiamine, calcium pantothenate, ascorbic acid, or pyridoxine hydrochloride; optionally, when there are two vitamins, the vitamins are ascorbic acid and pyridoxine hydrochloride, or thiamine and calcium pantothenate; when there are three vitamins, the vitamins are thiamine, calcium pantothenate, and pyridoxine hydrochloride.

[0013] By adding appropriate amounts and types of amino acids and vitamins, the expression level of recombinant type A collagen can be significantly improved, reducing production costs. When at least two amino acids or vitamins are selected, the proportions of each component can be formulated as needed, either identically or differently. For example, when glycine, proline, isoleucine, and arginine are added simultaneously, their respective concentrations can all be 200 mg / L. When thiamine, calcium pantothenate, ascorbic acid, and pyridoxine hydrochloride are added simultaneously, an optional ratio is: thiamine 50 mg / L, calcium pantothenate 100 mg / L, ascorbic acid 150 mg / L, and pyridoxine hydrochloride 200 mg / L.

[0014] In a further example of the invention, the culture medium, by total volume, comprises: 26.95–35.6 g / L disodium hydrogen phosphate dihydrate, 13.75–15.6 g / L citric acid, 11.9–15.84 g / L ammonium sulfate, 0.93–3.16 g / L calcium sulfate dihydrate, 15.7–22.6 g / L potassium sulfate, 14.9–17.1 g / L magnesium sulfate heptahydrate, 40–50 g / L glycerol, 0.8–1.2 g / L amino acids, 0.5–0.6 g / L vitamins, and 3.2–4.95 ml / L PTM1 solution; the pH of the culture medium is 5.0–6.0.

[0015] In a further example of the invention, the culture medium comprises, by total volume, 26.95 g / L disodium hydrogen phosphate dihydrate, 13.75 g / L citric acid, 11.9 g / L ammonium sulfate, 0.93 g / L calcium sulfate dihydrate, 15.7 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 40 g / L glycerol, 0.8 g / L amino acids, 0.5 g / L vitamins, and 3.2 ml / L PTM1 solution; the pH of the culture medium is 5.0.

[0016] Preferably, the 0.8 g / L amino acid is composed of 200 mg / L glycine, 200 mg / L proline, 200 mg / L isoleucine and 200 mg / L arginine, and the 0.5 g / L vitamin is composed of 50 mg / L thiamine, 100 mg / L calcium pantothenate, 150 mg / L ascorbic acid and 200 mg / L pyridoxine hydrochloride.

[0017] Secondly, the present invention also provides a method for preparing the above-mentioned fermentation culture medium, wherein a mixed system comprising disodium hydrogen phosphate dihydrate, citric acid, ammonium sulfate, calcium sulfate dihydrate, potassium sulfate, magnesium sulfate heptahydrate and glycerol is subjected to volume adjustment and high-temperature sterilization, and then cooled to 25-35°C and amino acids, vitamins and PTM1 solution are added to obtain the fermentation culture medium.

[0018] Traditional BSM culture uses a strong acid-base system of phosphate and potassium hydroxide. After sterilization, the pH deviates significantly from the optimal fermentation range, requiring large amounts of ammonia for drastic adjustment to meet inoculation requirements. However, this invention fundamentally reconstructs the ion balance and buffering capacity of the culture medium by introducing a disodium hydrogen phosphate dihydrate-citric acid buffer system and replacing the potassium salt as the primary component. After high-temperature sterilization, the pH of the culture medium stabilizes within the target range of 4.6–6.9, allowing for direct fermentation without the need for ammonia adjustment.

[0019] It should be noted that the high-temperature sterilization described in the preparation method preferably adopts a conventional moist heat sterilization process, with the sterilization temperature controlled at 121℃.

[0020] Thirdly, the present invention also provides a fermentation method for recombinant type A collagen Pichia pastoris engineered strain, comprising the following steps: (1) Preparation of culture medium: Prepare the fermentation culture medium described in the first aspect or prepare the fermentation culture medium, glycerol culture medium and methanol culture medium by the preparation method described in the second aspect respectively; (2) Seed culture preparation: Recombinant type A collagen Pichia pastoris engineered strains were transferred to YPD medium, activated, and cultured to obtain seed culture. The OD of the seed culture was... 600 It is 6~10; (3) Fermentation culture: The seed liquid described in step (2) is transferred to a fermenter containing 20L of fermentation medium at an inoculation rate of 5-10%. Fermentation culture is carried out under aeration and stirring conditions, and the dissolved oxygen content is controlled to be greater than 20%. When the dissolved oxygen content rises to greater than 80%, the glycerol medium is added. When the wet weight of the cells reaches 200-240g / L, the addition is stopped. When the dissolved oxygen content rises to greater than 90%, it is maintained for 10-30min. The pH is adjusted to 5.0-6.0. The dissolved oxygen content of the fermentation system is made to be greater than 20% by adjusting the flow rate of the methanol medium. After fermentation culture for 84-108h, recombinant type A collagen is obtained by solid-liquid separation.

[0021] The fermentation method of this invention uses dissolved oxygen content as a real-time monitoring indicator to precisely control the fed-batch strategy of glycerol and methanol media: During the growth phase, when the dissolved oxygen content rises above 80%, glycerol media is fed in, which avoids growth stagnation due to carbon source limitation and prevents excessive feeding from causing metabolic burden or a sudden drop in dissolved oxygen, thereby efficiently promoting the accumulation of cell biomass; During the induction phase, the methanol feeding rate is controlled by maintaining a dissolved oxygen content above 20%, allowing the cells to gradually adapt to methanol metabolism, avoiding sudden metabolic stress or toxicity accumulation, while ensuring metabolic activity and protein synthesis efficiency. This dissolved oxygen feedback regulation method significantly improves the expression level of recombinant type A collagen.

[0022] It should be noted that the dissolved oxygen content is determined using conventional methods in the art, such as online monitoring via a dissolved oxygen electrode.

[0023] The wet weight of the bacterial cells was determined using conventional methods in the art. For example, the mass M1 of the empty centrifuge tube was weighed using an electronic balance. After the fermentation broth to be tested was mixed evenly, 10 ml of the sample was taken into the centrifuge tube. The centrifuge tube was balanced and an equal weight of purified water was added. The two centrifuge tubes were placed symmetrically into the centrifuge and centrifuged at 10,000 rpm and 20°C for 5 min. After centrifugation, the supernatant was discarded, and the mass M2 of the centrifuge tube containing the wet bacterial cells was weighed. The wet weight of the bacterial cells was calculated according to the formula (M2-M1)g / 0.0lL.

[0024] In a further example of the present invention, the flow rate of the glycerol culture medium in step (3) is 3.2 to 10.5 ml / min; And / or, the flow rate of the methanol medium in step (3) is 1.1 to 5.5 ml / min.

[0025] In a further example of the present invention, the amino acid sequence of the recombinant type A collagen in step (2) is SEQ ID NO:1.

[0026] The engineered strain is a recombinant Pichia pastoris strain (named rhcol1A235 / X-33) that secretes and expresses humanized collagen. The target protein expressed by this strain is derived from the LPXTG-anchored collagen-like adhesin Scl2 / SclB fragment 499–759 aa of the human type I collagen α1 chain (NCBI reference sequence number: NP_000079.2). This collagen fragment is a surface adhesin anchored by LPXTG, containing a variable-length triple-helix region composed of collagen-like Gly-Xaa-Xaa repeat sequences, which may form a stable structure that is not easily degraded in vitro. This specific fragment is composed of human sequences and does not contain any non-human collagen amino acid sequences. The amino acid sequence is shown in SEQ ID NO:1: DGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAG ERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGPKGADGSPGK.

[0027] In a further example of the present invention, the stirring speed in step (3) is 300~1000 rpm; And / or, the ventilation is the introduction of air, and the flow rate of the introduced air is 0.5 to 2 vvm.

[0028] In a further example of the present invention, the initial temperature of the fermentation culture in step (3) is 28~30℃; And / or, after the glycerol culture medium is stopped from being fed, the temperature is lowered to 25-28°C.

[0029] In a further example of the invention, the glycerol culture medium contains 44-55% w / v glycerol and 5-15 ml / L PTM1 solution, based on the total volume of the glycerol culture medium. In a further example of the invention, the methanol culture medium contains 985-995 ml / L methanol and 5-15 ml / L PTM1 solution, based on the total volume of the methanol culture medium.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) The recombinant Pichia pastoris fermentation medium of the present invention, by introducing a disodium hydrogen phosphate dihydrate-citric acid buffer system, unexpectedly found that it can significantly reduce the amount of ammonia water used and significantly reduce ammonia nitrogen emissions. On this basis, by precisely controlling the phosphate concentration and replacing potassium salt with sodium salt, total phosphorus emission reduction and product safety improvement were simultaneously achieved. Ultimately, this overall synergistic design, while ensuring efficient protein expression, unexpectedly achieved a dual optimization of environmental friendliness and safety of use.

[0032] (2) The fermentation medium preparation method of the present invention introduces a disodium hydrogen phosphate dihydrate-citric acid buffer system and replaces potassium salt as the main component, fundamentally reconstructing the ion balance and buffering capacity of the medium. After high-temperature sterilization, the pH value of the medium is stabilized within the target range of 4.6 to 6.9, and it can be directly used for fermentation culture without the need for ammonia water adjustment.

[0033] (3) The fermentation method of Pichia pastoris engineered strain for recombinant type A collagen in this invention uses dissolved oxygen content as a real-time monitoring indicator and precisely controls the feeding strategy of glycerol culture medium and methanol culture medium to achieve efficient expression of recombinant type A collagen. Attached Figure Description

[0034] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0035] Figure 1This shows a single colony of recombinant Pichia pastoris engineered strain during the construction of recombinant type A collagen;

[0036] Figure 2 The SDS-PAGE spectra of the recombinant type A collagen Pichia pastoris engineered strain rhcol1A235 / X-33 after shake-flask screening are shown. Lane 1 is the supernatant of shake-flask fermentation of strain #1, lane 2 is the supernatant of shake-flask fermentation of strain #2, lane 3 is the supernatant of shake-flask fermentation of strain #3, and lane M is Broad Multi Color Pre-Stained Protein Standard (Nanjing Genscript Biotech Co., Ltd.).

[0037] Figure 3 The SDS-PAGE spectra of samples collected during the fermentation process of fermentation medium 1 in Application Example 1 are shown. Lane 1 is the supernatant of fermentation induced by methanol for 24 hours, lane 2 is the supernatant of fermentation induced by methanol for 48 hours, lane 3 is the supernatant of fermentation induced by methanol for 72 hours, and lane M is Broad Multi Color Pre-Stained Protein Standard (Nanjing Genscript Biotech Co., Ltd.).

[0038] Figure 4 The SDS-PAGE spectra of samples collected during the fermentation process of fermentation medium 3 in Application Example 3 are shown. Lane 1 is the supernatant of fermentation induced by methanol for 24 hours, lane 2 is the supernatant of fermentation induced by methanol for 48 hours, lane 3 is the supernatant of fermentation induced by methanol for 72 hours, and lane M is Broad Multi Color Pre-Stained Protein Standard (Nanjing Genscript Biotech Co., Ltd.).

[0039] Figure 5 The SDS-PAGE spectra of samples collected from fermentation medium 1 in Application Example 6 after optimizing the fermentation process are shown. Lane 1 is the supernatant of methanol-induced fermentation for 24 hours, lane 2 is the supernatant of methanol-induced fermentation for 48 hours, lane 3 is the supernatant of methanol-induced fermentation for 72 hours, and lane M is Broad Multi Color Pre-Stained Protein Standard (Nanjing Genscript Biotech Co., Ltd.). Detailed Implementation

[0040] To facilitate understanding of the present invention, a more comprehensive description will be provided below, along with preferred embodiments. However, it should be understood that these embodiments are merely for more detailed explanation and should not be construed as limiting the invention in any way, i.e., not intended to limit the scope of protection of the invention.

[0041] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0042] It should be noted that the PTM1 solution in the embodiments of the present invention is a PTM1 trace element solution, which, by total volume, includes 6 g / L copper sulfate pentahydrate, 0.08 g / L sodium iodide, 3 g / L magnesium sulfate monohydrate, 0.2 g / L sodium molybdate dihydrate, 0.02 g / L boric acid, 0.5 g / L cobalt chloride, 20 g / L zinc chloride, 65 g / L ferrous sulfate heptahydrate, 0.2 g / L biotin, and 5 ml / L sulfuric acid.

[0043] It should be noted that the preparation method of the glycerol culture medium in the embodiments of the present invention is as follows: 50% (w / v) glycerol solution, sterilized at 121°C for 20 minutes, and after cooling to room temperature, 12 ml of PTM1 solution is added per liter to obtain the glycerol culture medium.

[0044] It should be noted that the preparation method of the methanol culture medium in the embodiments of the present invention is as follows: 100% anhydrous methanol is used directly as raw material, 12 mL of PTM1 solution is added per liter, and after thorough mixing, the medium is filtered through a 0.22 μm filter membrane to sterilize and then autoclaved in a feed bottle to obtain the methanol culture medium.

[0045] It should be noted that the method for constructing the recombinant type A collagen Pichia pastoris engineered strain in this embodiment of the invention is as follows:

[0046] (1) Gene synthesis and construction of recombinant plasmids Based on the human type I collagen α1 chain (NP_000079.2) sequence from the NCBI database, a fragment containing a variable-length triple helix region of 499–759 amino acids composed of collagen-like Gly-Xaa-Xaa repeat sequences was selected. The amino acid sequence is shown in SEQ ID NO:1. DGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAG ERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPPGKGDRGDAGPKGADGSPGK.

[0047] Without altering the original amino acid sequence, the gene was synthesized by codon optimization based on the Pichia pastoris preference, and the recombinant plasmid pPICZαA-rhcol1A235 was constructed. The gene sequence with optimized codon synthesis is shown in SEQ ID NO:2: GATGGCGTTGCCGGACCAAAAGGACCTGCCGGAGAGAGAGGAAGTCCTGGTCCAGCCGGCCCAAAGGGTTCTCCTGGAGAAGCAGGTAGACCAGGTGAAGCTGGTCTTCCCGGTGCTAAGGGTCTTACCGGTTCTCCCGGTAGTCCAGGCCCTGATGGTAAAACTGGACCCCCAGGTCCCGCTGGTCAAGACGGTCGACCAGGTCCACCCGGACCTCCTGGTGCCCGAGGTCAGGCCGGCGTGATGGGTTTTCCCGGTCCAAAGGGAGCTGCCGGTGAGCCTGGTAAAGCTGGAGAGCGTGGTGTTCCTGGACCCCCCGGTGCTGTGGGACCAGCCGGAAAGGATGGAGAGGCTGGTGCTCAAGGTCCACCTGGTCCAGCCGGACCAGCTGGTGAAAGAGGTGAGCAAGGTCCTGCTGGATCTCCTGGTTTCCAAGGTTTGCCTGGTCCAGCAGGTCCCCCTGGTGAAGCTGGTAAACCAGGTGAGCAAGGAGTTCCAGGAGACTTAGGAGCTCCAGGTCCAAGTGGAGCCCGTGGAGAACGTGGTTTCCCAGGTGAGAGAGGTGTTCAGGGTCCTCCAGGACCAGCTGGTCCAAGAGGAGCTAATGGAGCACCAGGCAACGATGGCGCAAAGGGTGATGCTGGAGCCCCTGGAGCTCCAGGTTCTCAGGGTGCTCCAGGATTGCAAGGAATGCCTGGCGAACGAGGAGCCGCAGGTTTACCTGGACCAAAGGGAGATCGTGGTGATGCCGGACCAAAGGGTGCAGATGGATCACCAGGTAAG。

[0048] (2) Construction of recombinant Pichia pastoris engineering bacteria expressing type A collagen The recombinant plasmid pPICZαA-rhcol1A235 was linearized with the restriction enzyme PmeI. 1-10 μg of the linearized plasmid was added to 80 μL of Pichia pastoris X-33 competent cells, mixed thoroughly, and transferred to a 2 mm electroporation cuvette. The cuvette was incubated on ice for 5 min at 1.5 kV, 200 Ω, and 25 μF. Immediately after electroporation, 1 mL of pre-chilled 1 M sorbitol solution was added to the cuvette and mixed well. The mixture was then transferred to a sterile centrifuge tube and incubated at 30°C for 1 h. 200 μL of the bacterial culture was then plated on a Zeocin-resistant (100 μg / ml) YPDS plate and incubated at 30°C for 2-5 days until single colonies appeared. Figure 1 As shown, three morphologically typical single colonies were randomly selected and numbered 1#, 2# and 3# respectively.

[0049] (3) Induction of expression and screening Single colonies #1, #2, and #3 were inoculated into 25 ml of BMGY medium and cultured at 30°C and 200 rpm until OD500. 600 The bacterial cells were collected by centrifugation at 2000 rpm for 5 min at room temperature, and resuspended in 200 ml of fresh culture medium in a 1 L shake flask to allow the OD to reach 2-6. 600 The protein concentration was approximately 1.0. The culture was continued at 30℃ and 200 rpm, with 1% methanol added every 24 hours for induction, for a total of 72 hours. After induction, the supernatant was collected by centrifugation, and the expression of the recombinant protein was detected by SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown, all tested strains secreted a target band of about 30 kDa and weak degradation bands of about 16 kDa and 6.5 kDa. The strain with the highest expression level, #2, was named rhcol1A235 / X-33, which is the recombinant type A collagen Pichia pastoris engineered strain.

[0050] Example 1

[0051] A recombinant Pichia pastoris fermentation medium, comprising, by total volume: 26.95 g / L disodium hydrogen phosphate dihydrate, 13.75 g / L citric acid, 11.9 g / L ammonium sulfate, 0.93 g / L calcium sulfate dihydrate, 15.7 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 40 g / L glycerol, 0.8 g / L amino acids, 0.5 g / L vitamins, and 3.2 ml / L LPTM1 solution, wherein the amino acids are 200 mg / L each of glycine, proline, isoleucine, and arginine, and the vitamins are 50 mg / L thiamine, 100 mg / L calcium pantothenate, 150 mg / L ascorbic acid, and 200 mg / L pyridoxine hydrochloride.

[0052] The fermentation medium was prepared as follows: First, disodium hydrogen phosphate dihydrate (26.95 g / L), citric acid (13.75 g / L), ammonium sulfate (11.9 g / L), calcium sulfate dihydrate (0.93 g / L), potassium sulfate (15.7 g / L), magnesium sulfate heptahydrate (14.9 g / L), and glycerol (40 g / L) were dissolved in an appropriate amount of water. After mixing thoroughly, the mixture was brought to a total volume of 20 L to obtain the basal culture medium solution. Subsequently, the culture medium solution was sterilized at 121℃ for 20 minutes to complete the aseptic treatment. After the culture medium temperature dropped to 30℃, PTM1 solution, the predetermined amino acid components, and vitamin components were added sequentially. After mixing thoroughly, the pH was adjusted to 5.0, thus obtaining fermentation medium 1.

[0053] Example 2

[0054] A recombinant Pichia pastoris fermentation medium, comprising, by total volume: 35.6 g / L disodium hydrogen phosphate dihydrate, 15.6 g / L citric acid, 15.84 g / L ammonium sulfate, 3.16 g / L calcium sulfate dihydrate, 22.6 g / L potassium sulfate, 17.1 g / L magnesium sulfate heptahydrate, 50 g / L glycerol, 1.2 g / L amino acids, 0.6 g / L vitamins, and 4.95 ml / L PTM1 solution, wherein the amino acids are 300 mg / L each of glycine, proline, isoleucine, and arginine, and the vitamins are 150 mg / L thiamine, 100 mg / L calcium pantothenate, 150 mg / L ascorbic acid, and 200 mg / L pyridoxine hydrochloride.

[0055] The fermentation medium was prepared as follows: First, disodium hydrogen phosphate dihydrate (35.6 g / L), citric acid (15.6 g / L), ammonium sulfate (15.84 g / L), calcium sulfate dihydrate (3.16 g / L), potassium sulfate (22.6 g / L), magnesium sulfate heptahydrate (17.1 g / L), and glycerol (50 g / L) were dissolved in an appropriate amount of water. After mixing thoroughly, the volume was adjusted to 20 L to obtain the basal culture medium solution. Then, the culture medium solution was sterilized at 121℃ for 20 minutes to complete the aseptic treatment. After the culture medium temperature dropped to 30℃, PTM1 solution, the predetermined amino acid components, and vitamin components were added sequentially. After mixing thoroughly, the pH was 5.0, thus obtaining fermentation medium 2.

[0056] Comparative Example 1

[0057] A BSM fermentation medium, based on the total volume of the medium, comprises: 26.7 ml / L 85% phosphate, 0.93 g / L calcium sulfate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate, 4.13 g / L potassium hydroxide, 40 g / L glycerol, and 4 ml / L PTM1 solution.

[0058] The preparation method of the above BSM fermentation medium is as follows: First, dissolve 85% phosphate (26.7 ml / L), calcium sulfate (0.93 g / L), potassium sulfate (18.2 g / L), magnesium sulfate heptahydrate (14.9 g / L), potassium hydroxide (4.13 g / L), and glycerol (40 g / L) in an appropriate amount of water, mix thoroughly, and bring the volume to 20 L. Sterilize at 121℃ for 20 minutes. After the medium cools to 30℃, add 80 ml of PTM1 solution. Finally, before inoculation, adjust the pH of the medium to 5.0 using 25% ammonia water to obtain fermentation medium 3.

[0059] Comparative Example 2

[0060] A fermentation medium, by total volume, comprises 26.95 g / L disodium hydrogen phosphate dihydrate, 17.2 g / L sodium dihydrogen phosphate, 11.9 g / L ammonium sulfate, 0.93 g / L calcium sulfate dihydrate, 15.7 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 40 g / L glycerol, 0.8 g / L amino acids, 0.5 g / L vitamins, and 3.2 ml / L PTM1 solution, wherein the amino acids are 200 mg / L each of glycine, proline, isoleucine, and arginine, and the vitamins are 50 mg / L thiamine, 100 mg / L calcium pantothenate, 150 mg / L ascorbic acid, and 200 mg / L pyridoxine hydrochloride.

[0061] The preparation method of the above fermentation medium is as follows: First, disodium hydrogen phosphate dihydrate (26.95 g / L), sodium dihydrogen phosphate (17.2 g / L), ammonium sulfate (11.9 g / L), calcium sulfate dihydrate (0.93 g / L), potassium sulfate (15.7 g / L), magnesium sulfate heptahydrate (14.9 g / L), and glycerol (40 g / L) are dissolved in an appropriate amount of water, mixed thoroughly, and then brought to a total volume of 20 L to obtain the basal culture medium solution. Subsequently, the culture medium solution is sterilized at 121℃ for 20 minutes to complete the aseptic treatment. After the culture medium temperature drops to 30℃, PTM1 solution, the predetermined amino acid components, and vitamin components are added sequentially, mixed thoroughly, and the pH is adjusted to 5.0 to obtain fermentation medium 4.

[0062] Comparative Example 3

[0063] A fermentation medium, by total volume, comprises: 27.6 g / L disodium hydrogen phosphate dihydrate, 23.7 g / L citric acid, 11.9 g / L ammonium sulfate, 0.93 g / L calcium sulfate dihydrate, 15.7 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 40 g / L glycerol, 0.8 g / L amino acids, 0.5 g / L vitamins, and 3.2 ml / L PTM1 solution, wherein the amino acids are 200 mg / L each of glycine, proline, isoleucine, and arginine, and the vitamins are 50 mg / L thiamine, 100 mg / L calcium pantothenate, 150 mg / L ascorbic acid, and 200 mg / L pyridoxine hydrochloride.

[0064] The preparation method of the above fermentation medium is as follows: First, disodium hydrogen phosphate dihydrate (27.6 g / L), citric acid (23.7 g / L), ammonium sulfate (11.9 g / L), calcium sulfate dihydrate (0.93 g / L), potassium sulfate (15.7 g / L), magnesium sulfate heptahydrate (14.9 g / L), and glycerol (40 g / L) are dissolved in an appropriate amount of water, mixed evenly, and then brought to a total volume of 20 L to obtain the basic culture medium solution. Subsequently, the culture medium solution is sterilized at 121℃ for 20 minutes to complete the aseptic treatment. After the culture medium temperature drops to 30℃, PTM1 solution, the predetermined amino acid components, and vitamin components are added sequentially, stirred evenly until the pH is 4.0, and then the pH of the culture medium is adjusted to 5.0 using 25% ammonia water to obtain fermentation medium 5.

[0065] Application Example 1

[0066] A fermentation method for a recombinant type A collagen-producing Pichia pastoris engineered strain, comprising the following steps: (1) Preparation of culture medium: Fermentation medium 1, glycerol medium and methanol medium in Example 1 were prepared for use. (2) Seed culture preparation: The recombinant type A collagen Pichia pastoris engineered strain was transferred to a 50ml YPD medium Erlenmeyer flask for activation and cultured at 30℃ and 200rpm for 20h; then, at an inoculum rate of 2%, it was transferred to 2000ml of fresh YPD medium and cultured for another 20h under the same conditions to obtain OD. 600 The seed solution was 7.0. (3) Fermentation culture: The seed liquid in step (2) was transferred to a fermenter containing 20L of fermentation medium from Example 1 at an inoculation rate of 8%. The initial fermentation conditions were set as follows: temperature 30℃, pH 5.0, stirring speed 300rpm, and air flow rate 1.2vvm. The dissolved oxygen content was controlled to be greater than 20%. After 18h of culture, the glycerol in fermentation medium 1 was depleted and the dissolved oxygen rapidly rose to more than 80%. At this time, glycerol medium was added at a rate of 6.7ml / min until the wet weight of the cells reached 220g / L of fermentation liquid and then the addition of glycerol was stopped.

[0067] After stopping the glycerol feeding, the fermentation conditions were adjusted to pH 5.5 and temperature 26℃, followed by a 1-hour starvation period before methanol induction. A gradient methanol feeding strategy was employed: a rate of 1.3 mL / min was used for the first 2 hours of induction, increasing to 2.6 mL / min from hours 2 to 4, and further increasing to 3.9 mL / min after 4 hours and maintaining this rate until the end of fermentation. Throughout the fermentation process, pH was adjusted by adding ammonia, maintaining a pH of 5.0 during the cell growth phase, and setting the pH to 5.5 after methanol induction. Simultaneously, the dissolved oxygen level was ensured to remain above 20% throughout the entire process by dynamically adjusting the stirring speed (300–900 rpm), the air flow rate (0.5–2 vvm), and the flow rates of glycerol and methanol.

[0068] Fermentation was terminated 72 hours after induction, and the expression during fermentation was detected by SDS-PAGE electrophoresis. Figure 3 As shown. 910 ml of ammonia was used in this batch of fermentation. The collagen expression level at the end of fermentation, determined by the Coomassie Brilliant Blue method, was 10.16 g / L.

[0069] Application Examples 2-5

[0070] Fermentation media 2-5 prepared based on Example 2 and Comparative Examples 1-3 were used to produce recombinant type A collagen. The fermentation method was the same as in Application Example 1. The collagen expression level and ammonia amount in fermentation media 1-5 are shown in Table 1.

[0071] Table 1. Collagen expression levels and ammonia dosage in different fermentation media

[0072] As shown in Table 1, when using the fermentation media (fermentation medium 1 and fermentation medium 2) provided in Examples 1 and 2 of this invention for the fermentation production of recombinant type A collagen, the collagen expression level reached 9.33~10.16 g / L, and the ammonia water volume was 910~920 mL, which was significantly better than the comparative examples. This result indicates that the buffer system composed of disodium hydrogen phosphate dihydrate and citric acid in the fermentation media of this invention can effectively maintain pH stability during fermentation, reduce drastic pH fluctuations caused by the accumulation of cell metabolites, and thus provide a more suitable environment for cell growth and product synthesis; at the same time, the added citric acid can participate in cell energy metabolism as a metabolic intermediate, further improving the expression efficiency of the target protein.

[0073] In contrast, although the conventional BSM medium (fermentation medium 3) used in Application Example 3 is a commonly used formulation in the field, its collagen expression level was only 4.67 g / L, while the amount of ammonia water used was as high as 1670 mL, showing obvious pH instability and low metabolic efficiency. Furthermore, during the fermentation process, after adding methanol, 1 ml samples were taken every 24 hours to prepare protein samples. SDS-PAGE electrophoresis was used to detect the expression during the fermentation process. Figure 4 As shown, with the extension of methanol induction time, the expression of the target protein gradually increased, but the number of non-specific bands also increased. In Application Example 4, the fermentation process used disodium hydrogen phosphate and sodium dihydrogen phosphate as a buffer system (fermentation medium 4), and the collagen expression level was only 8.27 g / L, with an ammonia volume of 950 ml. Furthermore, the residual phosphorus content in the fermentation broth was high, which was detrimental to subsequent separation, purification, and process economy. In Application Example 5, although a combination of disodium hydrogen phosphate dihydrate and citric acid was also used (fermentation medium 5), the excessive addition of citric acid led to an imbalance in the buffer system, resulting in a decrease in collagen expression to 5.05 g / L, and an increase in ammonia volume to 1350 mL. The above comparative results indicate that the fermentation medium provided by this invention can effectively improve the expression level of recombinant type A collagen while significantly reducing the amount of ammonia used in the fermentation process, combining environmental friendliness and high production efficiency.

[0074] Application Example 6

[0075] Based on the fermentation method of Application Example 1, this application example replaces the fed-batch scheme of glycerol and methanol media, while other conditions remain the same as in Application Example 1. The specific steps are as follows: (1) Preparation of culture medium: Fermentation medium 1, glycerol medium and methanol medium in Example 1 were prepared for use. (2) Seed culture preparation: The preserved recombinant type A collagen Pichia pastoris engineered strain was transferred to a 50ml YPD medium Erlenmeyer flask for activation and cultured at 30℃ and 200rpm for 20h; then, at an inoculum rate of 2%, it was transferred to 2000ml of fresh YPD medium and cultured for another 20h under the same conditions to obtain OD. 600 The seed solution was 7.0. (3) Fermentation culture: The seed culture from step (2) was transferred to a fermenter containing 20L of fermentation medium from Example 1 at an inoculation rate of 8%. The initial fermentation conditions were set as follows: temperature 30℃, pH 5.0, stirring speed 300rpm. Fermentation culture was carried out under the conditions of stirring speed 300rpm and air flow rate of 1.2vvm, and the dissolved oxygen content was controlled to be greater than 20%. After 18h of culture, the glycerol in fermentation medium 1 was depleted, and the dissolved oxygen rapidly rose to over 80%. At this time, the timer was set to 0, and glycerol medium was added continuously until the wet weight of the cells reached 220g / L of fermentation broth. The rate of glycerol medium addition is shown in Table 2.

[0076] Table 2. Glycerol culture medium replenishment rate

[0077] After stopping the glycerol feeding, the fermentation conditions were adjusted to pH 5.5 and temperature 26℃, followed by a 1-hour starvation period before methanol induction. A gradient addition strategy was employed: 1.1 mL / min was added for the first 2 hours of induction, increasing to 1.4 mL / min from 2 to 4 hours, further increasing to 1.9 mL / min from 4 to 6 hours, 2.4 mL / min from 6 to 8 hours, 3.0 mL / min from 8 to 10 hours, 3.7 mL / min from 10 to 12 hours, and finally reaching 4.6 mL / min after 12 hours and maintaining this rate until the end of fermentation. The methanol feeding rates are shown in Table 3. Throughout the fermentation process, pH was adjusted by adding ammonia, maintaining a pH of 5.0 during the cell growth phase, and setting the pH to 5.5 after methanol induction. Simultaneously, the dissolved oxygen level was ensured to remain above 20% throughout the entire process by dynamically adjusting the stirring speed, air flow rate, and the flow rate of glycerol and methanol.

[0078] Table 3. Methanol culture medium replenishment rate table

[0079] Fermentation was terminated after 72 hours of induction in methanol medium. SDS-PAGE electrophoresis was used to detect the expression of the enzymes during fermentation. Figure 5 As shown. 910 ml of ammonia was used in this batch of fermentation. The collagen expression level at the end of fermentation, determined by the Coomassie Brilliant Blue method, was 13.45 g / L.

[0080] Application Examples 1 and 6 together demonstrate that the fermentation medium 1 prepared in Example 1 of this invention can achieve high levels of recombinant type A collagen expression under both conventional fed-batch and finely controlled variable-rate fed-batch strategies, with ammonia usage effectively controlled at low levels. Application Example 6 further refined the feeding strategies for glycerol and methanol. The glycerol replenishment rate was increased stepwise from 3.9 mL / min to 8.9 mL / min over 0-7 h, with a final pullback (see Table 2). During the methanol induction phase, the feeding rate of the methanol medium was adjusted according to dissolved oxygen content, achieving a closer dynamic match between carbon source supply and cell metabolic needs, resulting in a greater increase in product expression. This fully demonstrates the good adaptability and robust performance of the fermentation medium of this invention to upstream process optimization.

[0081] Test Example 1: Determination of Ammonia Nitrogen Concentration by Nessler's Reagent Spectrophotometric Method

[0082] To assess the residual ammonia nitrogen during fermentation, samples of the supernatant from Application Examples 1, 3, and 6 were taken after fermentation. The ammonia nitrogen concentration was determined using Nessler's reagent spectrophotometry. The specific steps are as follows: Accurately transfer 1.0 mL of fermentation supernatant into a 10 mL colorimetric tube, and dilute to the mark with deionized water. Add 1.0 mL of potassium sodium tartrate solution, mix well, then add 1.5 mL of Nessler's reagent and mix thoroughly again. After standing for 10 minutes for color development, measure the absorbance of the solution at a wavelength of 420 nm using a 20 mm path length cuvette and deionized water as a reference. Based on the pre-plotted ammonia nitrogen standard curve, convert the measured absorbance values ​​into the corresponding ammonia nitrogen mass concentration, and finally calculate the ammonia nitrogen concentration of each group of fermentation broth at the time of discharge. The results are shown in Table 4.

[0083] Table 4. Ammonia nitrogen concentrations during fermentation under different conditions

[0084] Table 4 shows that the fermentation medium of the present invention can significantly reduce the residual ammonia nitrogen concentration in the system at the end of fermentation. Specifically, in Application Example 3, which used conventional BSM medium, the ammonia nitrogen concentration at the time of fermentation was 173.6 mg / L. In Application Examples 1 and 6, which used the medium of the present invention, the ammonia nitrogen concentrations decreased to 76.1 mg / L and 73.9 mg / L, respectively, representing reductions of 56.2% and 57.4%. Notably, Application Example 6, by precisely controlling the feed rates of glycerol and methanol, achieved a lower ammonia nitrogen concentration at fermentation compared to Application Example 1, while maintaining the same ammonia consumption during fermentation. This was due to the higher expression level of the target protein, which consumed more nitrogen.

[0085] This result directly verifies one of the core effects of the technical solution of this invention. The significant reduction in ammonia nitrogen concentration is mainly due to the introduction of the disodium hydrogen phosphate dihydrate-citric acid buffer system in the culture medium of this invention and its unexpected technical effects. This system significantly enhances the pH buffering capacity of the fermentation broth, thereby greatly reducing the amount of ammonia water added solely for neutralizing metabolic acids, thus reducing the input of ammonia nitrogen at the source. At the same time, the optimized ammonium sulfate in the culture medium, as a basic nitrogen source, effectively replaces part of the function of ammonia water, further synergistically reducing the total ammonia nitrogen load.

[0086] Test Example 2: Determination of Total Phosphorus Concentration by Ammonium Molybdate Spectrophotometry

[0087] To assess the total phosphorus residue level in the culture system after fermentation, the total phosphorus concentration was determined from the fermentation supernatant samples corresponding to Application Example 1, Application Example 3, and Application Example 6. The specific steps are as follows: Accurately measure 25.00 mL of fermentation supernatant sample and place it in a 50 mL colorimetric tube with a PTFE stopcock. Add 4 mL of potassium persulfate solution, tighten the cap, and place the tube in an autoclave at 120°C for 30 minutes for digestion. After digestion, allow the colorimetric tube to cool to room temperature and dilute to the 50 mL mark with deionized water. Then, add 1 mL of ascorbic acid solution to the digestion solution, mix well, let stand for 30 seconds, and then add 2 mL of molybdate solution. Mix thoroughly and develop color for 15 minutes. Using deionized water as a reference, measure the absorbance of the solution at a wavelength of 700 nm. Based on the pre-established phosphorus content standard curve, convert the absorbance values ​​to the corresponding phosphorus mass concentration, and finally calculate the total phosphorus concentration in each group of fermentation broth. The results are recorded in Table 5.

[0088] Table 5 Total phosphorus concentration during fermentation under different conditions

[0089] Table 5 shows that the fermentation medium described in this invention has a significant effect on reducing total phosphorus emissions from the fermentation system. Specifically, in Application Example 3, which used conventional BSM medium, the total phosphorus concentration at the end of fermentation was as high as 807.5 mg / L. In contrast, in Application Examples 1 and 6, which used the medium of this invention, the total phosphorus concentration was significantly reduced to 199.5 mg / L and 196.3 mg / L, respectively, representing reductions of 75.3% and 75.7%.

[0090] This result is entirely consistent with the aforementioned optimization goals for the culture medium design. The significant reduction in total phosphorus concentration is directly attributed to the precise optimization of phosphate concentration in the culture medium by this invention. By controlling the phosphorus supply within the minimum effective concentration range required for cell growth and product synthesis, excessive phosphorus addition is avoided at the source, thereby achieving a substantial reduction in total phosphorus emissions at the end.

[0091] In summary, this invention not only improves operational safety and product compatibility by changing the type of phosphorus source (such as using disodium hydrogen phosphate dihydrate), but more importantly, through systematic formulation optimization, it achieves a significant reduction in total phosphorus emissions while ensuring fermentation efficiency, demonstrating the important value of this technology in promoting the greening of recombinant protein production.

[0092] It should be noted that the above description is a further detailed explanation of the present invention in conjunction with specific embodiments, and it should not be considered that the specific implementation of the present invention is limited to these descriptions; for those skilled in the art, several simple improvements and modifications can be made without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

Claims

1. A recombinant Pichia pastoris fermentation medium, characterized in that, The culture medium, by total volume, comprises: 10.7–35.6 g / L disodium hydrogen phosphate dihydrate, 5.1–15.6 g / L citric acid, 3.96–15.84 g / L ammonium sulfate, 0.63–3.16 g / L calcium sulfate dihydrate, 10.4–22.6 g / L potassium sulfate, 3.3–17.1 g / L magnesium sulfate heptahydrate, 20–50 g / L glycerol, 0.3–1.2 g / L amino acids, 0.1–0.6 g / L vitamins, and 2.65–4.95 ml / L PTM1 solution; the pH of the culture medium is 4.6–6.

9.

2. The recombinant Pichia pastoris fermentation medium according to claim 1, characterized in that, The amino acid includes at least two of glycine, proline, isoleucine, or arginine. And / or, the vitamins include at least two of thiamine, calcium pantothenate, ascorbic acid, or pyridoxine hydrochloride.

3. The recombinant Pichia pastoris fermentation medium according to claim 1, characterized in that, The culture medium, by total volume, comprises: 26.95–35.6 g / L disodium hydrogen phosphate dihydrate, 13.75–15.6 g / L citric acid, 11.9–15.84 g / L ammonium sulfate, 0.93–3.16 g / L calcium sulfate dihydrate, 15.7–22.6 g / L potassium sulfate, 14.9–17.1 g / L magnesium sulfate heptahydrate, 40–50 g / L glycerol, 0.8–1.2 g / L amino acids, 0.5–0.6 g / L vitamins, and 3.2–4.95 ml / L PTM1 solution; the pH of the culture medium is 5.0–6.

0.

4. The recombinant Pichia pastoris fermentation medium according to claim 3, characterized in that, The culture medium, by total volume, comprises: 26.95 g / L disodium hydrogen phosphate dihydrate, 13.75 g / L citric acid, 11.9 g / L ammonium sulfate, 0.93 g / L calcium sulfate dihydrate, 15.7 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 40 g / L glycerol, 0.8 g / L amino acids, 0.5 g / L vitamins, and 3.2 ml / L PTM1 solution; the pH of the culture medium is 5.

0.

5. A method for preparing a fermentation medium as described in any one of claims 1-4, characterized in that, A mixture containing disodium hydrogen phosphate dihydrate, citric acid, ammonium sulfate, calcium sulfate dihydrate, potassium sulfate, magnesium sulfate heptahydrate, and glycerol was brought to a constant volume and sterilized at high temperature. Then, the mixture was cooled to 25-35°C and amino acids, vitamins, and PTM1 solution were added to obtain the fermentation medium.

6. A fermentation method for a recombinant type A collagen-producing Pichia pastoris engineered strain, characterized in that, Includes the following steps: (1) Culture medium preparation: Prepare the recombinant Pichia pastoris fermentation culture medium according to any one of claims 1-4, or prepare the fermentation culture medium, glycerol culture medium and methanol culture medium by the preparation method according to claim 5; (2) Seed culture preparation: Recombinant type A collagen Pichia pastoris engineered strains were transferred to YPD medium, activated, and cultured to obtain seed culture. The OD of the seed culture was... 600 It is 6~10; (3) Fermentation culture: The seed liquid described in step (2) is transferred to a fermenter containing 20L of fermentation medium at an inoculation rate of 5-10%. Fermentation culture is carried out under aeration and stirring conditions, and the dissolved oxygen content is controlled to be greater than 20%. When the dissolved oxygen content rises to greater than 80%, the glycerol medium is added. When the wet weight of the cells reaches 200-240g / L, the addition is stopped. When the dissolved oxygen content rises to greater than 90%, it is maintained for 10-30min. The pH is adjusted to 5.0-6.

0. The dissolved oxygen content of the fermentation system is made to be greater than 20% by adjusting the flow rate of the methanol medium. After fermentation culture for 84-108h, recombinant type A collagen is obtained by solid-liquid separation.

7. The fermentation method of the recombinant type A collagen Pichia pastoris engineered strain according to claim 6, characterized in that, The flow rate of the glycerol culture medium in step (3) is 3.2~10.5 ml / min; And / or, the flow rate of the methanol medium in step (3) is 1.1~5.5 ml / min.

8. The fermentation method of the recombinant type A collagen Pichia pastoris engineered strain according to claim 6, characterized in that, The amino acid sequence of the recombinant type A collagen in step (2) is SEQ ID NO:

1.

9. The fermentation method of the recombinant type A collagen Pichia pastoris engineered strain according to claim 6, characterized in that, The stirring speed in step (3) is 300~1000 rpm; And / or, the ventilation is the introduction of air, and the flow rate of the introduced air is 0.5 to 2 vvm.

10. The fermentation method of the recombinant type A collagen Pichia pastoris engineered strain according to claim 6, characterized in that, The initial temperature for fermentation culture in step (3) is 28~30℃; And / or, after the glycerol culture medium is stopped from being fed, the temperature is lowered to 25-28°C.

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