Recombinant III-type human collagen expression strain with full-chain length and triple-helix structure as well as preparation method and application of recombinant III-type human collagen expression strain

Abstract

The invention relates to a recombinant III-type human collagen expression strain with a whole chain length and a triple helix structure as well as a preparation method and application thereof, and belongs to the technical field of genetic engineering bacteria. The recombinant pichia pastoris genetically engineered bacterium expresses recombinant III-type human collagen with a full chain length and a triple helix structure, and a ku70 gene of the recombinant pichia pastoris genetically engineered bacterium is knocked out and knocked in a recombinant 4-proline hydroxylase coding gene and an FKBP22 coding gene. The recombinant pichia pastoris genetically engineered bacterium provided by the invention can be used for obtaining the recombinant full-chain-length triple-helix recombinant III-type human collagen with a higher expression level through high-density fermentation, does not contain heterologous sequences, has higher hydroxyproline content and thermal stability, is close to natural human collagen, has a stable triple-helix structure, is high in thermal stability, and can be used for preparing the recombinant III-type human collagen. The application in the fields of medical health and medical beauty such as medical beauty filling and tissue repair can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of genetically engineered bacteria, and particularly relates to a recombinant type III human collagen-expressing strain with a full-chain length and a triple-helix structure, and a preparation method and application thereof. Background Art

[0002] In recent years, the market demand for collagen products has increased sharply and shown strong growth, and the proportion of recombinant collagen materials has been increasing year by year. Human type III collagen has excellent performance in skin rejuvenation, supporting skin elasticity and repair functions, and has currently become a mainstream choice for industrial research and terminal applications. In particular, human-derived type III collagen has received extensive attention and applications in fields such as beauty skin care and healthcare.

[0003] The core competitiveness of natural collagen lies in the triple-helix structure (formed by three α-chains), which endows it with mechanical stability and biological activity. Collagen with a triple-helix structure not only has obvious activity advantages, but also shows a significant improvement in anti-degradation performance. In addition, based on the basic structure of the triple helix, it can be assembled into a high-dimensional structure (collagen fiber), further enhancing the support of the product, and thus more effectively resisting the degradation process. It can be said that the triple-helix structure is the basis of the biological activity of collagen, and only collagen with a triple-helix structure can be regarded as true collagen; and the ability of triple-helix collagen molecules to assemble into supramolecular structures is the basis for the commercial application of collagen in the medical field (such as medical aesthetic filling, tissue repair). Therefore, how to make recombinant collagen form a triple-helix structure has also become the focus of attention in the industry.

[0004] However, the natural source of human type III collagen is extremely scarce and cannot be obtained commercially on a large scale. At present, the vast majority of human type III collagen on the market is produced by genetic recombination technology. From a structural point of view, such recombinant collagen is mainly truncated polypeptide fragments with the same amino acid sequence as natural human collagen, or a combination of multiple functional fragments, with sizes ranging from a few kDa to dozens of kDa. Such recombinant collagen products have deficiencies in the stability of the three-dimensional helix structure and molecular weight, which may affect their support effect and efficacy in clinical applications. For example, most recombinant collagens have a small molecular weight, lack the triple-helix structure of natural human collagen, and do not have complete biological activity. Secondly, existing recombinant collagen products generally face challenges such as poor stability, fast degradation in vivo, short maintenance time, and lack of mechanical support, which cannot meet the long-term application requirements of medical aesthetic injection filling, tissue repair, etc.

[0005] Current recombinant technologies still have difficulty in achieving the stable expression of full-chain-length triple-helix structures and face technical bottlenecks such as low expression levels and high production costs. As common chassis cells (Escherichia coli, yeast, insect cells, and mammalian cells) in current recombinant protein expression and production research, they have certain potential in synthesizing full-chain-length triple-helix collagen, but their limitations are also obvious. For example, although the Escherichia coli expression system has a short production cycle and relatively high production capacity, its system itself lacks endogenous hydroxylation modification, resulting in its inability to effectively hydroxylate proline or lysine on the collagen chain, and thus unable to form a stable triple-helix structure. Existing eukaryotic systems (such as insect and mammalian cells), although being the most human-like modification and expression systems and theoretically being the easiest to obtain active recombinant human collagen with a complete triple-helix structure close to human origin, generally have disadvantages such as long production cycles, low yields, high costs, and difficulty in scale-up in the recombinant expression of human collagen. Pichia pastoris, as an efficient eukaryotic expression system, compared with the Escherichia coli prokaryotic system, has certain post-translational modification and processing capabilities. Although its natural hydroxylase activity is insufficient, exogenous hydroxylase genes (such as human prolyl-4-hydroxylase, P4H) can be co-expressed through genetic engineering technology, significantly improving the hydroxylation levels of proline and lysine and enhancing triple-helix stability; secondly, Pichia pastoris mainly uses cheap carbon sources such as glycerol and methanol, and its growth and culture conditions are relatively simple, with costs much lower than those of mammalian cell or insect cell culture systems; in addition, Pichia pastoris has a relatively mature fermentation process and has been widely used in biopharmaceuticals, facilitating rapid scale-up of production.

[0006] Patents CN118440975A, CN118834284A, CN119331079A, and CN119431554A also disclose different preparation methods of recombinant humanized type III collagen with triple-helix structure characteristics, but the molecular weights of the recombinant collagens are much smaller than those of natural full-length collagen and the hydroxylation levels are limited, and there may be significant challenges in their biological function integrity and mechanical support. Patent CN118652924A achieved the preparation of triple-helix structure type III recombinant human collagen by co-expressing human type III collagen α1 chain and P4H in yeast cells, but the stability and expression level of this recombinant collagen are unknown. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a recombinant type III human collagen expression strain with full-chain length and triple-helix structure, its preparation method, and its application.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a recombinant Pichia pastoris genetic engineering bacterium, which expresses recombinant type III human collagen with full chain length and triple helix structure, and the ku70 gene of the recombinant Pichia pastoris genetic engineering bacterium is knocked out and the recombinant 4-prolyl hydroxylase coding gene and FKBP22 coding gene are knocked in; The amino acid sequence of the recombinant type III human collagen with full chain length and triple helix structure is as shown in SEQ ID NO: 3; The number of the ku70 gene on NCBI is Gene ID: 8199462; The recombinant 4-prolyl hydroxylase includes a recombinant 4-prolyl hydroxylase α1 subunit and a recombinant 4-prolyl hydroxylase β subunit. The nucleotide sequence of the coding gene of the recombinant 4-prolyl hydroxylase α1 subunit is as shown in SEQ ID NO: 5, and the nucleotide sequence of the coding gene of the recombinant 4-prolyl hydroxylase β subunit is as shown in SEQ ID NO: 6; The nucleotide sequence of the FKBP22 coding gene is as shown in SEQ ID NO: 10.

[0009] In the present invention, a special signal peptide sequence sPC1 and a folding domain sequence Foldon are respectively fused to the N-terminus and C-terminus of the full chain length type III human collagen α1 chain sequence to promote the formation of the triple helix structure.

[0010] The ku70 gene encodes a key protein of a non-homologous end joining (NHEJ) repair pathway and is involved in the direct ligation of broken DNA ends. By knocking out the ku70 gene in the present invention, the NHEJ repair pathway is inhibited, forcing DNA damage repair to rely on the homologous recombination pathway, significantly improving the accuracy and efficiency of gene editing, and thus enabling precise knockout or integration of target genes.

[0011] By knocking in the recombinant 4-prolyl hydroxylase coding gene, hydroxylated modified recombinant collagen can be obtained, making the proline of the recombinant full chain length humanized collagen 4-hydroxyproline, and endowing the recombinant Pichia pastoris genetic engineering bacterium with hydroxylation modification ability, which is beneficial to improving the stability of collagen during fermentation.

[0012] FKBP22 is a peptidyl-prolyl cis-trans isomerase, which can catalyze the folding of type III collagen and act as a molecular chaperone of type III collagen.

[0013] In the present invention, by transforming the coding gene of recombinant type III human collagen with full chain length and triple helix structure into a modified chassis strain, an engineering strain co-expressing a molecular chaperone, a hydroxylase and recombinant full chain length collagen is constructed.

[0014] Furthermore, the FKBP22 coding gene is knocked into the lnt39 gene locus of the Pichia pastoris chassis strain. The nucleotide sequence of the lnt39 gene is shown in SEQ ID NO: 40. The Pichia pastoris chassis strain includes at least one of Pichia pastoris X33, Pichia pastoris GS115, Pichia pastoris KM71H, and Pichia pastoris SMD1168.

[0015] In a specific embodiment of the present invention, Pichia pastoris X33 is used as an example of the Pichia pastoris chassis strain for experiments.

[0016] Most preferably, the recombinant Pichia pastoris genetic engineering bacterium is Komagataella phaffii XF / 3A1ncF-68#. It was deposited at the Guangdong Provincial Microbial Culture Collection Center on April 21, 2025, and its deposit number is: GDMCC No: 66181.

[0017] In a second aspect, the present invention provides the application of the recombinant Pichia pastoris genetic engineering bacterium in the preparation of collagen.

[0018] In a third aspect, the present invention provides a method for preparing the recombinant Pichia pastoris genetic engineering bacterium, including the following steps: S1: Knock out the ku70 gene of Pichia pastoris X33 to obtain the recombinant Pichia pastoris genetic engineering bacterium X33△ku70; S2: Knock in the FKBP22 coding gene in the recombinant Pichia pastoris genetic engineering bacterium X33△ku70 obtained in step S1 to obtain the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22; S3: Knock in the recombinant 4-prolyl hydroxylase coding gene in the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 obtained in step S2 to obtain the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 / α1β; S4: Knock in the recombinant type III human collagen coding gene with full chain length and triple helix structure in the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 / α1β obtained in step S2 to obtain the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 / α1β-3A1ncF. The nucleotide sequence of the recombinant type III human collagen coding gene with full chain length and triple helix structure is shown in SEQ ID NO: 4.

[0019] Furthermore, in step S1, the ku70 gene in Pichia pastoris X33 was knocked out using the CRISPR / Cas9 gene editing technology. Pichia pastoris X33, the CRISPR / Cas9 gene editing vector containing the sgRNA1 target sequence, and the △ku70-Donor fragment fused with the upstream and downstream homologous arms of the ku70 gene were mixed and subjected to electroporation transformation to obtain the recombinant Pichia pastoris genetic engineering bacterium X33△ku70; The nucleotide sequence of the sgRNA2 target sequence is as shown in SEQ ID NO: 29; The nucleotide sequence of the △ku70-Donor fragment is as shown in SEQ ID NO: 39.

[0020] Furthermore, during the electroporation transformation process, the ratio of Pichia pastoris X33 to the CRISPR / Cas9 gene editing vector containing the sgRNA1 target sequence and the △ku70-Donor fragment is 50 - 100 μL: 2 - 4 μg: 2 - 4 μg.

[0021] In the specific embodiment of the present invention, the ratio of Pichia pastoris X33 to the CRISPR / Cas9 gene editing vector containing the sgRNA1 target sequence and the △ku70-Donor fragment is 50 μL: 2 μg: 2 μg, and the OD of Pichia pastoris X33 600 is 40 - 100.

[0022] Furthermore, during the electroporation transformation process, the voltage is 1.2 - 1.8 kV, and the electroporation time is 4 - 6 mSec; preferably, the voltage is 1.5 kV.

[0023] Furthermore, in step S2, the recombinant Pichia pastoris genetic engineering bacterium X33△ku70 obtained in step S1, the CRISPR / Cas9 gene editing vector containing the sgRNA2 target sequence, and the △lnt39-P AOX1 -FKBP22-Donor fragment containing the upstream and downstream homologous arm fragments of the lnt39 gene and the expression cassette fragment of the FKBP22 gene were mixed and subjected to electroporation transformation to obtain the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22; The nucleotide sequence of the sgRNA2 target sequence is as shown in SEQ ID NO: 41; The △lnt39-P AOX1 -FKBP22-Donor fragment has a nucleotide sequence as shown in SEQ ID NO: 54.

[0024] Furthermore, during the electroporation transformation process, the ratio of the recombinant Pichia pastoris genetic engineering bacterium X33△ku70 to the CRISPR / Cas9 gene editing vector containing the sgRNA2 target sequence and the △lnt39-P AOX1 -FKBP22-Donor fragment is 50 - 100 μL : 2 - 4 μg : 2 - 4 μg.

[0025] In a specific embodiment of the present invention, the ratio of the recombinant Pichia pastoris genetic engineering bacterium X33△ku70 to the CRISPR / Cas9 gene editing vector containing the sgRNA2 target sequence and the △lnt39-P AOX1 -FKBP22-Donor fragment is 50 μL : 2 μg : 2 μg, and the OD 600 of the recombinant Pichia pastoris genetic engineering bacterium X33△ku70 is 40 - 100.

[0026] Further, in step S3, the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 described in step S2 and the recombinant vector containing the recombinant 4-prolyl hydroxylase encoding gene are mixed and subjected to electroporation transformation to obtain the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 / α1β.

[0027] Further, the recombinant vector containing the recombinant 4-prolyl hydroxylase encoding gene is the pPIC9K-P4Hα1β recombinant vector containing the recombinant 4-prolyl hydroxylase α1 subunit encoding gene and the P4Hβ subunit expression cassette; the nucleotide sequence of the P4Hβ subunit expression cassette is as shown in SEQ ID NO: 7; the backbone vector of the pPIC9K-P4Hα1β recombinant vector is the pPIC9K vector.

[0028] Furthermore, the pPIC9K plasmid is double digested with BamH I and EcoR I enzymes to obtain a linearized pPIC9K vector; the recombinant 4-prolyl hydroxylase α1 subunit encoding gene is ligated between the BamH I and EcoR I enzyme digestion sites of the linearized pPIC9K vector to obtain the pPIC9K-P4Hα1 recombinant vector; the pPIC9K-P4Hα1 recombinant vector is digested with the AatII endonuclease to obtain a linearized pPIC9K-P4Hα1 recombinant vector; the P4Hβ subunit expression cassette is ligated to the linearized pPIC9K-P4Hα1 recombinant vector to obtain the pPIC9K-P4Hα1β recombinant vector.

[0029] Furthermore, during the electroporation transformation process, the ratio of the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 to the recombinant vector containing the recombinant 4-prolyl hydroxylase encoding gene is 50 - 100 μL : 2 - 4 μg.

[0030] In a specific embodiment of the present invention, the ratio of the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 to the recombinant vector containing the recombinant 4-prolyl hydroxylase-encoding gene is 100 μL: 4 μg, and the OD of the recombinant Pichia pastoris genetic engineering X33△ku70:FKBP22 600 is 40 to 100.

[0031] Further, in step S4, the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 / α1β and the recombinant vector containing the recombinant type III human collagen-encoding gene with full chain length and triple helix structure are mixed, and electrotransformation is carried out to obtain the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 / α1β-3A1ncF. The backbone vector of the recombinant vector containing the recombinant type III human collagen-encoding gene with full chain length and triple helix structure is the pPICZαA vector.

[0032] Furthermore, during the electrotransformation process, the ratio of the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 / α1β to the recombinant vector containing the recombinant type III human collagen-encoding gene with full chain length and triple helix structure is 50 to 100 μL: 2 to 4 μg.

[0033] In a specific embodiment of the present invention, the ratio of the recombinant Pichia pastoris genetic engineering bacterium X33△ku70:FKBP22 / α1β to the recombinant type III human collagen-encoding gene with full chain length and triple helix structure is 100 μL: 4 μg, and the OD of the recombinant Pichia pastoris genetic engineering X33△ku70:FKBP22 / α1β 600 is 40 to 100.

[0034] Fourthly, the present invention provides a method for preparing collagen, culturing the recombinant Pichia pastoris genetic engineering bacterium to express collagen, and the collagen is recombinant type III human collagen with full chain length and triple helix structure, and the amino acid sequence of the recombinant type III human collagen with full chain length and triple helix structure is as shown in SEQ ID NO: 3. Due to the modification of 4-prolyl hydroxylase, the proline of the recombinant full-chain length humanized collagen is 4-hydroxyproline.

[0035] Further, the method for culturing the recombinant Pichia pastoris genetic engineering bacterium to express collagen includes the following steps: S41: Culturing the recombinant Pichia pastoris genetic engineering bacterium with YPD medium until the OD of the recombinant Pichia pastoris genetic engineering bacterium 600 reaches 3 to 5 to obtain the primary seed liquid mother liquor; S42: Inoculate the primary seed liquid mother liquor described in step S41 into BMGY medium and culture until the OD of the recombinant Pichia pastoris genetic engineering bacteria reaches 4 - 6 to obtain the secondary seed liquid; 600 S43: Inoculate the secondary seed liquid described in step S42 into the inorganic salt medium and culture until the dissolved oxygen content in the culture system reaches over 70%, then add feeding solution 1 for fed-batch culture until the wet cell weight reaches 300 - 350 g / L. The feeding solution 1 contains PTM1 and glycerol; S44: After the culture in step S43, when the dissolved oxygen content in the culture system reaches over 80%, add feeding solution 2 and feeding solution 3 for fed-batch culture. The feeding solution 2 contains PTM1 and methanol; the feeding solution 3 contains peptone, L-ascorbic acid, and α-ketoglutaric acid.

[0036] Furthermore, in step S42, the inoculation amount of the primary seed liquid mother liquor is 1 - 10%, and the culture conditions are: culture at 25 - 30°C, 200 - 300 rpm for 16 - 20 h. Preferably, the inoculation amount of the primary seed liquid mother liquor is 1%, and the culture conditions are: culture at 29°C, 250 rpm for 16 - 20 h.

[0037] Furthermore, in step S43, the inoculation amount of the secondary seed liquid is 2 - 10%, and the culture conditions are: culture at 23 - 30°C, pH 5 - 6, 300 - 1000 rpm, aeration rate 0.5 - 2 vvm, and dissolved oxygen content 20% - 30%. Preferably, the inoculation amount of the secondary seed liquid is 10%, at 28°C, pH 5.5, 300 rpm, aeration rate 1 vvm, and dissolved oxygen content 20% - 30%.

[0038] Furthermore, in step S43, the inorganic salt medium contains KH2PO4, K2HPO4, NH4H2PO4, CaSO4, MgSO4·7H2O, glycerol, and PTM1 (trace element complex mother liquor).

[0039] Furthermore, in step S43, the inorganic salt medium contains 17 - 22 g / L KH2PO4, 5 - 10 g / L K2HPO4, 5 - 10 g / L NH4H2PO4, 1 - 5 g / L CaSO4, 5 - 10 g / L MgSO4·7H2O, 10 - 40 g / L glycerol, and 4 - 12 mL / L PTM1. Preferably, the inorganic salt medium contains 20 g / L KH2PO4, 6.85 g / L K2HPO4, 6.9 g / L NH4H2PO4, 1.2 g / L CaSO4, 12.5 g / L MgSO4·7H2O, 40 g / L glycerol, and 4.5 mL / L PTM1.

[0040] ​Furthermore, in step S43, the concentration of PTM1 in the feeding solution 1 is 4 - 12 mL / L, and the concentration of glycerol is 10 - 50% v / v. Preferably, the concentration of PTM1 in the feeding solution 1 is 12 mL / L, and the concentration of glycerol is 50% v / v.

[0041] Furthermore, in step S43, in fed-batch culture, the feeding rate is 6.5 - 10.2 g / min, the maintained pH is 5 - 6, and the dissolved oxygen is 20% - 30%. Preferably, the feeding rate is 8.4 g / min, the maintained pH is 5, and the dissolved oxygen is 30%.

[0042] Furthermore, in step S44, the feeding solution 3 is added at a constant rate of 1 - 12 mL / h throughout the culture in step S43. Preferably, the feeding solution 3 is added at a constant rate of 10 mL / h throughout the culture in step S43.

[0043] Furthermore, in step S44, the concentration of PTM1 in the feeding solution 2 is 5 - 20 mL / L, and the feeding solution 3 contains 10 - 30 g / L peptone, 2 - 20 g / L L-ascorbic acid, and 1 - 5 g / L α-ketoglutaric acid. Preferably, the concentration of PTM1 in the feeding solution 2 is 12 mL / L, and the feeding solution 3 contains 20 g / L peptone, 2 g / L L-ascorbic acid, and 1 g / L α-ketoglutaric acid.

[0044] Furthermore, in step S44, the feeding rate of the initial feeding solution 2 is 1 - 4 g / h. After 1 - 2 h of fed-batch culture, the feeding rate is adjusted to 2 - 8 g / h. After another 1 - 2 h of fed-batch culture, the feeding rate is adjusted to 3 - 9 g / h, and the culture lasts for 60 - 72 h. Preferably, the feeding rate of the initial feeding solution 2 is 3.6 g / h. After 1 h of fed-batch culture, the feeding rate is adjusted to 7.2 g / h. After another 1 h of fed-batch culture, the feeding rate is adjusted to 8.4 g / h, and the culture lasts for 60 - 72 h.

[0045] Further, the separation and purification of the collagen includes lysing the recombinant Pichia pastoris genetic engineering bacteria after the culture in step S44, acid re-dissolving, enzymatic hydrolysis, protein chromatography, and ultrafiltration desalting.

[0046] Furthermore, the lysis includes the following steps: centrifuging the fermentation broth after the culture in step S44, collecting the bacterial sludge, mixing the bacterial sludge with the homogenizing lysis solution, fully resuspending it. The ratio of the bacterial sludge to the homogenizing lysis solution is 10 - 30 g:100 mL. Homogenize and circulate and break it at 800 - 1000 bar for 2 - 5 times, then centrifuge and collect the precipitate, which is the lysed precipitate. The homogenizing lysis solution includes Tris / HCl buffer, NaCl, and glycerol.

[0047] Furthermore, the centrifugation conditions for the fermented broth after culturing in step S44 are as follows: below 10°C, 5000 - 8000 rpm, for 5 - 10 min. Preferably, the centrifugation conditions for the fermented broth after culturing in step S44 are: 8000 rpm, for 10 min.

[0048] Preferably, the ratio of the bacterial sludge to the homogenizing lysis solution is 20 g : 100 mL.

[0049] Preferably, homogenize and circulate at 900 bar for 3 times.

[0050] Furthermore, the centrifugation conditions after homogenization and circulation are as follows: below 10°C, 8000 - 12000 rpm, for 5 - 30 min. Preferably, the centrifugation conditions after homogenization and circulation are: 4°C, 10000 rpm, for 30 min.

[0051] Furthermore, the homogenizing lysis solution includes 10 - 50 mM Tris / HCl buffer, 0.1 - 0.3 M NaCl, and 5 - 20% v / v glycerol, with a pH of 7. Preferably, the homogenizing lysis solution includes 50 mM Tris / HCl buffer, 0.2 M NaCl, and 15% v / v glycerol.

[0052] Furthermore, the acidic re - dissolution includes the following steps: Mix the precipitate after lysis and the acidic re - dissolution solution, and resuspend thoroughly to obtain an acidic resuspension. The ratio of the precipitate to the acidic re - dissolution solution is 0.1 - 1 g : 10 - 50 mL, and the acidic re - dissolution solution contains 0.1 - 0.5 M acetic acid and 0.1 - 0.3 M NaCl.

[0053] Preferably, the ratio of the precipitate to the acidic re - dissolution solution is 1 g : 50 mL, and the acidic re - dissolution solution contains 0.1 M acetic acid and 0.15 M NaCl.

[0054] Furthermore, the enzymatic hydrolysis includes the following steps: Mix the acidic resuspension with pepsin, with the final concentration of pepsin being 0.1 - 1 g / 100 mL, below 10°C, digest for 12 - 16 h to obtain an enzymatic hydrolysate.

[0055] Preferably, the final concentration of pepsin is 0.2 g / 100 mL, and the enzymatic hydrolysate is digested at 4°C.

[0056] Furthermore, the protein chromatography includes the following steps: Centrifuge the enzymatic hydrolysate at room temperature, collect the supernatant, adjust the pH of the supernatant to 3 - 5, add NaCl to a final concentration of 0.35 - 0.5 M to obtain a clarified sample solution for chromatography; Rinse and equilibrate the chromatography column with buffer A, elute with eluent B, and collect the eluate; Buffer A contains KH2PO4 and NaCl, adjust the pH to 3 - 5 with acetic acid, eluent B contains KH2PO4, adjust the pH to 3 - 5 with acetic acid, and the elution volume is 10 - 20 CV.

[0057] Furthermore, the conditions for centrifuging the enzymatic hydrolysate at room temperature are 3000 - 8000 rpm for 5 - 10 min. Preferably, it is 5000 rpm for 5 min.

[0058] Furthermore, the pH of the supernatant is adjusted with 0.1 M KOH. The buffer A contains 10 - 50 mM KH₂PO₄ and 0.5 - 1 M NaCl; the eluent B contains 10 - 50 mM KH₂PO₄.

[0059] Preferably, the pH of the supernatant is adjusted to 5 with 0.1 M KOH, and NaCl is added to a final concentration of 0.5 M to obtain a clarified sample loading solution for chromatography; the chromatography column is rinsed and equilibrated with buffer A, and eluted with eluent B, and the eluate is collected; the buffer A contains 20 mM KH₂PO₄ and 0.5 M NaCl, and the pH is adjusted to 5 with acetic acid. The eluent B contains 20 mM KH₂PO₄, and the pH is adjusted to 5 with acetic acid, and the elution volume is 20 CV.

[0060] Furthermore, the molecular weight of the ultrafiltration membrane used for ultrafiltration desalting is 30 - 100 KDa, preferably 50 KDa.

[0061] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the present invention, a special signal peptide sequence sPC1 and a folding domain sequence Foldon are respectively fused to the N - terminal and C - terminal of the full - length type III human collagen α1 chain sequence to promote the formation of the triple - helix structure, thereby obtaining a recombinant full - length triple - helix recombinant type III human collagen and its coding gene fragment.

[0062] (2) The chassis host bacteria of the present invention are modified, including introducing the key molecular chaperone peptidyl - prolyl cis - trans isomerase FKBP22 gene and the human prolyl - 4 - hydroxylase gene P4Hα1β in the biosynthesis process of human - sourced collagen to enhance the formation of the triple - helix structure.

[0063] (3) The present invention transforms the recombinant full - length triple - helix recombinant type III human collagen coding gene fragment into the modified chassis strain, and constructs a recombinant Pichia pastoris genetic engineering bacterium co - expressing molecular chaperone, hydroxylase and recombinant full - length collagen.

[0064] (4) The recombinant Pichia pastoris genetic engineering bacterium provided by the present invention can obtain a relatively high expression level (>0.5 g / L) of recombinant full - length triple - helix recombinant type III human collagen through high - density fermentation; the purification process provided by the present invention is relatively simple. The triple - helix structure type III recombinant human collagen obtained after enzymatic hydrolysis and purification does not contain heterologous sequences, and has a relatively high hydroxyproline content and thermal stability, approaching natural human collagen.

[0065] (5) The recombinant full-length triple-helix recombinant type III human collagen provided by the present invention has an amino acid sequence 100% identical to that of natural human-derived type III collagen, and at the same time has a stable triple-helix structure, high thermal stability, and can meet the applications in the fields of medical beauty filling, tissue repair and other medical health and medical beauty fields, and is more suitable for developing into relevant class III medical device products. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 It is the map of the HHP-sgRNA plasmid.

[0067] Figure 2 It is the map of the recombinant editing plasmid pHTXp-sgRNA-HsCas9-CENARS-△ku70.

[0068] Figure 3 It is the map of the recombinant editing plasmid pHTXp-sgRNA-HsCas9-CENARS-lnt39.

[0069] Figure 4 It is the expression of the target protein in the shake flask fermentation of the recombinant bacterium X33△ku70:FKBP22 / α1β-3A1ncF-68#. Among them, A is the detection result of non-reducing SDS-PAGE; B is the detection result of non-reducing SDS-PAGE after treatment with pepsin; C is the detection result of reducing SDS-PAGE.

[0070] Figure 5 It is the expression of the target protein in the high-density fermentation of the recombinant bacterium X33△ku70:FKBP22 / α1β-3A1ncF-68# in a 5L fermenter and after separation and purification. Among them, A is the electrophoretogram of the target protein under the high-density fermentation conditions of a 5L fermenter; B is the electrophoretogram of the target protein after separation and purification.

[0071] Figure 6 CD detection map of the target protein.

[0072] Figure 7 Thermal stability detection map of the target protein.

[0073] Figure 8 It is the transmission electron microscope detection map of the target protein.

[0074] FIGURE LEGEND: Figures 1 to 3In the figure, ori: vector replication origin; PARS1: protease-activated receptor 1; CYC TT: cytochrome C protein terminator; HygB: hygromycin resistance gene; TEF promoter: transcription enhancer factor promoter; AOX1TT: alcohol oxidase 1 terminator; HDV: 3'-cut hepatitis delta virus nuclease sequence; gRNA scaffold: expressed together with N20 (recognition sequence of editing site) to form sgRNA (guide RNA) structure, which can bind to cas9 protein (Ishino nuclease 9) and target to the genome; N20-gRNA1: recognition sequence of editing site required for ku70 gene knockout; N20-gRNA2: recognition sequence of editing site required for lnt39 gene knockout; HH: 5'-cut hammerhead ribozyme sequence; HTXp: a bidirectional promoter for yeast expression; Cas: human codon-optimized Cas9 protein (Ishino nuclease 9) gene; SV40 NLS: a nuclear localization signal derived from simian virus 40 tumor antigen; DAS1t: yeast-derived terminator; AmpR promoter: ampicillin resistance gene promoter; AmpR: ampicillin resistance gene; SUP4 terminator: ochre mutation suppressor gene terminator; Factor Xa site: coagulation factor Xa cleavage site; pSER: phosphorylated serine. DETAILED DESCRIPTION

[0075] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0076] Example 1 Construction of a recombinant expression vector expressing the full-length collagen α1 chain 1. According to the human type III collagen α1 chain preprotein sequence included in the GenBank database (NCBI Reference Sequence: NP_000081.2), the mature collagen α1 chain sequence (amino acids 149-1221) containing the N-terminal telopeptide, the classic triple helical domain and the C-terminal telopeptide was selected as the amino acid sequence of the full-chain type III human collagen, and a mammalian secretory signal peptide sequence sPC1 (SEQ ID NO: 1) was connected to its N-terminus, and the folded structure sequence Foldon (SEQ ID NO: 2) derived from phage T4 fibrin was fused to its C-terminus to obtain a fusion protein (SEQ ID NO: 3); then the fusion protein sequence was codon optimized according to the codon usage preference of Pichia pastoris, and the coding gene fragment sPC1-3A1ncF (SEQ ID NO: 4) of the recombinant full-chain triple helical recombinant type III human collagen was synthesized.

[0077] 2. Double digest the pPICZαA plasmid with BstBI and Sal I enzymes to obtain the linearized pPICZαA vector 1. Connect the sPC1-3A1ncF fragment to the linearized pPICZαA vector 1 through seamless cloning technology to obtain the pPICZ-sPC1-3A1ncF recombinant vector.

[0078] Example 2 Construction of a recombinant prolyl-4-hydroxylase recombinant expression vector 1. According to the codon usage preference of Pichia pastoris, codon-optimize the α1 subunit (P4Hα1, NCBI Reference Sequence: NP_000908.2) and β subunit (P4Hβ, NCBI Reference Sequence: NP_000909.2) of human prolyl-4-hydroxylase to obtain the optimized P4Hα1 subunit coding gene fragment (SEQ ID NO: 5) and the optimized P4Hβ subunit coding gene fragment (SEQ ID NO: 6).

[0079] 2. Double digest the pPIC9K plasmid with BamH I and EcoR I enzymes to obtain the linearized pPIC9K vector 1; double digest the pPICZαA plasmid with EcoR I and Sal I enzymes to obtain the linearized pPICZαA vector 2.

[0080] 3. Use seamless cloning technology to connect the optimized P4Hα1 subunit coding gene fragment between the BamH I and EcoR I restriction sites of the linearized pPIC9K vector 1 to obtain the pPIC9K-P4Hα1 recombinant vector; connect the optimized P4Hβ subunit coding gene fragment between the EcoR I and Sal I restriction sites of the linearized pPICZαA vector 2 to obtain the pPICZαA-P4Hβ recombinant vector.

[0081] 4. Digest the pPIC9K-P4Hα1 recombinant vector with AatII endonuclease to obtain the linearized pPIC9K-P4Hα1 recombinant vector.

[0082] 5. Amplification of the P4Hβ subunit expression cassette fragment (SEQ ID NO: 7): Using the pPICZαA-P4Hβ recombinant vector as a template, PCR amplification was performed using primers P4Hβ-F (SEQ ID NO: 8) and P4Hβ-R (SEQ ID NO: 9). The PCR amplification system was: 2×Phanta Max Buffer 12.5μL, dNTP Mix 1μL, P4Hβ-F (concentration 10μM) 1μL, P4Hβ-R (concentration 10μM) 1μL, pPICZαA-P4Hβ recombinant vector 1μL and Phanta Max Super-Friendly DNA ploymerse 1μL, and the total PCR amplification system was supplemented with ddH2O to 25μL. The PCR amplification program was: 95℃3min; 95℃15s, 58℃30s, 72℃100s, 72℃5min, 30 cycles.

[0083] 6. Using seamless cloning technology, the P4Hβ subunit expression cassette fragment was connected to the linearized pPIC9K-P4Hα1 recombinant vector to obtain the pPIC9K-P4Hα1β recombinant vector carrying both the P4Hα1 subunit and the P4Hβ subunit expression cassettes.

[0084] Example 3 Construction of a recombinant expression vector expressing the molecular chaperone peptidylprolyl cis-trans isomerase FKBP22 1. According to the codon usage preference of Pichia pastoris, the molecular chaperone peptidylprolyl cis-trans isomerase FKBP22 (NCBI Reference Sequence: NP_060416.1) was codon optimized to obtain the optimized FKBP22 encoding gene fragment (SEQ ID NO: 10).

[0085] 2. The pPIC9K plasmid was double-digested with BamHI and Not I enzymes to obtain a linearized pPIC9K vector 2. The optimized FKBP22 encoding gene fragment was connected between the BamHI and Not I restriction sites of the linearized pPIC9K vector 2 by seamless cloning technology to obtain a pPIC9K-FKBP22 recombinant vector.

[0086] Example 4 Construction of Pichia pastoris X33 with ku70 gene knocked out (X33△ku70) 1. Construction of pHTXp-sgRNA-HsCas9-CENARS-△X recombinant vector (1) Synthesize the sgRAN expression cassette flanked by ribozymes (HTXp-N20-gRNA expression cassette, SEQ ID NO: 11). Connect the sgRAN expression cassette flanked by ribozymes between the EcoRI and HindIII sites of the pUC19 vector to obtain the pUC19-HTXp-N20-sgRNA recombinant vector.

[0087] (2) Amplify the HTXp-N20-gRNA expression cassette: Replace the template in the PCR amplification system in Example 2 with the pUC19-HTXp-N20-sgRNA recombinant vector, and replace the primers with HTXp-sgRNA-F (SEQ ID NO: 12) and AOX1t-sgRNA-R (SEQ ID NO: 13), with other components and contents remaining unchanged. The PCR amplification program is: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 60 s, 72°C for 5 min, for 30 cycles.

[0088] Amplify Figure 1 The pHHP-sgRNA backbone vector as shown (obtain the fragment between the AmpR promoter and HygB in the pHHP-sgRNA plasmid, excluding the entire sgRNA expression cassette sequence): Replace the template in the PCR amplification system in Example 2 with the pHHP-sgRNA plasmid, and replace the primers with pHHP-F (SEQ ID NO: 14) and pHHP-R (SEQ ID NO: 15), with other components and contents remaining unchanged. The PCR amplification program: 95°C for 3 min; 95°C for 15 s, 55°C for 15 s, 72°C for 80 s, 72°C for 5 min, for 30 cycles.

[0089] Use seamless cloning technology to connect the HTXp-N20-gRNA expression cassette between the AmpR promoter and TEF promoter of the pHHP-sgRNA backbone vector to obtain the pHTXp-sgRNA recombinant vector.

[0090] (3) Amplify the yeast-derived terminator DAS1t fragment (SEQ ID NO: 16): Replace the template in the PCR amplification system in Example 2 with Pichia pastoris genomic DNA, and replace the primers with DAS1t-F (SEQ ID NO: 17) and DAS1t-R (SEQ ID NO: 18), with other components and contents remaining unchanged. The PCR amplification program: 95°C for 3 min; 95°C for 15 s, 55°C for 15 s, 72°C for 20 s, 72°C for 5 min, for 30 cycles.

[0091] Amplify the HsCas9 fragment (SEQ ID NO: 19): Replace the template in the PCR amplification system in Example 2 with the pGAP-Cas9 plasmid, and replace the primers with HsCas9-F (SEQ ID NO: 20) and HsCas9-R (SEQ ID NO: 21). Keep other components and their contents unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 90 s, 72°C for 5 min, for 30 cycles.

[0092] Amplify the pHTXp-sgRNA backbone vector: Replace the template in the PCR amplification system in Example 2 with the pHTXp-sgRNA plasmid, and replace the primers with pHTXp-sgRNA-F (SEQ ID NO: 22) and pHTXp-sgRNA-R (SEQ ID NO: 23). Keep other components and their contents unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 90 s, 72°C for 5 min, for 30 cycles.

[0093] Using seamless cloning technology, ligate the DAS1t fragment, HsCas9 fragment and pHTXp-sgRNA backbone vector. The HsCas9 fragment is located counterclockwise downstream of the HTXp promoter of the pHTXp-sgRNA backbone vector, and the DAS1t fragment follows immediately, to obtain the pHTXp-sgRNA-HsCas9 recombinant vector.

[0094] (4) Amplify the linearized pHTXp-sgRNA-HsCas9 vector: Replace the template in the PCR amplification system in Example 2 with the pHTXp-sgRNA-HsCas9 recombinant vector, and replace the primers with pHTXp-sgRNA-HsCas9-F (SEQ ID NO: 24) and pHTXp-sgRNA-HsCas9-R (SEQ ID NO: 25). Keep other components and their contents unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 100 s, 72°C for 5 min, for 30 cycles.

[0095] Synthesize the CEN / ARS sequence (SEQ ID NO: 26) from Saccharomyces cerevisiae, and ligate the CEN / ARS sequence between the EcoRI and HindIII sites of the pUC19 vector to obtain the pUC19-CEN / ARS recombinant vector.

[0096] Amplify the CEN / ARS fragment: Replace the template in the PCR amplification system in Example 2 with the pUC19-CENARS recombinant vector, and replace the primers with CENARS-F (SEQ ID NO: 27) and CENARS-R (SEQ ID NO: 28), while keeping other components and their contents unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 10 s, 72°C for 5 min, for 30 cycles.

[0097] Using seamless cloning technology, ligate the linearized pHTXp-sgRNA-HsCas9 vector with the CEN / ARS fragment to obtain a gene editing tool plasmid that can target and edit the N20 sequence on the X gene (i.e., the target gene): pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector.

[0098] 2. Construct a knockout plasmid for the ku70 gene (Gene ID: 8199462) on the Pichia pastoris genome.

[0099] Using the online database at https: / / chopchop.cbu.uib.no / , design the recognition sequence N20-gRNA1 sequence (SEQ ID NO: 29) for the editing site required for ku70 gene knockout.

[0100] Amplify the Δku70-N20-sgRNA fragment (SEQ ID NO: 30) that can express and target the specific site on the ku70 gene: Replace the template in the PCR amplification system in Example 2 with the pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector, and replace the primers with ku70-N20-F (SEQ ID NO: 31) and Δku70-N20-R (SEQ ID NO: 32), while keeping other components and their contents unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 65°C for 20 s, 72°C for 15 s, 72°C for 3 min, for 30 cycles.

[0101] Digest the pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector with Not I and Pem I endonucleases to obtain the linearized pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector. Use seamless cloning technology to ligate the Δku70-N20-sgRNA fragment between the Not I and Pem I digestion sites of the linearized pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector to obtain the pHTXp-sgRNA-HsCas9-CENARS-Δku70 recombinant vector with the function of targeting and editing the ku70 gene ( Figure 2 )

[0102] Amplify the upstream homologous arm fragment △ku70-up (SEQ ID NO: 33) of the ku70 gene editing site: Replace the template in the PCR amplification system in Example 2 with the Pichia pastoris X33 genome, replace the primers with △ku70-up-F (SEQ ID NO: 34) and △ku70-up-R (SEQ ID NO: 35), and keep other components and their contents unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 30 s, 72°C for 20 s, 72°C for 3 min, 30 cycles.

[0103] Amplify the downstream homologous arm fragment △ku70-dw (SEQ ID NO: 36) of the ku70 gene editing site: Replace the template in the PCR amplification system in Example 2 with the Pichia pastoris X33 genome, replace the primers with △ku70-dw-F (SEQ ID NO: 37) and △ku70-dw-R (SEQ ID NO: 38), and keep other components and their contents unchanged. The PCR amplification program is the same as that for amplifying the upstream homologous arm fragment of the ku70 gene editing site.

[0104] Amplify the △ku70-Donor fragment (SEQ ID NO: 39) fused with the upstream and downstream homologous arms of the ku70 gene editing site: Replace the template in the PCR amplification system in Example 2 with the upstream homologous arm fragment and the downstream homologous arm fragment of the ku70 gene editing site (1 μL each), replace the primers with △ku70-up-F and △ku70-dw-R, and perform Overlap PCR amplification, keeping other components and their contents unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 30 s, 72°C for 30 s, 72°C for 3 min, 30 cycles.

[0105] 3. Prepare Pichia pastoris X33 competent cells: Pick a single colony of Pichia pastoris X33 and inoculate it into 5 mL of YPD liquid medium. Incubate overnight (16 - 24 h) at 29°C with shaking at 250 rpm to obtain a culture. Take 50 μL of the culture and inoculate it into 50 mL of YPD liquid medium. Incubate at 29°C with shaking at 250 rpm until the OD of Pichia pastoris X33 600The value reaches 1 - 1.5, centrifuge at 5000 rpm for 5 min, discard the supernatant, and collect the thalli; resuspend the thalli with 50 mL of ice-precooled sterile double-distilled water, centrifuge at 5000 rpm for 5 min, discard the supernatant, and collect the thalli. Resuspend the thalli with 25 mL of ice-precooled sterile double-distilled water again, centrifuge at 5000 rpm for 5 min, discard the supernatant, and collect the thalli; then resuspend the thalli with 20 mL of ice-precooled 1 M sorbitol solution, centrifuge at 5000 rpm for 5 min, discard the supernatant, and collect the thalli; finally, resuspend the thalli with 0.3 mL of ice-precooled 1 M sorbitol solution to obtain the Pichia pastoris X33 competent cell suspension with a final volume of 0.5 mL. Aliquot it into 80 μL per portion and store at -80 °C for later use.

[0106] 4. Electroporation: Take 50 μL of a tube of Pichia pastoris X33 competent cell suspension (OD 600 = 40 - 100) and place it on ice to thaw. Add 2 μg of the △ku70-Donor fragment and 2 μg of the pHTXp-sgRNA-HsCas9-CENARS-△ku70 recombinant vector, gently pipette and mix well, transfer it to a pre-cooled electroporation cuvette, and continue to pre-cool on ice for 5 min. Set the voltage for electroporation at 1.5 kV, and the electroporation time is 4 - 6 mSec. After electroporation, quickly add 1 mL of ice-precooled 1 M sorbitol solution, gently pipette and mix well, and transfer it to a 1.5 mL sterile centrifuge tube. Incubate it statically in a 30 °C incubator for 1 - 2 h to obtain the incubated electroporated bacterial suspension.

[0107] 5. Coating and culturing: Take 200 μL of the electroporated bacterial suspension and spread it on a YPD plate medium supplemented with hygromycin at a final concentration of 0.3 mg / mL. Incubate it inverted in a 30 °C incubator for 2 - 3 days until individual colonies appear. Through colony PCR identification, Pichia pastoris X33△ku70 with the ku70 gene successfully knocked out is obtained.

[0108] Example 5 Construction of Pichia pastoris X33 with the molecular chaperone FKBP22 gene knocked in and the ku70 gene knocked out 1. Using the online database at https: / / chopchop.cbu.uib.no / , the recognition site N20-gRNA2 sequence (SEQ ID NO: 41) required for gene editing at the lnt39 gene sequence (PAS_chr1-4_0294...PAS_chr1-4_0295, SEQ ID NO: 40) on the Pichia pastoris genome was designed.

[0109] Amplify the Δlnt39-N20-sgRNA fragment (SEQ ID NO: 42) that can express and specifically recognize a site on the lnt39 gene sequence: Replace the template in the PCR amplification system in Example 2 with the pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector, and replace the primers with lnt39-N20-F (SEQ ID NO: 43) and lnt39-N20-R (SEQ ID NO: 44), with other components and contents remaining unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 65°C for 20 s, 72°C for 15 s, 72°C for 3 min, for 30 cycles.

[0110] Use seamless cloning technology to ligate the lnt39-N20-sgRNA fragment between the Not I and Pem I restriction enzyme cleavage sites of the linearized pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector to obtain the pHTXp-sgRNA-HsCas9-CENARS-lnt39 recombinant vector with the function of targeted editing of the lnt39 gene sequence ( Figure 3 ).

[0111] 2. Amplify the upstream homologous arm fragment (SEQ ID NO: 45) of the lnt39 gene editing site: Replace the template in the PCR amplification system in Example 2 with the Pichia pastoris X33 genome, and replace the primers with lnt39-up-F (SEQ ID NO: 46) and lnt39-up-R (SEQ ID NO: 47), with other components and contents remaining unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 65°C for 20 s, 72°C for 15 s, 72°C for 5 min, for 30 cycles.

[0112] Amplify the downstream homologous arm fragment (SEQ ID NO: 48) of the lnt39 gene editing site: Replace the template in the PCR amplification system in Example 2 with the Pichia pastoris X33 genome, and replace the primers with lnt39-dw-F (SEQ ID NO: 49) and lnt39-dw-R (SEQ ID NO: 50), with other components and contents remaining unchanged. The PCR amplification program is the same as that for amplifying the upstream homologous arm fragment of the lnt39 gene editing site.

[0113] 3. Amplify the expression cassette fragment of the molecular chaperone FKBP22 gene (P AOX1 -FKBP22, SEQ ID NO: 51): Replace the template in the PCR amplification system in Example 2 with the pPIC9K-FKBP22 recombinant expression vector constructed in Example 3, and replace the primers with P AOX1 -FKBP22-F (SEQ ID NO: 52) and P AOX1-FKBP22-R (SEQ ID NO: 53), with other components and their contents remaining unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 55°C for 20 s, 72°C for 75 s, 72°C for 5 min, for 30 cycles.

[0114] Amplify Δlnt39-P AOX1 -FKBP22-Donor fragment (SEQ ID NO: 54): Replace the template in the PCR amplification system in Example 2 with the upstream and downstream homologous arm fragments of the lnt39 gene editing site and the expression cassette fragment of the FKBP22 gene (1 μL each), and replace the primers with lnt39-up-F and lnt39-dw-R, with other components and their contents remaining unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 30 s, 72°C for 90 s, 72°C for 5 min, for 30 cycles.

[0115] 4. According to the method of Example 4, replace Pichia pastoris X33 with Pichia pastoris X33Δku70 to prepare Pichia pastoris X33Δku70 competent cells. Take 50 μL of a tube of Pichia pastoris X33Δku70 competent cell suspension (OD 600 = 40 - 100) and thaw it on ice. Add 2 μg of Δlnt39-P AOX1 -FKBP22-Donor fragment and 2 μg of the pHTXp-sgRNA-HsCas9-CENARS-lnt39 recombinant vector, gently pipette and mix well, and perform electrotransformation, plating culture and identification according to the method of Example 4 to obtain Pichia pastoris X33Δku70:FKBP22 in which the expression cassette of the FKBP22 gene has been successfully knocked in.

[0116] Example 6 Construction of a recombinant strain co-expressing prolyl-4-hydroxylase and full-length collagen and its culture 1. Construction of a recombinant strain co-expressing prolyl-4-hydroxylase and full-length collagen (1) Digest the pPIC9K-P4Hα1β recombinant vector in Example 2 with BspE I endonuclease to obtain a linearized pPIC9K-P4Hα1β recombinant vector. According to the method of Example 4, replace Pichia pastoris X33 with Pichia pastoris X33Δku70:FKBP22 to prepare Pichia pastoris X33Δku70:FKBP22 competent cells. Take 100 μL of a tube of Pichia pastoris X33Δku70:FKBP22 competent cell suspension (OD 600=40~100) was thawed on ice, 4 μg of linearized pPIC9K-P4Hα1β recombinant vector was added, and the mixture was mixed by gently blowing, and electroporation, plating culture and identification were performed according to the method of Example 4 to obtain Pichia pastoris X33△ku70:FKBP22 / α1β with single copy insertion of P4Hα1 subunit and P4Hβ subunit expression cassette. Among them, 0.3 mg / mL hygromycin in the plating culture medium was replaced with 0.5 mg / mL neomycin G418.

[0117] (2) The pPICZ-sPC1-3A1ncF recombinant vector of Example 1 was digested with Sac I endonuclease to obtain a linearized pPICZ-sPC1-3A1ncF recombinant vector. According to the method of Example 4, Pichia pastoris X33 was replaced with Pichia pastoris X33△ku70:FKBP22 / α1β competent cells. Take 100 μL of a tube of Pichia pastoris X33△ku70:FKBP22 / α1β competent cell suspension (OD 600 =40~100) was placed on ice to thaw, 4 μg of the linearized pPICZ-sPC1-3A1ncF recombinant vector was added, and the mixture was gently pipetted and mixed. The electroporation and plating culture were performed according to the method of Example 4 until a single colony appeared. The plating culture medium was a medium containing Zeocin with a final concentration of 0.1 mg / mL. TM YPDS plates.

[0118] (3) Use a 10 μL pipette to carefully pick up the single colonies with good growth in step (2) and resuspend and mix them in 5 μL of sterile water to obtain a resuspended bacterial solution; then take 1 μL of the resuspended bacterial solution and copy it on the plate with different final concentrations of Zeocin (0.5, 1 and 1.5 mg / mL). TM YPDS plates were placed in a 30°C incubator and inverted for 2-3 days until a single colony appeared. Colony PCR was performed to confirm that the full-chain collagen gene was successfully integrated into the Pichia pastoris genome, thereby screening the Pichia pastoris X33△ku70:FKBP22 / α1β-3A1ncF that co-expressed prolyl-4-hydroxylase and full-chain collagen gene. 5 mL of overnight culture was taken to detect its copy number and OD 600 .

[0119] The results are shown in Table 1. A total of 5 recombinant strains were obtained, among which the X33△ku70:FKBP22 / α1β-3A1ncF-68# recombinant strain (No. 68) had a better expression effect.

[0120] Table 1 2. Shake flask fermentation and expression identification (1) Pick a single colony of the identified X33△ku70:FKBP22 / α1β-3A1ncF and inoculate it into 10 mL of YPD medium. Incubate overnight at 29°C and 250 rpm until OD 600 = 3 - 5 to obtain an overnight culture.

[0121] (2) Inoculate the overnight culture into 30 mL of BMGY medium at an inoculation ratio of 1%. Incubate at 29°C and 250 rpm until OD 600 = 4 - 6. Centrifuge the cells at 5000 rpm for 5 min at room temperature to collect the cell pellet. Resuspend the cell pellet in BMMY medium to an OD600 of 1 to obtain a resuspended cell suspension. Transfer 30 mL of the resuspended cell suspension to a 250 mL conical flask, and add L-ascorbic acid at a final concentration of 100 mg / L and FeSO4 at 0.6 mM as cofactors for the hydroxylase. Add methanol at a final concentration of 1% (v / v) and L-ascorbic acid at 100 mg / mL to the culture system every 24 h and continue the induction culture. After the induction culture is completed, collect the cell suspension and detect the expression of the target protein by reducing and non-reducing SDS-PAGE. Digest the cell suspension with pepsin and detect its pepsin tolerance by non-reducing SDS-PAGE.

[0122] The electrophoresis results are shown in Figure 4 A, Figure 4 B in Figure 4 and C in

[0123] The obtained X33△ku70:FKBP22 / α1β-3A1ncF-68# was named Komagataella phaffii XF / 3A1ncF-68# and was deposited at the Guangdong Provincial Microbial Culture Collection Center on April 21, 2025. The deposit number is: GDMCC No: 66181, and the deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0124] Example 7 High-density fermentation of the co-expression engineering bacteria (1) Streak the recombinant bacteria of X33△ku70:FKBP22 / α1β-3A1ncF-68# from Example 6 on a YPD slant medium and culture in a 29°C constant temperature incubator for 3 days; (2) Preparation of the primary seed liquid mother liquor: Scrape the strain cultured on the slant culture medium in step (1) and transfer it to 10 mL of YPD liquid medium. Culture at 29 °C and 250 rpm for 16 - 20 h until the OD of the recombinant strain 600 reaches 3 - 5 to obtain the primary seed liquid mother liquor.

[0125] (3) Preparation of the secondary seed liquid: According to an inoculation amount of 1% (v / v) (i.e., the volume concentration of the primary seed liquid mother liquor in the secondary seed liquid), take the primary seed liquid mother liquor and transfer it to 200 mL of BMGY liquid medium. Culture at 29 °C and 250 rpm for 16 - 24 h until the OD of the recombinant strain 600 reaches 4 - 6.

[0126] (4) Inoculation and glycerol culture stage: Add 200 mL of the secondary seed liquid to a 5 L fermenter containing 1.8 L of inorganic salt medium according to an inoculation amount of 10% (v / v) (i.e., the volume concentration of the secondary seed liquid in the 1.8 L inorganic salt medium). Culture at 28 °C, pH 5.5, an initial rotation speed of 300 rpm, and an aeration rate of 1 vvm, and control the dissolved oxygen content at 20% - 30%. The inorganic salt medium consists of 20 g / L KH2PO4, 6.85 g / L K2HPO4, 6.9 g / L NH4H2PO4, 1.2 g / L CaSO4, 12.5 g / L MgSO4·7H2O, 40 g / L glycerol, and 4.5 ml / L PTM1 (trace element composite mother liquor).

[0127] (5) Glycerol fed-batch culture stage: After the dissolved oxygen content in step (4) rises to 70%, start feeding with the glycerol feed solution at a feeding rate of 8.4 g / min, maintain the pH at 5, and the dissolved oxygen content at 30%; stop feeding when the wet weight of the bacteria reaches 300 - 350 g / L; the glycerol feed solution consists of 12 mL / L PTM1 and 50% (v / v) sterile glycerol.

[0128] (6) Methanol fed-batch induction culture stage: After the dissolved oxygen content in step (5) rises to 80%, start feeding and culturing with the methanol feed solution and the accessory feed solution. Set the initial rate of the methanol feed solution at 3.6 g / h, adjust the feeding rate to 7.2 g / h after 1 h of fed-batch culture, and then adjust the feeding rate to 8.4 g / h after another 1 h of fed-batch culture. End the fermentation after 60 - 72 h of methanol fed-batch induction culture. The methanol feed solution consists of PTM1 and methanol, and the concentration of PTM1 is 12 mL / L. The accessory feed solution consists of 20 g / L casein peptone, 2 g / L L-ascorbic acid, and 1 g / L α-ketoglutaric acid, and it is fed at a constant rate of 10 mL / h throughout the fed-batch culture process. Observe the change in dissolved oxygen during the whole process and control the dissolved oxygen content at 20 - 40%.

[0129] (7) The SDS-PAGE electrophoresis results of the target protein after cultivation in a 5L fermenter are as follows Figure 5 As shown in A, the expression level of the obtained recombinant type III human collagen with a full-chain triple-helix structure exceeds 0.5 g / L.

[0130] Example 8 Isolation, purification and preparation of recombinant triple helical collagen (1) After the culture of Example 7 was completed, the fermentation broth was cooled (below 10°C) and centrifuged (8000 rpm, 10 min) to collect the bacterial sludge.

[0131] (2) The bacterial sludge from step (1) was mixed with a homogenized lysis buffer (50 mM Tris / HCl buffer, pH 7, containing 0.2 M NaCl and 15% v / v glycerol), and the mixture was fully resuspended in a ratio of 20 g of bacterial sludge to 100 mL of homogenized lysis buffer. The mixture was then crushed three times at 4°C and 900 bar. After the crushing, the mixture was centrifuged at 10,000 rpm and 4°C for 30 min, and the precipitate was collected.

[0132] (3) The precipitate from step (2) was mixed with an acidic resolution solution (0.1 M acetic acid and 0.15 M NaCl) and fully resuspended. The ratio of precipitate to acidic resolution solution was 1 g:50 mL. Pepsin was then added at a final concentration of 0.2 mg / mL and digested at 4°C for 12-16 h to obtain an enzymatic hydrolyzate.

[0133] (4) Centrifuge the enzymatic hydrolyzate of step (3) at 5000 rpm for 5 min at room temperature, collect the supernatant, adjust the pH of the supernatant to 5 with 0.1 M KOH, and add NaCl to a final concentration of 0.5 M to obtain a chromatographic loading clear solution; (5) Protein chromatography: First, equilibrate a hydrophobic chromatography column (brand: Bio-LINK, model: Maxtar Butyl HR) with 5CV buffer A (20mM KH2PO4, 0.5M NaCl, pH adjusted to 5 with acetic acid); after equilibration, load the chromatographic loading clarified liquid from step (4), and after loading, rinse and equilibrate the column with buffer A until A220 drops to the baseline and the conductivity is stable; set a linear gradient from 0 to 100% eluent B (20mM KH2PO4, pH adjusted to 5 with acetic acid) for elution, the elution volume is 20CV, and collect the eluate during the elution; use SDS-PAGE electrophoresis to detect the eluate, the results are as follows: Figure 5 As shown in B, the purity of the target protein is significantly improved after chromatography purification.

[0134] (6) Desalting the target protein in step (5) using an ultrafiltration membrane with a molecular weight of 50 KDa, collecting the concentrate, and freeze-drying it using a freeze dryer to obtain a recombinant type III human collagen freeze-dried powder with a full-chain triple helix structure and a purity greater than 95%.

[0135] Example 9 Structural Characterization of Recombinant Triple-Helix Collagen (1)Mass spectrometry detection: Amino acid composition analysis was performed on the purified full-length triple-helix recombinant type III human collagen obtained in Example 8. The results showed that the amino acid coverage of the full-length triple-helix recombinant type III human collagen reached 100%, containing 9.55% 4-hydroxyproline.

[0136] (2)Circular dichroism detection: Set the parameters: starting wavelength = 190 nm, ending wavelength = 260 nm, step size = 1 nm, repeat 1 time, collect at room temperature, recording time = 1 s / point, cuvette width 0.1 cm. The blank control solution (0.2 M acetic acid solution) and the sample solution (recombinant type III human collagen with different concentrations of full-length triple-helix structure prepared with 0.2 M acetic acid solution) were loaded in sequence, and each sample was measured three times repeatedly, and the average value of the three scans was calculated. The results are as Figure 6 shown. The recombinant type III human collagen with full-length triple-helix structure prepared in the present invention has a negative peak at 195 nm and a maximum positive absorption peak at 221 nm, which conforms to the CD characteristics of triple-helix collagen. Therefore, it is judged that the recombinant type III human collagen with full-length triple-helix structure prepared in Example 8 has a triple-helix structure.

[0137] (3)Thermal stability detection: The recombinant type III human collagen with full-length triple-helix structure prepared in Example 8 was dissolved in 20 mM PB buffer (pH 7.4), and the final protein concentration was 2 mg / mL. It was incubated at different temperatures in a metal bath (25℃, 27℃, 30℃, 32℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 42℃ and 45℃) for 5 min; samples were taken for circular dichroism spectroscopy (CD) detection. The results are as Figure 7 shown. In the range of 25℃ to 36℃, there is no obvious denaturation trend, and the protein has good heat stability, approaching the thermal stability of natural human type III collagen (37 - 40℃).

[0138] (4)Fibroblast-forming ability detection: The recombinant type III human collagen with full-length triple-helix structure in Example 8 was redissolved in 20 mM phosphate buffer (pH 7.4, containing 0.15 M NaCl and 1 mM CaCl2) to obtain a 1 mg / mL sample solution, and incubated at 4℃ for 24 h; 20 μL of the sample solution was dropped on a clean copper mesh, left standing for 5 min, the sample was carefully sucked away with a clean filter paper, then negatively stained with 2 g / 100 mL uranyl acetate for 5 min, the excess dye was sucked away with a filter paper, and finally rinsed three times with pure water and air-dried naturally to prepare a TEM sample, and the sample was detected using a transmission electron microscope. The results are as Figure 8As indicated by the black arrow, the prepared recombinant type III human collagen can form a typical structural feature of collagen microfibrils with alternating light and dark patterns, which is consistent with the collagen characteristics reported in the mainstream literature (Holmes D F, Graham H K, Trotter J A, et al. STEM / TEM studies of collagen fibril assembly[J]. Micron, 2001, 32(3):273-285. DOI:10.1016 / S0968-4328(00)00040-8.). The length of the collagen fiber can exceed 1μm, and the width is between 100nm and 300nm. The collagen fiber has alternating light and dark cross striations, which are generated by the lateral aggregation of collagen molecules with a triple helix structure. The transmission electron microscopy results indicate that the collagen of the present invention has a triple helix structure and the ability to self-assemble into collagen fibers.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A recombinant Pichia pastoris genetically engineered bacterium, characterized in that: The recombinant Pichia pastoris genetically engineered bacteria expresses recombinant type III human collagen with a full chain length and a triple helical structure, and the ku70 gene of the recombinant Pichia pastoris genetically engineered bacteria is knocked out and a recombinant 4-proline hydroxylase encoding gene and a FKBP22 encoding gene are knocked in; The amino acid sequence of the recombinant type III human collagen with full chain length and triple helix structure is shown in SEQ ID NO: 3; The ku70 gene is numbered as Gene ID: 8199462 in NCBI; The recombinant 4-proline hydroxylase comprises a recombinant 4-proline hydroxylase α1 subunit and a recombinant 4-proline hydroxylase β subunit, the nucleotide sequence of the recombinant 4-proline hydroxylase α1 subunit encoding gene is shown in SEQ ID NO: 5, and the nucleotide sequence of the recombinant 4-proline hydroxylase β subunit encoding gene is shown in SEQ ID NO: 6; The nucleotide sequence of the FKBP22 encoding gene is shown in SEQ ID NO:

10.

2. The recombinant Pichia pastoris genetically engineered bacterium according to claim 1, characterized in that: The FKBP22 encoding gene is knocked into the lnt39 gene site of the Pichia pastoris chassis strain, the nucleotide sequence of the lnt39 gene is shown in SEQ ID NO: 40, and the Pichia pastoris chassis strain includes at least one of Pichia pastoris X33, Pichia pastoris GS115, Pichia pastoris KM71H and Pichia pastoris SMD1168.

3. The recombinant Pichia pastoris genetically engineered bacterium according to claim 2, characterized in that: The recombinant Pichia pastoris genetically engineered bacterium is Pichia pastoris komagataella phaffiiXF / 3A1ncF-68#, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 21, 2025, and its preservation number is: GDMCC No: 66181.

4. Use of the recombinant Pichia pastoris genetically engineered bacteria according to any one of claims 1 to 3 in the preparation of collagen.

5. The method for preparing the recombinant Pichia pastoris genetically engineered bacteria according to any one of claims 1 to 3, comprising the following steps: S1: Knock out the ku70 gene of Pichia pastoris X33 to obtain the recombinant Pichia pastoris genetically engineered bacteria X33△ku70; S2: knocking in the FKBP22 encoding gene into the recombinant Pichia genetically engineered bacteria X33Δku70 in step S1 to obtain the recombinant Pichia genetically engineered bacteria X33Δku70:FKBP22; S3: knocking in the recombinant 4-proline hydroxylase encoding gene into the recombinant Pichia genetically engineered bacteria X33△ku70:FKBP22 in step S2 to obtain the recombinant Pichia genetically engineered bacteria X33△ku70:FKBP22 / α1β; S4: Knock-in a recombinant human type III collagen encoding gene with a full chain length and a triple helix structure into the recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22 / α1β described in step S2 to obtain the recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22 / α1β-3A1ncF, wherein the nucleotide sequence of the recombinant human type III collagen encoding gene with a full chain length and a triple helix structure is shown in SEQ ID NO:

4.

6. A method for preparing collagen, characterized in that: Cultivating the recombinant Pichia pastoris genetically engineered bacteria according to any one of claims 1 to 3 to express collagen, wherein the collagen is a recombinant type III human collagen with a full chain length and a triple helix structure, and the amino acid sequence of the recombinant type III human collagen with a full chain length and a triple helix structure is shown in SEQ ID NO:

3.

7. The preparation method according to claim 6, characterized in that: The method for culturing the recombinant Pichia pastoris genetically engineered bacteria to express collagen comprises the following steps: S41: Cultivate the recombinant Pichia pastoris genetically engineered bacteria in a yeast extract peptone glucose medium until the OD of the recombinant Pichia pastoris genetically engineered bacteria reaches 0. 600 When the temperature reaches 3-5, the first-grade seed solution mother solution is obtained; S42: Inoculate the primary seed solution in step S41 into the BMGY medium and culture until the OD value of the recombinant Pichia pastoris genetically engineered bacteria reaches 600 When the temperature reaches 4-6, the secondary seed solution is obtained; S43: inoculating the secondary seed solution in step S42 into an inorganic salt culture medium for cultivation until the dissolved oxygen content of the culture system reaches more than 70%, adding feed solution 1 for feed culture until the wet weight of the bacteria reaches 300-350 g / L, wherein the feed solution 1 contains PTM1 and glycerol; S44: After culturing in step S43, the dissolved oxygen content of the culture system reaches more than 80%, and feeding solution 2 and feeding solution 3 are added for fed-batch culture, wherein the feeding solution 2 contains PTM1 and methanol; the feeding solution 3 contains casein peptone, L-ascorbic acid and α-ketoglutaric acid.

8. The preparation method according to claim 7, characterized in that: In step S43, the PTM1 concentration in the feed solution 1 is 4-12 mL / L, and the glycerol concentration is 10-50% v / v.

9. The preparation method according to claim 7, characterized in that: In step S44, the concentration of PTM1 in feed solution 2 is 5-20 mL / L, and feed solution 3 contains 10-30 g / L casein peptone, 2-20 g / L L-ascorbic acid, and 1-5 g / L α-ketoglutaric acid.

10. The preparation method according to claim 7, characterized in that: The collagen is separated and purified, including lysing the recombinant Pichia pastoris genetically engineered bacteria cultured in step S44, acid resolubilization, enzymatic hydrolysis, protein chromatography and ultrafiltration desalination.

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