E.coli engineering bacterium for efficiently expressing hydroxylated collagen and establishment method of E.coli engineering bacterium

Abstract

The invention discloses an E. coli engineering bacterium capable of efficiently expressing hydroxylated collagen. The E. coli engineering bacterium is prepared by knocking out a gene proA / proB of a proline metabolic pathway in an E. coli strain; meanwhile, the P4H gene is knocked into an E. coli strain body to supplement or regulate hydroxylation modification of proline, and a strain which is expected to realize large-scale stable mass production of high-hydroxylation modified collagen is obtained. The problem that the recombinant collagen cannot be hydroxylated or is insufficient in hydroxylation in a traditional prokaryotic expression system is solved, a stable hydroxylated collagen expression strain and a culture method are constructed, possibility is provided for obtaining more ideal triple-helix stable and functional collagen products, and stable and large-scale production of the recombinant collagen is promoted. Furthermore, rapid development of the medical field, beauty and skin care, food supplements, cosmetics and other industries related to the recombinant collagen can be promoted to a great extent.

Description

Technical Field

[0001] The present invention relates to an E. coli The invention relates to engineering bacteria and a method for establishing the same, belonging to the technical field of bioengineering. Background Art

[0002] Collagen is one of the most abundant proteins in animals and humans. In the human body, human collagen accounts for 25% to 30% of the total protein and is widely found in the skin, bones, tendons, ligaments, cornea, various organs and blood vessels. It is a fibrous protein known for its excellent biocompatibility, low antigenicity, biological activity and degradability, and is essential for maintaining tissue structure and function. The commercial applications of collagen are very wide, including: medical fields (used in wound dressings, tissue engineering, drug delivery systems, etc.), beauty and skin care (as an active ingredient for anti-aging and skin repair), food supplements (as nutritional supplements that help joint health and skin health), cosmetics (as moisturizing and anti-aging ingredients), etc. At present, most of the sources of collagen on the market come from two ways: one is to extract and separate directly from animals (pigs, cattle, marine fish, etc.). Although the yield is considerable and active, there are risks of viruses and allogeneic rejection; the other is obtained by heterologous recombinant expression. In recent years, collagen obtained by recombinant expression has received widespread attention in the field of medical health. It has many advantages that traditional animal-derived collagen does not have, such as no viral risks, low immunogenicity, high water solubility and easy processing.

[0003] However, the research and application of recombinantly expressed collagen still face some technical bottlenecks and challenges. First, there are technical challenges in the expression system: different expression systems such as Escherichia coli, yeast, insect cells and mammalian cells have their own advantages and limitations. For example, although the expression systems of Escherichia coli and yeast have short production cycles and relatively high production capacity, the systems themselves lack endogenous hydroxylation modification, and the hydroxylation level of exogenously introduced P4H modification is not high, especially for the hydroxylation of the Y site in the GXY repeat sequence of collagen, so it is impossible to effectively hydroxylate the proline or lysine of collagen, and thus cannot form a stable triple helix structure. Although the existing eukaryotic systems (such as insects and mammalian cells) are the modification and expression systems closest to human sources, and theoretically the easiest to obtain active recombinant human collagen with a complete triple helix structure close to human sources, their recombinant expression of human collagen generally has the disadvantages of long production cycles, low yields, high costs and difficulty in scale-up. Second, there are problems of biological activity and stability: the biological activity and stability of recombinant collagen are the key to its application. So far, there has been no report on the efficient modification of recombinant human collagen by a natural high-performance P4H enzyme in a heterologous host. In addition, current recombinant collagen products also have deficiencies in the stability and molecular weight of the three-dimensional helical structure, which may affect their support effect and efficacy in clinical applications. Summary of the invention

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: A highly efficient expression of hydroxylated collagen in E. coli Engineered bacteria, E. coli The strains were transformed as follows: a. Knockout E. coli Genes of proline metabolic pathway in strains pro A or pro B, or pro A and pro B is knocked out simultaneously so that the host can efficiently and reasonably utilize exogenous proline and / or hydroxyproline; b. Knock the P4H gene into E. coli The strain can express P4H protein in vivo to supplement or regulate the hydroxylation modification of proline; c. In E. coli The collagen expression gene was introduced into the strain.

[0005] Preferably, the P4H gene is selected from P4H genes from the following sources: Homo sapiens human-P4H, Parameciumbursaria Chlorella virus-1 Chlorella virus type 1-P4H, Drosophila melanogaster-P4H Drosophila melanogaster-P4H, Caenorhabditis elegans-P4H Caenorhabditis elegans-P4H, Bacillus anthracis-P4H anthrax Bacillus-P4H, Chlamydomonas reinhardtii-P4H Clemidomonas-P4H, Mus musculus-P4H mice-P4H, Dactylosporangiumsp RH1, and uncultured bacterium-P4H. The P4H gene can be selected from the following genes: Homo sapiens human-P4H (Accession number P13674 / O15460), Paramecium bursaria Chlorella virus-1 Chlorella virus type 1-P4H (NP_048433.1), Drosophila melanogaster-P4H Drosophila melanogaster (Q9I7H9), Caenorhabditis elegans-P4H Caenorhabditis elegans (Q20065), Bacillus santhracis-P4H Bacillus anthracis (Q81LZ8), Chlamydomonas reinhardtii-P4H Clemydomonas reinhardtii (A8IDI8), Mus musculus-P4H mouse (Q60715, Q60716), Dactylosporangium sp RH1-P4H (GenbankID: D78338.1), uncultured bacterium-P4H (AGS49339.1).

[0006] Preferably, the P4H gene is Dactylosporangium sp. RH1-P4H.

[0007] Preferably, the pro The gene of A is shown in SEQ ID NO.8, pro The gene of B is shown in SEQ ID NO.9.

[0008] Preferably, the expression gene of the collagen encodes an amino acid sequence as shown in any one of SEQ ID NO.3 or SEQ ID NO.10-14. The technical solution provided by the present invention can be used for collagen and recombinant collagen from various species, such as the amino acid sequence shown in any one of SEQ ID NO.3 or SEQ ID NO.10-14 is human collagen; or an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9% identity with the amino acid sequence shown in any one of SEQ ID NO.3 or SEQ ID NO.10-14.

[0009] Preferably, the gene knockout method is selected from 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, TALEN gene targeting or RNA interference.

[0010] Preferably, the E. coli The strain was selected from the following strains: BL21(DE3), Rosetta(DE3), Origami(DE3), JM109, HSM174(DE3), AS1.357, TOP10 or DH5α strains.

[0011] Preferably, a strong expression promoter is knocked in at the same time for expressing the P4H protein, and the strong expression promoter is selected from Ptac, Plac, PT7, PT7lac, pL, and Ptrp, and more preferably is the PT7 promoter.

[0012] Preferably, P4H is in E. coli A non-essential gene site is knocked in the strain, wherein the non-essential gene site is lpxM, lacZ, phoP, Lpp, ompA or Lpp-ompA, and more preferably the lpxM gene site.

[0013] Among them, knocking out E. coli The method used for knocking out the genes of the proline metabolic pathway in the strain was CRISPR / Cas9-mediated gene knockout. The specific method was to transform the plasmid pEcCas carrying the Cas9 nuclease gene into the E. coli strains, and based on this, the genes containing the proline metabolic pathway were pro A and / or proB. The plasmid pEcgRNA at the PAM site is transformed into the bacteria; then, the monoclonal bacteria with successful gene editing are screened; the pEcgRNA plasmid is induced to be eliminated in the cells to obtain the gene knocking out the proline metabolic pathway pro A and / or pro B of E coli strains.

[0014] In some embodiments of the present invention, in CRISPR / Cas9-mediated gene knockout, the knockout pro The sgRNA expression element used in A is selected from the following nucleotide sequences: 5'-gtcctaggtataatactagtatcgacatgctgatcccgcggttttagagctagaaatag-3'; 5'-Tcaaaaaaagcaccgactcgg-3'; Knockout pro A The homologous recombination arm primers used are selected from: 5'-cgagtcggtgctttttttgaaagatgtttacggcattg-3'; 5'-tccacttatcagcctgctccttaacggg-3'; 5'-gagcaggctgataagtggattcgtccgctgtttacgttaacgcc-3'; 5′-atggcggcagcaattggctt-3′; Knockout pro The sgRNA expression element used in B is selected from the following nucleotide sequences: 5'-gtcctaggtataatactagtGCTACGGCAGAGATTAAGGTgttttagagctagaaatag-3'; 5′-tcaaaaaaagcaccgactcgg-3′; Knockout pro The homologous recombination arm primers used in B are selected from: 5'-cgagtcggtgctttttttgatctggtcgccatctttac-3'; 5'-ccttcgagattctctgccattcaatttt-3'; 5'-tggcagagaatctcgaaggccgcgatatcgcccacggcgtcagt-3'; 5′-tcaagacgcaggccgctgtc-3′; Knockout pro A and pro The sgRNA expression element used in B is selected from the following nucleotide sequences: 5'-gtcctaggtataatactagtAACGGGTAATCATGTCATCAgttttagagctagaaatag-3'; 5'-tcaaaaaaagcaccgactcgg-3'; Knockout pro A and pro The homologous recombination arm primers used in B are selected from: 5'-cgagtcggtgctttttttgaaaattgaatggcagagaa-3'; 5'-caggattaacctttttgatcggtcagca-3'; 5'-atcaaaaaggttaatcctgcggcttaccggcgggtgccgtgcag-3'; 5′-cgggtcaggatcgcatcgga-3′.

[0015] The P4H gene was knocked into E. coli The specific method in vivo is to use E. coli The lpxM gene of the strain was used as the insertion site, the P4H knock-in sequence was connected to the plasmid pEcgRNA to construct a targeting plasmid, and the plasmid pEcCas carrying the Cas9 nuclease gene was transformed into the E. coli The targeting plasmid was then transferred into the strain, and the monoclonal bacteria with successful gene editing were screened out, and the plasmid pECgRNA and plasmid pEcCas were induced to remove, and the proline metabolic pathway genes were knocked out and the P4H gene was knocked in. coli strains.

[0016] In CRISPR / Cas9-mediated P4H gene knock-in, the sgRNA expression element used is selected from the following nucleotide sequences: 5'-gtcctaggtataatactagtacagattgattaacgcacgagttttagagctagaaatag-3'; 5'-tcaaaaaaagcaccgactcgg-3'; The primers for the P4H knock-in sequence were selected from the following nucleotide sequences: 5′-tgtcggcgatatagtcgcca-3′; 5′-caaaaaacccctcaagaccc-3′; The homologous recombination arm primers used for P4H knock-in are selected from: 5'-cgagtcggtgctttttttgattagtatccaggtgtatt-3'; 5'-tggcgcctatatcgccgacacgttgactggcaagggctgg-3'; 5'-gggtcttgaggggttttttgcccagtagcgcgggtggcga-3'; 5'-gcaggtcgactctagagaatctggtctggcgaaaggattc-3'.

[0017] In E. coli The specific method of introducing the collagen expression gene into the strain is to recombinantly transform the collagen expression gene into the pET28A plasmid, and transform the recombinant pET28A plasmid into the E. coli strains to obtain the desired E. coli Engineered bacteria.

[0018] The present invention also discloses the above-mentioned E. coli Application of engineered bacteria in expressing hydroxylated collagen.

[0019] The present invention also discloses a method for preparing hydroxylated collagen, which comprises culturing the above-mentioned E. coli Engineered bacteria, induced to express collagen and purified.

[0020] In the method for preparing hydroxylated collagen, the modified E. coli The concentration of proline in the culture medium used for culturing the strain before induction of expression is less than 1mM; during the induction of expression, sodium chloride, proline and / or hydroxyproline are added; the concentration of sodium chloride in the culture medium is 20 to 40g / L, the concentration of proline is 5 to 100mM, and / or the final concentration of hydroxyproline is 10 to 100mM. The method of supplementing sodium chloride, proline and / or hydroxyproline in the present invention can be directly added to the original culture medium; it can also be carried out by replacing the culture medium, as long as the concentration of sodium chloride in the culture medium is 20 to 40g / L, the concentration of proline is 5 to 100mM, and / or the final concentration of hydroxyproline is 10 to 100mM. In some embodiments of the present invention, proline and hydroxyproline are both L-type amino acids.

[0021] Preferably, the transformed E. coli The medium used for culturing the strain before inducing expression is a low-free proline medium, and the components of the medium are: 10g of trypticase peptone containing low free proline, 5g of low free proline yeast extract, 20g to 40g of sodium chloride per liter of medium, and L-proline and / or hydroxyproline are added, so that the concentration of L-proline in the medium is 5 to 100mM, and the concentration of hydroxyproline is 10 to 100mM. In some embodiments of the present invention, L-proline is supplemented, or L-proline and hydroxyproline are supplemented.

[0022] In the present invention, a medium with low free proline refers to a medium in which the final concentration of free proline is less than 1 mM. In some embodiments of the present invention, the medium with low free proline is prepared by using trypticase peptone with low free proline and yeast extract with low free proline.

[0023] The present invention knocks out the endogenous pro A / B genes are used to construct a strain with a certain proline dependence or deficiency so that it can efficiently utilize exogenously added Pro / Hyp; exogenous culture uses a specific minimal medium LB (Na+) and adds Pro / Hyp; the P4H gene is knocked into the genome to supplement or regulate the hydroxylation modification degree of Pro. The resulting engineered bacterial genome does not contain pro A / B gene, and knock-in P4H gene, thus systematically solving the problem of E. coli The engineered bacteria cannot hydroxylate exogenous recombinant proteins, directly utilize hydroxyproline in the culture medium, and solve the problem of low modification efficiency when traditional strains co-express P4H. The engineered host bacteria and culture method provided by the present invention can be used to recombinantly express and quantitatively produce various types of human collagen and short peptides with high hydroxylation degrees, and recombinant collagen with high hydroxylation is more conducive to the structure and stability of collagen (for example, forming a triple helix structure) and maintaining effective functions (for example, platelet adhesion and activation).

[0024] The present invention solves the problem that recombinant collagen cannot be hydroxylated or is insufficiently hydroxylated in traditional prokaryotic expression systems, constructs a stable hydroxylated collagen expression strain and culture method, and provides the possibility of obtaining more ideal triple helical stable and functional collagen products, which will promote the stability and large-scale production of recombinant collagen. Furthermore, it will greatly promote the rapid development of recombinant collagen-related medical fields, beauty and skin care, food supplements, cosmetics and other industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the scheme of the present invention.

[0026] Figure 2 Schematic diagram of the glutamate (Glu) pathway for proline production in bacteria.

[0027] Figure 3 pro A. PCR verification results of gene knockout.

[0028] Figure 4 pro B. PCR verification results of gene knockout.

[0029] Figure 5 pro PCR verification results of AB double gene knockout.

[0030] Figure 6 Knock-in of the proline hydroxylase gene.

[0031] Figure 7 Plasmid map of pEcCas protein vector.

[0032] Figure 8 pro A. Gene knockout targeting sgRNA vector plasmid map.

[0033] Fig. 9 pro B. Gene knockout targeting sgRNA vector plasmid map.

[0034] Fig.10 pro AB gene knockout targeting sgRNA vector plasmid map.

[0035] Fig.11 P4H gene knock-in targeting sgRNA vector plasmid map.

[0036] Fig.12 Plasmid map of recombinant expression rhCol vector.

[0037] Fig.13 Plasmid map of recombinant expression P4H vector.

[0038] Fig.14 SDS-PAGE identification results of rhCol expression in different strains.

[0039] Fig.15 SDS-PAGE results of rhCol expression under different NaCl addition conditions.

[0040] Fig.16 SDS-PAGE results of rhCol expression under different Pro addition conditions.

[0041] Fig.17SDS-PAGE results of rhCol expression under different Hyp addition conditions.

[0042] Fig.18 A SDS-PAGE result of rhCol protein purification.

[0043] Fig.19 A hydroxyproline detection result of rhCol bacterial cell disruption fluid.

[0044] Fig. 20 Hydroxyproline detection results of a purified rhCol sample solution.

[0045] Fig.21 Hydroxyproline test results of various rhCollagens. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present application more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not intended to limit the scope of the present invention.

[0047] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art, and the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0048] In order to further understand the present invention, the present invention is further described in detail below in conjunction with the best embodiment.

[0049] like Figure 1 As shown, the present invention knocks out pro A / pro B can effectively inhibit the synthesis of proline in bacteria, causing them to form a certain degree of proline dependence or deficiency, and this type of strain can better utilize proline (Pro) or hydroxyproline (Hyp) in the exogenous culture medium. At present, there are two main proline synthesis pathways reported, which are divided into glutamate (Glu) pathway and ornithine (Orn) pathway according to the different starting materials. It is currently believed that the Glu pathway is the main pathway for bacterial proline biosynthesis. Figure 2 As shown in FIG. 1 , the synthesis process is as follows: L-glutamate (L-glutamate) in γ-glutamyl kinase (γ-GK, pro B) to generate γ-glutamyl phosphate (γ-GP); γ-GP is then phosphorylated by γ-glutamyl phosphate reductase (γ-GPR, pro A) catalyzes the production of γ-glutamic semialdehyde (GSA); GSA undergoes spontaneous cyclization and removes a water molecule to produce Δ1-pyrroline-5-carboxylate (P5C); P5C generates L-proline under the action of Δ1-pyrroline-5-carboxylate reductase (P5CR).

[0050] Example 1: E . coli BL21(DE3) genome pro A / B gene knockout 1. Primer Design According to E . coli BL21(DE3) genome pro B and pro The upstream and downstream sequences of gene A were used to design upstream and downstream homologous recombination arm primers, upstream and downstream homologous recombination arm connection primers, and identification primers. The primer sequences are shown in Table 1.

[0051] Table 1 Gene knockout and identification primers 2. Amplification of targeting sequences and construction of targeting vectors Use the genome extraction kit to extract E . coli The genome of BL21 (DE3) was used as a PCR template, and the primers were upstream and downstream homologous recombination arm primers (pEcgRNA-3 to pEcgRNA-6). High-fidelity DNA polymerase was used to amplify the upstream and downstream homologous recombination arms; pEcgRNA was used as a template, pEcgRNA-1 / 2 was used as primers to amplify the expression element, and pEcgRNA-7 / 8 was used as primers to amplify the vector. Use a homologous recombination kit to connect "upstream and downstream homologous recombination arms" + "expression element" + "vector" to obtain the targeting construction vector. Inoculate DH5α strains (Shanghai Sangon Biotechnology), extract plasmids, and sequence correctly to obtain the targeting vector: pECgRNA-Δ pro A, pECgRNA-Δ pro B, pECgRNA-Δ pro AB (plasmid maps are as follows Figures 8-10 as shown).

[0052] 3. Preparation of pECcas competent cells 1) Preheat a water bath to 42°C.

[0053] 2) Take the competent cell EP tube from the -80℃ refrigerator and place it on ice to thaw for 5-10 minutes.

[0054] 3) Add the pEcCas plasmid for transformation ( Figure 7 ) 1-10uL (≥10ng) in 100uL BL21 (DE3) competent cells, pipette 3-5 times to mix.

[0055] 4) Place in ice bath for 30 minutes.

[0056] 5) Heat in a 42℃ water bath for 90 seconds and immediately remove from the ice bath.

[0057] 6) Place in ice bath for 2 to 3 minutes.

[0058] 7) Add 800 μL of sterile LB medium (without antibodies) into the EP tube.

[0059] 8) The EP tube was placed at an angle and fixed in the incubator, and cultured at 37°C with shaking (180-200 rpm) for 1 hour.

[0060] 9) Depending on the concentration of the bacterial solution, spread about 150 μL of the bacterial solution on LB medium containing Kan antibiotic (50 μg / ml) and culture overnight.

[0061] 10) The next day, pick a single clone and inoculate it into 100mL LB medium with Kan resistance (50µg / ml), and add 0.2% arabinose to induce the expression of RED recombinase on the pECcas plasmid. After culturing at 37℃ and 220 rpm for 2 hours, monitor the bacterial concentration every 20-30 minutes. Cultivate at 37℃ and 220 rpm until the OD600 value is about 0.4-0.5, then stop culturing and place the conical flask on ice for 30 minutes; 11) Pour the bacterial solution into a 50 mL centrifuge tube, centrifuge at 4°C, 4000 rpm for 10 min, discard the supernatant, and collect the bacteria; 12) Add 50 mL of pre-cooled ultrapure water to the centrifuge tube, resuspend the cells, centrifuge at 4000 rpm for 10 min at 4°C, discard the supernatant, and wash twice; 13) Add 1 mL of 10% glycerol solution to the centrifuge tube to resuspend the cells, and quickly dispense 100 μL / tube into pre-cooled 1.5 mL centrifuge tubes and place on ice for later use.

[0062] 4.pECgRNA electroporation targeting: 1) Clean the electric rotating cup, dry it and pre-cool it on ice; 2) Take 100-200 ng of the targeting vector pECgRNA plasmid and add it to the pECcas-BL21(DE3) competent cells, flick to mix, and place on ice for 10 min; 3) Transfer the competent mixture to a pre-cooled electroporation cup, wipe off the moisture on the outer wall, and perform electroporation transformation; 4) After the electroporation is completed, quickly add 1 mL of LB medium, mix well with a pipette, and transfer to a 1.5 mL centrifuge tube; 5) Resuscitate the cells in a shaking incubator at 37°C and 200 rpm for 1 hour, then apply the plates to LB plates containing Kan (50 µg / ml) and SmR (50 µg / ml) dual antibodies.

[0063] 5. Screening and identification of positive clones 1) Pick the plaques on the plate and place them in 200 μL LB liquid medium containing Kan (50 μg / ml) and SmR (50 μg / ml) dual antibodies, and culture them at 37°C and 220 rpm in a shaking incubator for 2 h.

[0064] 2) Bacterial liquid PCR verification, identification results are shown in Figures 3-5 .

[0065] 6. Elimination of pECgRNA Plasmid Rhamnose-induced excision of pECgRNA plasmid and spectinomycin resistance screening and verification 1) Inoculate a clone containing pEcCas and pEcgRNA into 2 ml LB medium containing rhamnose (10 mM) and kanamycin (50 µg / ml).

[0066] 2) The culture was grown overnight with shaking at 220 rpm, then diluted and spread on solid LB medium containing kanamycin (50 µg / ml) and incubated at 37°C overnight.

[0067] 3) Screening was performed on LB culture plates containing kanamycin (50 µg / ml) and spectinomycin (50 µg / ml). The single clone sensitive to spectinomycin was the strain in which the pEcgRNA plasmid was eliminated.

[0068] from Figure 3-5 It can be seen that Figure 3 pro A knockout (SEQ ID NO. 4): "Corridor 2, 7, 8" is a negative clone amplification product of 1.7 kb, "Corridor 3-6, 9-15" is a positive clone amplification product of 1.1 kb (deletion type), and the deleted gene length is 0.6 kb; Figure 4 pro B knockout (SEQ ID NO.5): the amplification product of the negative clone is 2.0 kb, and all 5 lanes are amplification products of the positive clone 1.1 kb (deletion type), and the length of the deleted gene is 0.9 kb; Figure 5 proAB knockout (SEQ ID NO.6): "Channel 2" is a negative clone amplification product of 2.3kb, "Channels 1, 3, 4" are positive clone amplification products of 1.2kb (deletion type), and the deleted gene length is 1.1kb; "Channel 5" is non-specific amplification; The products amplified by the above primers were sent for sequencing, and the sequencing results confirmed that it was a gene deletion clone, named BL21(DE3) / Δ pro A. BL21(DE3) / Δ pro B. BL21(DE3) / Δ pro AB; the corresponding strains were knocked out pro A gene, pro B gene, pro A gene and pro B gene.

[0069] Example 2: E . coli BL21(DE3),Δ pro Knock-in of P4H in strains 1. Primer Design BL21(DE3) / Δ pro Strain A was used as an example to knock in the P4H gene. The lpxM gene of BL21(DE3) was selected as the insertion site, and upstream and downstream homologous recombination arm primers, upstream and downstream homologous recombination arm connection primers, and identification primers were designed according to the upstream and downstream sequences of the lpxM gene of the BL21(DE3) genome. The primer sequences are shown in Table 2.

[0070] Table 2 Gene knock-in and identification primers 2. Amplification of targeting sequences and construction of targeting vectors The synthetic pET-P4H plasmid was used as a template and pEcgRNA5 / 6 as primers to amplify the P4H knock-in sequence; the genome BL21 (DE3), Δ pro A's genome was used as a PCR template, and the primers were upstream and downstream homologous recombination arm primers (pEcgRNA-3 / 4 and pEcgRNA-7 / 8). High-fidelity DNA polymerase was used to amplify the upstream and downstream homologous recombination arms; pEcgRNA was used as a template, pEcgRNA-1 / 2 was used as primers to amplify the expression element, and pEcgRNA-9 / 10 was used as primers to amplify the vector. Use a homologous recombination kit to connect "upstream and downstream homologous recombination arms" + "expression element" + "knock-in sequence" + "vector" to obtain the targeting plasmid construction. Inoculate DH5α bacteria, extract the plasmid, and the correct sequencing is the targeting vector: pEcgRNA-(lxpm) P4H (plasmid map as shown in Fig.11 as shown).

[0071] The specific knock-in method is the same as in Example 1. pEcCas and pEcgRNA-P4H are successively transferred into the above BL21 (DE3)Δ pro A strain, PCR verification (such as Figure 6 As shown). In addition to the pEcgRNA plasmid elimination, the pEcCas plasmid elimination operation needs to be added as follows: 1)-3). Finally, BL21(DE3) / Δ pro A, P4H strain.

[0072] 1) Incubate the cells in liquid LB medium containing glucose (5 g / L) at 37°C with shaking at 220 rpm overnight.

[0073] 2) Approximately 10 µL of cells were plated on LB plates containing glucose (5 g / L) and sucrose (10 g / L) and incubated at 37°C overnight.

[0074] 3) Randomly select single colonies and screen them on LB plates with or without kanamycin (50 µg / ml). Colonies sensitive to kanamycin are strains that have eliminated the pEcCas plasmid.

[0075] The results are as follows Figure 6 As shown, "lanes 1, 2, 3, 5" are positive clones with amplification products of 1.3 kb (knock-in type), and "lane 4" is a negative clone with amplification products of 480 bp (wild type). The amplified products were sent for sequencing, and the sequencing results confirmed that it was a P4H gene knock-in clone, named BL21(DE3) / Δ pro A, P4H; this strain has knocked out pro A gene and knocked in the P4H gene.

[0076] Example 3: Construction and fermentation of recombinant expression strains 1. Gene Synthesis The P4H and Col nucleotide sequences were synthesized by Nanjing GenScript Biotech Co., Ltd. The expression cassette coding sequences were selected from SEQ ID NO: 1 and SEQ ID NO: 2, and the above sequences were all optimized for large intestine codons. In this embodiment, the P4H expression cassette consists of RH1-TPH and C-terminal his tag sequence ( Fig.13 , SEQ ID NO: 1), fused to Col ( Fig.12 , SEQ ID NO: 2) is composed of TrxA, DDDDK, Col C-terminal histag (SEQ ID NO: 3) sequences, and the Nco I site is homologously recombined into the 5' end of the coding region of the above expression cassette, and the Xho I restriction site is added to the 3' end.

[0077] 2. Construction of recombinant expression strain pET21b-P4H / pET28A-rhCol / BL21 (DE3), pET28A-rhCol / BL21 (DE3) / Δ pro A. pET28A-rhCol / BL21 (DE3) / Δ pro P4H and Col in A / P4H were cloned into the Nco I / Xho I restriction sites of pET21b and pET28A respectively by homologous recombination. The products were transformed into competent E. coli DH5a and spread on LB plates with Amp / Kan double resistance, Kan resistance, and Kan resistance to screen recombinants. Positive clones were selected for sequencing verification. BL21(DE3), BL21 (DE3) / Δ were prepared by CaCl2 method. pro A (prepared in Example 1) and BL21 (DE3) / Δ pro A / P4H (prepared in Example 2) competent cells, the recombinant expression vector verified by sequencing was transformed into the corresponding competent cells, that is, the above three recombinant expression strains were obtained, and the obtained recombinant expression strains were labeled as pET21b-P4H / pET28A-rhCol / BL21 (DE3), pET28A-rhCol / BL21 (DE3) / Δ pro A. pET28A-rhCol / BL21 (DE3) / Δ pro A / P4H: In the expression strain pET21b-P4H / pET28A-rhCol / BL21 (DE3), the P4H gene was constructed on the pET21b plasmid and rhCol was constructed on the pET28A plasmid; pET28A-rhCol / BL21 (DE3) / Δ pro In the A expression strain, the rhCol gene was constructed on the pET28A plasmid; the resulting pET28A-rhCol / BL21 (DE3) / Δ pro A / P4H, rhCol gene was constructed on pET28A plasmid.

[0078] 3. Induced expression of recombinant expression strains in LB medium shake flasks pET21b-P4H / pET28A-rhCol / BL21 (DE3), pET28A-rhCol / BL21 (DE3) / Δ pro A. pET28A-rhCol / BL21 (DE3) / Δ proThe positive clones verified by A / P4H sequencing were inoculated into 5 mL of LB medium containing 2% glucose and cultured to the logarithmic phase. The 2% ratio was transferred into 50 mL of LB medium containing 0.5% glucose and cultured at 37°C until OD600 = 0.8. The temperature was lowered to 30°C to adapt to OD600 = 1.0 (about 30 minutes), and IPTG was added to induce at a final concentration of 0.5 mM. The cells were cultured at 30°C in a constant temperature shaker at 220 rpm for 4 hours. After the expression was completed, the supernatant was centrifuged at 6000 rpm for 5 minutes and identified by SDS-PAGE (see the results). Fig.14 ), lanes 1-4 represent the electrophoresis results of the supernatants of the following strains: BL21 control, pET21b-P4H / pET28A-rhCol / BL21 (DE3), pET28A-rhCol / BL21 (DE3) / Δ pro A. pET28A-rhCol / BL21 (DE3) / Δ pro A / P4H.

[0079] 4. Shake flask induced expression of mutant strains in medium supplemented with different concentrations of NaCl pET28A-rhCol / BL21 (DE3) / Δ pro The positive clones verified by A / P4H sequencing were inoculated into 5mL LB medium containing 2% glucose and cultured to the logarithmic phase. The 2% ratio was transferred into 50mL LB medium and cultured separately at 37℃ until OD600 = 0.8. The temperature was lowered to 30℃ to adapt to OD600 = 1.0 (about 30min), and NaCl gradient (5~40g / L) and final concentration of 0.5mM IPTG were added for induction. The cells were cultured at 30℃ constant temperature shaker at 220rpm for 4 hours. After the expression was completed, the supernatant was centrifuged at 6000rpm for 5 minutes and identified by SDS-PAGE (see the results). Fig.15 ). 10g~40 / LNaCl input is called LB (Na+).

[0080] 6. Shake flask induced expression of mutant strains in medium supplemented with different concentrations of Pro / Hyp Considering that hypertonicity has a better pumping effect on Pro / Hyp, LB (Na+) medium with a final concentration of 30g / L NaCl was selected.

[0081] LB (Pro↓) is different from LB in that it is prepared by using casein trypsin hydrolyzate with low free proline components of non-animal origin (e.g. Aladdin tryptone T139519, 0.02% free Proline) and yeast extract with low free proline (e.g. Aladdin yeast extract powder Y278674, 0.38% free Proline).

[0082] 1) Pro addition: pET28A-rhCol / BL21 (DE3) / Δ pro The positive clones verified by A / P4H sequencing were inoculated into 5mL LB medium and cultured to the logarithmic phase. 5% of the clones were transferred into 50mL LB (Pro↓) and cultured at 37℃ constant temperature shaker at 220rpm until OD600=0.8; the temperature was lowered to 30℃ and adapted to OD600=1.0 (about 30min), the NaCl concentration was supplemented to a final concentration of 30g / L, a Pro gradient (5~100mM) was set, and 0.5mM IPTG was added for induction. The clones were cultured at 30℃ constant temperature shaker at 220rpm for 4 hours. After the expression was completed, the supernatant was centrifuged at 6000rpm for 5 minutes and identified by SDS-PAGE (see the results). Fig.16 ).

[0083] 2) Hyp addition: pET28A-rhCol / BL21 (DE3) / Δ pro The positive clones verified by A / P4H sequencing were inoculated into 5mL LB medium and cultured to the logarithmic phase. 5% of the clones were transferred into 50mL LB (Pro↓) and cultured at 37℃ constant temperature shaker at 220rpm until OD600=0.8; the temperature was lowered to 30℃ to adapt to OD600=1.0 (about 30min), and the NaCl concentration was supplemented to 30g / L final concentration, and the Pro concentration was supplemented to 5mM final concentration. A Hyp gradient (5~100mM) was set and 0.5mM IPTG was added for induction. The clones were cultured at 30℃ constant temperature shaker at 220rpm for 4 hours. After the expression was completed, the supernatant was centrifuged at 6000rpm for 5 minutes and identified by SDS-PAGE (see the results). Fig.17 ).

[0084] from Figure 14-17 It can be seen that Fig.14 The SDS-PAGE results of different expressions in the LB medium shown showed that rhCol was significantly expressed in the three strains, and the expression levels were basically the same. Figure 15-17 The results of adding different culture media show that NaCl concentrations above 30 g / L have a certain inhibitory effect on the expression of the target protein but still have a high expression. When Pro is added above 5 mM, the expression tends to be stable. When Hyp is added above 10 mM, the expression of rhCol tends to be stable.

[0085] Example 4 Purification and preparation of rhCol 1) Bacteria collection: After the above steps of amplification fermentation and expression in the corresponding shake flask, collect bacteria ≥ 10g by centrifugation at 6000g*30min.

[0086] 2) Cell disruption: The harvested cells (1:10) were suspended in disruption buffer (20mM Tris-HCl, 500mMNaCl, 5%Glycerol, pH 7.5) and subjected to ultrasonic disruption (ice bath φ10, 60%, ultrasonic 3' stop 5', 15min / time*2), and the supernatant was collected by centrifugation at 12000g*30min.

[0087] 3) Protein purification: Use nickel column (Ni-NTA) for affinity capture of target protein, linearly eluted in 200-500mM imidazole buffer; add enterokinase (EK) to the target protein elution sample identified by SDS-PAGE; then further purify with anion DEAE to remove EK enzyme and other impurities to obtain the target protein penetration solution; dialyze (40mM NaH2PO4, 150mMNaCl, pH 7.5) at a volume ratio of 1:500; ultrafiltration and concentration to a target protein concentration of 5-6mg / mL.

[0088] The experimental results are shown in Fig.18 "Lane 1" is a protein marker (10-180KD), "Lane 2" is a bacterial cell fragment, "Lane 3" is a sample purified by NI column, "Lane 4" is a sample digested by enterokinase EK, and "Lane 5" is a sample concentrated by ultrafiltration after DEAE purification. The final concentration of rhCol protein stock solution by PAGE is ~5.0mg / mL, and the purity is Lane 5 (>95%)>Lane 4>Lane 3>Lane 2.

[0089] Example 5 Comparison of hydroxylation detection of rhCol obtained by different expression methods rhCol Proline Hydroxylation Detection Kit Analysis Method: Chloramine T method to detect free Hyp: The sample is acid-lyzed to produce free Hyp, which 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 sample color change. There is a characteristic absorption peak at 560nm. By measuring the absorbance of the sample hydrolyzate at 560nm, the Hyp content can be compared or calculated. (For details, see the "Hydroxyproline (HYP) Content Detection Kit Instructions") 1. Sample processing Required P4H / rhCol-BL21(DE3)-LB, rhCol-BL21(DE3) / Δ pro A-LB(NaCl+,Hyp), rhCol-BL21(DE3) / Δ proA / P4H-LB (NaCl+, Hyp) disrupted liquid was obtained according to the corresponding culture conditions in Example 3; the purified stock solution was obtained according to the steps in Example 4. Take 100μL (100μL of disrupted liquid, 5μL / sample of purified stock solution diluted to 100μL) of liquid sample in a glass tube, add 100μL of extract (if the measured value is too small, the ratio of the two can be adjusted), boil at 100℃ for 1 hour to digest until no large clumps are visible, and adjust the pH value to 6-8 with NaOH solution after cooling. Finally, centrifuge at 12000rpm, 25℃ for 20min (if there are still impurities after centrifugation, they can be removed by filtration), and take the supernatant for testing.

[0090] 2. Activity Assay Observe the color reaction.

[0091] 3. The microplate reader reads and records the 560nm wavelength data.

[0092] The results are as follows Figure 19-20 As shown, "Channel 1" is the negative control, and "Channel 2" is the free proline in the kit ( Fig.19 :0.25μg, Fig. 20 :2.5μg) hydroxylation positive control, "channel 3" is the P4H and rhCol co-expression BL21 (DE3) group, "channel 4" is rhCol-BL21 (DE3), Δ pro Group A, “Channel 5” is rhCol-BL21(DE3), Δ pro A, P4H group. Fig.19 The results of the broken liquid showed that the rhCol expressed by the three strains was hydroxylated, and the degree of protein modification in the modified culture strain was slightly higher than that of the co-expression strain. Fig. 20 It can be seen that the hydroxylation degree of the purified rhCol sample BL21(DE3)Δ pro A,P4H > BL21(DE3)Δ pro A>BL21(DE3).

[0093] Example 6 Application and detection of various types of recombinantly expressed human hydroxylated collagen rhCol According to the above method, human collagen I, III, IV, VII, XVII (SEQ ID NO. 10-14) sequences were recombinantly expressed in BL21 (DE3) / Δ pro A / P4H engineered strain, and finally detected the proline hydroxylation of the purified sample of its recombinantly expressed collagen.

[0094] The results are as follows Fig.21As shown, "Channel 1" is a negative control, "Channel 2" is a positive control for free proline (3 μg) hydroxylation in the kit, "Channel 3" is a control for rhCol (SEQ ID NO. 2) expressed in rhCol-BL21 (DE3) strain, and "Channels 4-8" are modified engineering bacteria BL21 (DE3) / Δ pro A / P4H expressed ColⅠ, Ⅲ, Ⅳ, Ⅶ, and XVII experimental groups respectively. The results showed that the above different types of rhCol can all undergo efficient hydroxyproline integration or proline hydroxylation modification.

Claims

1. An E. coli The engineered bacteria are characterized by: To E. coli The strains were transformed as follows: a. Knockout E. coli Proline metabolic pathway genes in strains pro A or pro B, or pro A and pro B knocked out simultaneously; b. Knock the P4H gene into E. coli The strain was used to express the P4H protein; c. In E. coli The collagen expression gene was introduced into the strain.

2. E according to claim 1. coli The engineered bacteria are characterized by: The P4H gene is selected from P4H genes from the following sources: Homo sapiens human-P4H, Paramecium bursaria Chlorella virus-1 Chlorella virus type 1-P4H, Drosophila melanogaster-P4H Drosophila melanogaster-P4H, Caenorhabditis elegans-P4H Caenorhabditis elegans-P4H, Bacillus anthracis-P4H anthrax Bacillus-P4H, Chlamydomonas reinhardtii-P4H Clemidomonas-P4H, Mus musculus-P4H mice-P4H, Dactylosporangiumsp cyst fungus RH1-P4H, and uncultured bacterium-P4H.

3. E according to claim 2. coli The engineered bacteria are characterized by: The P4H gene is Dactylosporangiumsp RH1-P4H.

4. E according to claim 1. coli The engineered bacteria are characterized by: Said pro The gene of A is shown in SEQ ID NO.8, pro The gene of B is shown in SEQ ID NO.

9.

5. E according to claim 4. coli The engineered bacteria are characterized by: The gene knockout method is selected from CRISPR / Cas9-mediated gene knockout, suicide plasmid pCVD-mediated gene knockout, Red / ET recombination-mediated gene knockout, group II intron insertion-mediated gene knockout, Cre-LoxP recombination-mediated gene knockout, TALEN gene targeting or RNA interference methods.

6. E according to claim 1. coli The engineered bacteria are characterized by: The collagen expression gene encodes an amino acid sequence as shown in SEQ ID NO.3 or any one of SEQ ID NOs.10-14.

7. E according to any one of claims 1 to 6. coli The engineered bacteria are characterized by: Said E. coli The strain was selected from the following strains: BL21(DE3), Rosetta(DE3), Origami(DE3), JM109, HSM174(DE3), AS1.357, TOP10 or DH5α strains.

8. E according to any one of claims 1 to 6. coli The engineered bacteria are characterized by: At the same time, a strong expression promoter was knocked in to express P4H protein. The strong expression promoter was derived from P tac , P lac , P T7 , P T7lac , pL, P trp Select in.

9. E according to any one of claims 1 to 6. coli The engineered bacteria are characterized by: The P4H gene is expressed in E. coli The knock-in site in the strain is selected from lpxM, lacZ, phoP, Lpp, ompA or Lpp-ompA.

10. E according to any one of claims 1 to 9. coli Application of engineered bacteria in expressing hydroxylated collagen.

11. A method for preparing hydroxylated collagen, characterized in that: It comprises culturing the E. coli Engineered bacteria, induced to express collagen and purified.

12. The method according to claim 11, characterized in that include: The transformed E. coli The proline concentration in the culture medium used for culturing the strain before induction of expression is less than 1 mM; during the induction of expression, sodium chloride, proline and / or hydroxyproline are supplemented to make the sodium chloride concentration in the culture medium 20 ~ 40g / L, the proline concentration 5~100mM, and / or the final hydroxyproline concentration 10~100mM.

13. Use of the hydroxylated collagen prepared by the method according to claim 11 or 12 in the preparation of bioengineering materials, food supplements and cosmetics.

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