Escherichia coli capable of efficiently expressing hydroxylated human collagen and construction method of escherichia coli

Abstract

The invention discloses Escherichia coli capable of efficiently expressing hydroxylated human collagen and a construction method of the Escherichia coli, and belongs to the technical field of biological engineering. According to the technical scheme, the engineered escherichia coli capable of efficiently expressing hydroxylated collagen is characterized in that an escherichia coli strain is modified as follows: at least one of proline metabolic pathway genes proA and proB in the escherichia coli strain is knocked out; and a collagen expression gene is introduced into a knocked-out Escherichia coli strain body. The invention develops a method for efficiently expressing hydroxylated human collagen, and the problems that an escherichia coli system cannot perform hydroxylation modification on exogenous recombinant protein, hydroxyproline in a culture medium is directly utilized, and the modification efficiency is low when a traditional strain co-expresses P4H are systematically solved.

Description

Escherichia coli that efficiently expresses hydroxylated human collagen and its construction method Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to Escherichia coli that efficiently expresses hydroxylated human collagen and its construction method. Background Technology

[0002] Collagen, a fibrous protein, is abundant in the human and animal bodies, accounting for approximately 25% to 30% of total protein. More than 30 members of the collagen family have been identified. Type I, II, and III collagen are the main components of human collagen fibers. Collagen molecules have a characteristic triple helix structure, and their polypeptide chains contain a typical Gly-XY repeating amino acid sequence, usually with proline (Pro) at the X site and hydroxyproline (Hyp) at the Y site. The formation of hydroxyproline plays a crucial role in maintaining the stability of the triple helix structure.

[0003] In living organisms, collagen is widely distributed in skin, bones, tendons, ligaments, cornea, blood vessels, and various organs and tissues. It is a key component of the extracellular matrix and is crucial for maintaining the integrity and mechanical stability of tissue structures. Type I collagen forms highly ordered fiber bundles, providing support for tissues such as skin; Type II collagen is mainly found in cartilage, forming its intricate network structure; Type III collagen often coexists with Type I in connective tissue, participating in the formation of the network matrix and playing an important role in cell attachment, migration, and tissue repair. Due to its excellent biocompatibility, low immunogenicity, biodegradability, and diverse biological activities, collagen has broad application value in medical materials, skincare, functional foods, and cosmetics.

[0004] Currently, industrial-scale collagen production methods mainly fall into two categories: The first is extraction from animal tissues such as pig skin, cow skin, and fish skin using acid, alkali, or enzymatic methods. This method is technically mature and can yield collagen with certain biological activity, but it also has significant drawbacks, including potential viral contamination and immunogenicity risks, large batch-to-batch quality fluctuations, and limited raw material sources, making it difficult to meet the growing market demand. The second method involves preparation through recombinant gene expression technology. This approach has advantages such as a short production cycle, no risk of animal-derived viruses, low immunogenicity, good solubility, and ease of large-scale production, and is considered an important direction for solving collagen source limitations and promoting industrial upgrading.

[0005] However, the large-scale production of recombinant collagen still faces significant technical bottlenecks, hindering its industrial promotion and competitiveness. The core issues are concentrated in the performance limitations of expression systems and the low efficiency of hydroxylation modification: First, existing expression systems suffer from performance imbalances. While eukaryotic systems such as insect cells and mammalian cells can perform post-translational modifications similar to those in humans, theoretically yielding highly bioactive recombinant human collagen, they generally suffer from long culture cycles, low expression levels, high costs, and difficulties in process scale-up, making it difficult to achieve economical and efficient large-scale production. Prokaryotic expression systems such as E. coli and yeast, although fast-growing, high-yield, and low-cost, lack a proline hydroxylase (P4H) system, making it impossible to effectively hydroxylate proline in collagen. This results in the product failing to form a stable triple helix structure, limiting its bioactivity and application performance.

[0006] Secondly, the problem of low efficiency in hydroxylation modification has not been effectively solved. Hydroxylation of proline is key to the formation of a stable triple helix structure in collagen and the maintenance of its biological functions (such as platelet adhesion activity). At present, the hydroxylation modification strategies for prokaryotic systems still have obvious defects: (1) When proline hydroxylase (P4H) is introduced exogenously for co-expression, the hydroxylation efficiency is generally low, especially the modification of proline at the Y site in the Gly-XY sequence is insufficient; (2) P4H from natural sources has a complex structure and often has low expression activity in prokaryotic hosts, and so far no ideal natural P4H that can be efficiently expressed in prokaryotic systems and achieve efficient hydroxylation of collagen has been found.

[0007] The aforementioned technological bottlenecks have resulted in persistently high production costs for recombinant collagen, weakening its market competitiveness. This has led many application areas to continue using lower-cost but potentially unsafe animal-derived collagen, severely hindering the healthy development of the recombinant collagen industry. Therefore, developing a simple, cost-effective, highly efficient, and stable prokaryotic expression technology for recombinant human collagen has become an urgent technical problem to be solved in this field.

[0008] Relevant patent literature retrieved: Publication country: China, Publication number: CN119506180A, Publication date: February 25, 2025. This literature discloses an engineered E. coli strain that efficiently expresses hydroxylated collagen and its establishment method. The core technical solution is an engineered E. coli strain that efficiently expresses hydroxylated collagen. This is achieved by knocking out the proline metabolism pathway genes proA / proB in the E. coli strain; simultaneously, the P4H gene is knocked into the E. coli strain to supplement or regulate the hydroxylation modification of proline, resulting in a strain that is expected to achieve large-scale, stable production of highly hydroxylated modified collagen. This invention solves the problem of insufficient or no hydroxylation of recombinant collagen in traditional prokaryotic expression systems, constructing a stable hydroxylated collagen expression strain and cultivation method. This provides the possibility of obtaining more ideal triple-helix stable and functional collagen products, and will promote the stable and large-scale production of recombinant collagen.

[0009] This document, published in China (CN118240735B) on June 25, 2024, discloses a bacterial strain capable of expressing exogenous proteins, recombinant human collagen, its synthesis method, and its applications. The invention first provides a proline-auxotrophic *Escherichia coli* strain capable of efficiently expressing exogenous proteins. Using this strain as a host to express recombinant human collagen, proline and hydroxyproline are exogenously incorporated. By controlling the ratio of proline to hydroxyproline in the culture medium, the hydroxylation rate of collagen can be precisely controlled. This synthesis method yields recombinant collagen with a hydroxylation rate close to that of natural human collagen, exhibiting better cell adhesion and protein stability.

[0010] Relevant non-patent literature retrieved: Journal title: *Applied Microbiology and Biotechnology*, Article title: "Recombinant expression of hydroxylated human collagen in *Escherichia coli*", Volume: 98, pp. 4445–4455, Publication date: December 21, 2013. This article discloses a method for generating hydroxylated collagen by characterizing novel prolyl and lysine hydroxylase genes encoded by the mimivirus. In *E. coli*, co-expression of human type III collagen with mimivirus prolyl and lysine hydroxylases yielded up to 90 mg of hydroxylated collagen per liter of culture. The prolyl and lysine hydroxylation levels reached 25% and 26%, respectively, similar to the hydroxylation levels of natural human type III collagen. The distribution of hydroxyproline and hydroxyethylamine along the recombinant collagen was also similar to that of natural collagen as determined by tryptophan peptide mass spectrometry analysis. The triple-helix characteristic of recombinant hydroxyl collagen was confirmed by circular dichroism, a process that also demonstrated that hydroxylation enhances the thermal stability of the recombinant collagen structure. Recombinant hydroxyl collagen produced by *E. coli* supported the growth of human umbilical cord endothelial cells, highlighting the biocompatibility of the recombinant protein as an extracellular matrix. The high yield of recombinant protein expression and the extensive levels of proline and lysyl hydroxylation indicate that recombinant hydroxyl collagen can be mass-produced in biomaterials engineering for biomedical applications.

[0011] The existing technologies represented by the aforementioned literature have at least the following unresolved technical problems or defects: the strain construction process is cumbersome, requiring multiple gene knockouts and relying on dual-vector co-transformation, resulting in high operational complexity and increased costs. Relevant evidence is as follows: CN118240735B requires the knockout of the proline synthesis pathway gene (ProC) and the ompT and Lon protease genes (a total of 3 genes), and requires the co-transformation of the recombinant collagen expression vector (pET28a-rhCOL) and the T7 RNA polymerase expression vector (pBAD33-T7RNAP) into the host bacteria at a 1:1 molar ratio. The multi-step gene editing and dual-vector transformation not only prolong the strain construction cycle, but also increase the screening difficulty and reagent costs; at the same time, this scheme requires the use of a specific knockout plasmid (pTargetF) in conjunction with the Cas9 plasmid, further raising the technical threshold. Summary of the Invention

[0012] The purpose of this invention is to provide: an Escherichia coli strain that efficiently expresses hydroxylated human collagen and its construction method, as well as related technologies, to solve the technical problems or combinations thereof in the prior art, such as cumbersome strain construction process, complex culture process and poor adaptability to large-scale production.

[0013] Terminology: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions provided in this chapter shall prevail.

[0014] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0015] Definitions of standard terms can be found in the reference books “Molecular Cloning: A Laboratory Manual (4th Edition), Science Press, authors: (US) MR. Green and J. Sambrook, 2013.10”, “Genetic Engineering (2nd Edition), Chemical Industry Press, author: Yuan Wuzhou, 2019.09”, and “Protein Purification: A Manual (Chinese 2nd Edition), Science Press, author: (US) RR. Burgess, 2013.08”.

[0016] Unless otherwise stated, conventional methods within the scope of the art, such as SDS-PAGE electrophoresis, are used. Unless specifically defined, the use of various commercially available products used herein employs standard techniques. For example, they may be performed using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein are generally performed according to conventional methods well known in the art, based on the descriptions in the various general and more specific documents cited and discussed in this specification.

[0017] The terms "optional" or "arbitrarily" mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: "The collagen includes, but is not limited to, any one or more of: recombinant humanized type III collagen, recombinant humanized type I collagen, recombinant humanized type IV collagen, and recombinant humanized type VII collagen," for example: it means "The collagen includes, but is not limited to: recombinant humanized type III collagen"; or "The collagen includes, but is not limited to: recombinant humanized type VII collagen"; or "The collagen includes, but is not limited to: recombinant humanized type III collagen and recombinant humanized type I collagen"; or "The collagen includes, but is not limited to: recombinant humanized type III collagen, recombinant humanized type I collagen, recombinant humanized type IV collagen, and recombinant humanized type VII collagen."

[0018] The term "collagen" used in this article refers to a type of fibrous protein that is widely found in animal connective tissues. It consists of three polypeptide chains intertwined to form a triple helix structure and is a major structural protein that makes up tissues such as skin, bones, and tendons. It has the functions of supporting, protecting, and connecting tissues.

[0019] The term "hydroxylated collagen" as used in this article refers to the protein product formed after the proline or lysine residues in the collagen peptide chain are hydroxylated under the catalysis of the corresponding hydroxylase. Hydroxylation modification can enhance the cross-linking between collagen molecules, thereby improving its structural stability and biological activity.

[0020] The term "engineered Escherichia coli" as used in this article refers to genetically engineered strains that use Escherichia coli as a host strain and modify its genome through genetic engineering techniques (such as gene knockout, introduction of exogenous genes, etc.) to enable it to have specific functions (such as efficient expression of exogenous proteins, synthesis of specific metabolites, etc.).

[0021] The term "proline metabolic pathway" used in this article refers to a series of enzymatic reaction pathways in organisms that catalyze the synthesis and degradation of proline, including the biosynthetic pathway that synthesizes proline from precursors such as glutamate, and the degradation pathway that breaks down proline into products such as glutamate. This pathway regulates the homeostasis of proline in cells.

[0022] The term "proA" used in this article refers to the gene encoding γ-glutamyl phosphate reductase (γ-GPR), whose expression product catalyzes the conversion of γ-glutamyl phosphate (γ-GP) to γ-glutamic acid semialdehyde (GSA). It is a key functional gene in the bacterial proline biosynthesis of glutamate (Glu). Knocking out this gene can inhibit the endogenous synthesis of proline in bacteria, making the strain proline-dependent or defective.

[0023] The term "proB" used in this article refers to the gene encoding γ-glutamyl kinase (γ-GK), whose expression product catalyzes the phosphorylation of L-glutamate to γ-glutamyl phosphate (γ-GP), which is a key gene for initiating the bacterial proline biosynthesis of glutamate (Glu). Knocking out this gene can block the endogenous proline synthesis pathway, enabling the strain to better utilize proline (Pro) or hydroxyproline (Hyp) in the exogenous culture medium.

[0024] The term "proline hydroxylase" as used in this article refers to a class of metalloproteinases that catalyze the hydroxylation of proline residues in the peptide chains of proteins such as collagen. It can convert proline into hydroxyproline and is essential for maintaining the triple helix structure and biological function of collagen.

[0025] The term "BL21(DE3)" used in this article refers to a commonly used Escherichia coli protein expression strain that integrates the DE3 region of λ phage on its chromosome. It can induce the expression of T7 RNA polymerase, is suitable for expression vectors containing the T7 promoter, and can efficiently express exogenous recombinant proteins.

[0026] The term "Rosetta (DE3)" used in this article refers to a codon-biased Escherichia coli expression strain that supplements the tRNA genes corresponding to rare codons in E. coli, effectively solving the problem of low expression levels of exogenous proteins caused by rare codons.

[0027] The term “Origami (DE3)” as used in this article refers to an Escherichia coli strain used to express exogenous proteins with correctly folded disulfide bonds. The metabolic pathways of reduced and oxidized glutathione in its genome have been modified, and the cytoplasmic environment is more conducive to the formation of disulfide bonds, making it suitable for expressing proteins that require disulfide bonds to maintain their activity.

[0028] The term "HSM174(DE3)" as used in this article refers to an Escherichia coli strain suitable for the expression of exogenous proteins, which usually has specific genetic background modifications (such as protease deficiency) to reduce the degradation of exogenous proteins and improve the expression stability of target proteins.

[0029] The term "JM109" as used in this article refers to a commonly used Escherichia coli clone strain, belonging to the recombinant-deficient strain (recA). - This can reduce the probability of homologous recombination of exogenous plasmids and improve the stability of plasmids. It is often used in molecular biology experiments such as gene cloning and vector construction.

[0030] The term "AS1.357" as used in this article refers to an Escherichia coli strain commonly used in microbial metabolism research or enzymatic experiments, possessing specific physiological and biochemical characteristics, suitable for the synthesis of specific metabolites or the analysis of enzyme activity.

[0031] The term "TOP10" as used in this article refers to a strain of *E. coli* widely used in gene cloning, characterized by high transformation efficiency and good plasmid stability, whose genotype is typically recA. - endA - This avoids the degradation of exogenous DNA and is suitable for plasmid amplification and preservation.

[0032] The term "DH5α" used in this article refers to: a classic Escherichia coli clone strain that is deficient in endonuclease (endA). - And recombinase deficiency (recA) - It has high transformation efficiency and good plasmid extraction quality, and is often used for plasmid cloning, gene library construction and amplification of exogenous genes.

[0033] The term “CRISPR / Cas9” as used in this article refers to a gene editing technology derived from the bacterial acquired immune system, consisting of guide RNA (sgRNA) and Cas9 protein. The sgRNA can guide the Cas9 protein to target and cut specific DNA sequences in the genome, enabling precise editing such as gene knockout, insertion, or replacement.

[0034] The term “suicide plasmid pCVD” used in this article refers to a type of plasmid vector that cannot replicate autonomously in a host strain. It typically carries a homologous recombination arm, a selection marker gene, and a suicide gene, and can integrate the target sequence into the host genome through homologous recombination. It is suitable for gene knockout or gene replacement operations.

[0035] The term "Red / ET recombination" as used in this article refers to a homologous recombination technology based on λ phage Red recombinase or Rac phage ET recombinase, which can mediate efficient recombination between linear DNA fragments and homologous sequences of the host genome without the need for restriction endonucleases and ligases, and is widely used for rapid modification of bacterial genomes.

[0036] The term “type II intron insertion” used in this article refers to a gene editing technique that utilizes the homing properties of type II introns. Type II introns are a type of mobile genetic element that can specifically insert into the target gene sequence, causing the target gene to become inactive and thus achieving gene knockout.

[0037] The term “Cre-LoxP recombination” as used in this article refers to a site-specific recombination technology based on Cre recombinase and LoxP sites. Cre recombinase can recognize and catalyze the deletion, inversion or translocation of DNA sequences between two LoxP sites, and is often used for conditional gene knockout and gene expression regulation.

[0038] The term "TALEN gene targeting" used in this article refers to a gene editing technology based on transcription activator-like effector nucleases. TALEN consists of a specific DNA-binding domain and a nuclease domain, which can target and cut specific gene sequences, inducing host cells to repair DNA through non-homologous end joining or homologous recombination, thereby achieving gene editing.

[0039] The term "homologous recombination arm" used in this article refers to a DNA fragment that is highly homologous to the target sequence at both ends of the host genome. During homologous recombination, it can mediate the precise pairing and exchange between exogenous DNA and the target sequence of the host genome, and is a key element for gene knockout, replacement and other operations.

[0040] The term "sgRNA" used in this article refers to single guide RNA, which is one of the core components of the CRISPR / Cas9 gene editing system. It consists of a target sequence and a backbone sequence. The target sequence can be complementary to the genomic target DNA to guide the Cas9 protein to cut the target sequence.

[0041] The term "RED recombinase" as used in this article refers to a group of recombinases (including Exo, Beta, and Gamma proteins) derived from λ phage. Among them, the Exo protein has 5'→3' exonuclease activity, the Beta protein can bind single-stranded DNA and mediate homologous pairing, and the Gamma protein can inhibit the activity of host nucleases. The three work together to efficiently mediate homologous recombination reactions.

[0042] The term "IPTG" used in this article refers to isopropyl-β-D-thiogalactoside, a synthetic lactose analog that can bind to the repressor protein of the Escherichia coli lactose operon and relieve its inhibition of the promoter. It is often used to induce expression vectors containing lactose promoters or T7 promoters to synthesize exogenous proteins.

[0043] The term "SDS-PAGE" used in this article refers to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an electrophoresis technique that separates proteins based on their molecular weight. SDS denatures proteins and imparts a uniform negative charge. During electrophoresis, the migration rate of proteins in the gel is only related to their molecular weight. It is commonly used for the separation, identification, and expression analysis of proteins.

[0044] The term “Ni-NTA affinity chromatography” as used in this article refers to a protein purification technique based on metal chelate affinity. Ni-NTA (nickel ion-nitrotriacetic acid) resin can specifically bind to proteins with histidine tags, and high-purity target proteins can be obtained by elution with imidazole solution.

[0045] The term "enterokinase (EK)" as used in this article refers to a serine protease derived from the mammalian intestine that can specifically recognize and cleave the carboxyl terminus of the Asp-Asp-Asp-Asp-Lys sequence in proteins. It is often used to remove fusion sequences such as histidine tags from recombinant proteins to obtain target proteins with native structures.

[0046] The term "DEAE anion exchange chromatography" used in this article refers to a protein separation and purification technique based on charge-electric interaction. DEAE (diethylaminoethyl) is an anion exchange group that can bind to negatively charged proteins. By changing the ionic strength of the eluent, proteins with different charges can be separated.

[0047] The term "P4H" used in this article refers to prolyl-4-hydroxylase, a type of metalloproteinase located in the endoplasmic reticulum, which usually exists in the form of α2β2 tetramers. It can specifically recognize and catalyze the introduction of a hydroxyl group at the C4 carbon atom of proline residues in the -X-Pro-Gly- repeat sequence of proteins such as collagen, converting them into 4-hydroxyproline. This hydroxylation modification can stabilize the triple helix structure of collagen and is a key post-translational modification step in maintaining the biological activity of collagen.

[0048] The term "rhCol" used in this article refers to recombinant human collagen, which is a recombinant protein obtained by introducing the human collagen gene into a host strain (such as Escherichia coli) through genetic engineering, followed by induced expression and purification.

[0049] The term "chloramine-T method" used in this article refers to a chemical method for detecting the content of hydroxyproline in a sample. Chloramine-T is used to oxidize hydroxyproline, and the oxidation product reacts with p-dimethylaminobenzaldehyde to generate a red compound. The content of hydroxyproline can be quantitatively analyzed by measuring the absorbance value, which indirectly reflects the hydroxylation level of collagen.

[0050] The term "Pro" used in this article refers to proline, an α-amino acid that is a nonpolar amino acid and one of the basic building blocks of proteins. It is also a substrate for the hydroxylation modification of proteins such as collagen.

[0051] The term "Hyp" used in this article refers to hydroxyproline, which is the product of proline undergoing hydroxylation modification under the catalysis of proline hydroxylase. It is a characteristic amino acid of collagen, and its content can reflect the degree of hydroxylation and structural stability of collagen.

[0052] In a first aspect, the present invention provides: an engineered strain of *Escherichia coli* that efficiently expresses hydroxylated collagen.

[0053] This includes engineered Escherichia coli.

[0054] Specifically, the following modifications were made to the Escherichia coli strain: a. knocking out at least one of the proline metabolic pathway genes proA and proB in the Escherichia coli strain; b. introducing a collagen expression gene into the knocked-out Escherichia coli strain.

[0055] Specifically, the Escherichia coli strains include, but are not limited to: BL21(DE3), Rosetta(DE3), Origami(DE3), HSM174(DE3), JM109, AS1.357, TOP10, or DH5α strains.

[0056] Preferably, the Escherichia coli strain includes BL21(DE3) or DH5α strain.

[0057] More preferably, the Escherichia coli strain is BL21(DE3).

[0058] Specifically, the collagen includes, but is not limited to, any one or more of the following: recombinant humanized type III collagen, recombinant humanized type I collagen, recombinant humanized type IV collagen, and recombinant humanized type VII collagen.

[0059] Preferably, the collagen is recombinant humanized type III collagen.

[0060] More specifically, the amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID NO:35.

[0061] Specifically, the knockout methods include, but are not limited to: CRISPR / Cas9-mediated gene knockout, suicide plasmid pCVD-mediated gene knockout, Red / ET recombination-mediated gene knockout, type II intron insertion-mediated gene knockout, Cre-LoxP recombination-mediated gene knockout, or TALEN gene targeting.

[0062] Preferably, the knockout method is CRISPR / Cas9-mediated gene knockout.

[0063] Specifically, the nucleotide sequence of the proA gene is shown in SEQ ID NO:1.

[0064]

[0065] Specifically, the nucleotide sequence of the proB gene is shown in SEQ ID NO:2.

[0066]

[0067] Based on further solutions to the technical problems of the present invention, or simultaneously solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: A first preferred solution: an engineered *E. coli* strain that efficiently expresses hydroxylated collagen, comprising the following modifications to the *E. coli* strain: a. knocking out at least one of the proline metabolic pathway genes proA and proB within the *E. coli* strain; b. introducing a collagen expression gene into the knocked-out *E. coli* strain. This technical solution, while solving the technical problem of "the cumbersome strain construction process, complex culture procedure, and poor scalability in the prior art," further solves the technical problem of "providing an engineered *E. coli* strain with a simple construction process."

[0068] Secondly, the present invention provides the application of the above-mentioned engineered Escherichia coli.

[0069] This includes: applications.

[0070] The application is selected from: the application of the above-mentioned engineered Escherichia coli in the expression of hydroxylated collagen.

[0071] Based on further solutions to the technical problems of the present invention, or simultaneously solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes: A first preferred solution: the application of the above-mentioned engineered Escherichia coli strain in the expression of hydroxylated collagen. This technical solution, while solving the technical problem of "the cumbersome strain construction process, complex culture procedure, and poor scalability in the prior art," further solves the technical problem of "providing an application of engineered Escherichia coli strains with a simple construction process."

[0072] Thirdly, the present invention provides a method for preparing hydroxylated collagen.

[0073] This includes: preparation method.

[0074] Specifically, the preparation method includes the following steps: S1, culturing the above-mentioned engineered Escherichia coli in a culture medium; S2, inducing collagen expression; S3, separating and purifying the expression product to obtain hydroxylated collagen.

[0075] Specifically, the culture medium described in step S1 is a culture medium with low free proline.

[0076] Specifically, the concentration of proline in the culture medium described in step S1 is less than 1 mM.

[0077] Specifically, the components of the culture medium described in step S1 include, but are not limited to: 10g of casein peptone containing low free proline, 5g of yeast extract containing low free proline, and 20-40g of sodium chloride per liter of culture medium.

[0078] Specifically, the final concentration of the low free proline is <1 mM.

[0079] Specifically, sodium chloride, proline, and / or hydroxyproline are added during the induction process described in step S2.

[0080] Specifically, the final concentration of sodium chloride is 20-40 g / L, the final concentration of proline is 5-100 mM, and the final concentration of hydroxyproline is 10-100 mM.

[0081] Preferably, the final concentration of sodium chloride is 30 g / L.

[0082] Preferably, the proline is L-proline, and the hydroxyproline is L-hydroxyproline.

[0083] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the third aspect of the present invention includes: a first preferred solution: a method for preparing hydroxylated collagen, the preparation method comprising the following steps: S1, culturing the above-mentioned engineered Escherichia coli in a culture medium; S2, inducing collagen expression; S3, separating and purifying the expression product to obtain hydroxylated collagen. This technical solution, while solving the technical problem of "the cumbersome strain construction process, complex culture procedure, and poor scalability in the prior art," further solves the technical problem of "providing a method for preparing hydroxylated collagen with strong scalability."

[0084] Fourthly, the present invention provides the application of the hydroxylated collagen obtained by the above preparation method.

[0085] This includes: applications.

[0086] The applications are selected from: the use of hydroxylated collagen prepared by the above preparation method in the preparation of bioengineering materials, food supplements, and cosmetics.

[0087] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the fourth aspect of the present invention includes: A first preferred solution: the application of hydroxylated collagen obtained by the above-mentioned preparation method in the preparation of bioengineering materials, food supplements, and cosmetics. This technical solution, while solving the technical problem of "the cumbersome strain construction process, complex culture procedure, and poor scalability in the prior art," further solves the technical problem of "providing the application of hydroxylated collagen expressed by engineered Escherichia coli strains with a simple construction process."

[0088] Examples 1-2 of this invention at least support the protection scope of "engineered Escherichia coli that efficiently expresses hydroxylated collagen".

[0089] The term "engineered Escherichia coli strain that efficiently expresses hydroxylated collagen" is derived from the aforementioned explanation and / or the corresponding "knockout of the proA / B gene" and "the correctly deleted strains being named BL21(DE3) / ΔproA, BL21(DE3) / ΔproB, and BL21(DE3) / ΔproAB," etc., summarized by the common characteristic of "engineered Escherichia coli strain that efficiently expresses hydroxylated collagen" in Examples 1-2. Therefore, those skilled in the art can reasonably infer that "engineered Escherichia coli strain that efficiently expresses hydroxylated collagen," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing technical level, should all fall within the protection scope of "engineered Escherichia coli strain that efficiently expresses hydroxylated collagen." Replacing "engineered Escherichia coli strain that efficiently expresses hydroxylated collagen" with "genetically engineered Escherichia coli that can efficiently secrete active hydroxylated collagen" still falls within the protection scope of this invention.

[0090] Examples 1-4 of this invention at least support the protection scope of "hydroxylated collagen".

[0091] The term "hydroxylated collagen" is derived from the aforementioned explanations and / or the corresponding statements in Examples 1-4, such as "rhCol expression tends to stabilize when Hyp is added at a level of 20 mM or higher" and "rhCol protein," and is summarized by the common characteristic of "hydroxylated collagen." Therefore, those skilled in the art can reasonably infer that "hydroxylated collagen," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level, should all fall within the protection scope of "hydroxylated collagen." Replacing "hydroxylated collagen" with "recombinant hydroxylated collagen," "enzymatic modification of hydroxylated collagen," etc., still falls within the protection scope of this invention.

[0092] Examples 1-4 of this invention at least support the protection scope of "method for preparing hydroxylated collagen".

[0093] The "method for preparing hydroxylated collagen" is summarized from the foregoing explanation and / or the corresponding steps in Examples 1-4, such as "inoculating positive clones verified by pET28A-rhCol / BL21(DE3) / ΔproA sequencing into 5 mL of LB medium containing Kan (50 μg / mL) and 2% glucose," "culturing at 37°C and 220 rpm to the logarithmic phase," and "loading the supernatant onto a well-equilibrated Ni-NTA affinity chromatography column," all of which are derived from the common characteristic of "method for preparing hydroxylated collagen." Therefore, those skilled in the art can reasonably infer that "method for preparing hydroxylated collagen," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of "method for preparing hydroxylated collagen." Replacing "hydroxylated collagen" with "method for preparing recombinant hydroxylated collagen," "method for preparing hydroxylated collagen," etc., still falls within the protection scope of this invention.

[0094] Examples 1-4 of this invention at least support the protection scope of "the application of hydroxylated collagen".

[0095] The term "application of hydroxylated collagen" is derived from the aforementioned explanation and / or the corresponding statements in Examples 1-4, such as "collagen has a wide range of applications in medical materials, skincare, functional foods, and cosmetics," and is summarized by the common characteristic of "application of hydroxylated collagen." Therefore, those skilled in the art can reasonably presume that "application of hydroxylated collagen," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of "application of hydroxylated collagen."

[0096] The present invention has at least the following beneficial effects: compared with the prior art, the present invention has better technical effects in terms of the hydroxylation level of recombinant human collagen (rhCol).

[0097] According to experimental tests, this invention reduces the hydroxylation level of recombinant human collagen (rhCol) from the absorbance (OD) value of existing technologies. 560 The value was increased from 0.06-0.75 to over 0.86. Attached Figure Description

[0098] Figure 1 is a schematic diagram of the technical solution of the present invention.

[0099] Figure 2 is a schematic diagram of the glutamate pathway for proline production in bacteria.

[0100] Figure 3 shows the plasmid map of the proA gene knockout targeting sgRNA vector.

[0101] Figure 4 shows the plasmid map of the proB gene knockout targeting sgRNA vector.

[0102] Figure 5 shows the plasmid map of the proAB gene knockout targeting sgRNA vector.

[0103] Figure 6 shows the pEcCas protein vector plasmid map.

[0104] Figure 7 shows the electrophoresis diagram for PCR verification of proA gene knockout; in the figure, M represents DNA marker, WT represents negative clone amplification product control, 1 represents positive clone amplification product-1, 2 represents negative clone amplification product-1, 3 represents positive clone amplification product-2, 4 represents positive clone amplification product-3, 5 represents positive clone amplification product-4, 6 represents positive clone amplification product-5, 7 represents negative clone amplification product-2, 8 represents negative clone amplification product-3, 9 represents positive clone amplification product-6, 10 represents positive clone amplification product-7, 11 represents positive clone amplification product-8, 12 represents positive clone amplification product-9, 13 represents positive clone amplification product-10, 14 represents positive clone amplification product-11, and 15 represents positive clone amplification product-12; the size of the positive amplification product is 1.2kb, and the size of the negative amplification product is 1.7kb.

[0105] Figure 8 shows the PCR verification electrophoresis diagram of proB gene knockout; in the figure, M represents DNA marker, WT represents negative clone amplification product control, 1 represents positive clone amplification product-1, 2 represents positive clone amplification product-2, 3 represents positive clone amplification product-3, 4 represents positive clone amplification product-4, and 5 represents positive clone amplification product-5; the size of the positive amplification product is 1.1kb.

[0106] Figure 9 shows the PCR verification electrophoresis diagram of proAB gene knockout; in the figure, M represents DNA marker, WT represents negative clone amplification product control, 1 represents positive clone amplification product-1, 2 represents negative clone amplification product-1, 3 represents positive clone amplification product-2, 4 represents positive clone amplification product-3, and 5 represents non-specific amplification product; the size of the positive amplification product is 1.2kb, and the size of the negative amplification product is 2.3kb.

[0107] Figure 10 shows the plasmid map of the recombinant expression P4H vector.

[0108] Figure 11 shows the plasmid map of the recombinant rhCol expression vector.

[0109] Figure 12 shows the SDS-PAGE electrophoresis diagram of rhCol expression in different strains.

[0110] Figure 13 shows the SDS-PAGE electrophoresis diagram of rhCol expression under different NaCl additions.

[0111] Figure 14 shows the SDS-PAGE electrophoresis diagram of rhCol expression under different Pro additions.

[0112] Figure 15 shows the SDS-PAGE electrophoresis diagram of rhCol expression under different Hyp additions.

[0113] Figure 16 shows the SDS-PAGE electrophoresis diagram of rhCol protein purification; in the figure, M represents protein marker, lysate represents bacterial cell lysate, NI represents sample purified by nickel column, EK represents sample digested by enterokinase EK, and Col represents ultrafiltration concentrated sample purified by DEAE.

[0114] Figure 17 shows the hydroxyproline detection results of rhCol cell lysate.

[0115] Figure 18 shows the hydroxyproline detection results of the ultrafiltration concentrated sample solution after rhColDEAE purification. Detailed Implementation

[0116] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0117] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0118] As shown in Figure 1, this invention effectively inhibits bacterial proline synthesis by knocking out proA / proB, leading to a certain proline dependence or deficiency. Furthermore, this type of strain can better utilize proline (Pro) or hydroxyproline (Hyp) in the exogenous culture medium. Currently, there are two main reported proline synthesis pathways, classified according to the starting material as the glutamate (Glu) pathway and the ornithine (Orn) pathway. The Glu pathway is currently considered the main pathway for bacterial proline biosynthesis. As shown in Figure 2, the synthesis process is as follows: L Glutamic acid (L glutamate) in γ Glutamine kinase (γ-) Phosphorylation under the action of GK,proB) generates γ Glutamyl phosphate (γ) glutamyl phosphate, γ GP); then γ GP is γ Glutamyl phosphate reductase (γ) GPR, proA) catalyzes the generation of γ Glutamic acid semialdehyde (γ Glutamic semialdehyde (GSA); GSA undergoes spontaneous cyclization, losing one molecule of water to form Δ1. Pyrrolidine 5 Carboxylic acid (Δ1) pyrroline 5 carboxylate, P5C); P5C in Δ1 Pyrrolidine-5-carboxylic acid reductase (Δ1) pyrroline 5 L is generated under the action of carboxylate reductase (P5CR). Proline (L) (proline).

[0119] Example 1 Knockout of proA / B genes on BL21(DE3) genome 1. Primer design Based on the upstream and downstream sequences of proB and proA genes on the BL21(DE3) genome, primers for upstream and downstream homologous recombination arms, ligation primers for upstream and downstream homologous recombination arms, and identification primers were designed. The primer sequences are shown in Table 1.

[0120] Table 1. Primers for gene knockout and identification

[0121] 2. Amplification of the targeting sequence and construction of the targeting vector: The genome of BL21(DE3) was extracted using the MolPure® Bacterial DNA Kit (manufacturer: YEASEN, catalog number: 18806ES70) as a PCR template. The primers were upstream and downstream homologous recombination arm primers (pEcgRNA-3 to pEcgRNA-6). The upstream and downstream homologous recombination arms were amplified using high-fidelity DNA polymerase. Using pEcgRNA (manufacturer: Aono Gene, catalog number: HG-VCA1139) as a template, pEcgRNA-1 / 2 were used as primers to amplify the expression element, and pEcgRNA-7 / 8 were used as primers to amplify the vector. Using the homologous recombination kit: Hieff Clone® Plus Multi OneStep Cloning Kit (manufacturer: YEASEN, catalog number: 10912ES10), the upstream and downstream homologous recombination arms + expression element + vector were ligated to obtain the targeting vector. After inoculation with DH5α bacteria (Shanghai Sangon Biotech), the extracted plasmids were correctly sequenced, and these were the targeting vectors: pECgRNA-ΔproA, pECgRNA-ΔproB, and pECgRNA-ΔproAB. The plasmid maps of these targeting vectors are shown in Figures 3-5, respectively.

[0122] 3. Preparation of pECcas competent cells (1) Preheat a water bath at 42°C.

[0123] (2) Take BL21(DE3) competent cells (purchased from YEASEN) in an EP tube and place it on ice for 5 minutes to thaw.

[0124] (3) Add 1 μL (≥10 ng) of pECcas plasmid (purchased from Ono Gene, catalog number: HG-VCA1138) to 100 μL of BL21(DE3) competent cells and mix gently by blowing 3 times.

[0125] (4) Let it stand in an ice bath for 30 minutes.

[0126] (5) After heat shock in a 42℃ water bath for 90 seconds, immediately remove the water bath and place it in an ice bath.

[0127] (6) Ice bath in ice for 2 minutes.

[0128] (7) Add 800 μL of sterile LB medium (antibiotic-free) to the EP tube.

[0129] (8) The EP tube was placed at an angle and fixed in the incubator, and incubated at 37°C with shaking for 1 hour (200 rpm).

[0130] (9) Based on the bacterial concentration, take about 150 μL of bacterial solution and spread it onto LB medium containing Kan antibiotic (50 µg / mL) and incubate overnight.

[0131] (10) The next day, single clones were picked and inoculated into 100 mL LB medium containing Kan resistance (50 µg / mL), and 0.2% arabinose was added to induce RED recombinase expression on the pECcas plasmid. After culturing at 37°C and 220 rpm for 2 h, the bacterial concentration was monitored every 20 min. The culture was continued at 37°C and 220 rpm until the OD value was reached. 600 Stop culturing when the value is about 0.5, and place the conical flask on ice to pre-cool for 30 min; (11) Pour the bacterial solution into a 50 mL centrifuge tube, centrifuge at 4000 rpm for 10 min at 4℃, discard the supernatant and collect the bacterial cells; (12) Add 50 mL of pre-cooled ultrapure water to the centrifuge tube, resuspend the bacterial cells, centrifuge at 4000 rpm for 10 min at 4℃, discard the supernatant, and repeat this washing operation twice; (13) Add 1 mL of 10% glycerol solution to the centrifuge tube to resuspend the bacterial cells, and take 100 μL / vial and quickly dispense it into a pre-cooled 1.5 mL centrifuge tube, and place it on ice for later use.

[0132] The plasmid map of the pECcas plasmid is shown in Figure 6.

[0133] 4. pECgRNA electroporation targeting (1) Clean the electroporation cup, air dry and pre-cool on ice; (2) Take 100ng of the prepared target vectors pECgRNA-ΔproA, pECgRNA-ΔproB and pECgRNA-ΔproAB plasmids, add them to pECcas-BL21(DE3) competent cells, gently tap to mix, and stand on ice for 10min; (3) Transfer the competent mixture to the pre-cooled electroporation cup, wipe off the water on the outer wall, and electroporate for transformation. Program: electroporation 1mm, voltage 1.8kV, capacitance 25μF, resistance 200Ω, electroporation once; (4) After electroporation, quickly add 1mL of LB medium, mix by pipetting and transfer to a 1.5mL centrifuge tube; (5) Recover at 37℃ and 200rpm on a shaker for 1h, take it out and spread it on an LB plate containing Kan (50µg / mL) and SmR (50µg / mL) double antibodies.

[0134] 5. Screening and identification of positive clones (1) Pick the plaques on the plate into 200 μL of LB liquid medium containing double antibodies Kan (50 µg / mL) and SmR (50 µg / mL) and incubate at 37℃ and 220 rpm for 2 h.

[0135] (2) Using bacterial culture as a template, PCR verification was performed using the corresponding identification primers.

[0136] The results are shown in Figures 7-9: the amplification product of the proA knockout positive clone (nucleotide sequence as shown in SEQ ID NO:31) is 1.2kb (deletion type), the length of the deleted gene is 0.6kb, and the negative clone is 1.7kb; the amplification product of the proB knockout positive clone (nucleotide sequence as shown in SEQ ID NO:32) is 1.1kb (deletion type), and the negative clone is 2.0kb; the amplification product of the proAB double knockout positive clone (nucleotide sequence as shown in SEQ ID NO:33) is 1.2kb (deletion type), and the negative clone is 2.3kb.

[0137] (3) The positive clones that were verified by PCR were sent for sequencing. The sequencing results confirmed that the gene deletion was correct. The strains were named BL21(DE3) / ΔproA, BL21(DE3) / ΔproB, and BL21(DE3) / ΔproAB, respectively.

[0138]

[0139] 6. pECgRNA plasmid elimination: rhamnose induction excision of pECgRNA plasmid, spectinomycin resistance screening verification (1) Inoculate clonal colonies containing pEcCas and pEcgRNA (pECgRNA-ΔproA, pECgRNA-ΔproB or pECgRNA-ΔproAB) into 2 mL of LB medium containing rhamnose (10 mM) and kanamycin (50 µg / mL).

[0140] (2) The culture was shaken at 220 rpm overnight, then diluted and spread on solid LB medium containing kanamycin (50 µg / mL) and incubated at 37°C overnight.

[0141] (3) Screening was performed on LB culture dishes containing kanamycin (50µg / mL) and spectinomycin (50µg / mL). The single clones sensitive to spectinomycin were the strains with pEcgRNA plasmid eliminated.

[0142] Example 2 Construction and fermentation culture of recombinant expression strain 1. Gene synthesis The nucleotide sequences of P4H and rhCol were synthesized by Nanjing Genscript Biotech Co., Ltd., and both sequences were optimized by E. coli. The amino acid sequences of the P4H and rhCol expression cassettes are shown in SEQ ID NO:34 and SEQ ID NO:35, respectively.

[0143] SEQ ID NO:34:MLTPTELKQYREAGYLLIEDGLGPREVDCLRRAAAALYAQDSPDRTLEKDGRTVRAVHGCHRRDPVCRDLVRHPRLLGPAMQILSGDVYVHQFKINAKAPMTGDVWPWHQDYIFWAREDGMDRPHVVNVAVLLDEATHL SEQ ID NO:35:MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDL GTDDDDKAMADIGSEFELRRPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

[0144] In this embodiment, as shown in Figure 10, the P4H expression cassette consists of RH1-TPH and a C-terminal his tag sequence; as shown in Figure 11, the fusion rhCol consists of TrxA, DDDDK, Col Ⅲ (amino acid sequence as shown in SEQ ID NO:36) and a C-terminal his tag sequence. The coding region of the above expression cassette is introduced with an NcoI site at the 5' end and an XhoI restriction site at the 3' end by homologous recombination.

[0145] SEQ ID NO: 36: AMADIGSEFELRRPPGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLDGFPGPKGNDGAPGKNGERGGPGGPGPQGPPLEHHHHHH.

[0146] 2. Construction of recombinant expression strains: The pET28A vector was double-digested with NcoI and XhoI, and the vector backbone fragment was recovered. The synthesized rhCol gene fragment was homologously recombinated with the digested pET28A vector to construct the recombinant expression vector pET28A-rhCol. Similarly, the pET21b-P4H vector was constructed.

[0147] The recombinant vector was transformed into DH5α competent cells. The pET28A-rhCol transformant was plated on LB plates containing Kan (50 μg / mL). The pET21b-P4H / pET28A-rhCol co-expression transformant was plated on plates containing Amp (100 μg / mL) and Kan (50 μg / mL). Plasmids were extracted from single clones and sequenced to verify their correctness. The recombinant vectors were then transformed into competent cells of BL21(DE3), BL21(DE3) / ΔproA, BL21(DE3) / ΔproB, and BL21(DE3) / ΔproAB using the CaCl2 method, resulting in five recombinant expression strains: pET28A-rhCol / BL21(DE3), pET21b-P4H / pET28A-rhCol / BL21(DE3), pET28A-rhCol / BL21(DE3) / ΔproA, pET28A-rhCol / BL21(DE3) / ΔproB, and pET28A-rhCol / BL21(DE3) / ΔproAB.

[0148] 3. Shake-flask induction of recombinant expression strains in LB medium: Positive clones of pET28A-rhCol / BL21(DE3), pET21b-P4H / pET28A-rhCol / BL21(DE3), pET28A-rhCol / BL21(DE3) / ΔproA, pET28A-rhCol / BL21(DE3) / ΔproB, and pET28A-rhCol / BL21(DE3) / ΔproAB, which were verified by sequencing, were inoculated into 5 mL of LB medium containing the corresponding antibiotic and 2% glucose and cultured at 37°C and 220 rpm until the logarithmic growth phase.

[0149] Transferred 2% (v / v) into 50 mL of LB medium containing the corresponding antibiotic and 2% glucose, and incubated at 37°C until OD500. 600 = Approximately 0.8. Cool down to 30℃ to adapt to OD. 600 = 1.0 (about 30 min), add 0.2 mM IPTG to induce expression, culture at 30℃ and 220 rpm for 4 h with shaking. After expression, centrifuge at 6000 rpm for 5 min, collect the supernatant, and perform SDS-PAGE electrophoresis for identification.

[0150] The results are shown in Figure 12. It can be seen that rhCol is clearly expressed in all five strains, and the expression levels are basically the same.

[0151] 4. Shake-flask induction of mutant strains with different concentrations of NaCl added to the medium: The positive clones of pET28A-rhCol / BL21(DE3) / ΔproA that were verified by sequencing were inoculated into 5 mL of LB medium containing Kan (50 μg / mL) and 2% glucose, and cultured at 37℃ and 220 rpm to the logarithmic phase.

[0152] Transferred at a ratio of 2% to 50 mL LB medium and cultured separately at 37°C until OD... 600 = Approximately 0.8. Cool down to 30℃ to adapt to OD. 600 =1.0 (about 30 min), add NaCl gradient (5-40 g / L) and IPTG to a final concentration of 0.5 mM for induction, culture at 30℃ and 220 rpm for 4 h with shaking. After expression, centrifuge at 6000 rpm for 5 min and collect the supernatant for SDS-PAGE electrophoresis identification.

[0153] The results are shown in Figure 13, where the 10-40 g / L NaCl addition group is defined as LB (Na + The culture medium was tested, and the results showed that NaCl concentrations above 30 g / L slightly inhibited the expression of the target protein, but still maintained a high expression level. Given that hyperosmolarity has a better pumping effect on Pro / Hyp, subsequent experiments used LB (NaCl-rich LB) culture medium with a final NaCl concentration of 30 g / L. + Culture medium.

[0154] 5. Shake-flask induction of expression of mutant strains with different concentrations of Pro / Hyp-added medium. LB (Pro↓) medium was prepared with casein trypsin hydrolysate (Aladdin T139519) with low free proline (<0.1%), 5 g / L yeast extract was added, and NaCl was added to a final concentration of 30 g / L.

[0155] (1) Pro addition experiment: Positive clones that were correctly sequenced by pET28A-rhCol / BL21(DE3) / ΔproA were inoculated into 5 mL of LB medium and cultured at 37℃ and 220 rpm until the logarithmic phase. Then, they were transferred at a ratio of 5% to 50 mL of LB (Pro↓) medium and cultured until the OD phase. 600 =0.8; Cool down to 30℃ to adapt to OD 600=1.0 (approximately 30 min), add NaCl to a final concentration of 30 g / L, set a Pro gradient (10-100 mM), and add 0.5 mM IPTG for induction. Incubate at 30℃ and 220 rpm with shaking for 4 h. After expression, centrifuge at 6000 rpm for 5 min, collect the supernatant, and perform SDS-PAGE electrophoresis for identification.

[0156] As shown in Figure 14, the expression of the target protein tended to stabilize when Pro was added at a level of 5 mM or more.

[0157] (2) Hyp addition experiment: Positive clones of pET28A-rhCol / BL21(DE3) / ΔproA that were verified by sequencing were inoculated into 5 mL of LB medium and cultured at 37℃ and 220 rpm until the logarithmic phase. Then, they were transferred to 50 mL of LB (Pro↓) medium at a ratio of 5% and cultured until the OD phase. 600 =0.8; Cool down to 30℃ to adapt to OD 600 =1.0 (approximately 30 min), add NaCl to a final concentration of 30 g / L, set a Hyp gradient (10-100 mM), and add 0.5 mM IPTG for induction. Incubate at 30℃ and 220 rpm with shaking for 4 h. After expression, centrifuge at 6000 rpm for 5 min, collect the supernatant, and perform SDS-PAGE electrophoresis for identification.

[0158] As shown in Figure 15, rhCol expression tends to stabilize when Hyp is added at a level of 20 mM or higher.

[0159] Example 3 Purification and preparation of rhCol 1. Collection of bacterial cells: Expand the bacterial culture after shake-flask fermentation in Example 2, collect the bacterial cells by centrifugation at 6000g for 30min, and ensure that the wet weight of the bacterial cells is ≥10g.

[0160] 2. Cell disruption: The bacterial cells were suspended in disruption buffer (20 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, pH 7.5) at a ratio of 1:10 (w / v) and disrupted by sonication in an ice bath (φ10 probe, 60% power, 3 seconds over, 5 seconds off, 15 min / cycle, 2 cycles). After disruption, the supernatant was collected by centrifugation at 12000g for 30 min.

[0161] 3. Protein purification: (1) Load the supernatant onto a well-equilibrated Ni-NTA affinity chromatography column, elute linearly with a buffer containing 200-500 mM imidazole, collect the elution peak of the target protein, and identify the elution buffer by SDS-PAGE.

[0162] (2) Add enterokinase (EK) to the target protein elution buffer, mix at an enzyme to protein ratio of 1:100, and digest at 25°C for 120 min.

[0163] (3) Load the enzyme-digested sample onto a DEAE anion exchange chromatography column and elute with a buffer gradient containing 20 mmol / L Tris-HCl (pH 7.5) and NaCl concentration linearly increasing from 0 mol / L to 1 mol / L to further purify and remove EK enzyme and other impurities, and collect the target protein permeate.

[0164] (4) Dialyze the target protein permeation solution overnight in dialysis buffer (40mM NaH2PO4, 150mM NaCl, pH 7.5) at a volume ratio of 1:500 using a dialysis bag (molecular weight cutoff 14kDa). Then concentrate the solution to a target protein concentration of 5-6mg / mL using an ultrafiltration centrifuge tube.

[0165] The SDS-PAGE electrophoresis results are shown in Figure 16. The purity of the purified rhCol protein is >95%, and the original concentration is about 5.0 mg / mL, which meets the requirements for subsequent experiments and applications.

[0166] Example 4 Comparison of rhCol hydroxylation detection using different expression methods 1. rhCol proline hydroxylation detection kit (manufacturer: Solarbio, catalog number: BC0255) Analytical method: The chloramine T method is used to detect free Hyp: Free Hyp is generated by acid hydrolysis of the sample. After Hyp is oxidized by chloramine T, the oxide reacts with p-dimethylaminobenzaldehyde to produce a red compound. The degree of proline hydroxylation can be determined by directly observing the intensity of the color change in the sample. This red compound has a characteristic absorption peak at 560 nm. By measuring the absorbance of the sample hydrolysate at 560 nm, the Hyp content can be compared or calculated.

[0167] 2. Sample processing (1) Collect fermentation lysates of strains pET28A-rhCol / BL21(DE3), pET21b-P4H / pET28A-rhCol / BL21(DE3), pET28A-rhCol / BL21(DE3) / ΔproA, pET28A-rhCol / BL21(DE3) / ΔproB, and pET28A-rhCol / BL21(DE3) / ΔproAB respectively. The lysates were obtained according to the corresponding culture conditions in Example 2, and 100 μL of each was taken for later use.

[0168] (2) The purified stock solutions were obtained according to the steps in Example 3. Take 5 μL of the rhCol purified stock solution corresponding to each strain and dilute it to 100 μL.

[0169] (3) Take 100 μL of liquid sample (100 μL of the lysate, and 5 μL of the purified stock solution diluted to 100 μL) into a glass tube, add 100 μL of the extract (adjust the ratio if the measured value is too small), boil at 100℃ for 1 h to digest until no large clumps are visible (wrap the tube with a sealing film to prevent the cap from bursting), and adjust the pH to 7.0 with NaOH solution after cooling (do not make it too acidic or too alkaline). Finally, centrifuge at 12000 rpm and 25℃ for 20 min (if there are still impurities after centrifugation, they can be removed by filtration), and take the supernatant for testing.

[0170] 3. Activity Assay: Add the reaction reagents to the supernatant according to the kit instructions and Table 2, and incubate at room temperature for color development. Read and record the absorbance value at 560 nm using a microplate reader.

[0171] Table 2

[0172] 4. Detection results (1) The detection results of the lysate are shown in Figure 17: the rhCol single expression BL21(DE3) group had no hydroxylation modification (absorbance value 0.05), the P4H and rhCol co-expression group and the three Δpro modified strain groups all had hydroxylation modification, and the modification degree of the modified strain group was slightly higher than that of the co-expression group.

[0173] (2) The results of the purified sample test are shown in Figure 18: the degree of hydroxylation is ranked as BL21(DE3)ΔproA>BL21(DE3)ΔproB≈BL21(DE3)ΔproAB>BL21(DE3)-P4H, which confirms that the proline-deficient strain constructed in this invention, combined with optimized culture conditions, can significantly improve the hydroxylation level of rhCol.

[0174] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An engineered Escherichia coli strain that efficiently expresses hydroxylated collagen, characterized in that, The following modifications were made to the Escherichia coli strain: a. at least one of the proline metabolic pathway genes proA and proB in the Escherichia coli strain was knocked out; b. a collagen expression gene was introduced into the knocked-out Escherichia coli strain.

2. The engineered Escherichia coli strain according to claim 1, characterized in that, The Escherichia coli strains include BL21(DE3), Rosetta(DE3), Origami(DE3), HSM174(DE3), JM109, AS1.357, TOP10, or DH5α strains.

3. The engineered Escherichia coli strain according to claim 2, characterized in that, The Escherichia coli strain is BL21(DE3).

4. The engineered Escherichia coli strain according to claim 1, characterized in that, The collagen includes any one or more of recombinant humanized type III collagen, recombinant humanized type I collagen, recombinant humanized type IV collagen, and recombinant humanized type VII collagen.

5. The engineered Escherichia coli according to claim 4, characterized in that, The collagen is recombinant humanized type III collagen.

6. The engineered Escherichia coli strain according to claim 5, characterized in that, The amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID NO:

35.

7. The engineered Escherichia coli strain according to claim 1, characterized in that, The knockout methods include CRISPR / Cas9-mediated gene knockout, suicide plasmid pCVD-mediated gene knockout, Red / ET recombination-mediated gene knockout, type II intron insertion-mediated gene knockout, Cre-LoxP recombination-mediated gene knockout, or TALEN gene targeting.

8. The use of the engineered Escherichia coli according to any one of claims 1-7 in the expression of hydroxylated collagen.

9. A method for preparing hydroxylated collagen, characterized in that, Includes the following steps: S1. Culture the engineered Escherichia coli strain according to any one of claims 1-7 in a culture medium; S2. Induce collagen expression; S3. Separate and purify the expression product to obtain hydroxylated collagen.

10. The preparation method according to claim 9, characterized in that, The concentration of proline in the culture medium described in step S1 is <1 mM; sodium chloride, proline, and / or hydroxyproline are added during the induction process described in step S2; the final concentration of sodium chloride is 20-40 g / L, the final concentration of proline is 5-100 mM, and the final concentration of hydroxyproline is 10-100 mM.

11. The application of the hydroxylated collagen prepared by the preparation method according to claim 9 or 10 in the preparation of bioengineering materials, food supplements, and cosmetics.

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