Method for efficiently producing recombinant human collagen and product and application thereof

Abstract

The present application relates to a kind of recombinant human collagen efficient production method and its product and application.The genetically engineered bacteria are introduced into human collagen gene XVII-4 in the E.coli of knockout dacB gene, and the nucleotide sequence of human collagen gene XVII-4 is as shown in SEQ ID NO.4.The present application also provides the application of the genetically engineered bacteria for efficiently producing recombinant human collagen in the preparation of recombinant human collagen, and the efficient production method of recombinant human collagen.The recombinant human collagen yield of the genetically engineered bacteria E.coli BL21-△dacB-pRSFDuet-1-XVII-4 provided by the present application reaches 96mg / L, and after optimizing production method, the recombinant human collagen yield can reach 150.7mg / L.

Description

Technical Field

[0001] This invention relates to a highly efficient production method for recombinant human collagen, its products, and their applications, belonging to the field of genetic engineering technology. Background Technology

[0002] Collagen is the earliest discovered and most abundant extracellular matrix protein, widely present in the skin, muscles, bones, and internal organs of humans and animals. It plays a vital role in maintaining the normal physiological functions of cells, tissues, and organs, and in repairing damage. Due to its excellent physicochemical properties, biological efficacy, biocompatibility, and biodegradability, collagen is widely used in food, cosmetics, and nutritional supplements.

[0003] Recombinant human collagen has a structure identical to human-derived collagen, but it has been optimized to improve its hydrophilicity and activity. It contains abundant hydrophilic groups, exhibiting excellent film-forming properties and maintaining moisture in the stratum corneum. Furthermore, it can increase fibroblast adhesion and proliferation, replenish collagen in wounds and promote its deposition, reducing the likelihood of scarring. Its tropism-guided action can guide epithelial cells to rapidly enter the damaged area, effectively improving skin regeneration speed, shortening wound healing time, and thus restoring skin barrier function. It is a protein with significant therapeutic potential for wound healing and can be used in any area with epithelial tissue damage. It can be applied as an adjunct treatment for burns, scalds, external injuries, surgical wounds, chronic ulcers, ulcerative colitis, and other diseases. Based on the important role of recombinant human collagen, researchers have developed various preparation and purification methods.

[0004] Recombinant collagen expression systems mainly include prokaryotic (e.g., E. coli), yeast, plant, baculovirus, and mammalian cell expression systems. Among them, type XVII collagen (Collagen XVII, also known as COL17) is a transmembrane protein that plays a crucial role in maintaining the connections between intracellular and extracellular structural elements involved in epidermal adhesion. Type XVII collagen is a transmembrane protein located in the epidermal basement membrane region and plays an important role in maintaining the homeostasis of related stem cells in the skin. Due to its safety, controllability, and production flexibility, recombinant collagen is gradually becoming a powerful alternative to natural collagen.

[0005] However, type XVII collagen is present in small amounts in the human body, and is also extremely rare in animals. Extraction is very difficult, and relying solely on extraction from biological tissues is inefficient and costly, making mass production impossible. Furthermore, there are no reports of recombinant expression and preparation of type XVII collagen in E. coli.

[0006] Therefore, how to develop a simple and efficient production method for recombinant human collagen has become an urgent problem to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a highly efficient method for producing recombinant human collagen, along with its products and applications.

[0008] The technical solution of the present invention is as follows:

[0009] A genetically engineered bacterium that efficiently produces recombinant human collagen is created by introducing the human collagen gene XVII-4 into Escherichia coli with the dacB gene knocked out.

[0010] The nucleotide sequence of the human collagen gene XVII-4 is shown in SEQ ID NO.4.

[0011] According to a preferred embodiment of the present invention, the dacB gene expresses D-carboxypeptidase, the nucleotide sequence of which is shown in SEQ ID NO. 11.

[0012] The method for constructing the above-mentioned genetically engineered bacteria for efficient production of recombinant human collagen includes the following steps:

[0013] (1) Using plasmid pKD13 as a template and dacB-F / R as primers, PCR amplification was performed to obtain a knockout frame fragment containing the resistance gene and FRT site; plasmid pKD46 was introduced into E. coli to obtain recombinant E. coli containing pKD46 plasmid; the knockout frame fragment was transformed into recombinant E. coli containing pKD46 plasmid, homologous recombination was performed under the action of pKD46 plasmid, and the kan gene was eliminated using pCP20 helper plasmid. Positive transformants were selected to obtain E. coli BL21ΔdacB with the dacB gene knocked out;

[0014] (2) The human collagen gene XVII-4 was ligated into the plasmid vector pRSFDuet-1 to obtain the recombinant plasmid pRSFDuet-1-XVII-4; then the recombinant plasmid pRSFDuet-1-XVII-4 was transformed into the E. coli BL21ΔdacB obtained in step (1) with the dacB gene knocked out, and positive transformants were selected to obtain the genetically engineered bacterium E. coli BL21-ΔdacB-pRSFDuet-1-XVII-4 that produces recombinant human collagen efficiently.

[0015] According to a preferred embodiment of the present invention, in step (1), the sequence of dacB-F / R is as follows:

[0016] dacB-F: 5′-GATTACCACAGTCAGCAGATGGCGCAGCCCGCCAGTACGCAGAAAGTGATGTGTAGGCTGGAGCTGCTTC-3′;

[0017] dacB-R: 5′-CATCCACGCCCGCCTGATGCAGACCTGCACGGTACTGCAAAGAGCCGTCAATTCCGGGGATCCGTCGACC-3′.

[0018] The above-mentioned genetically engineered bacteria that efficiently produce recombinant human collagen are used in the preparation of recombinant human collagen.

[0019] A highly efficient method for producing recombinant human collagen includes the following steps:

[0020] The genetically engineered strain *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4, which efficiently produces recombinant human collagen, was streaked onto LB agar and incubated statically at 35–40°C for 10–15 h. Single colonies were picked and inoculated into LB liquid medium and cultured at 35–40°C and 180–220 rpm for 12 h to obtain seed culture. The seed culture was added to the fermentation medium at a volume ratio of 1.5–2.5% and cultured at 35–40°C and 180–220 rpm for 1.5–2.5 h to allow OD to develop. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 22–28 °C and 180–220 r / min for 20–30 h to obtain a fermentation product containing recombinant human collagen.

[0021] According to a preferred embodiment of the present invention, the fermentation medium comprises a carbon source, a nitrogen source, metal ions, and a surfactant.

[0022] Further preferably, the carbon source is selected from one or more of glucose, glycerol, maltose, lactose, dextrin, and sucrose; the nitrogen source is selected from one or more of yeast powder, beef extract, corn syrup powder, peptone, and ammonium sulfate; the metal ion is selected from one or more of zinc sulfate, copper sulfate, and magnesium sulfate; and the surfactant is Tween-80.

[0023] In a further preferred embodiment, the fermentation medium is formulated as follows: glycerol 4-6 g / L, yeast extract 20-30 g / L, peptone 12-15 g / L, KH2PO4 2-4 g / L, K2HPO4 10-14 g / L, with an additional 0.2-1% of Tween-80 by weight of the remaining components.

[0024] Most preferably, the fermentation medium is formulated with 5 g / L glycerol, 24 g / L yeast extract, 12 g / L peptone, 1 g / L magnesium sulfate, 2.32 g / L KH2PO4, 12.54 g / L K2HPO4, and an additional 0.5% Tween-80.

[0025] A recombinant human collagen protein, characterized in that the recombinant human collagen protein is prepared using the above-mentioned efficient production method for recombinant human collagen protein.

[0026] The beneficial effects of this invention are as follows:

[0027] 1. This invention uses *Escherichia coli* as the starting strain. The dacB gene expressing D-carboxypeptidase was knocked out in *E. coli* BL21, and the human collagen gene XVII-4 was introduced, resulting in the genetically engineered bacterium *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4. Knocking out the dacB gene increases the extracellular secretion level of recombinant human collagen. The human collagen gene XVII-4 is a recombinant human collagen gene fragment with high expression levels obtained by the applicant through screening and expression of 10 different human collagen fragments XVII-1, XVII-2, XVII-3, XVII-4, XVII-5, XVII-6, XVII-7, XVII-8, XVII-9, and XVII-10. Due to its specific sequence composition, the recombinant human collagen gene fragment XVII-4 can be efficiently expressed in *E. coli*. The protein expression levels of other gene fragments are significantly lower than those of XVII-4, rendering them impractical for future industrialization. The genetically engineered *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4 provided in this invention can effectively increase the expression level of recombinant human collagen, resulting in a recombinant human collagen yield of 96 mg / L.

[0028] 2. This invention also provides a method for efficiently producing recombinant human collagen using the genetically engineered bacterium *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4. The fermentation conditions of the genetically engineered bacterium were investigated, and it was found that the bacterium could produce high yields of human collagen and achieve efficient extracellular expression in a culture medium with glycerol as the carbon source. Furthermore, the concentrations of magnesium sulfate and Tween-80 both affected the yield of human collagen. Therefore, fermentation in the culture medium provided by this invention, which uses glycerol as the carbon source and contains magnesium sulfate and Tween-80, can further and significantly increase the yield of recombinant human collagen, achieving a yield of 150.7 mg / L. Attached Figure Description

[0029] Figure 1 These are the results of recombinant collagen expression and mass spectrometry identification;

[0030] In the figure, a is the electrophoresis diagram of recombinant collagen expression; b is the Mascot Search Results diagram after mass spectrometry identification.

[0031] Figure 2 The effect of different culture media on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0032] In the figure, a: the effect of different culture media on the growth and protein concentration of genetically engineered bacteria; b: SDS-PAGE image of recombinant human collagen. Each culture medium group has two corresponding electrophoresis bands below it. Among them, odd-numbered bands are whole-cell electrophoresis bands, and even-numbered bands are extracellular supernatant bands of fermentation broth. The horizontal arrow on the right side of the SDS-PAGE image indicates the theoretical electrophoresis band position of recombinant human collagen.

[0033] Figure 3 The effect of different carbon sources on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0034] In the figure, a: the effect of different carbon sources on the growth and protein concentration of genetically engineered bacteria; b: SDS-PAGE image of recombinant human collagen, with two electrophoretic bands below each carbon source group, where odd-numbered bands are whole-cell electrophoresis bands and even-numbered bands are extracellular supernatant bands from fermentation broth. The horizontal arrows on the right side of the SDS-PAGE image indicate the theoretical electrophoretic band positions of recombinant human collagen.

[0035] Figure 4 The effect of different nitrogen sources on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0036] In the figure, a: the effect of different nitrogen sources on the growth and protein concentration of genetically engineered bacteria; b: SDS-PAGE image of recombinant human collagen, with two electrophoretic bands below each nitrogen source group, where odd-numbered bands are whole-cell electrophoresis bands and even-numbered bands are extracellular supernatant bands from fermentation broth. The horizontal arrows on the right side of the SDS-PAGE image indicate the theoretical electrophoretic band positions of recombinant human collagen.

[0037] Figure 5 The effect of different inorganic salts on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0038] In the figure, a: the effect of different inorganic salts on the growth and protein concentration of genetically engineered bacteria; b: SDS-PAGE image of recombinant human collagen, with two electrophoretic bands below each inorganic salt group, where odd-numbered bands are whole-cell electrophoresis bands and even-numbered bands are extracellular supernatant bands from fermentation broth. The horizontal arrows on the right side of the SDS-PAGE image indicate the theoretical electrophoretic band positions of recombinant human collagen.

[0039] Figure 6 The effect of different metal ions on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0040] In the figure, a: the effect of different metal ions on the growth and protein concentration of genetically engineered bacteria; b: SDS-PAGE image of recombinant human collagen, with two electrophoretic bands below each metal ion group. Odd-numbered bands represent whole-cell electrophoresis bands, and even-numbered bands represent extracellular supernatant bands from the fermentation broth. The horizontal arrows on the right side of the SDS-PAGE image indicate the theoretical electrophoretic band positions of the recombinant human collagen.

[0041] Figure 7 The effect of different inducer concentrations on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0042] In the figure, a: the effect of different inducer concentrations on the growth and protein concentration of genetically engineered bacteria; b: SDS-PAGE image of recombinant human collagen, with two electrophoretic bands corresponding to each nitrogen source group below, where odd-numbered bands are whole-cell electrophoresis bands, and even-numbered bands are extracellular supernatant bands from fermentation broth. The horizontal arrows on the right side of the SDS-PAGE image indicate the theoretical electrophoretic band positions of recombinant human collagen.

[0043] Figure 8 The effect of different induction culture temperatures on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0044] In the figure, a: the effect of different induction culture temperatures on the growth and protein concentration of genetically engineered bacteria; b: SDS-PAGE image of recombinant human collagen, with two electrophoretic bands corresponding to each group of different induction culture temperatures. Odd-numbered bands represent whole-cell electrophoresis bands, and even-numbered bands represent extracellular supernatant bands from the fermentation broth. The horizontal arrows on the right side of the SDS-PAGE image indicate the theoretical electrophoretic band positions of the recombinant human collagen.

[0045] Figure 9 The effect of different Tween-80 concentrations on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0046] In the figure, a: the effect of different Tween-80 concentrations on the growth and total protein concentration of genetically engineered bacteria; b: SDS-PAGE image of recombinant human collagen, with two electrophoretic bands corresponding to each Tween-80 concentration group below. Odd-numbered bands represent whole-cell electrophoresis bands, and even-numbered bands represent extracellular supernatant bands from the fermentation broth. The horizontal arrows on the right side of the SDS-PAGE image indicate the theoretical electrophoretic band positions of the recombinant human collagen.

[0047] Figure 10 The combined effects of different induction culture temperatures, inducer concentrations, and Tween-80 concentrations on the synthesis of recombinant human collagen by genetically engineered bacteria;

[0048] In the figure, a: L9(3) was prepared by selecting three factors at three levels: induction culture temperature, Tween-80 concentration, and magnesium sulfate addition. 3 a: Orthogonal array; b: Growth status and total protein concentration of genetically engineered bacteria in the orthogonal experiment, including intracellular and extracellular protein concentrations; c: SDS-PAGE image of whole cells of genetically engineered bacteria producing recombinant human collagen in the orthogonal experiment; d: SDS-PAGE image of extracellular supernatant of genetically engineered bacteria producing recombinant human collagen in the orthogonal experiment. Detailed Implementation

[0049] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art. Unless otherwise specified, the reagents and pharmaceuticals involved in the present invention are all commercially available products.

[0050] LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L.

[0051] TB liquid culture medium: yeast extract 24 g / L, peptone 12 g / L, glycerol 5 g / L, KH2PO4 2.32 g / L, K2HPO4 12.54 g / L.

[0052] SOB liquid medium: yeast extract 5 g / L, peptone 20 g / L, NaCl 0.5 g / L, KCl 0.19 g / L, MgCl2 0.95 g / L.

[0053] SOC liquid culture medium: yeast extract 5g / L, peptone 20g / L, glucose 3.6g / L, NaCl 0.5g / L, KCl 0.19g / L, MgCl2 0.95g / L.

[0054] SB liquid medium: yeast extract 20 g / L, peptone 35 g / L, NaCl 5 g / L.

[0055] M9 liquid culture medium: glucose 4 g / L, MgSO4·7H2O 0.49 g / L, CaCl2·6H2O 0.022 g / L, Na2HPO4·7H2O 12.8 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, NH4Cl 1.0 g / L.

[0056] For solid culture media, add 2g of agar per 100mL of liquid culture media.

[0057] Method for determining E. coli biomass: After appropriately diluting the fermentation broth with sterile water, the absorbance at a wavelength of 600 nm was measured using a spectrophotometer. 600 This allows us to determine the growth status of recombinant Escherichia coli.

[0058] Protein concentration was determined using the Bradford method. After appropriate dilution, 50 μL of the fermentation sample was mixed thoroughly with 250 μL of Coomassie Brilliant Blue solution, and the absorbance (A) was measured at 595 nm. 595 .

[0059] Example 1: Construction of E. coli BL21ΔdacB gene knockout

[0060] The carboxypeptidase gene was knocked out using Red homologous recombination. Red homologous recombination knockout is a novel genetic engineering technique that has emerged in recent years, based on λ phage Red recombinase and in vivo homologous recombination reactions. This technique mainly relies on helper plasmids pKD46 and pCP20. Plasmid pKD46 is a low-copy, temperature-sensitive plasmid that expresses three recombinant proteases, Exo, Bet, and Gam, under the control of the arabinose promoter PBAD to promote homologous recombination of the gene fragment in vivo. Plasmid pCP20 is also a temperature-sensitive plasmid that expresses FLP recombinase, causing FRT (FLP recombinase recognition sites) on both sides of the resistance gene to recombine, thereby eliminating the resistance gene. The recombination assisted by helper plasmid pKD46 is also known as Wanner recombination, and its specific construction process is as follows:

[0061] 1. Using plasmid pKD13 as a template and dacB-F / R as primers, PCR amplification was performed to obtain a knockout frame fragment containing the resistance gene and FRT site.

[0062] The sequence of dacB-F / R is as follows:

[0063] dacB-F: 5′-GATTACCACAGTCAGCAGATGGCGCAGCCCGCCAGTACGCAGAAAGTGATGTGTAGGCTGGAGCTGCTTC-3′;

[0064] dacB-R: 5′-CATCCACGCCCGCCTGATGCAGACCTGCACGGTACTGCAAAGAGCCGTCAATTCCGGGGATCCGTCGACC-3′.

[0065] 2. Electroporate plasmid pKD46 into E. coli BL21 competent cells and culture overnight at 30°C. Inoculate the overnight culture into a 50 mL Erlenmeyer flask containing LB liquid medium at a volume ratio of 1% and culture at 30°C until the OD600 reaches approximately 0.2. Add 10 mM arabinose for induction. When the OD600 reaches 0.6, remove the flask from the shaker and cool it on ice for 20 min. Transfer the culture to a pre-chilled 50 mL sterile centrifuge tube in a clean bench and centrifuge at 4°C and 5000 rpm for 10 min. Discard the supernatant, add 30 mL of pre-chilled sterile water, resuspend the cells, and centrifuge at 4°C and 5000 rpm for 10 min. Repeat the operation once. Wash once with pre-cooled 10% glycerol, centrifuge at 4°C and 5000 rpm for 10 min and discard the supernatant. Resuspend the bacterial cells in 0.5 mL of 10% glycerol and aliquot 50 μL of competent cells into 1.5 mL centrifuge tubes to obtain recombinant Escherichia coli containing the pKD46 plasmid.

[0066] 3. Add 2 μL of the knockout frame fragment to recombinant *E. coli* containing the pKD46 plasmid and mix thoroughly. Transfer the mixture to a pre-chilled 2 mm electroporation cuvette and pre-chill on ice for 10 min. Turn on the electroporator and set the parameters to 2.5 kV, 25 μF, 200 Ω, and a pulse duration of 4–5 ms. Immediately after pulse, add 1 mL of LB liquid medium and incubate at 30°C and 200 rpm for 2 h. After incubation, plate the transformed *E. coli* onto ampicillin and kanamycin LB solid medium and incubate at 30°C for 12 h to obtain the positive transformant *E. coli* BL21ΔdacB::kan / pKD46.

[0067] 4. Inoculate E. coli BL21ΔdacB::kan / pKD46 into antibiotic-free LB liquid medium and incubate overnight at 37°C. Streak the bacterial culture onto LB solid medium and spot single colonies onto kanamycin-resistant and ampicillin-resistant plates. Select the strain that grows on the kanamycin plate but not on the ampicillin-resistant plate as strain E. coli BL21ΔdacB::kan.

[0068] Plasmid pCP20 was electroporated into *E. coli* BL21ΔdacB::kan competent cells and plated on LB solid medium resistant to both ampicillin and chloramphenicol. The cells were cultured overnight at 30°C to obtain positive transformants. During this process, the FLP recombinase expressed by the pCP20 plasmid induced recombination at the FRT site, thereby eliminating the kanamycin resistance gene. Subsequently, the helper plasmid pCP20 was eliminated by culturing in antibiotic-free LB liquid medium at 37°C. The bacterial cells were then diluted and plated on antibiotic-free LB solid medium. Single colonies were picked and inoculated into antibiotic-free, chloramphenicol-resistant, and kanamycin-resistant solid media. The *E. coli* BL21ΔdacB cells that only grew on antibiotic-free LB solid medium were identified as having the dacB gene knocked out (SEQ ID NO. 11).

[0069] Example 2: Construction of a genetically engineered bacterium, E. coli BL21-△dacB-pRSFDuet-1-XVII-4, for efficient production of recombinant human collagen.

[0070] This application selected 10 human collagen genes, namely XVII-1, XVII-2, XVII-3, XVII-4, XVII-5, XVII-6, XVII-7, XVII-8, XVII-9, and XVII-10, with nucleotide sequences as shown in SEQ ID NO.1 to 10, respectively.

[0071] Genes XVII-1 to XVII-10 were artificially synthesized by GenScript Genetics Co., Ltd. according to their sequence information, and then constructed into plasmid pRSFDuet-1 (with BamHI and HindIII restriction sites) to obtain recombinant plasmid pRSFDuet-1-XVII-1 to XVII-10. These plasmids were then transformed into E. coli BL21-ΔdacB to construct recombinant E. coli E. coli BL21-ΔdacB-pRSFDuet-1-XVII-1 to XVII-10.

[0072] Meanwhile, the plasmid pRSFDuet-1 was transformed into E. coli BL21-△dacB using the same method to construct the control strain E. coli BL21-△dacB-pRSFDuet-1.

[0073] Recombinant *E. coli* and the control strain *E. coli* BL21-△dacB-pRSFDuet-1 were streaked onto LB solid medium and incubated statically at 37°C for 12 h. Single colonies of the recombinant *E. coli* were picked and inoculated into LB liquid medium (20 mL / 150 mL) and incubated at 37°C and 200 rpm for 12 h to obtain the seed culture. 1 mL of the seed culture was added to fermentation medium (TB medium, 50 mL / 250 mL) and cultured in a shake flask at 37°C and 200 rpm for 2 h to allow OD to develop. 600 The concentration reached 0.6. IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 25 °C and 200 r / min for 24 h to obtain the fermentation broth of recombinant Escherichia coli BL21-△dacB-pRSFDuet-1-XVII-1~10.

[0074] The fermentation broth was then centrifuged at 8000 rpm for 10 min to collect the cells, which were resuspended in equilibration buffer. The cells were then sonicated on ice (15 s on, 45 s off, 33% amplitude, 1500 KJ energy, 4℃) for 20 min. The disrupted cells were centrifuged at 12000 rpm for 40 min (4℃), and the supernatant was retained and filtered through a 0.22 μm filter. Purification was performed using a nickel column with histidine tagging: the nickel column was first equilibrated to 5–10 column volumes with equilibration buffer. The filtered supernatant was then passed through the nickel column for loading. After loading, the column was equilibrated to 3–5 column volumes with equilibration buffer, followed by washing with washing buffer to remove contaminating proteins. Finally, the target proteins XVII-1–10 were eluted with elution buffer. The purified proteins were stored in 20% glycerol, aliquoted into tubes, and stored at -80℃.

[0075] The target proteins XVII-1 to XVII-10 were subjected to SDS-PAGE protein electrophoresis, and GC-MS was used to detect and analyze them. The results are as follows: Figure 1 As shown.

[0076] Depend on Figure 1 It can be seen that the recombinant collagen genes were all successfully expressed heterologously, consistent with the theoretical molecular weight. Furthermore, compared with recombinant collagen XVII-1~3 and 5~10, the band of recombinant collagen XVII-4 was thicker and more obvious, with a higher expression concentration, greater expression level, and more efficient expression.

[0077] Therefore, recombinant Escherichia coli E. coli BL21-△dacB-pRSFDuet-1-XVII-4 was screened from recombinant E. coli BL21-△dacB-pRSFDuet-1-XVII-1 to 10 as a strain for efficient production of recombinant human collagen, and was named genetically engineered strain E. coli BL21-△dacB-pRSFDuet-1-XVII-4.

[0078] Example 3: Effects of different culture media on the synthesis of recombinant human collagen by genetically engineered bacteria

[0079] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of fermentation medium (50 mL / 250 mL), and the flask was shake-cultured at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 25 °C and 200 r / min for 24 h to obtain a fermentation product containing recombinant human collagen.

[0080] Following this method, fermentation products containing recombinant human collagen were prepared using LB liquid medium, TB liquid medium, SOB liquid medium, SOC liquid medium, SB liquid medium, and M9 liquid medium as fermentation media, respectively. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4. The results are as follows: Figure 2 As shown in figure a. The fermentation products (whole-cell fermentation broth and extracellular supernatant) were identified by SDS-PAGE, and the results are shown in figure a. Figure 2 As shown in b.

[0081] Depend on Figure 2As shown in Figure a, the TB liquid medium exhibited the best growth performance, with the biomass and total protein content of the genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 significantly higher than other groups. The nutrient-rich SB liquid medium was the second best. Because the M9 liquid medium contains only inorganic salts and glucose, lacking essential organic components such as amino acids and vitamins, the growth rate of E. coli was significantly lower than that of the other nutrient-rich media. Compared to the TB liquid medium, the M9 liquid medium lacks a pH buffer system, and the metabolic products of the recombinant E. coli resulted in poor pH stability, further exacerbating growth inhibition.

[0082] Depend on Figure 2 As shown in b, the whole-cell fermentation broth clearly contains recombinant human collagen, while the extracellular supernatant of the fermentation broth contains almost no recombinant human collagen. This indicates that the recombinant human collagen is mainly located intracellularly, and the genetically engineered strain E.coli BL21-△dacB-pRSFDuet-1-XVII-4 has a high intracellular recombinant human collagen yield.

[0083] Example 4: Effects of different carbon sources on the synthesis of recombinant human collagen by genetically engineered bacteria

[0084] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of TB liquid medium (50 mL / 250 mL), and the flask was shaken at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 25 °C and 200 r / min for 24 h to obtain a fermentation product containing recombinant human collagen.

[0085] Following this method, glucose, glycerol, maltose, lactose, dextrin, and sucrose were used respectively to replace yeast extract, peptone, and glycerol in TB liquid culture medium as the sole carbon source (concentration 5 g / L) to prepare fermentation products containing recombinant human collagen. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain E. coli BL21-△dacB-pRSFDuet-1-XVII-4. The results are as follows: Figure 3 As shown in figure a. The fermentation products (whole-cell fermentation broth and extracellular supernatant) were identified by SDS-PAGE, and the results are shown in figure a. Figure 3As shown in b.

[0086] Depend on Figure 3 It can be seen that the genetically engineered bacterium E. coli BL21-△dacB-pRSFDuet-1-XVII-4 grows fastest in fermentation medium with maltose as the sole carbon source, and its OD value reaches a peak at 24 h. 600 The highest concentration indicates active cell proliferation. However, the protein concentration ( Figure 3 a) and SDS-PAGE Figure 3 (b) This indicates low production of recombinant human collagen. When maltose is the sole carbon source, the metabolic resources in the genetically engineered bacteria may be primarily used for cell growth rather than protein synthesis. The growth and protein concentration of the genetically engineered bacteria are similar when glucose and glycerol are the sole carbon sources, but SDS-PAGE analysis shows that the amount of recombinant human collagen produced by the bacteria when glycerol is the sole carbon source is higher than that when glucose is the sole carbon source. Both glucose and glycerol can supply cell growth and protein synthesis, but the synthesis of recombinant human collagen may prefer glycerol as a carbon source. Dextrin, as the sole carbon source, results in rapid growth, but lower total protein concentration and recombinant human collagen yield. When lactose and sucrose are used as the sole carbon sources in the fermentation medium, cell growth and protein synthesis do not reach high levels, and cell growth is slow, indicating limited utilization efficiency of these carbon sources in the genetically engineered bacteria.

[0087] Example 5: Effects of different nitrogen sources on the synthesis of recombinant human collagen by genetically engineered bacteria

[0088] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of TB liquid medium (50 mL / 250 mL), and the flask was shaken at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 25 °C and 200 r / min for 24 h to obtain a fermentation product containing recombinant human collagen.

[0089] Following this method, yeast extract, beef extract, corn steep liquor powder, peptone, and ammonium sulfate were used as the sole nitrogen source (concentration 36 g / L) to replace yeast extract, peptone, and glycerol in TB liquid culture medium, respectively, to prepare fermentation products containing recombinant human collagen. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain E. coli BL21-△dacB-pRSFDuet-1-XVII-4. The results are as follows: Figure 4 As shown in figure a. The fermentation products (whole-cell fermentation broth and extracellular supernatant) were identified by SDS-PAGE, and the results are shown in figure a. Figure 4 As shown in b.

[0090] Depend on Figure 4 It was found that under a complex nitrogen source, the genetically engineered bacterium *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4 grew fastest, with the highest OD value at 24 hours, and accumulated the most intracellular and extracellular protein. When yeast extract, beef extract, and corn steep liquor were used as nitrogen sources, the genetically engineered bacteria grew well, and protein accumulation was also achieved. The peptone group showed particularly high protein levels in the later stages. However, when ammonium sulfate was used as the sole nitrogen source, the genetically engineered bacteria grew slowly, with the lowest OD value and protein content. Overall, this indicates that organic nitrogen sources (such as complex nitrogen sources and yeast extract) are more conducive to the growth and protein synthesis of *E. coli*, while the effect of the inorganic nitrogen source ammonium sulfate is weaker.

[0091] Example 6: Effects of different inorganic salts on the synthesis of recombinant human collagen by genetically engineered bacteria

[0092] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of TB liquid medium (50 mL / 250 mL), and the flask was shaken at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 25 °C and 200 r / min for 24 h to obtain a fermentation product containing recombinant human collagen.

[0093] Following this method, sodium chloride, potassium chloride, and calcium chloride were added to TB liquid culture medium as supplementary inorganic salts (concentration 1 g / L) to prepare a fermentation product containing recombinant human collagen. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4. A control without added inorganic salts was used. The results are as follows: Figure 5 As shown in Figure a. The fermentation products (whole-cell fermentation broth and extracellular supernatant) were identified by SDS-PAGE, with a control of no added inorganic salts. The results are shown in Figure a. Figure 5 As shown in b.

[0094] Depend on Figure 5 It was found that the addition of sodium chloride, calcium chloride, and potassium chloride increased the extracellular protein concentration, but the expression level of the genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 was worse than that without the addition of inorganic salts. The addition of inorganic salts inhibited cell growth and reduced protein concentration. None of the three inorganic salts increased the yield of recombinant human collagen.

[0095] Example 7: Effects of different metal ions on the synthesis of recombinant human collagen by genetically engineered bacteria

[0096] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of TB liquid medium (50 mL / 250 mL), and the flask was shaken at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 25 °C and 200 r / min for 24 h to obtain a fermentation product containing recombinant human collagen.

[0097] Following this method, ferrous sulfate, ferric sulfate, zinc sulfate, copper sulfate, and magnesium sulfate were added to TB liquid culture medium as supplementary metal ions (concentration 1 g / L) to prepare a fermentation product containing recombinant human collagen. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4. A control without added metal ions was used. The results are as follows: Figure 6As shown in Figure a. The fermentation products (whole-cell fermentation broth and extracellular supernatant) were identified by SDS-PAGE, with a control of no added metal ions. The results are shown in Figure a. Figure 6 As shown in b.

[0098] Depend on Figure 6 It was found that after adding zinc sulfate, the cell growth rate of the genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 was close to that without metal ions, but the protein concentration was significantly lower than that of recombinant human collagen without metal ions. Adding ferrous sulfate, ferric sulfate, manganese sulfate, and copper sulfate resulted in slow cell growth and low protein concentration, which were unsuitable for the expression of recombinant human collagen. Adding magnesium sulfate inhibited cell growth to some extent, but the recombinant human collagen concentration was close to that without metal ions.

[0099] Example 8: Effect of different inducer concentrations on the synthesis of recombinant human collagen by genetically engineered bacteria

[0100] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of TB liquid medium (50 mL / 250 mL), and the flask was shaken at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then IPTG was added, and the mixture was induced and cultured at 25℃ and 200r / min for 24h to obtain a fermentation product containing recombinant human collagen.

[0101] Following this method, the concentrations of the inducer IPTG were adjusted to 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM to prepare fermentation products containing recombinant human collagen. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain E. coli BL21-△dacB-pRSFDuet-1-XVII-4. The results are as follows: Figure 7 As shown in figure a. The fermentation products (whole-cell fermentation broth and extracellular supernatant) were identified by SDS-PAGE, and the results are shown in figure a. Figure 7 As shown in b.

[0102] Depend on Figure 7It can be seen that with the increase of IPTG addition, the protein concentration of the genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 tends to increase. When the IPTG concentration is 1mM, it has no significant inhibitory effect on the growth of recombinant E. coli and is more conducive to the expression of recombinant human collagen.

[0103] Example 9: Effect of different induction culture temperatures on the synthesis of recombinant human collagen by genetically engineered bacteria

[0104] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of TB liquid medium (50 mL / 250 mL), and the flask was shaken at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 0.1M, and the mixture was induced and cultured at 200r / min for 24h to obtain a fermentation product containing recombinant human collagen.

[0105] Following this method, the induction culture temperatures were adjusted to 20℃, 25℃, 30℃, and 37℃ to prepare fermentation products containing recombinant human collagen. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4. The results are as follows: Figure 8 As shown in figure a. The fermentation products (whole-cell fermentation broth and extracellular supernatant) were identified by SDS-PAGE, and the results are shown in figure a. Figure 8 As shown in b.

[0106] Depend on Figure 8 It was found that the genetically engineered bacterium *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4 grew fastest at 37℃, but protein accumulation was low. 37℃ is the optimal growth temperature for the genetically engineered bacterium. The metabolic byproducts accumulated during fermentation negatively impacted the expression of recombinant human collagen. At 30℃, the genetically engineered bacterium grew slowly and accumulated little protein; at this temperature, it could neither grow rapidly nor achieve efficient expression of the target protein. At 25℃ and 20℃, the growth of the genetically engineered bacterium was similar, and protein accumulation was relatively high.

[0107] Example 10: Effect of different Tween-80 concentrations on the synthesis of recombinant human collagen by genetically engineered bacteria

[0108] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of TB liquid medium (50 mL / 250 mL), and the flask was shaken at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 25 °C and 200 r / min for 24 h to obtain a fermentation product containing recombinant human collagen.

[0109] Following this method, Tween-80 at concentrations of 1%, 0.5%, and 0.2% (w / w) was added to TB liquid culture medium as a surfactant to prepare fermentation products containing recombinant human collagen. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4. A control without Tween-80 was used. The results are as follows: Figure 9 As shown in figure a. The fermentation products (whole-cell fermentation broth and extracellular supernatant) were identified by SDS-PAGE, with a control of no Tween-80 added. The results are shown in figure a. Figure 9 As shown in b.

[0110] Depend on Figure 9 It was found that the addition of Tween-80 had little effect on the growth of the genetically engineered bacterium *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4, and the growth of the genetically engineered bacterium was similar at different addition levels. Without the addition of Tween-80, the extracellular protein concentration was low. With increasing Tween-80 concentration, the amount of extracellular protein increased, and the target protein band appeared on extracellular protein electrophoresis, indicating an increase in the extracellular secretion of the genetically engineered bacterium.

[0111] Example 11: Effects of different induction culture temperatures, magnesium sulfate concentrations, and Tween-80 concentrations on the synthesis of recombinant human collagen by genetically engineered bacteria.

[0112] The genetically engineered E. coli BL21-△dacB-pRSFDuet-1-XVII-4 constructed in Example 2 was streaked onto LB solid medium and incubated statically at 37°C for 12 h. A single colony of the genetically engineered bacterium was picked and inoculated into an Erlenmeyer flask containing LB liquid medium (20 mL / 150 mL), and cultured at 37°C and 200 rpm for 12 h as the seed culture. 1 mL of the seed culture was added to an Erlenmeyer flask containing 50 mL of TB liquid medium (50 mL / 250 mL), and the flask was shaken at 37°C and 200 rpm for 2 h to allow the OD to adjust. 600 The concentration was increased to 0.6; then magnesium sulfate was added, and the mixture was induced and cultured at 200 r / min for 24 h to obtain a fermentation product containing recombinant human collagen.

[0113] An orthogonal experimental design was performed using three factors at three levels: induction culture temperature, Tween-80 concentration, and magnesium sulfate addition. An L9(3)2(3)2(4)3(1)3(2)3)3(1)3 ...203)203)203)"" 3)3)""" 3)""" 3.5) 3) 3 3 Orthogonal arrays Figure 10 a) Following an orthogonal array, Tween-80 was added to TB liquid culture medium, and the induction culture temperature and magnesium sulfate dosage were adjusted to prepare a fermentation product containing recombinant human collagen. Samples were taken every 8 hours during the induction culture to determine the biomass, intracellular protein concentration, and extracellular protein concentration of the genetically engineered strain E. coli BL21-△dacB-pRSFDuet-1-XVII-4. The results are as follows: Figure 10 As shown in b. SDS-PAGE analysis was performed on the whole-cell fermentation products and the extracellular supernatant within the fermentation products, and the results are shown in Figure 1. Figure 10 As shown in c and 10d.

[0114] Depend on Figure 10 From b to d, it can be seen that the extracellular protein content is high when the induction culture temperature is 25℃, the Tween-80 addition is 0.5%, and the magnesium sulfate addition is 1g / L. Figure 9 b), the electrophoretic bands are most obvious ( Figure 10 c and Figure 10 d) The genetically engineered bacterium *E. coli* BL21-△dacB-pRSFDuet-1-XVII-4 showed high extracellular recombinant human collagen expression. At 25℃, the genetically engineered bacterium achieved efficient synthesis of recombinant human collagen. The addition of 1 g / L magnesium sulfate increased the growth rate and biomass of the genetically engineered bacterium, while the addition of 0.5% Tween-80 promoted the extracellular secretion of recombinant human collagen. Under these conditions, the total extracellular protein yield reached 150.7 mg / L, indicating that the extracellular production of recombinant human collagen by the genetically engineered bacterium reached its optimal level.

Claims

1. A genetically engineered bacterium for the efficient production of recombinant human collagen, characterized in that, It is knocking out dacB Human collagen gene introduced into E. coli XVII-4 ; The human collagen gene XVII-4 The nucleotide sequence is shown in SEQ ID NO.

4.

2. The genetically engineered bacteria for high-efficiency production of recombinant human collagen as described in claim 1, characterized in that, The dacB The gene expresses D-carboxypeptidase, the nucleotide sequence of which is shown in SEQ ID NO.

11.

3. The method for constructing genetically engineered bacteria for efficient production of recombinant human collagen as described in claim 1, characterized in that, The steps include the following: (1) Using plasmid pKD13 as a template and dacB-F / R as primers, PCR amplification was performed to obtain a knockout frame fragment containing the resistance gene and FRT site; plasmid pKD46 was introduced into E. coli to obtain recombinant E. coli containing pKD46 plasmid; the knockout frame fragment was transformed into recombinant E. coli containing pKD46 plasmid, homologous recombination was performed under the action of pKD46 plasmid, and the kan gene was eliminated using pCP20 helper plasmid. Positive transformants were selected to obtain the knockout gene. dacB E. coli with genetic traits E . coli BL21Δ dacB ; (2) Human collagen gene XVII-4 The plasmid was ligated into the plasmid vector pRSFDuet-1 to obtain the recombinant plasmid pRSFDuet-1-XVII-4; then the recombinant plasmid pRSFDuet-1-XVII-4 was transformed into the knockout plasmid obtained in step (1). dacB E. coli with genetic traits E . coli BL21Δ dacB In the process, positive transformants were selected to obtain genetically engineered bacteria that efficiently produce recombinant human collagen. E . coli BL21-△ dacB -pRSFDuet-1-XVII-4.

4. The application of the genetically engineered bacteria for producing recombinant human collagen as described in claim 1 in the preparation of recombinant human collagen.

5. A highly efficient method for producing recombinant human collagen, characterized in that, The steps include the following: The genetically engineered bacteria described in claim 1 were streaked on LB solid medium and incubated statically at 35-40°C for 10-15 hours; a single colony was picked and inoculated into LB liquid medium and incubated at 35-40°C and 180-220 r / min for 12 hours to obtain a seed culture. Add the seed culture to the fermentation medium at a volume ratio of 1.5–2.5%, and incubate at 35–40°C and 180–220 rpm for 1.5–2.5 h to allow the OD to rise. 600 The concentration was increased to 0.6; then IPTG was added to a final concentration of 1 mM, and the mixture was induced and cultured at 22-28℃ and 180-220 r / min for 20-30 h to obtain a fermentation product containing recombinant human collagen.

6. The efficient production method of recombinant human collagen as described in claim 5, characterized in that, The fermentation medium includes a carbon source, a nitrogen source, metal ions, and a surfactant; The carbon source is selected from one or more of glucose, glycerol, maltose, lactose, dextrin, and sucrose; The nitrogen source is selected from one or more of yeast powder, beef extract, corn syrup powder, peptone, and ammonium sulfate; The metal ion is magnesium sulfate; The surfactant is Tween-80.

7. The efficient production method of recombinant human collagen as described in claim 5, characterized in that, The fermentation medium is formulated with 4-6 g / L glycerol, 20-30 g / L yeast extract, 12-15 g / L peptone, 2-4 g / L KH2PO4, 10-14 g / L K2HPO4, and additionally 0.2-1% of the total mass of the remaining components, Tween-80 and 1 g / L magnesium sulfate.

8. The efficient production method of recombinant human collagen as described in claim 7, characterized in that, The fermentation medium is formulated with 5 g / L glycerol, 24 g / L yeast extract, 12 g / L peptone, 2.32 g / L KH2PO4, 12.54 g / L K2HPO4, and additionally 0.5% Tween-80 and 1 g / L magnesium sulfate by weight of the remaining components.

9. A recombinant human collagen, characterized in that, The recombinant human collagen is prepared using the efficient production method for recombinant human collagen as described in any one of claims 5 to 8.

10. The efficient production method of recombinant human collagen according to any one of claims 5 to 8, or the application of recombinant human collagen according to claim 9 in the preparation of skin care products.

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