Recombinant hydroxylated small molecule collagen and its expression system and preparation method

Abstract

The application provides a kind of recombinant hydroxylated small molecule collagen and its expression system and preparation method, belong to the field of bioengineering technology;The application utilizes synthetic biology, genetic engineering and biotechnology to co-express small molecule collagen and P4H, then carries out amino acid modification, hydroxylation is carried out at G-X-Y theory hydroxylation site, post-translational modification is carried out, and the recombinant hydroxylated small molecule collagen is obtained;The recombinant hydroxylated small molecule collagen of the application is stable in molecular structure by amino acid post-expression modification, and is more controllable in comparison with small molecule self-assembly after expression, which can balance transdermal absorption and biological structure stability;The recombinant hydroxylated small molecule collagen can realize industrial production, and has good application in the fields of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products and the like.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant hydroxylated small molecule collagen, its expression system, and preparation method. Background Technology

[0002] Collagen is an important biomaterial, a major component of the extracellular matrix (ECM). It can be produced by expressing recombinant collagen in insect cells or other mammalian cells, using genetic engineering to increase expression levels. However, this method is costly, has low yields, and produces large-molecule collagen. Large-molecule collagen is expensive and difficult to synthesize and separate; it is also prone to degradation, has poor stability, low yields, and is easily lost during production and separation, making the process complex. Furthermore, its applications are limited, making large-scale production difficult.

[0003] In recent years, mammalian cell culture has been costly, has a slow growth cycle, and produces low expression levels. Although secreted into the supernatant, the purification process is complex, resulting in significant product loss and hindering industrial-scale production. While colonic expression systems have improved yields, they have not yet reached commercial-scale production. Therefore, both of these expression methods result in low yields, poor protein stability, and easy degradation, limiting their application to laboratory research. Thus, there is a need to develop a more stable recombinant small-molecule collagen protein.

[0004] The triple helix of collagen is composed of repeating glycine-XY motifs, where X and Y positions can be any amino acid, typically proline or 4-hydroxyproline, respectively. Collagen is a procollagen molecule consisting of an amino-terminal propeptide, a short, non-helical n-terminal peptide, a central triple helix, a c-terminal peptide, and a carboxyl-terminal propeptide. Post-translational modifications of individual proa chains (hydroxylation of proline and lysine residues, glycosylation of lysine and hydroxylysine residues, and sulfation of tyrosine residues) cease due to triple helix formation. Hydroxylation of collagen proline residues catalyzed by collagen proline 4-hydroxylase (C-P4H) is crucial for the stability of the collagen triple helix.

[0005] Hydroxylation mainly relies on proline hydroxylase (P4H). Currently, there are no studies on the co-expression system of recombinant small molecule collagen with P4H enzyme. Therefore, it is necessary to develop a recombinant hydroxylated small molecule collagen to improve the stability and activity of recombinant small molecule collagen. Summary of the Invention

[0006] To address some shortcomings in existing technologies, this invention provides a recombinant hydroxylated small molecule collagen, its expression system, and preparation method. This invention utilizes synthetic biology, genetic engineering, and biotechnology to co-express small molecule collagen with P4H, followed by amino acid modification, hydroxylation at the theoretical GXY hydroxylation site, and post-translational modification to obtain the recombinant hydroxylated small molecule collagen. The recombinant hydroxylated small molecule collagen of this invention achieves molecular structural stability through amino acid post-expression modification, offering more controllable self-assembly compared to small molecule post-expression, and achieving a balance between transdermal absorption and biological structural stability. This recombinant hydroxylated small molecule collagen can be industrially produced and has excellent applications in pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics, and health products.

[0007] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0008] The present invention first provides a recombinant hydroxylated small molecule collagen, wherein the recombinant small molecule collagen is a hydroxylated modified recombinant small molecule collagen, and the recombinant small molecule collagen includes an amino acid sequence containing hydroxylation sites on the triple helix region of human type VII collagen or the α1 chain of human type III collagen.

[0009] Preferably, the amino acid sequence is as shown in i, ii, or iii:

[0010] i. The amino acid sequence is as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or the amino acid sequence is obtained by repeating the sequence shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 in series multiple times as the basic unit;

[0011] ii. The amino acid sequence is shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6;

[0012] iii. A protein derived from i or ii that has human collagen activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in i or ii, or an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the same amino acid sequence as the sequence shown in i or ii.

[0013] The present invention also provides a nucleic acid encoding the above-mentioned recombinant hydroxylated small molecule collagen.

[0014] Preferably, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO:7 to SEQ ID NO:9, or a degenerate sequence thereof.

[0015] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid.

[0016] The present invention also provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the above-mentioned nucleic acid, or the above-mentioned recombinant expression vector, or a recombinant engineered bacterium comprising a recombinant plasmid expressing P4H enzyme capable of expressing the above-mentioned recombinant hydroxylated small molecule collagen.

[0017] Preferably, the recombinant engineered bacteria includes a chassis strain for expressing the above-mentioned recombinant hydroxylated small molecule collagen, wherein the chassis strain expresses P4H enzyme.

[0018] Preferably, the chassis strain contains nucleic acid encoding the amino acids shown in SEQ ID No. 10 and SEQ ID No. 13.

[0019] Preferably, the recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC NO.33067, CGMCC NO.33068, and CGMCC NO.33069.

[0020] The present invention also provides a chassis strain for expressing the above-mentioned recombinant hydroxylated small molecule collagen, wherein the chassis strain expresses P4H enzyme.

[0021] Preferably, the host strain of the chassis strain includes one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha; preferably Pichia pastoris; more preferably Pichia pastoris X33 or Pichia pastoris KM71H.

[0022] This invention also provides a method for preparing the above-mentioned recombinant hydroxylated small molecule collagen, the preparation method comprising:

[0023] (1) Select and design the sequence of recombinant hydroxylated small molecule collagen, and then construct collagen tandem repeat sequence based on the recombinant hydroxylated small molecule collagen as the basic unit;

[0024] (2) Construct a recombinant expression vector for expressing collagen tandem repeat sequences;

[0025] Construct a recombinant vector expressing P4H enzyme;

[0026] (3) The recombinant expression vector expressing collagen tandem repeat sequence and the recombinant vector expressing P4H enzyme were electroporated into the host bacteria, screened and verified, and recombinant engineered bacteria expressing high copy number of recombinant hydroxylated small molecule collagen were obtained.

[0027] (4) The obtained recombinant engineered bacteria were fermented and induced to express the recombinant hydroxylated small molecule collagen.

[0028] Preferably, in step (1), the recombinant hydroxylated small molecule collagen is a hydroxylated modified recombinant small molecule collagen, and the recombinant small molecule collagen includes an amino acid sequence containing hydroxylation sites on the triple helix region of human type VII collagen or the α1 chain of human type III collagen.

[0029] The amino acid sequence is shown as i, ii, or iii:

[0030] i. The amino acid sequence is as shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or the amino acid sequence is obtained by repeating the sequence shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5 in series multiple times as the basic unit;

[0031] ii. The amino acid sequence is shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6;

[0032] iii. A protein derived from i or ii that has human collagen activity by substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in i or ii, or an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the same amino acid sequence as the sequence shown in i or ii.

[0033] Preferably, when the amino acid sequence of the recombinant hydroxylated small molecule collagen is as shown in i, the collagen tandem sequence contains 8 to 10 basic units of recombinant type VII collagen; there are sites for recognition and cleavage by Kex2, Ste13 or Kex1 enzymes between two adjacent basic units.

[0034] Preferably, the recognition and cleavage site includes KR or RR dual-basic amino acid residues, followed by EA, EAEA, or other amino acid residues that facilitate cleavage by Kex2, Ste13, or Kex1 enzymes.

[0035] Preferably, in step (1), the nucleic acid of the collagen tandem sequence includes the nucleotide sequence shown in SEQ ID NO:7 to SEQ ID NO:9, or its degenerate sequence.

[0036] Preferably, in step (2), the vectors of the recombinant expression vector and the recombinant plasmid include pPICZαB, pFLDα, and pPIC9K.

[0037] Preferably, the carrier is pPIC9K or pPICZαB.

[0038] Preferably, in step (2), the recombinant plasmid expressing the P4H enzyme contains nucleic acid encoding the amino acids shown in SEQ ID No. 10 and SEQ ID No. 13, a PTEF promoter element, and a PEM7 promoter element.

[0039] Preferably, in step (3), the host bacteria includes one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha.

[0040] Preferably, the host bacteria is Pichia pastoris, and more preferably Pichia pastoris X33 or Pichia pastoris KM71H.

[0041] Preferably, the host bacteria are CGMCC No.29601, CGMCC No.25815, or CGMCC No.258199.

[0042] Preferably, in step (3), the recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC NO.33067, CGMCC NO.33068, and CGMCC NO.33069.

[0043] The present invention also provides recombinant hydroxylated small molecule collagen prepared by the above method.

[0044] The present invention also provides an expression system for the above-mentioned recombinant hydroxylated small molecule collagen, the expression system comprising: a recombinant engineered bacterium expressing a recombinant plasmid of P4H enzyme and a collagen tandem sequence constructed based on the recombinant hydroxylated small molecule collagen as a basic unit.

[0045] The present invention also provides a composition comprising the above-mentioned recombinant hydroxylated small molecule collagen, or the above-mentioned recombinant hydroxylated small molecule collagen encoded by nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered bacteria, or the above-mentioned recombinant hydroxylated small molecule collagen prepared by the above-mentioned method; the composition comprises medical devices, biomaterials, tissue engineering products, and cosmetics.

[0046] The present invention also provides an article comprising the above-mentioned recombinant hydroxylated small molecule collagen, or the above-mentioned recombinant hydroxylated small molecule collagen encoded by nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered bacteria, or the above-mentioned recombinant hydroxylated small molecule collagen prepared by the above-mentioned method, or a composition; the article comprises medical devices, biomaterials, tissue engineering products, and cosmetics.

[0047] The present invention also provides the use of the above-mentioned recombinant hydroxylated small molecule collagen, or the above-mentioned recombinant hydroxylated small molecule collagen encoded by nucleic acid, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered bacteria, or the above-mentioned recombinant hydroxylated small molecule collagen prepared by the above-mentioned method, or the above-mentioned composition, or the above-mentioned product in the preparation of medical devices, biomaterials, tissue engineering products, and cosmetics.

[0048] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0049] (1) The recombinant hydroxylated small molecule collagen of this invention is selected from approximately 50 amino acid sequences with hydroxylation sites on the triple helix region of human type VII collagen or the α1 chain of human type III collagen. This recombinant hydroxylated small molecule collagen can be expressed in Pichia pastoris, enabling efficient extracellular secretion. It is less prone to degradation during purification, thus reducing the difficulty of purification. The expression method of this invention avoids the costs and risks of exogenous protein residue associated with in vitro protease digestion, while also shortening the time and cost of subsequent purification processes.

[0050] (2) The recombinant hydroxylated small molecule collagen and P4H enzyme described in this invention are co-expressed in Pichia pastoris. Based on the expression of the target sequence, the amino acid sequence is modified by hydroxylation at the theoretical GXY hydroxylation sites, followed by post-translational modification. This makes the small molecule collagen structure more stable, achieving a balance between transdermal absorption and biological structural stability. The post-expression modification of amino acids in this invention stabilizes the molecular structure and provides more controllable self-assembly compared to post-expression self-assembly of small molecules.

[0051] (3) The recombinant hydroxylated small molecule collagen of the present invention has similar physicochemical properties and biological functions to the original sequence. It has a smaller molecular weight, better absorption by tissues, and better cell adhesion and migration effects than unhydroxylated small molecule collagen. Attached Figure Description

[0052] Figure 1 The results are validation results of Pichia pastoris colony PCR after co-transformation with P4H using 705, 721, and 3A5D2NT.

[0053] Figure 2 This is the validation result of Pichia pastoris colony PCR after co-transformation with P4H using KM71H / P4H.

[0054] Figure 3 The results of Western blot (WB) analysis after co-transformation with P4H in the recombinant engineered bacteria are shown. In the figure, a is the WB detection image of P4HA1; b is the WB detection image of P4HB.

[0055] Figure 4The image shows the supernatant of recombinant hydroxylated small molecule collagen expressed in shake flasks after 72 hours of induction. In the figure, a is X33 / pPICZαB-705-P4H; b is KM71H / pPICZαB-721-P4H; and c is GS115-HCPB-PPKEX2 / 3A5D2NT-P4H.

[0056] Figure 5 Electrophoresis images of the supernatant (a) and purified (b) from the fermentation of recombinant hydroxylated small molecule collagen in a 5L tank.

[0057] Figure 6 The graph shows the cell adhesion activity assay results. a represents the NIH / 3T3 cell viscosity ratio, and b represents the HaCat cell adhesion ratio.

[0058] Figure 7 The graph shows the results of cell migration area ratio detection. a represents the migration rate of NIH / 3T3 cells, and b represents the migration rate of HaCat cells.

[0059] Figure 8 Images show cell migration; in the images, (a) shows NIH / 3T3 cell migration; and (b) shows HaCat cell migration.

[0060] Figure 9 The hydroxylation modification sites (a) and secondary mass spectrum (b) of recombinant hydroxylated small molecule collagen 705 are shown.

[0061] Figure 10 The hydroxylation modification sites (a) and secondary mass spectra (b) of recombinant hydroxylated small molecule collagen 721 are shown.

[0062] Figure 11 (a) shows the hydroxylation modification site of recombinant hydroxylated small molecule collagen 3A5D2NT, and (b) shows its secondary mass spectrum.

[0063] Figure 12 The results are for the cytotoxicity assay of recombinant hydroxylated small molecules. Detailed Implementation

[0064] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the embodiments of the present invention, unless otherwise described, conventional experimental methods are used. The processes involved in the embodiments, unless otherwise described, are those that can be understood and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0065] Example 1:

[0066] S1. Design of amino acid sequences for recombinant hydroxylated small molecule collagen:

[0067] A sequence of approximately 50 amino acids from the triple helix region of human type VII collagen (Q02388·CO7A1_HUMAN) or the α1 chain of human type III collagen (P02461) was selected. KR was added to the C-terminus, and EA was added to the N-terminus. After tandem copying 6-10 times, the target sequence size was approximately 50 kDa, suitable for the optimal expression range in Pichia pastoris. After expression in Pichia pastoris, the kex2 enzyme naturally present in Pichia pastoris cleaves the KR at the C-terminus of KREA at the amino acid linking site, ste13 cleaves the N-terminal EA, and Kex1 cleaves the remaining KR residues, resulting in secretion into the extracellular space via a secretory signal peptide. The recombinant hydroxylated small molecule collagen consists of multiple repeating polypeptide sequences, each beginning with EA, with two monomers linked by a KEX2 restriction site, ending with GLEKR or GLEKR.

[0068] (1) Select 2156-2207AA from Q02388.CO7A1-HUMAN, add LE to the C-terminus to obtain recombinant hydroxylated small molecule collagen, named 705, with the sequence shown in SEQ ID NO:1. Based on SEQ ID NO:1, add EA to the N-terminus and KR to the C-terminus to form a monomer. Repeat the monomer 6 times to form the recombinant collagen sequence shown in SEQ ID NO:2.

[0069] SEQ ID NO:1:

[0070] GERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLE;

[0071] SEQ ID NO:2:

[0072] KREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEK REAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKREAGERGMAGPEGKPGLQGPRGPPGPVGGHGDPGPPGAPGLAGPAGPQGPSGLKGLEKR.

[0073] (2) Select 2656-2701AA from Q02388.CO7A1-HUMAN, add LE to the C-terminus to obtain recombinant hydroxylated small molecule collagen, named 721, with the sequence shown in SEQ ID NO:3. Based on SEQ ID NO:3, add EA to the N-terminus and KR to the C-terminus to form a monomer. Repeat the monomer 10 times to form the recombinant collagen sequence shown in SEQ ID NO:4.

[0074] SEQ ID NO:3:

[0075] GHKGEMGEPGVPGQSGAPGKEGLIGPKGDRGFDGQPGPKGDQGEKGLE;

[0076] SEQ ID NO:4:

[0077] *

[0078] (3) Select the amino acid sequence from position 1036 to 1085 of the human type III collagen α1 chain: P02461, to construct a recombinant hydroxylated small molecule collagen with a length of 50 amino acids, named 3A5D2NT, as shown in SEQ ID NO:5. Based on SEQ ID NO:5, add the amino acid "RGLE" as a linker sequence to its C-terminus, add EA to the N-terminus, and add KR to the C-terminus to form a monomer. Repeat the monomer 9 times to form the recombinant collagen sequence as shown in SEQ ID NO:6.

[0079] SEQ ID NO:5:

[0080] GKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGH;

[0081] SEQ ID NO:6:

[0082] .

[0083] S2. DNA sequence synthesis and recombinant plasmid construction:

[0084] S21. Construction of a recombinant expression vector for recombinant hydroxylated small collagen:

[0085] The nucleic acid sequences encoding the above-mentioned amino acid sequences SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 were synthesized by Nanjing Genscript Biotech Co., Ltd., and the corresponding nucleic acid sequences are shown as SEQ ID NO:7-SEQ ID NO:9 respectively.

[0086] The gene fragments shown in SEQ ID NO:7 and SEQ ID NO:8 were cloned into pPICZαB, and the gene fragment shown in SEQ ID NO:9 was cloned into the pPIC9K vector. The exogenous target protein sequence was cloned using XhoI and NotI as restriction sites, so that the target fragment was accurately inserted into the reading frame of the secretory vector containing the secretion signal α-factor, and the recombinant expression vectors expressing SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 were obtained and named pPICZαB-705, pPICZαB-721 and pPIC9K-3A5D2NT, respectively.

[0087] SEQ ID NO:7-SEQ ID NO:9 are shown below, where “CTCGAG” is the XhoⅠ restriction site and “GCGGCCGC” is the NotⅠ restriction site.

[0088] SEQ ID NO:7:

[0089]

[0090] SEQ ID NO:8:

[0091] CTCGAGAAAAGGGAAGCCGGACATAAGGGCGAGATGGGCGAACCAGGAGTCCCA

[0092] GGCCAATCCGGTGCGCCTGGCAAGGAAGGCTTAATTGGTCCCAAGGGGGACAGGGGCT

[0093] TCGATGGTCAGCCTGGTCCAAAGGTGATCAAGGAGAGAAGGGACTAGAGAAACGCGA

[0094] GGCTGGCCATAAGGGTGAGATGGGAGAACCAGGCGTTCCAGGTCAGTCTGGCGCCCCC

[0095] GGAAAAGAAGGGCTCATCGGGCCGAAGGGCGATCGCGGTTTTGACGGGCAGCCAGGA

[0096] CCGAAAGGGGATCAGGGTGAGAAGGGCTTGGAGAAGCGGGAAGCCGGTCACAAGGGGG

[0097] GAAATGGGTGAACCTGGGGTCCCCGGGCAATCGGGTGCTCCTGGAAAGGAGGGGTTGA

[0098] TTGGGCCAAAAGGCGACCGAGGATTCGACGGGCAGCCTGGCCCTAAGGGAGATCAAGG

[0099] GGAAAAGGGCCTGGAGAAACGCGAGGCTGGTCACAAGGGTGAGATGGGGGAACCGGG

[0100] CGTACCTGGACAAAGCGGGGCCCCGGGAAAAGAGGGTCTTATAGGTCCAAAGGGCGAC

[0101] AGAGGATTCGACGGCCAACCCGGACCCAAGGGTGATCAGGGCGAAAAGGCTTAGAG

[0102] AAGCGAGAGGCAGGGCACAAAGGGGAGATGGGGGAACCCGGTGTACCTGGACAATCG

[0103] GGAGCACCCGGCAAGGAGGGACTCATTGGGCCGAAAGGGGATAGGGGTTTTGATGGAC

[0104] AACCTGGGCCGAAAGGGGACCAAGGGGAAAAGGGGCTAGAGAAACGAGAAGCAGGA

[0105] CACAAAGGTGAAATGGGTGAGCCAGGAGTGCCTGGACAAAGCGGGGCTCCCGGGAAA

[0106] GAAGGGCTGATAGGCCCGAAGGGTGACAGAGGCTTCGATGGCCAGCCGGGCCCAAAAG

[0107] GAGATCAGGGCGAAAAAGGTCTGGAGAAACGGGAGGCAGGACACAAAGGAGAGATGG

[0108] GGGAGCCTGGCGTCCCCGGGCAATCTGGTGCTCCCGGCAAGGAAGGACTAATAGGACC

[0109] CAAGGGCGATCGTGGTTTTGATGGCCAACCCGGGCCAAAAGGTGATCAAGGTGAAAAA

[0110] GGGTTGGAAAAACGTGAAGCAGGACATAAAGGTGAAATGGGCGAACCGGGAGTACCA

[0111] GGCCAGTCAGGGGCTCCAGGAAAGGAGGGCCTTATCGGACCGAAGGGAGATCGGGGCT

[0112] TCGACGGCCAGCCCGGGCCTAAAGGTGACCAGGGGGAAAAAGGATTAGAGAAGCGTG

[0113] AGGCGGGTCATAAGGGTGAGATGGGCGAGCCTGGTGTTCCTGGGCAGAGTGGAGCCCC

[0114] AGGGAAAGAAGGGCTTATTGGTCCTAAGGGAGACAGGGGGTTTGACGGTCAACCCGGG

[0115] CCGAAAGGAGATCAGGGGGAAAAGGGCTTAGAAAAACGAGAAGCGGGTCATAAAGGC

[0116] GAGATGGGTGAGCCGGGAGTGCCGGGTCAATCAGGAGCGCCGGGCAAGGAAGGTCTG

[0117] ATCGGACCTAAGGGTGATCGTGGTTTTGATGGCCAACCAGGTCCGAAGGGTGACCAGGGGGAGAAAGGTTTAGAAAAGAGATGAGCGGCCGC。

[0118] SEQ ID NO:9:

[0119] CTCGAGGAAGCTGGAAAGTCCGGCGACCGGGGAGAGTCAGGGCCTGCAGGGCCT

[0120] GCTGGTGCCCCAGGACCGGCCGGCAGTAGAGGTGCTCCTGGTCCTCAAGGACCACGGG

[0121] GCGACAAAGGTGAAACAGGTGAACGAGGTGCGGCAGGTATAAAAGGGCACAGAGGGC

[0122] TAGAAAAGCGGGAAGCAGGAAAAAGTGGCGATCGCGGGGAATCAGGTCCCGCGGGGC

[0123] CGGCGGGTGCGCCCGGACCGGCGGGGTCCAGAGGTGCCCCTGGACCGCAGGGACCTC

[0124] GGGGTGACAAAGGTGAAACTGGGGAAAGGGGGGCTGCGGGCATAAAGGGACACCGCG

[0125] GACTCGAGAAGAGAGAGGCAGGTAAAAGTGGGGATAGAGGCGAATCTGGTCCGGCAG

[0126] GACCAGCGGGCGCCCCCGGTCCTGCTGGATCACGAGGTGCCCCGGGTCCGCAAGGTCC

[0127] TAGGGGTGACAAAGGCGAGACGGGCGAACGAGGGGCAGCAGGAATTAAAGGCCACCG

[0128] AGGGCTGGAGAAGCGGGAAGCAGGTAAATCAGGAGATAGGGGGGAAAGCGGACCGGC

[0129] CGGACCCGCTGGTGCGCCGGGACCTGCTGGATCGAGAGGTGCACCGGGTCCCCAAGGG

[0130] CCTCGTGGAGATAAAGGGGAGACCGGGGAGCGCGGGGCTGCAGGGATTAAGGGACAC

[0131] CGCGGCTTGGAGAAGCGAGAGGCTGGTAAAAGCGGCGACCGCGGCGAATCGGGTCCG

[0132] GCGGGTCCCGCGGGCGCACCTGGGCCTGCTGGGTCTAGGGGAGCGCCAGGGCCTCAAG

[0133] GTCCTCGCGGTGACAAGGGAGAAACAGGCGAGCGTGGGGCAGCGGGCATTAAAGGTCA

[0134] TCGTGGGTTAGAAAAGCGTGAGGCTGGCAAATCTGGCGATCGCGGGGAGAGCGGTCCA

[0135] GCAGGTCCGGCTGGTGCTCCAGGTCCAGCTGGTAGCCGTGGCGCCCCAGGTCCTCAAG

[0136] GTCCTCGTGGCGACAAAGGGGAGACCGGCGAGCGTGGAGCGGCAGGCATCAAAGGTC

[0137] ATCGTGGACTTGAAAAGAGGGAGGCGGGAAAGTCGGGCGATCGGGGTGAATCCGGCCC

[0138] AGCGGGTCCTGCTGGAGCCCCAGGCCCCGCTGGATCGCGGGGCGCACCTGGCCCCCAA

[0139] GGACCGCGTGGGGATAAAGGGGAGACTGGCGAGAGAGGCGCCGCCGGAATCAAAGGT

[0140] CACCGGGGATTAGAAAAGAGGGAGGCAGGGAAGTCGGGTGATCGAGGCGAATCAGGT

[0141] CCTGCTGGACCCGCTGGCGCGCCGGGTCCTGCTGGTAGCAGAGGAGCCCCTGGGCCTC

[0142] AAGGCCCGAGGGGTGATAAGGGTGAAACCGGAGAACGAGGCGCCGCTGGTATAAAGG

[0143] GACATCGTGGACTCGAGAAGCGAGAAGCGGGGAAAAGTGGGGACAGAGGAGAGTCTG

[0144] GTCCTGCCGGACCCGCCGGTGCTCCTGGTCCAGCAGGCTCCCGCGGCGCGCCTGGTCC

[0145] GCAAGGTCCTAGGGGAGACAAAGGAGAAACGGGCGAGCGAGGCGCCGCCGGGATCAAGGGGCATCGGGGGTTGGAGAAGAGGTAAGCGGCCGC。

[0146] S22. Construction of recombinant plasmid expressing P4H enzyme:

[0147] A. Construction of human P4H alpha subunit (abbreviated as P4HA) vector:

[0148] The amino acids from P13674.P4HA1_HUMAN were selected and named the human P4H alpha subunit (abbreviated as P4HA), the sequence of which is shown in SEQ ID NO:10. The nucleic acid sequence encoding P4HA was synthesized by Nanjing Genscript Biotech Co., Ltd. Primers P4HA Forward and P4HA Reverse were designed to amplify the P4HA fragment, resulting in an amplified fragment containing BamHI at the 5' end and EcoRI at the 3' end. The amplified fragment and pPIC9K plasmid were digested with restriction endonucleases BamHI and EcoRI. The digested DNA was recovered and purified. The amplified fragment and pPIC9K plasmid were ligated overnight at 16°C using T4 ligase to obtain ligated samples. The ligated samples were then transformed into competent Escherichia coli DH5α using the CaCl2 heat shock method, plated, cultured, screened, and sequenced to verify that the sequence was P4HA. The human P4H alpha subunit (named P4HA) vector was successfully obtained and designated as P4HA 9K plasmid (abbreviated as P4H 9K).

[0149] SEQ ID NO:10:

[0150] ;

[0151] P4HA Forward (SEQ ID NO:11): AGATATCGGGTCCCCGGGAT;

[0152] P4HA Reverse (SEQ ID NO: 12): CGTGCCCCGGAATTCCTATTA.

[0153] B. Construction of a human P4H enzyme β subunit (PDI) vector:

[0154] The amino acids from P07237.PDIA1_HUMAN 18-508 were selected and named the human P4H enzyme β subunit (PDI), whose sequence is shown in SEQ ID NO:13. The nucleic acid sequence encoding PDI was synthesized by Nanjing Genscript Biotech Co., Ltd. Primers PDI Forward and PDI Reverse were designed to amplify the PDI fragment, resulting in an amplified fragment containing BamHI at the 5' end and EcoRI at the 3' end. The amplified fragment and pPIC9K plasmid were digested with restriction endonucleases BamHI and EcoRI, and the digested DNA was recovered and purified. The amplified fragment and pPIC9K plasmid were ligated overnight at 16℃ using T4 ligase to obtain the ligated sample. The ligated sample was transformed into competent E. coli DH5α using the CaCl2 heat shock method, plated, screened, and sequenced for verification, confirming that the sequence was the PDI sequence, thus successfully obtaining the human P4H enzyme β subunit (PDI) vector.

[0155] SEQ ID NO:13:

[0156] DAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTL PDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPK SVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKK NVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL.

[0157] PDI Forward (SEQ ID NO:14):

[0158] CCGCTCGAGGTTGCTATTGAAAAAAGAGATGCTCCAGAAGAAGAAGATCA;

[0159] PDI Reverse (SEQ ID NO:15):

[0160] GCTCTAGACCTATTATTACAATTCATCTTTAAC.

[0161] C. Construction of the P4H plasmid pPIC9K-P4H(DP) with dual promoters:

[0162] Primers Forward and Reverse were designed to amplify the PDI, promoter, and terminator fragments on the human P4H enzyme β subunit (PDI) vector by PCR, yielding a PCR product that is complementary to pPIC9K at both ends after linearization with AtaII. The plasmid P4HA 9K was digested with the restriction endonuclease AtaII to obtain a linearized plasmid. The PCR product and the linearized plasmid were recovered and purified for later use.

[0163] The PCR product and the obtained linearized plasmid were ligated at 50°C for 30 min using a ready-to-use seamless cloning kit, and the sample was transformed into competent *E. coli* DH5α using the CaCl2 heat shock method. The transformed *E. coli* were plated on LB agar plates containing ampicillin and cultured for 24 hours. Colony PCR was performed on the obtained *E. coli* colonies to screen for *E. coli* colonies containing the cloned PDI and related elements into the P4HA 9K plasmid (P4H 9K). Plasmids extracted from colony PCR-positive strains were identified by BamHI and BspEI restriction enzyme digestion. DNA sequencing of the plasmids showed that their sequences contained the PDI, promoter, and terminator sequences. The obtained P4H plasmid with dual promoters was named pPIC9K-P4H(DP).

[0164] Forward (SEQ ID NO:16): CCGAAAAGTGCCACCTGACGTAGATCTAACATCCAAAGA;

[0165] Reverse (SEQ ID NO:17):

[0166] AGGTTAATGTCATGATAATAATGGTTTCTTAGACGTGTCTCACTTAATCTCTGTACT C.

[0167] Construction of the D.P4H expression vector pPIC9K-P4H(DP)-1:

[0168] Nucleic acid sequences expressing the promoters PTEF (SEQ ID NO:18) and PEM7 (SEQ ID NO:19) were synthesized, and the 5' and 3' ends of the pPIC9K-P4H(DP) plasmid KanR were modified by adding PTEF and PEM7 to the 5' end of the KanR selection marker (in the direction of the pPIC9K plasmid) and adding CYC1tt to the 3' end to obtain the P4H expression vector pPIC9K-P4H(DP)-1.

[0169] PTEF (SEQ ID NO:18):

[0170] cttagattagattgctatgctttctttctaatgaacaagaagtaaaaaaagttgtaatagaacaagaaaaatgaaactgaaacttgagaaattgaagaccgtt tattaacttaaatatcaatggaggtcactgaaagagaaaaaaactaaaaaaaaaaatttcaagaaaaagaaacgtgataaaaatttttattgcctttttcgac gaagaaaaagaaacgaggcggtctcttttttcttttccaaacctttagtacgggtaattaacgacaccctagaggaagaaagagggaaaatttagtatgctgt gcttgggtgttttgaagtggtacggcgatgcgcggagtccgagaaaatctggaagagtaaaaaaggagtagaaacattttgaagctatggtgtgtgggggatc;

[0171] PEM7 (SEQ ID NO: 19): gtcgtattatactatgccgatatactatgccgatgattaattgtcaac.

[0172] S3. Construction of recombinant engineered strains:

[0173] 10 μg of recombinant expression vectors pPICZαB-705, pPICZαB-721, and pPIC9K-3A5D2NT were linearized by digestion with Sal I (purchased from TaKaRa, Dalian; specific procedures were performed according to the kit instructions) at 37°C. The recombinant plasmid pPIC9K-P4H(DP) was linearized by digestion with BspEI (purchased from TaKaRa, Dalian; specific procedures were performed according to the kit instructions) at 37°C. The linearized plasmids were then recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), with the volume controlled at approximately 10 μL, to obtain linearized plasmids pPICZαB-705, pPICZαB-721, pPIC9K-3A5D2NT, and pPIC9K-P4H(DP)-1.

[0174] Linearized plasmid pPIC9K-P4H(DP)-1 was electroporated into competent Pichia pastoris KM71H cells (purchased from Thermo Fisher Scientific). The electroporated bacterial culture was plated on YPDG (500 μg / mL, G418) plates, 200 μL per plate. The plates were incubated at room temperature for 10 min, then inverted at 30°C for 2-5 days until a single colony (positive transformant) appeared. The obtained transformant was named KM71H / P4H. Linearized plasmid pPICZαB-721 was electroporated into the constructed KM71H / P4H. The electroporated bacterial culture was plated on YPDZ (100 μg / mL, Zeocin) plates, 200 μL per plate. The plates were incubated at room temperature for 10 min, then inverted at 30°C for 2-5 days until a single colony (positive transformant) appeared. The obtained transformant was named KM71H / pPICZαB-721-P4H, or simply KM71H-721-Hyp.

[0175] The linearized plasmid pPICZαB-705 was electroporated into competent Pichia pastoris X33 cells (purchased from Thermo Fisher Scientific). The electroporated bacterial culture was spread on YPDZ (100 μg / mL, Zeocin) plates, with 200 μL per plate. The plates were incubated at room temperature for 10 min and then inverted at 30°C for 2-5 days until a single colony (positive transformant) appeared. The obtained transformant was named X33 / pPICZαB-705 (from strain CGMCC NO.29601 in patent CN118146354A). The linearized plasmid pPIC9K-P4H(DP) was electroporated into the constructed X33 / pPICZαB-705. The electroporated bacterial culture was plated on YPDG (500 μg / mL, G418) plates, with 200 μL per plate. The plates were incubated at room temperature for 10 min, then inverted at 30°C for 2-5 days until single colonies (positive transformants) appeared. The obtained transformant was named X33 / pPICZαB-705-P4H, or X33-705-Hyp for short.

[0176] The linearized plasmid pPIC9K-3A5D2NT was electroporated into competent Pichia pastoris GS115-HCPB-PPKEX2 cells (from patent CN116948013A, strain number: CGMCC No. 25815). The electroporated bacterial culture was plated on YPDG (500 μg / mL, G418) plates, 200 μL per plate. After incubation at room temperature for 10 min, the plates were incubated upside down at 30°C for 2-5 days until single colonies (positive transformants) appeared. The obtained transformants were named...

[0177] GS115-HCPB-PPKEX2 / 3A5D2NT (from strain CGMCC No. 25819 in patent CN116948013A). The linearized plasmid pPIC9K-P4H(DP)-1 was electroporated into the constructed GS115-HCPB-PPKEX2 / 3A5D2NT. The electroporated bacterial culture was plated on YPDG (500 μg / mL, G418) plates, with 200 μL per plate. The plates were incubated at room temperature for 10 min and then inverted at 30°C for 2-5 days until a single colony (positive transformant) appeared. The obtained transformant was named GS115-HCPB-PPKEX2 / 3A5D2NT-P4H, abbreviated as TTA03-Hyp.

[0178] S4. Verification of target gene transfer:

[0179] Resistance plate screening can accelerate the experimental process and identify strains containing the target gene. To further verify this at the gene level, polymerase chain reaction (PCR) was used as a technique to extract the genome from Pichia pastoris as a template. The specific steps are as follows:

[0180] S41.X33 / pPICZαB-705-P4H, KM71H / pPICZαB-721-P4H,

[0181] Verification of GS115-HCPB-PPKEX2 / 3A5D2NT-P4H:

[0182] Add 200 μL of YPD medium to a 96-well plate, and pick 16 single colonies of X33 / pPICZαB-705-P4H, KM71H / pPICZαB-721-P4H, and GS115-HCPB-PPKEX2 / 3A5D2NT-P4H respectively. Incubate in a 96-well plate at 30°C for 24 h. After shaking well, take 50 μL of bacterial cells from each well into an 8-tube bundle, centrifuge for 5 min, discard the supernatant, add 80 μL of sterile resuspended bacterial cells, centrifuge for 5 min, discard the supernatant, and then add 10 μL of 0.02M NaOH solution to vortex and suspend the bacterial cells to obtain the bacterial suspension.

[0183] Place the mixed bacterial culture on a PCR instrument, boil it at 99℃ for 10 min, let it cool to room temperature, centrifuge it for 5 min, take 2 μL of the supernatant as a template, use rTaq as PCR polymerase, and perform polymerase chain reaction by PCR. After the reaction, verify the target band by nucleic acid electrophoresis.

[0184] The primers used in the above PCR reaction are:

[0185] 5'AOX:GACTGGTTCCAATTGACAAGC (SEQ ID NO:20);

[0186] 3'AOX:GCAAATGGCATTCTGACATCC (SEQ ID NO:21);

[0187] P4HB veri R: CCTTAAAAGATTCAGCAGCAGTC (SEQ ID NO: 22);

[0188] P4HA veri F: CAAGAAGATGAGTGGGATAAGC (SEQ ID NO: 23);

[0189] The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 50 s, 30 cycles. Primers SEQ ID NO:19 and SEQ ID NO:21 were used to amplify the P4HA target fragment using polymerase chain reaction under the above reaction system and conditions. The target fragment size was 756 bp. The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min 12 s, 30 cycles. Primers SEQ ID NO:18 and SEQ ID NO:20 were used to amplify the P4HB target fragment using polymerase chain reaction under the above reaction system and conditions. The target fragment size was 1152 bp.

[0190] The verification results are as follows Figure 1 As shown in the figure, the nucleic acid size is in line with the expected PCR results. Strains that are positive for both P4HA1 and P4HB fragments in the PCR results were selected for shake-flask induction expression.

[0191] Validation of the target sequence S42.KM71H / pPICZαB-721-P4H:

[0192] Sixteen single colonies of KM71H / pPICZαB-721-P4H were picked and placed in 200 mL of YPD medium in a 96-well plate. The plates were incubated at 30 °C for 24 h. 80 μL of the bacterial culture was used to extract the template, and 2 μL of the extracted template was added to a PCR tube. Polymerase chain reaction was performed by PCR. After the reaction, the target band was verified by nucleic acid electrophoresis.

[0193] The primers used in the above PCR reaction are:

[0194] 705BB1-23 F: ggtctcaCATGaacgatgagatttccttcaatt (SEQ ID NO: 24);

[0195] 707BB1 R: ggtctcaAAGCttaattcgcggccgctca (SEQ ID NO: 25);

[0196] The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min, 30 cycles.

[0197] The verification results are as follows Figure 2 As shown in the figure, the target band size is 1837bp, as indicated by the band within the red box. Positive strains were selected for subsequent shake-flask induction expression.

[0198] If Pichia pastoris transformants can grow on plates containing high concentrations of Zeocin, it indicates that the transformants contain multiple copies of the target gene, meaning that multiple recombinant fragments have entered the yeast and integrated into the yeast chromosome through homologous recombination. This screening step yields high-copy, high-efficiency recombinant yeast engineered strains. The strain KM71H / pPICZαB-721-P4H was used for subsequent experiments after antibiotic plate screening and Pichia pastoris colony PCR verification.

[0199] The recombinant engineered strains KM71H-721-Hyp, X33-705-Hyp, and TTA03-Hyp were sent to the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCC NO.33067, CGMCC NO.33068, and CGMCC NO.33069. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Accession date: December 13, 2024; Classification and name: *Pichia pastoris* *Komagataella phaffii*.

[0200] S43.WB identification of P4H:

[0201] Cell disruption: After inducing bacterial expression, the fermentation broth was collected, centrifuged, and the precipitate was placed on ice. An equal volume of glass beads, three times the volume of cell disruption buffer (1 mM EDTA, 5% glycerol, pH adjusted to 7.4 with NaOH), and 10 μL of 1 mM PMSF were added. The mixture was then shaken on a benchtop shaker for 2 min, placed on ice for 2 min, and repeated three times. The fourth disruption was performed for 4 min. After disruption, the mixture was centrifuged at 10,000 rpm at 4°C for 10 min. The supernatant was collected, and 5× protein loading buffer was added. The mixture was then boiled at 99°C for 10 min. SDS-PAGE precast gel (12%, GenScript) was used to verify the expression of P4HA1 and P4HB. After SDS-PAGE electrophoresis of P4HA1 and P4HB, the samples were transferred to membranes. The experimental methods and procedures are described in "Molecular Cloning: A Laboratory Manual". The experimental equipment was from GenScript, and the operation was performed according to the equipment requirements. P4HA1: Incubate with primary antibody P4HA1 (mouse-derived, 66101-1-Ig, 1:2000 dilution) for 2 hours. Secondary antibody: Incubate with goat anti-mouse antibody (1:5000 dilution) for 2 hours. Target protein size of P4HA1: 61 kDa. P4HB: Primary antibody P4HB1 (rabbit-derived, ET7110-92), 1:2000 dilution; Secondary antibody: Goat anti-rabbit antibody (1:5000). Molecular weight of P4HB: 52.2 kDa.

[0202] Experimental results are as follows Figure 3 As shown, after incubation with specific P4HA1 and P4HB antibodies, the target band sizes were consistent: P4HA1 61 kDa and P4HB 52.2 kDa, with clear target bands. Other target bands were present in the results of X33 / pPICZαB-705-P4H and KM71H / pPICZαB-721-P4HWB, but the antibodies were specific and identified as P4H enzymes. It is speculated that degradation or aggregation may have occurred during cell disruption or storage. Positive strains were selected for subsequent experiments.

[0203] S5. Induction of recombinant engineered strain expression and identification of recombinant collagen:

[0204] Engineered bacterial strains X33 / pPICZαB-705-P4H, KM71H / pPICZαB-721-P4H, and GS115-HCPB-PPKEX2 / 3A5D2NT-P4H, which tested positive by PCR and Western blotting, were placed in 100mL Erlenmeyer flasks containing 10mL of BMGY medium and incubated at 28-30℃ and 220rpm until the OD600 reached 6-10 (16-18h). The cells were then centrifuged at 1500-3000g for 5min at room temperature, collected, and resuspended in BMGY medium until the OD600 reached 6-10. 600The sample was approximately 10 μL. It was then cultured for 3 days at 28-30℃ and 220 rpm in a biochemical incubator, with 1% methanol added every 12 hours. Samples were taken after 48 and 72 hours of induction, and the supernatant was collected by centrifugation at 1500-3000g for 5 minutes at room temperature. The collected expression supernatant was mixed with 2× loading buffer (Coomassie Brilliant Blue, DTT denaturant, and SDS buffer), heated in a 100℃ metal bath for 5 minutes, and then subjected to Tricine protein gel assay using a precast GLASS gel.

[0205] The results are as follows Figure 4 As shown in the figure, each target band can be secreted into the culture medium supernatant, indicating that the target bands with the 705, 721 and 3A5D2NT sequences are secreted and expressed extracellularly. The molecular weight is consistent with the epigenetic migration characteristics of theoretical collagen and there is no degradation. Protein gel mass spectrometry identification of the target bands shows that the amino acid sequence is the same as the theoretical amino acid sequence.

[0206] S6. High-density fermentation and purification experiment:

[0207] (1) The selected recombinant engineered bacteria X33 / pPICZαB-705-P4H, KM71H / pPICZαB-721-P4H, and GS115-HCPB-PPKEX2 / 3A5D2NT-P4H were subjected to high-density fermentation experiments to express and produce recombinant hydroxylated small molecule collagen on a large scale, and to obtain fermentation broth containing recombinant hydroxylated small molecule collagen.

[0208] The seed culture medium YPG consisted of 10 g / L yeast extract, 20 g / L peptone, and 10 g / L glycerol.

[0209] Fermentation medium: NH4H2PO4 190.4g / L, KH2PO4 10.06g / L, CaSO4·2H2O 1.18g / L, K2SO4 18.2g / L, MgSO4·7H2O 14.9g / L, glycerol 40g / L; After the fermentation medium was sterilized at high temperature, PTM1 was added after the temperature dropped to room temperature, and the pH was adjusted to 5.0 with ammonia water.

[0210] Feeding medium: 50% w / v glycerol, with 12 mL PTM1 micronutrients per liter;

[0211] Induction medium: 100% methanol, with 12 mL of trace elements added per liter;

[0212] PTM1: Sterilize by filtration through a 0.22μm filter membrane and store at 4℃.

[0213] The batch culture and induction expression conditions for the engineered strain were as follows: A fed-batch culture method was used, with a culture temperature of 30℃. The engineered strain was inoculated into a 1L shake flask containing 200mL of seed culture medium YPG, and cultured at 220rpm and 30℃ for 18-20h until OD600 = 2-10. A 5L fermenter (Baoxing Biotechnology) was used, containing 2L of fermentation medium. 2% glycerol was sterilized separately. Before inoculation, the fermentation speed was adjusted to 300rpm, the aeration rate to 4L / min, and the temperature to 30℃. The pH was adjusted to 4.5 using a concentrated ammonia solution. Then, 0.9mL of PTM1 was inoculated first, followed by 200mL of the prepared seed culture (flame ring inoculation). The dissolved oxygen electrode was then calibrated, and fermentation began after calibration.

[0214] Induction culture: When the dissolved oxygen level drops to 30% for the first time during growth, use the dissolved oxygen cascade rotation function to maintain it at 30%; wait for the glycerol to be depleted and the dissolved oxygen to rebound and be greater than 70% (OD). 600 (Value approximately 20), cancel dissolved oxygen cascade speed, increase stirring speed to 650 rpm, and use 30% dissolved oxygen linked feeding, adding 80 mL of feeding medium. Stop adding feeding medium when dissolved oxygen rebounds to above 70%, set pH to 5.0 and temperature to 30℃, and perform induction culture by feeding induction medium: manually add 5 mL of induction medium. After dissolved oxygen rebounds to above 70%, set the feeding parameters as follows: feed induction medium at a rate of 8 mL / h for 1 hour, then at a rate of 10 mL / h for 1 hour, then at a rate of 20 mL / h until the dissolved oxygen value is below 30%. Wait for dissolved oxygen to rebound; once dissolved oxygen rises back to 30%, resume linked feeding of induction medium. Induce culture for a total of 40–60 hours, measuring protein concentration with UV. Once the protein concentration increase is no longer significant or decreases, the culture can be removed from the tank.

[0215] UV protein quantification formula: C(mg / mL)=0.144*(A215-A225), A215<1.5.

[0216] The principle behind UV 215nm and 225nm protein content detection is based on the difference in light absorption of proteins at wavelengths of 215nm and 225nm. The difference in absorbance between 215nm and 225nm corresponds to the protein's concentration within a certain range, following Beer's Law. Therefore, the protein concentration can be quantitatively determined by measuring the difference in absorbance at these two wavelengths.

[0217] After fermentation, the mixture was centrifuged at 9000g for 5 minutes, and the fermentation supernatant was collected for protein electrophoresis. The results are as follows: Figure 5As shown in figure a, under high-density fermentation conditions, collagen sequences 705, 721, and 3A5D2NT showed almost only the target band after induction for 24 hours or more, and the expression level increased with increasing induction time. Density analysis showed that the proportion of the main band exceeded 85% in all sequences. This indicates that 705, 721, and 3A5D2NT can express the target protein and secrete it extracellularly under high-density fermentation conditions.

[0218] (2) Purification:

[0219] Buffer A: 20 ml M KH2PO4, pH 4.0;

[0220] Buffer B: 20mM KH2PO4, 1M NaCl, pH 4.0.

[0221] Collect the fermentation broth from step (1), and centrifuge at 2000g, 30min, and 4℃ to separate the bacterial cells and fermentation supernatant. Equilibrate the cation exchange medium (UniGel-80sp from Suzhou Nanomicro, loaded onto an XK50 / 30 column from Lishui Technology, using a GE AKTAPure protein separation chromatography purification system) with buffer A until the A215 absorbance and conductivity remain constant. Then, set the flow rate to 100us / cm and load the sample at a volume of 0.5L / time. Detect the UV A215 absorbance; when it rises, start loading the sample. When the A215 absorbance decreases, stop loading the sample when the UV and conductivity reach their lowest levels and no longer change. After loading, close the loading circuit and equilibrate the cation exchange medium with buffer A again. Collect the eluent, and after identifying the components, dialyze them (dialysate is ultrapure water), then concentrate and freeze-dry to collect the lyophilized collagen sponge.

[0222] The purified lyophilized sponge was dissolved in ultrapure water and subjected to protein electrophoresis. The electrophoresis results are as follows: Figure 5 As shown in b, the collagen bands of 705, 721, and 3A5D2NT are clear and undegraded, indicating that the target protein can be efficiently separated after purification.

[0223] Example 2: Cell adhesion activity experiment of recombinant hydroxylated small molecule collagen

[0224] In this embodiment, natural collagen was used as a control, and recombinant 705, 721 and 3A5D2NT collagen (recombinant hydroxylated small molecule collagen with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5) were used as examples to examine the cell adhesion activity of the recombinant hydroxylated small molecule collagen described in this invention.

[0225] The specific steps are as follows:

[0226] (1) Material preparation: mouse embryonic fibroblasts NIH / 3T3 (cell line from SCSP-515 of Chinese Academy of Sciences Cell Bank, culture and passage methods were performed according to the cell instructions), human immortalized keratinocytes (HaCaT, cell line from SCSP-5091 of Chinese Academy of Sciences Cell Bank), recombinant 705, 721 and 3A5D2NT collagen lyophilized sponge, positive natural collagen (Sigma, catalog number C7774-5MG).

[0227] The positive control, natural collagen, was prepared as follows: A 5mL sterile centrifuge tube was weighed, and human collagen was added to it in a laminar flow hood. The weight was then reduced to obtain the sample volume. The sample was dissolved in ultrapure water, and acetic acid was added to bring the pH to 3.0 (concentration 5mg / mL), resulting in a milky white solution. Protein concentration was determined using the empirical formula for UV protein quantification: C(mg / mL) = 0.144*(A215-A225). Before use, the concentration was diluted to 0.5mg / mL with serum-free DMEM medium, and the solution was filtered through a 0.22μm sterile filter for sterilization.

[0228] (2) Coating preparation:

[0229] Add 100 μL of sample (standard / sample / blank control) to each well of a 96-well plate. Standard: Positive control: 0.5 mg / mL natural collagen (Sigma, catalog number C7774-5MG); Sample: 0.5 mg / mL recombinant hydroxylated small molecule collagen of 705, 721 and 3A5D2NT; Blank control: D-PBS phosphate buffer.

[0230] Prepare four wells for each sample and add the samples to a 96-well plate. Incubate at 37°C, 5% CO2 (v / v) for 1-4 hours. Remove excess solution from the wells to complete the coating. Add 100 μL of 1% BSA-PBS solution and incubate at 37°C, 5% CO2 for 1 hour. After removing the liquid from the wells, wash three times with D-PBS and discard the washing solution. Premix Hoechst-33342 fluorescent staining agent (10%) with complete culture medium and dilute NIH / 3T3 cells or HaCaT cells to 5 × 10⁻⁶ cells / well using the above solution. 4 Cells / mL. Add 100 μL of cells to the wells, cover with aluminum foil, and incubate at 37°C and 5% CO2 for 1 h.

[0231] The specific detection steps are as follows: Capture at least 4×4 digital tiled images (fluorescence) for each of the three wells using an inverted microscope. Fill each well with D-PBS to form an "inverted meniscus," purge air bubbles, and cover with sealing film. Centrifuge the plate (inverted) at 22°C for 5 min at a relative centrifugation force (RCF) of 300 g. After centrifugation, discard the sealing film, remove the supernatant from the wells, wash once with D-PBS, and then add 100 μL of D-PBS. For each of the three wells, capture a total of 25 fluorescent tiled digital images (at least a 4×4 matrix is ​​recommended, with 10% tile overlap). Calculate approximately 2400 to 3600 cells per sample (800–1200 cells / well × 3 wells). Measure three replicate samples. The fourth well is used to adjust microscope parameters and its measurement value is not used. The measurement results are as follows: Figure 6 As shown.

[0232] from Figure 6 As can be seen, the coated samples exhibit stronger adhesion than the blank control group. This superiority is even more pronounced in NIH / 3T3 cells compared to the blank control group. The adhesion effect of the hydroxylated sample is significantly better than that of the unhydroxylated sample, as evidenced by the 705 hydroxylated and unhydroxylated raw materials. Therefore, the recombinant hydroxylated small molecule collagen described in this invention possesses cell adhesion similar to natural collagen, and hydroxylation promotes the bioactivity of small molecule collagen.

[0233] Example 3: Recombinant collagen cell migration experiment

[0234] In this embodiment, NIH / 3T3 cells and HaCaT cells, consistent with those in Example 2, were used to examine the cell migration ability of recombinant 705, 721, and 3A5D2NT recombinant hydroxylated small molecule collagen lyophilized sponges and positive control natural collagen (Sigma, catalog number C7774-5MG).

[0235] (1) Experimental preparation: First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate, approximately every 0.5cm to 1cm, passing through the holes. Three lines should pass through each hole. Add approximately 5×10⁻⁶ ppm of the mixture into the holes. 5 Each cell.

[0236] (2) Scratch assay: On the second day of cell culture, use a pipette tip to make a scratch as perpendicular as possible to the horizontal line on the back of the cell, ensuring the pipette tip is vertical and not tilted. Then wash the cells three times with PBS to remove the scratched cells. Add serum-free culture medium containing the test sample as the experimental group, with a concentration of 0.05% (w / v). Incubate at 37°C in a 5% CO2 incubator. Take samples and photographs at 0 and 24 hours of culture.

[0237] (3) Data Processing: The scratch area of ​​each image was calculated using ImageJ image processing software. The cell migration rate of each group was calculated by dividing the total area of ​​migrating cells in the fixed scratch area by the initial area of ​​the fixed scratch area. A graph was plotted with time on the horizontal axis and the migration area ratio on the vertical axis (unit: %), and the photos of the experimental group and the control group at the initial 0 and the end of the experiment were compared. One-way ANOVA was used to analyze the differences in data between the experimental groups, and the chi-square test was used. The experimental results are as follows: Figure 7 and Figure 8 As shown.

[0238] Combination Figure 7 and Figure 8 As can be seen, the migration experiments of NIH / 3T3 and HaCat cells after 24 hours showed the same results as the cell adhesion experiments. Hydroxylation increased collagen stability and strengthened its binding to cell surface sites. This indicates that as cells continue to grow over time, the experimental group samples exhibited a more pronounced cell migration-promoting effect under physiological conditions, effectively enhancing the migration of cells to biomaterials.

[0239] Example 4. Detection of hydroxylation modification

[0240] (1) Detection of hydroxyproline content:

[0241] In this embodiment, samples of 705, 721, and 3A5D2NT recombinant hydroxylated small molecule collagen after co-transferring with P4H enzyme, before freeze-drying, were hydrolyzed. The hydroxyproline content was then detected using a hydroxylation detection kit (Sangon Biotech: D799573-0050) to examine the hydroxylation rate and stability of the recombinant hydroxylated small molecule collagen.

[0242] Testing steps:

[0243] S1. Sample and reagent preparation:

[0244] Extraction solution: prepared with 6M HCl; the volume ratio of 6mol / L hydrochloric acid, concentrated hydrochloric acid (37%), and H2O is 1:1.

[0245] Hydroxyproline standard solution dilution: Prepare according to the kit instructions.

[0246] S2. Experimental Procedure:

[0247] After purifying the fermentation broth of the engineered bacteria, 100 μL of the solution obtained before freeze-drying was added to the sample. 1 mL of the extract was added, and the mixture was digested in a 110℃ oven for 4 hours until transparent. The solution was then centrifuged at 16000 rpm and 25℃ for 20 min. 1 mL of the supernatant was taken, and the pH was adjusted to 6–8 with approximately 0.5 mL of 10 mol / L NaOH. If the pH was too high, it was corrected with 6 mol / L hydrochloric acid. The volume was then brought to 2 mL with distilled water to obtain the test solution. The spectrophotometer was preheated for at least 30 min, the wavelength was adjusted to 560 nm, and the instrument was zeroed with distilled water. Hydroxyproline standards were diluted to prepare standard solutions of 7.5, 3.75, 1.875, 0.938, 0.469, 0.234, and 0.117 μg / mL. The solutions were added according to the kit instructions for hydroxylation detection.

[0248] A standard curve was plotted with the concentration of the standard solution on the x-axis and ΔA (standard A = A standard tube - blank A tube) on the y-axis, yielding the equation y = kx + b. Substituting the measured ΔA (measurement A = A measurement tube - blank A tube) into the equation, we obtained x (μg / mL). Based on the sample protein concentration, the tissue hydroxyproline content (μg / mg prot) was calculated as: x × V sample ÷ (Cpr × V sample) = x ÷ Cpr. The original experimental results of the standards are shown in Table 1. A standard curve was constructed using the results in Table 1, with the equation: y = 0.0321x + 0.004, R². 2 =0.9986, indicating a good fit. The absorbance values ​​of the experimental groups were substituted into the standard curve to calculate the hydroxyproline content. The results are shown in Table 2. The results show that 705, 721 and 3A5D2NT are hydroxylated.

[0249] Table 1: Absorbance of Hydroxyproline Standard

[0250]

[0251] Table 2: Hydroxyproline Detection Results

[0252]

[0253] Hydroxylation was detected in the samples using a kit, and the hydroxyproline content in the test solutions was measured. The results showed that sample 705 had a hydroxyproline content of 0.917 μg / mg prot, sample 721 had a hydroxyproline content of 0.854 μg / mg prot, and 3A5D2NT had a content of 1.414 μg / mg prot. The hydroxyproline content (μg / mg prot) results for 3A5D2NT were the highest, indicating that the hydroxylation rate per unit protein content is more representative. The lower protein content in samples 705 and 721 led to lower calculated results; however, 705 and 721 are human type VII collagen, which naturally has fewer hydroxylation sites in its original sequence. Although type III and type VII collagen have different functions, hydroxylation modification can stabilize the protein and improve its biological properties.

[0254] (2) Quantitative detection of amino acids

[0255] This step uses Waters ACQUITY UPLC I-CLASS ultra-high performance liquid chromatography to separate the 705, 721, and 3A5D2NT recombinant hydroxylated small molecule collagen proteins after co-transferring with P4H enzyme.

[0256] Chromatographic conditions:

[0257] Chromatographic column: Waters UPLC HSS T3 (1.8 μm, 2.1 mm × 150 mm);

[0258] Mobile phase: Phase A: water, 0.1% formic acid; Phase B: acetonitrile;

[0259] Elution gradient: conditions are shown in Table 4;

[0260] Flow rate: 0.5 mL / min;

[0261] Injection volume: 5.0 μL;

[0262] Column temperature: 50℃.

[0263] Mass spectrometry conditions: Mass spectrometry analysis was performed using a Waters XEVO TQ-S Micro tandem quadrupole mass spectrometer system. The positive ion source voltage was 1.5 kV and the cone voltage was 20 V; the desolventizing temperature was 600℃ and the desolventizing gas flow rate was 1000 L / h; the cone gas flow rate was 10 L / h.

[0264] Targeted data processing: The peak area was calculated using MassLynx quantitative software, with a retention time tolerance of 15 s. The quantitative results were obtained using the standard curve method, and the results are shown in Tables 3 and 4.

[0265] Table 3: Amino Acid Content

[0266]

[0267]

[0268] The hydroxylation rate was calculated using the data in Table 3. The calculation formula is: Target protein hydroxylation rate detection = C(L-hydroxyproline) / (C(L-hydroxyproline)+C(L-proline)). The calculation results are shown in Table 4.

[0269] Table 4: Hydroxylation rate of target protein (%)

[0270] Grouping 705 721 3A5D2NT Hydroxylation rate (%) 11.59％ 20.71％ 16.13％

[0271] As can be seen from Table 4, the collagen sequences 705, 721, and 3A5D2NT are hydroxylated.

[0272] (3) Hydroxylation modification analysis:

[0273] Protein samples were separated by chromatographic separation and identified by proline hydroxylation modification using high performance liquid chromatography-mass spectrometry (LC-MS).

[0274] S1. Sample preparation:

[0275] Take 200 μL of 705, 721, and 3A5D2NT into a glass bottle, add 0.8 mL of water and 1 mL of concentrated hydrochloric acid, and react at 110 °C for 20–24 h. After the reaction, acid hydrolyze the sample. Take 1 mL of the acid-hydrolyzed sample, concentrate it under vacuum, and then take 10 μL into an EP tube. Add water to bring the volume to 100 μL, and then add 1 μL of 1 M DTT solution to make the final DTT concentration 10 mmol / L. Then, reduce the sample in a 56 °C water bath for 1 h. After the reaction, add 2 μL of 1 M IAM solution to the reaction solution to make the final IAM concentration 20 mmol / L. React in the dark at room temperature for 40 min. After the reaction, add 1 μL of 1 M DTT solution to make the final DTT concentration 10 mmol / L to neutralize the unreacted IAM. Next, add 3 μL of Chymotrypsin enzyme (concentration: 0.5 μg / μL) and incubate in a 30℃ incubator for 16 h;

[0276] After enzyme digestion, the peptides were desalted using a self-filled desalting column and the solvent was evaporated in a vacuum centrifuge at 45°C to remove impurities other than the target peptides from the digested solution. The peptides were then dissolved in sample dissolving solution (0.1% formic acid, 2% acetonitrile), vortexed thoroughly, and centrifuged at 13200 rpm at 4°C for 10 min. The supernatant was transferred to a sample tube and awaited mass spectrometry analysis.

[0277] S2. Equipment Parameters:

[0278] Chromatographic separation equipment parameters: Mobile phase A is an aqueous solution containing 0.1% (v / v) formic acid; mobile phase B is an 80% (v / v) acetonitrile solution containing 0.1% (v / v) formic acid. Liquid phase gradient settings: 0-4 min, 1-8% B; 4-23 min, 8-28% B; 23-27 min, 28-99% B; 27-30 min, 99% B; 30-30.1 min, 99-1% B; 30.1-35 min, 1% B; flow rate 0.6 μL / min. Column: 75 μm id × 50 cm, NanoViper C18 2 μm, 100A.

[0279] Mass spectrometry acquisition: Primary mass spectrometry parameters: Resolution: 60,000; AGC target: Custom; Maximum IT: Custom; Scan range: 300 to 1800 m / z; Secondary mass spectrometry parameters: Resolution: 15,000; AGC target: Custom; Maximum IT: Custom; NCE / stepped NCE: 30.

[0280] S3. Software Analysis:

[0281] The data was analyzed using the Peptide Mapping analysis function of BioPharma Finder software. The analysis results are as follows: Figure 9 , 10 As shown in Figure 11.

[0282] Data processing parameters: Protease (Chymotrypsin); Static Modification (Carbamidomethylation); Variable Modification (Deamidation, Oxidation, Hyp); Mass Accuracy (10ppm).

[0283] Combination Figure 9 It can be seen that the sequence coverage of sample 705, as determined by mass spectrometry, is 100%. Proline hydroxylation-modified peptides were identified in the sample; hydroxylation modifications were detected at sites P8, P12, P17, P20, P21, P33, and P36, but the theoretical hydroxylation rates were higher at sites P12 and P36. Combined with... Figure 10It can be seen that sample 721, identified by mass spectrometry, had a sequence coverage of 93.8%. Hydroxylation modification of proline was detected at all six sites in the sample, namely P9, P12, P18, P26, P36, and P38. Sample 721 showed a higher modification ratio at the theoretical hydroxylation sites P18 and P36. Combined with... Figure 11 As can be seen from the mass spectrometry analysis, the sequence coverage of sample 3A5D2NT was 90.6%; the sample was identified as showing hydroxylation modification of proline (see [reference needed]). Figure 11 a) The sample underwent hydroxylation modification at sites P11, P14, P18, P20, P27, and P29. The secondary fragmentation matching diagram and secondary mass spectrum of the modified peptides are shown below. Figure 11 b.

[0284] In summary, 705, 721, and 3A5D2NT underwent hydroxylation at their theoretical hydroxylation sites, and also hydroxylated at other sites where proline was present. This may be related to the hydroxylation efficiency of the P4H enzyme, where proline was hydroxylated in the intracellular biological environment of Pichia pastoris.

[0285] Example 5:

[0286] This embodiment refers to GB / T16886.5-2017 in vitro cytotoxicity assay and GB / T16886.12-2017 sample preparation and reference materials. Taking recombinant 705, 721 and 3A5D2NT collagen as examples, the cytotoxicity of recombinant hydroxylated small molecule collagen was investigated by examining the cytotoxicity of recombinant 705, 721 and 3A5D2NT collagen on L-929 mammalian fibroblasts (cells from the Chinese Academy of Sciences Cell Bank, catalog number: SCSP-5039).

[0287] The specific steps are as follows:

[0288] Experimental group samples: Recombinant 705, 721 and 3A5D2NT collagen were dissolved and diluted in MEM medium to prepare concentrations of 10, 5, 2, 5, 1 and 25 mg / mL.

[0289] Negative control sample: 10% (v / v) DMSO.

[0290] Blank control sample: MEM culture medium.

[0291] (1) Cell preparation:

[0292] L-929 cells were cultured in MEM medium (containing 10% FBS and 1% penicillin-streptomycin) to the logarithmic growth phase at 37°C and 5% CO2. After culture, L-929 cells were digested with 0.25% (w / v) trypsin (containing EDTA). After digestion, the cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the centrifuged cells were resuspended in MEM medium and counted to obtain 1×10⁶ cells. 5 Cell suspension of cells / mL.

[0293] (2) Sample addition and incubation:

[0294] Cell suspension was seeded at 100 μL per well in 96-well plates and incubated in a cell culture incubator at 37°C, 5% CO2, and >90% humidity. Cell morphology was observed under a microscope. After 24 hours of culture, when the cells had adhered to the plate and grown to approximately 70% confluence, the original culture medium in the 96-well plates was discarded. 100 μL of sample working solution (blank control, negative control, and positive control) was added to each well of the 96-well plate to achieve a final concentration of 10 mg / mL. The 96-well plates were then placed in a cell culture incubator and cultured for 24 hours at 37°C, 5% CO2, and >90% humidity. Six replicates were set up for each group.

[0295] (3) Detection:

[0296] Cell morphology was observed under a microscope after 24 hours of culture in 96-well plates. The liquid was then removed, and 50 μL of MTT (final concentration 1 mg / mL) was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 2 hours. The supernatant was then removed, and 100 μL of isopropanol was added to each well to dissolve the crystals. The absorbance at 570 nm was measured using a microplate reader, and cytotoxicity was calculated. Results are as follows: Figure 12 As shown.

[0297] Data processing: mean ± standard deviation (X) 均值 ±S);

[0298] Cell viability % = OD570 of experimental sample group / OD570 of blank control group × 100%.

[0299] from Figure 12 It can be seen that the relative survival rate of recombinant 705, 721, and 3A5D2NT collagen at a concentration of 10 mg / mL is greater than 70%. The test substances showed no potential cytotoxicity to human fibroblasts. At a concentration of 1.25 mg / mL, they exhibited almost no toxicity, demonstrating potential application value in the future fields of biomaterials and cosmetics.

[0300] In summary, this invention utilizes synthetic biology, genetic engineering, and biotechnology to co-express small-molecule collagen with P4H, followed by amino acid modification, including hydroxylation at the theoretical GXY hydroxylation site, and post-translational modification to obtain the recombinant hydroxylated small-molecule collagen. The recombinant hydroxylated small-molecule collagen of this invention achieves molecular structural stability through amino acid post-expression modification, offering more controllable self-assembly compared to small-molecule collagen after expression. It achieves a balance between transdermal absorption and biological structural stability. This recombinant hydroxylated small-molecule collagen can be industrially produced and has excellent applications in pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics, and health products.

[0301] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A recombinant hydroxylated small molecule collagen, characterized in that, The recombinant hydroxylated small molecule collagen is a hydroxylated modified recombinant small molecule collagen, wherein the recombinant small molecule collagen contains an amino acid sequence with hydroxylation sites on the triple helix region of human type VII collagen or the α1 chain of human type III collagen; the amino acid sequence is shown in SEQ ID NO:

3.

2. The nucleic acid encoding the recombinant hydroxylated small molecule collagen of claim 1.

3. A tandemly recombinant hydroxylated small molecule collagen, characterized in that, The tandem recombinant hydroxylated small molecule collagen is obtained by repeating the sequence shown in SEQ ID NO:3 multiple times as the basic unit; the amino acid sequence of the tandem recombinant hydroxylated small molecule collagen is shown in SEQ ID NO:

4.

4. The nucleic acid encoding the tandemly recombinant hydroxylated small molecule collagen of claim 3.

5. The nucleic acid according to claim 4, characterized in that, The nucleic acid is selected from the nucleotide sequence shown in SEQ ID NO:8 or its degenerate sequence.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 2, or the nucleic acid as described in any one of claims 4 to 5.

7. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria comprises the nucleic acid of claim 2, or the nucleic acid of any one of claims 4 to 5, or comprises the recombinant expression vector of claim 6, or is a recombinant engineered bacteria containing a recombinant plasmid expressing P4H enzyme that can express the recombinant hydroxylated small molecule collagen of claim 1 or the tandem recombinant hydroxylated small molecule collagen of claim 3.

8. The recombinant engineered bacteria according to claim 7, characterized in that, The recombinant engineered bacteria include a chassis strain for expressing the recombinant hydroxylated small molecule collagen of claim 1 or the tandem recombinant hydroxylated small molecule collagen of claim 3, wherein the chassis strain expresses P4H enzyme.

9. The recombinant engineered bacteria according to claim 8, characterized in that, The chassis strain contains nucleic acids encoding the amino acids shown in SEQ ID No. 10 and SEQ ID No.

13.

10. The recombinant engineered bacteria according to claim 7, characterized in that, The recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.33067.

11. A method for preparing recombinant hydroxylated small molecule collagen, characterized in that, The preparation method includes: (1) Select and design the sequence of recombinant hydroxylated small molecule collagen, and then construct tandem recombinant hydroxylated small molecule collagen based on the recombinant hydroxylated small molecule collagen unit in tandem repeat; The recombinant hydroxylated small molecule collagen is a hydroxylated modified recombinant small molecule collagen, wherein the recombinant small molecule collagen is an amino acid sequence containing hydroxylation sites on the triple helix region of human type VII collagen or the α1 chain of human type III collagen. The amino acid sequence is shown in SEQ ID NO:4; (2) Construct a recombinant expression vector for expressing tandem recombinant hydroxylated small molecule collagen; Construct a recombinant vector expressing P4H enzyme; (3) The recombinant expression vector expressing tandem recombinant hydroxylated small molecule collagen and the recombinant vector expressing P4H enzyme were electroporated into the host bacteria, screened and verified, and recombinant engineered bacteria expressing high copy number of recombinant hydroxylated small molecule collagen were obtained. (4) The obtained recombinant engineered bacteria were fermented and induced to express the recombinant hydroxylated small molecule collagen.

12. The method for preparing recombinant hydroxylated small molecule collagen according to claim 11, characterized in that, In step (1), the nucleic acid of the tandemly recombinant hydroxylated small molecule collagen is selected from the nucleotide sequence shown in SEQ ID NO:8 or its degenerate sequence.

13. The method for preparing recombinant hydroxylated small molecule collagen according to claim 11, characterized in that, In step (2), the recombinant expression vector and the recombinant plasmid are selected from pPICZαB, pFLDα, and pPIC9K.

14. The method for preparing recombinant hydroxylated small molecule collagen according to claim 13, characterized in that, The carriers are pPIC9K and pPICZαB.

15. The method for preparing recombinant hydroxylated small molecule collagen according to claim 11, characterized in that, In step (2), the recombinant plasmid expressing P4H enzyme contains nucleic acid encoding the amino acids shown in SEQ ID No. 10 and SEQ ID No. 13, a PTEF promoter element, and a PEM7 promoter element.

16. The method for preparing recombinant hydroxylated small molecule collagen according to claim 11, characterized in that, In step (3), the host bacteria is selected from one of Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha.

17. The method for preparing recombinant hydroxylated small molecule collagen according to claim 16, characterized in that, The host organism is Pichia pastoris.

18. The method for preparing recombinant hydroxylated small molecule collagen according to claim 17, characterized in that, The host bacteria are Pichia pastoris X33 or Pichia pastoris KM71H.

19. The method for preparing recombinant hydroxylated small molecule collagen according to claim 18, characterized in that, The host bacteria are CGMCC No.29601, CGMCC No.25815, and CGMCC No.25819.

20. The method for preparing recombinant hydroxylated small molecule collagen according to claim 11, characterized in that, In step (3), the recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.33067.

21. Recombinant hydroxylated small molecule collagen prepared by the method according to any one of claims 11 to 20.

22. An expression system for recombinant hydroxylated small molecule collagen, characterized in that, The expression system comprises: the recombinant engineered bacteria expressing the recombinant plasmid of P4H enzyme as described in claim 7, and the tandem recombinant hydroxylated small molecule collagen constructed based on the recombinant hydroxylated small molecule collagen as described in claim 1; The amino acid sequence of the tandemly recombinant hydroxylated small molecule collagen is shown in SEQ ID NO:

4.

23. A composition, characterized in that, The composition comprises the recombinant hydroxylated small molecule collagen of claim 1, or the recombinant hydroxylated small molecule collagen encoded by nucleic acid of claim 2, or the tandem recombinant hydroxylated small molecule collagen of claim 3, or the tandem recombinant hydroxylated small molecule collagen encoded by nucleic acid of any one of claims 4-5, or the recombinant expression vector of claim 6, or the recombinant engineered bacteria of any one of claims 7-10, or the recombinant hydroxylated small molecule collagen prepared by any one of claims 11-20; the composition is selected from medical devices, biomaterials, tissue engineering products, and cosmetics.

24. Articles comprising the recombinant hydroxylated small molecule collagen of claim 1, or the nucleic acid-encoded recombinant hydroxylated small molecule collagen of claim 2, or the tandem recombinant hydroxylated small molecule collagen of claim 3, or the nucleic acid-encoded tandem recombinant hydroxylated small molecule collagen of any one of claims 4-5, or the recombinant expression vector of claim 6, or the recombinant engineered bacteria of any one of claims 7-10, or the recombinant hydroxylated small molecule collagen prepared by any one of claims 11-20, or the composition of claim 23; wherein the articles are selected from medical devices, biomaterials, tissue engineering products, and cosmetics.

25. The use of the recombinant hydroxylated small molecule collagen of claim 1, or the recombinant hydroxylated small molecule collagen encoded by nucleic acid of claim 2, or the tandem recombinant hydroxylated small molecule collagen of claim 3, or the tandem recombinant hydroxylated small molecule collagen encoded by nucleic acid of any one of claims 4-5, or the recombinant expression vector of claim 6, or the recombinant engineered bacteria of any one of claims 7-10, or the recombinant hydroxylated small molecule collagen prepared by the method of any one of claims 11-20, or the composition of claim 23, or the article of claim 24 in the preparation of medical devices, biomaterials, tissue engineering products, and cosmetics.

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