A recombinant Escherichia coli Nissle 1917 producing lysostaphin
By using a low-copy-number plasmid pSC101 in Escherichia coli Nissle 1917 to carry plasmid expression systems containing T7 RNA polymerase and lysostaphin genes, the problems of cumbersome operation and poor expression in existing technologies have been solved, achieving efficient lysostaphin expression and high enzyme activity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU XUE BAO DAILY CHEM CO
- Filing Date
- 2026-03-26
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, the integration of the T7 RNA polymerase gene into the genome using E. coli expression systems is cumbersome, time-consuming, and difficult to regulate in small amounts, making it hard to achieve efficient heterologous expression, especially for lysostaphylococcal enzyme expression.
The low-copy-number plasmid pSC101 was used to carry T7 RNA polymerase, and it and the lysostaphin gene were placed on two different plasmids to construct recombinant Escherichia coli Nissle 1917. Stable protein expression was achieved using the plasmid expression system.
This method achieves efficient expression of lysostaphin, increases protein concentration and enzyme activity, simplifies the operation process, and improves the controllability and expression effect of the expression system.
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Figure CN122104544A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant Escherichia coli Nissle1917 that produces lysostaphin. Background Technology
[0002] Escherichia coli expression systems are currently the most commonly used recombinant protein expression systems in genetic engineering. Due to their clear genetic background and simple genetic manipulation techniques, E. coli has become one of the most common model organisms in genetic engineering and molecular biology research. A typical E. coli expression system requires an expression vector and an expression strain. In addition to the properties of cloning vectors, the expression vector should also contain expression elements, i.e., the DNA sequences necessary for transcription and translation. The expression elements required for the specific function of the expression vector include the replication origin, multiple cloning site, transcription termination signal, fusion tag, resistance gene, and promoter. The promoter on the expression vector, as the recognition sequence for the transcription initiation of the heterologous gene, is one of the key genetic elements for heterologous gene expression. It can efficiently initiate the transcription process and directly affect the expression level of the target gene. Based on their regulatory characteristics, promoters can be divided into constitutive promoters and inducible promoters. Constitutive promoters can be continuously expressed and do not require specific inducers to induce their expression function, but their biggest drawback is that they cannot precisely regulate the response. Conversely, inducible promoters can precisely alter the expression intensity of the target gene through external stimuli, such as changes in ambient temperature, pH, the addition of specific inducers, and changes in their concentration. Among commercial pET series expression vectors, there exists a promoter derived from T7 phage RNA polymerase—the T7 promoter. As a typical representative of inducible promoters, the T7 promoter can be efficiently recognized and initiated by the specific T7 RNA polymerase (T7RNAP), achieving transcription levels 3-5 times higher than other promoters. Notably, the activity of T7RNAP is entirely dependent on the presence of the T7 promoter; without it, T7RNAP will not initiate transcription. This ensures high selectivity and controllability of the transcription process, avoiding non-specific transcription and potential gene expression interference. The T7 promoter and... T7 RNAP As a key functional element in the T7 expression system, it strictly controls the transcription process under the influence of other regulatory elements.
[0003] The selection of the expression strain is crucial for the expression of heterologous proteins. *Escherichia coli*, as a currently popular engineered strain, occupies a place in synthetic biology due to its high expression levels and high-purity proteins. (Strain) Escherichia coliNissle 1917 (EcN) is one of the few probiotic model organisms among Gram-negative bacteria. Traditionally used for the prevention and treatment of various inflammatory diseases, it is marketed as a commercial probiotic preparation. Due to its high biosafety, non-toxicity, biocompatibility, and good acceptability, probiotics possess enormous potential for application in synthetic biology and metabolic engineering.
[0004] The T7 system's highly efficient heterologous expression capability has allowed researchers to deepen their exploration of its applications, enabling efficient expression of heterologous genes in various host strains. Current techniques primarily involve integrating the T7 RNA polymerase gene into the genome for expression, a process that is overly cumbersome, time-consuming, and requires a massive amount of work in screening for genomic insertion sites. Furthermore, integration into the genome makes fine-tuning of T7 RNA polymerase expression inconvenient. Therefore, developing a simple and highly efficient E. coli expression system is crucial.
[0005] Lysostachysin, with its high specificity and safety, has demonstrated significant value in the control of drug-resistant bacteria, food preservation, and bioengineering. Lysostachysin specifically recognizes and hydrolyzes the glycine pentapeptide-bridging structure (found in the peptidoglycan layer) in the cell wall of Staphylococcus aureus, leading to cell wall rupture and bacterial death. In the food industry, it directly disrupts the cell wall of Staphylococcus aureus (such as Staphylococcus aureus), inhibiting its growth and reproduction, extending the shelf life of food, and overcoming the limited effectiveness of traditional preservatives (such as nitrites) against Gram-positive bacteria. In the medical field, lysostachysin is immobilized on the surface of medical devices such as catheters and implants to prevent iatrogenic infections caused by Staphylococcus aureus and reduce the risk of Staphylococcus aureus transmission. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides a recombinant Escherichia coli Nissle1917 that produces lysostaphin.
[0007] To rapidly industrialize EcN production, the inventors directly constructed a plasmid expression module. This module expresses protein independently of the genome, allowing for rapid and stable passage under the maintenance of resistance genes, and producing consistent protein expression. The inventors found that compared to high-copy-number plasmids such as pET and pRSF-Duet, using the low-copy-number plasmid pSC101 to express T7 RNA polymerase resulted in better expression of the lysostaphin gene controlled by the T7 promoter in strain EcN△P. Furthermore, compared to integrating the T7 RNA polymerase gene into the genome, plasmid expression of T7 RNA polymerase is more effective and allows for more efficient control of lysostaphin expression by the T7 promoter. Moreover, placing the T7 RNA polymerase and lysostaphin encoding genes in two separate plasmids resulted in even better expression. This study demonstrates the possibility that the probiotic Escherichia coli Nissle 1917 can serve as a heterologous protein expression vector, and that high production of lysostaphin can be achieved through expression of T7 RNA polymerase carried on the pSC101 plasmid.
[0008] The technical solution of the present invention is as follows: A recombinant Escherichia coli Nissle 1917 producing lysostaphin, comprising the expression strain EcN△P and plasmid pSC101- lacUV5-T7RNAP and contains genes encoding lysostaphin lys plasmid pET22b- lys ; The plasmid pSC101- lacUV5-T7RNAP The nucleotide sequence is SEQ ID NO.1; The amino acid sequence of the lysostaphin enzyme is SEQ ID NO.2; The plasmid pET22b- lys Gene encoding lysostaphin lys It was ligated into plasmid pET-22b(+); The expression strain EcN△P is composed of knockout plasmids pMUT1 and pMUT2. Escherichia coli Nissle 1917.
[0009] According to a preferred embodiment of the present invention, the nucleotide sequence encoding the lysostaphin gene is SEQ ID NO.3.
[0010] The method for constructing the above-mentioned recombinant Escherichia coli Nissle 1917 producing lysostaphin includes the following steps: (1) The gene encoding lysostaphin lys Linked to plasmid pET-22b(+) Nde I and XhoI site, plasmid pET22b- lys ; (2) Plasmid pSC101- lacUV5-T7RNAP And the plasmid pET22b- obtained in step (1) lys The strain was transformed into the expression strain EcN△P, and after screening, recombinant strain EcN△P / pSC101- was obtained. lacUV5-T7 RNAP / pET22b- lys .
[0011] According to a preferred embodiment of the present invention, the gene sequence encoding the lysostaphin enzyme is SEQ ID NO.3.
[0012] Further preferredly, the lysostaphin gene sequence was amplified using primers lys-F as shown in SEQ ID NO.13 and lys-R as shown in SEQ ID NO.14 to obtain the lysostaphin gene.
[0013] According to a preferred embodiment of the present invention, in step (2), the pRE112 plasmid is used to... Escherichia coli The plasmids pMUT1 and pMUT2 in Nissle1917 were knocked out to obtain the expression strain EcN△P.
[0014] The application of the above-mentioned recombinant Escherichia coli Nissle 1917 in the preparation of lysostaphin.
[0015] The method for preparing lysostaphin from recombinant Escherichia coli Nissle 1917 includes the following steps: Recombinant bacteria EcN△P / pSC101- lacUV5-T7RNAP / pET22b- lys Fermentation was carried out to prepare lysostaphin enzyme.
[0016] An Escherichia coli Nissle 1917 expression system includes the following: expression strain EcN△P, plasmid pSC101- lacUV5-T7RNAP、 Expression vector pET-22b(+); The plasmid pSC101- lacUV5-T7RNAP The nucleotide sequence is SEQ ID NO.1; The expression strain EcN△P is composed of knockout plasmids pMUT1 and pMUT2. Escherichia coli Nissle 1917.
[0017] The method for constructing the above-mentioned Escherichia coli Nissle 1917 expression system includes the following: Plasmid pSC101- lacUV5-T7RNAP The expression vector pET-22b(+) was transformed into the expression strain EcN△P.
[0018] According to a preferred embodiment of the present invention, the pRE112 plasmid is used to... Escherichia coli The plasmids pMUT1 and pMUT2 in Nissle 1917 were knocked out to obtain the expression strain EcN△P.
[0019] The above-mentioned Escherichia coli Nissle 1917 expression system is used in the preparation of organic acids, proteins, amino acids or polysaccharides.
[0020] The beneficial effects of the present invention include at least the following: 1. This invention provides an Escherichia coli Nissle 1917 expression system. The inventors discovered that by using the pSC101 plasmid to carry T7 RNA polymerase, exogenous genes can be expressed at a high level in the expression strain EcN△P.
[0021] 2. This invention provides a recombinant Escherichia coli Nissle 1917 that produces lysostaphin, and the recombinant bacteria produces a high concentration of lysostaphin protein. Attached Figure Description
[0022] Figure 1 This is a diagram showing the verification results of cryptic plasmid knockout.
[0023] Figure 2 For recombinant plasmid pSC101- lacUV5-T7RNAP A schematic diagram of the construction structure.
[0024] Figure 3 This is a graph showing the expression detection of T7 RNA polymerase.
[0025] Figure 4 This is a graph showing the expression detection of GFP (green fluorescent protein).
[0026] Figure 5 This is a graph showing the expression detection of lysostaphin; In the figure: 1 represents the final concentration of 2 g / L lactose induction. E. coli BL21 (DE3) / pET22b- lys Group 2: EcNΔP / pSC101- for a final concentration of 0.5 mM IPTG. lacUV5-T7RNAP / pET22b- lys Group 3 represents the EcNΔP / pSC101- induced by a final lactose concentration of 2 g / L. lacUV5-T7RNAP / pET22b- lys Group 4 represents the EcNΔP / pET28a- induced by a final lactose concentration of 2 g / L. lacUV5-T7RNAP / pET22b- lys Group 5 represents the EcNΔP / pRSF induced by a final lactose concentration of 2 g / L. lacUV5- T7RNAP / pET22b- lys Group.
[0027] Figure 6 This is a curve for the determination of enzyme activity of standard samples. Detailed Implementation
[0028] The present invention will now be described in detail with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.
[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0031] Reagents and materials: Plasmid extraction kits were purchased from Tiangen Biotech Co., Ltd., while 2×Phanta Master Mix, genome extraction kits, product purification and recovery kits, ClonExpress MultiS, and ClonExpress II one-step cloning kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.
[0032] Plasmid pRE112 (laboratory collection), strain Escherichia coli Nissle 1917 (EcN) (purchased from Beijing Bio-Tech Biotechnology Co., Ltd.), plasmid pMUT1 (extracted from EcN), plasmid pMUT2 (extracted from EcN), plasmid pSC101-TIMER (laboratory deposit), plasmid pET-22b(+) (laboratory deposit), plasmid pET-28a(+) (laboratory deposit), plasmid pRSF-Duet (laboratory deposit), plasmid pOSIP (laboratory deposit). E. coli BL21(DE3) (competent cells purchased from Sangon Biotech). All plasmids and strains used were commercially available products.
[0033] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.
[0034] 25 mg / ml chloramphenicol: Dissolve 250 mg of chloramphenicol in 10 mL of anhydrous ethanol at -20°C.
[0035] 100 mg / mL ampicillin: Dissolve 1.0 g of ampicillin sodium in 10 mL of deionized water and store at -20°C.
[0036] 50 mg / mL kanamycin: Dissolve 0.5 g kanamycin in 10 mL of deionized water and store at -20 ℃.
[0037] 1 M Isopropyl-β-D-thiogalactoside (IPTG): 2.38 g IPTG dissolved in 10 mL deionized water, stored at -20°C protected from light.
[0038] PBS buffer: 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, and 0.27 g potassium dihydrogen phosphate were dissolved in 1000 mL ddH2O, and the pH was adjusted to 7.0 with hydrochloric acid and sodium hydroxide.
[0039] CYT electroporation buffer: 10% (v / v) glycerol, 1.25 g / L yeast extract, 2.5 g / L peptone.
[0040] Example 1 1. Method for preparing electric shock-receptor states Pick a single EcN colony or other colonies from an LB agar plate and inoculate them into 5 mL of LB medium. Incubate at 37°C with shaking at 200 rpm for 8-10 h. After incubation, transfer the culture to 100 mL of fresh LB medium at a 1% (v / v) inoculation ratio and incubate again at 37°C with shaking at 200 rpm until OD (occurrence limit) is reached. 600 When the concentration reaches 0.2, place the bacterial culture in an ice bath for 30 minutes. Centrifuge at 6000 r / min for 5 min, discard the supernatant, and collect the bacterial cells. Resuspend the precipitated bacteria in pre-chilled and sterile CYT, wash, and centrifuge again at 6000 r / min for 5 min, discard the supernatant, and collect the bacterial cells. Repeat the above bacterial cell washing steps 3-4 times. Resuspend the competent cells in 40 mL of pre-chilled CYT, aliquot the competent cells into 40 μL tubes, and freeze at -80 ℃.
[0041] 2. Electroconversion methods Mix 40 μL of EcN competent cells or other competent cells with 10 μL of recombinant plasmid, add to a pre-chilled electroporation cuvette, gently tap the liquid to ensure the mixture is at the bottom of the cuvette, and incubate on ice for 10 min. After wiping away any condensation and mist from the outside of the cuvette, place it in an electroporator, set the parameters to 2500 V / mm / 25 μF / 200 Ω, and electroporate. Immediately after electroporation, place on ice and add 1 mL of fresh LB medium. Gently resuspend the cells and transfer to a 1.5 mL sterile centrifuge tube. Incubate at 37°C with shaking at 200 rpm for 1 h. Centrifuge the resuscitation solution at 6000 rpm for 10 min, remove 900 μL of supernatant, resuspend the cells, and spread onto LB agar plates containing 50 μg / mL chloramphenicol (or LB agar plates containing other antibiotics). Incubate overnight at 37°C.
[0042] 3. Thermal conversion method The bonding products were converted to thermal shock. E. coli In the DH5α competent state, the specific process is as follows: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] E. coli DH5α competent cells were removed from the -80 ℃ freezer and thawed on ice. 10 μL of ligation product was added, and the mixture was gently tapped into a 1.5 mL centrifuge tube to mix. The tubes were then incubated in an ice-water bath for 30 min. The competent cells were then heat-shocked at 42 ℃ for 90 s, immediately followed by an ice-water bath for 2 min. Immediately afterward, 900 μL of fresh LB medium was added, and the cells were incubated at 37 ℃ with shaking for 1 h. The revival solution was centrifuged at 6000 r / min for 3 min, and a portion of the supernatant was aspirated. The revival solution was then spread onto LB agar plates containing the appropriate antibiotic and incubated at 37 ℃ for 24 h. Transformants were picked and incubated in liquid LB containing the appropriate antibiotic at 37 ℃ for 12 h. Plasmids were then extracted using a plasmid extraction kit.
[0043] 4. Knockout of cryptic plasmids pMUT1 and pMUT2 in strain EcN The cryptic plasmid was knocked out using the knockout plasmid pRE112.
[0044] First, construct the pRE112-pMUT1 and pRE112-pMUT2 knockout plasmids.
[0045] The construction method is as follows: The pMUT1-F sequence is shown in SEQ ID NO.4; The pMUT1-R sequence is shown in SEQ ID NO.5; The pMUT2-F sequence is shown in SEQ ID NO.6; The pMUT2-R sequence is shown in SEQ ID NO.7; The pRE112-F sequence is shown in SEQ ID NO.8; The pRE112-R sequence is shown in SEQ ID NO.9.
[0046] Using primers pMUT1-F and pMUT1-R, the linear fragment pMUT1 was amplified using plasmid pMUT1 as a template. Using primers pMUT2-F and pMUT2-R, the linear fragment pMUT2 was amplified using plasmid pMUT2 as a template. Simultaneously, using primers pRE112-F and pRE112-R, the linear fragment pRE112 was amplified using plasmid pRE112 as a template after plasmid pRE112 was knocked out. All amplification products were purified using a product purification kit to obtain linear fragments of pMUT1 and pMUT2 containing homologous arms, as well as a linear fragment of pRE112. Using the ClonExpress II one-step cloning kit, the linear fragments of pMUT1 and pMUT2 were ligated to the linear fragment of pRE112, respectively, to obtain ligated fragments. The ligated fragments were then transformed using a heat shock method. E. coli In DH5α competent cells, knockout plasmids pRE112-pMUT1 and pRE112-pMUT2 were obtained.
[0047] The constructed recombinant plasmid pRE112-pMUT2 was electroporated into EcN electrocompetent cells and passaged in LB medium containing chloramphenicol (25 μg / mL) to screen for recombinant strain EcN△pMUT2 / pMUT2-pRE112, which had lost the pMUT2 plasmid. Strain EcN△pMUT2 / pRE112-pMUT2 was then inoculated into LB liquid medium containing 10% sucrose and passaged to screen for EcN△pMUT2 strain, which had lost the recombinant plasmid pMUT2-pRE112.
[0048] The recombinant plasmid pRE112-pMUT1 was again electroporated into EcN△pMUT2 electrocompetent cells and passaged in LB medium containing chloramphenicol (25 μg / mL) to screen for the recombinant strain EcN△pMUT1△pMUT2 / pRE112-pMUT1, which lost the pMUT1 plasmid. The strain EcN△pMUT1△pMUT2 / pRE112-pMUT1 was then inoculated into LB liquid medium containing 10% sucrose and passaged to screen for the EcN△P strain, which lost the recombinant plasmid pRE112-pMUT1. The knockout of the cryptic plasmid in the EcN△P strain was verified, and the results are shown in […]. Figure 1 ,Depend on Figure 1 It can be seen that the cryptic plasmids pMUT1 and pMUT2 have been knocked out in the EcN△P strain.
[0049] 5.pSC101- lacUV5-T7RNAP plasmid construction The pSC101-F sequence is shown in SEQ ID NO.10; The pSC101-R sequence is shown in SEQ ID NO.11.
[0050] The lac IF sequence is shown in SEQ ID NO.15; The lac IR sequence is shown in SEQ ID NO.16; The T7 RNA pol-F sequence is shown in SEQ ID NO.17; The T7 RNA pol-R sequence is shown in SEQ ID NO.18.
[0051] The linear pSC101 gene fragment was amplified using primers pSC101-F and pSC101-R, with plasmid pSC101-TIMER as a template. The lacI gene fragment was amplified using primers lac IF and lac IR, with plasmid pET-28a(+) as a template. Genome samples from *E. coli* BL21(DE3) were extracted using a genome extraction kit, and primers T7 RNApol-F and T7 RNApol-R were used to amplify the T7 RNA polymerase module (containing the promoter). lac UV5 and lac PCR amplification was performed using the O sequence. The lacI+T7 RNA polymerase base sequence and the pSC101 linear gene fragment were ligated using the ClonExpressMultiS one-step cloning kit to obtain the ligated fragment. The ligated fragment was then transformed into [a specific gene] using a heat shock method. E. coli In DH5α competent cells, the recombinant plasmid pSC101- was obtained. lacUV5-T7RNAP .
[0052] pSC101- lacUV5-T7RNAP The nucleotide sequence is shown in SEQ ID NO.1.
[0053] pSC101- lacUV5-T7RNAP See the schematic diagram of the construction structure. Figure 2 .
[0054] 6. Recombinant strain EcN△P / pSC- lacUV5-T7RNAP Construction The recombinant plasmid pSC101- lacUV5-T7RNAP Electrotransformation of plasmid pSC101 into EcNΔP electrocompetent cells yielded EcNΔP / pSC101- cells after screening on LB plates containing kanamycin (50 μg / mL). lacUV5- T7RNAP Recombinant strain and EcN△P / pSC101. Transferred to LB liquid medium containing kanamycin (50 ug / mL), and incubated at 37°C with shaking at 200 rpm until OD. 600When the concentration reached 0.5, IPTG (0.5 mM) was added for induction. After 24 h of induction, cells were collected and resuspended in PBS for cell lysis. Cells were centrifuged at 10000 r / min for 10 min, and the supernatant was used to prepare extracellular protein samples. SDS-PAGE protein electrophoresis results are shown in the image. Figure 3 .
[0055] Depend on Figure 3 The result is displayed: EcN△P / pSC101- lacUV5-T7RNAP T7 RNA polymerase was successfully expressed.
[0056] 7. Recombinant strain EcN△P / pSC101- lacUV5-T7RNAP / pET22b- GFP Construction The GFP-F sequence is shown in SEQ ID NO.19; The GFP-R sequence is shown in SEQ ID NO.20; The green fluorescent protein (GFP) gene sequence was amplified using primers GFP-F and GFP-R to obtain the GFP gene fragment. Nde I and Xho I. The plasmid pET-22b(+) was double-digested to obtain the linear pET-22b(+) gene fragment. Using the ClonExpress II one-step cloning kit, the green fluorescent protein gene fragment and the linear pET-22b(+) gene fragment were ligated to obtain the ligated fragment. The ligated fragment was then transformed into [a specific gene] using a heat shock method. E. coli In DH5α competent cells, the recombinant plasmid pET22b- was obtained. GFP The nucleotide sequence of the green fluorescent protein gene fragment is shown in SEQ ID NO.12.
[0057] Preparation of EcN△P / pSC101- lacUV5-T7RNAP Electroporation of competent cells, with recombinant plasmid pET22b- GFP Electroconversion EcN△P / pSC101- lacUV5-T7RNAP In the electroacupuncture competent state, EcN△P / pSC101- was obtained by screening on LB plates containing ampicillin (100 ug / mL) and kanamycin (50 ug / mL). lacUV5-T7RNAP / pET22b- GFP Recombinant strain. Transferred to LB liquid medium containing ampicillin (100 ug / mL) and kanamycin (50 ug / mL), and incubated at 37°C with shaking at 200 rpm until OD... 600When the concentration reached 0.5, IPTG (final concentration 0.5 mM) was added for induction. After 24 h of induction, cells were collected, resuspended in PBS, and subjected to cell lysis. Whole-cell protein samples were prepared and analyzed by SDS-PAGE protein electrophoresis. Figure 4 EcN△P / pSC101- lacUV5-T7RNAP Successfully performed pET22b- GFP The green fluorescent protein in the plasmid vector was expressed, and a clear protein band appeared.
[0058] 8. Recombinant strain EcN△P / pSC101- lacUV5-T7RNAP / pET22b- lys Construction The lys-F sequence is shown in SEQ ID NO.13; The lys-R sequence is shown in SEQ ID NO.14; The lysostaphin gene sequence was amplified using primers lys-F and lys-R to obtain the lysostaphin gene fragment. Nde I and Xho I. The plasmid pET-22b(+) was double-digested to obtain the linear pET-22b(+) gene fragment. Using the ClonExpress II one-step cloning kit, the lysostaphin gene fragment and the linear pET-22b(+) gene fragment were ligated to obtain the ligated fragment. The ligated fragment was then transformed into [a specific enzyme / technology] using a heat shock method. E. coli In DH5α competent cells, the recombinant plasmid pET22b- was obtained. lys .
[0059] The lysostaphylococcal enzyme gene sequence is shown in SEQ ID NO.3.
[0060] The recombinant plasmid pET22b- lys Electroconversion EcN△P / pSC101- lacUV5-T7RNAP In the electroacupuncture competent state, EcN△P / pSC101- was obtained by screening on LB plates containing ampicillin (100 ug / mL) and kanamycin (50 ug / mL). lacUV5-T7RNAP / pET22b- lys Recombinant strain. Transferred to LB liquid medium containing ampicillin (100 ug / mL) and kanamycin (50 ug / mL), and incubated at 37°C with shaking at 200 rpm until OD... 600 When the concentration reached 0.5, IPTG (final concentration 0.5 mM) or lactose (2 g / L) was added for induction. After 24 h of induction, the fermentation supernatant was obtained by centrifugation (10000 r / min for 5 min), and extracellular protein samples were prepared for SDS-PAGE protein electrophoresis analysis.Figure 5 .
[0061] control group E. coli BL21(DE3) / pET22b- lys For laboratory preservation of strains, the heat shock method was used directly (pET22b- lys Add competent cells E. coli In BL21(DE3), after a 30-minute ice-water bath, the sample was incubated at 42°C for 90 seconds, then immediately placed in an ice-water bath for 2 minutes. Fresh LB medium was added, and the sample was incubated at 37°C with shaking for recovery. After recovery, the sample was plated on LB agar plates containing 100 μg / mL ampicillin for screening.
[0062] The inventors used plasmids pET-28a(+) and pRSF-Duet instead of pSC101-TIMER to construct the corresponding recombinant bacteria EcN△P / pRSF- lacUV5-T7RNAP、 EcN△P / pET28a- lacUV5-T7RNAP / pET-22b- lys The fermentation supernatant was obtained as described above, and extracellular protein samples were prepared. SDS-PAGE protein electrophoresis analysis was performed (see attached image). Figure 5 .
[0063] The results showed that, compared with the control group, the recombinant bacteria EcN△P / pRSF- lacUV5-T7RNAP / pET-22b- lys、 EcN△P / pET28a- lacUV5-T7RNAP / pET-22b- lys The expression of lysostaphylococcal enzyme was not effective.
[0064] pRSF- lacUV5-T7RNAP The nucleotide sequence is shown in SEQ ID NO.27; pET28a- lacUV5-T7RNAP The nucleotide sequence is shown in SEQ ID NO.28.
[0065] EcN△P / pSC101- lacUV5-T7RNAP / pET22b- lys The strain exhibited good expression of lysostaphin, with high concentrations of lysostaphin protein induced by both IPTG and lactose.
[0066] Enzyme activity assay method: The lysozyme activity was determined spectrophotometrically. Staphylococcus aureus ATCC6538 (purchased from Beijing BioBio Biotechnology Co., Ltd.) strain was cultured overnight in LB broth. Cells were collected and washed once with PBS. The cells were then resuspended in PBS and the OD was diluted.600 Up to 1.0. The enzyme activity standard curve was determined using lysostaphin standard (Sangon Biotech, Shanghai, China). Different volumes (0, 0.1, 0.2, 0.3, 0.5, 0.7, 1.0, 2.0 μL) of lysostaphin standard dilution (1000 U / mL) were added to 1 mL of bacterial suspension. The suspension was incubated at 37℃ for 10 min, and the turbidity of the Staphylococcus aureus suspension was measured. The enzyme activity assay curve is shown below. Figure 6 As shown.
[0067] When determining the extracellular enzyme activity of the recombinant expression strain, 50 μL of extracellular protein sample was added to 1 mL of bacterial suspension, and the suspension was incubated at 37 ℃ for 10 min. The change in turbidity of the Staphylococcus aureus suspension was then measured, with PBS as a blank control. The actual enzyme activity was calculated using the measured lysostaphin activity standard curve. Lysostaphin activity was defined as 1 U required to reduce the turbidity of a Staphylococcus aureus suspension by 0.1 voltammetry after 10 min at 37 ℃ under pH 7.0 conditions.
[0068] The results of the detection of extracellular protein samples induced by the addition of IPTG (final concentration of 0.5 mM) are as follows: E . coli BL21 (DE3) / pET-22b- lys Enzyme activity: 161.2 U / mL; EcN△P / pSC- lacUV5-T7RNAP / pET-22b- lys Enzyme activity: 224.3 U / mL; EcN△P / pET28a- lacUV5-T7RNAP / pET-22b- lys Enzyme activity: 95.6 U / mL; EcN△P / pRSF- lacUV5-T7RNAP / pET-22b- lys Enzyme activity: 63.3 U / mL.
[0069] Comparative Example 1 1. Construction of the genome-integrated T7 RNA polymerase strain EcNT7 Using the pOSIP plasmid system, genome integration was performed via the λ phage attachment site (attB site) to construct an expression system strain named EcNT7, incorporating T7 RNA polymerase into the genome of *E. coli* Nissle 1917. The specific construction procedure is as follows: Using the BL21(DE3) strain genome as a template, the LacUV5-T7RNAP fusion gene fragment was obtained by PCR amplification using T7 RNA pol-F2 and T7 RNA pol-R2; EcoR I and Pst After double digestion with enzyme I, the plasmid was ligated with the pOSIP vector, which had undergone the same enzyme digestion. The successfully constructed recombinant plasmid was introduced into the EcNΔP strain via heat shock transformation. To obtain an antibiotic-free engineered strain, the antibiotic resistance genes flanking the FRT site of the pOSIP plasmid were removed using FLP recombinase. A three-step screening method was used to eliminate the marker-recombinant strain: first, the strain was cultured on kanamycin, ampicillin, and antibiotic-free LB agar plates; then, single colonies that could only grow on antibiotic-free plates were verified by colony PCR.
[0070] The T7 RNA pol-F2 sequence is shown in SEQ ID NO.21; The T7 RNA pol-R2 sequence is shown in SEQ ID NO.22.
[0071] 2. Construction of recombinant strain EcNT7 / pET22b-lys The recombinant plasmid pET22b- lys EcNT7 electrocompetent cells were electroconverted and screened on LB plates containing ampicillin (100 ug / mL) to obtain EcNT7 / pET22b-. lys Recombinant strain. Transferred to LB liquid medium containing ampicillin (100 ug / mL), and incubated at 37°C with shaking at 200 rpm until OD500. 600 When the concentration reached 0.5, IPTG (final concentration 0.5 mM) was added for induction. After 24 h of induction, the fermentation supernatant was obtained by centrifugation (10000 r / min for 5 min). Enzyme activity was measured to be 89.5 U / mL.
[0072] Comparative Example 2 1. pSC101- lacUV5-T7RNAP-lys plasmid construction The lys-F2 sequence is shown in SEQ ID NO.23; The lys-R2 sequence is shown in SEQ ID NO.24; The pSC101-T7 RNA pol-F sequence is shown in SEQ ID NO.25; The pSC101-T7 RNA pol-R sequence is shown in SEQ ID NO.26.
[0073] Using primers pSC101-T7 RNA pol-F and pSC101-T7 RNA pol-R, plasmid pSC101- lacUV5- T7RNAP A linear gene fragment was amplified using the plasmid pET22b-lys as a template. Using primers lys-F2 and lys-R2, the lysostaphin gene with a promoter was amplified. The gene fragment was ligated using the ClonExpress II one-step cloning kit to obtain the ligated fragment. The ligated fragment was then transformed into [a specific culture medium] using a heat shock method. E. coli In DH5α competent cells, the recombinant plasmid pSC101- was obtained. lacUV5-T7RNAP-lys This means achieving the construction of a single plasmid co-expressing the T7 RNA polymerase and lysostaphin genes.
[0074] 2. Recombinant strain EcN△P / pSC101- lacUV5-T7RNAP-lys Construction The recombinant plasmid pSC101- lacUV5-T7RNAP-lys Electroporation of EcNΔP electrocompetent cells was performed using LB plates containing kanamycin (50 ug / mL) to obtain EcNΔP / pSC101-. lacUV5-T7RNAP-lys Recombinant strain. Transferred to LB liquid medium containing kanamycin (50 μg / mL), and incubated at 37°C with shaking at 200 rpm until OD500. 600 When the concentration reached 0.5, IPTG (final concentration 0.5 mM) was added for induction. After 24 h of induction, the fermentation supernatant was obtained by centrifugation (10000 r / min for 5 min). Enzyme activity was measured to be 113.7 U / mL.
[0075] This invention provides a recombinant Escherichia coli Nissle 1917 that produces lysostaphin, which produces a high concentration of lysostaphin protein and also has high enzyme activity.
Claims
1. A recombinant Escherichia coli Nissle 1917 producing lysostaphin, characterized in that, Includes expression strain EcN△P, plasmid pSC101- lacUV5-T7RNAP and contains genes encoding lysostaphin lys plasmid pET-22b- lys ; The plasmid pSC101- lacUV5-T7RNAP The nucleotide sequence is SEQ ID NO.1; The amino acid sequence of the lysostaphin enzyme is SEQ ID NO.2; The plasmid pET-22b- lys Gene encoding lysostaphin lys It was ligated into plasmid pET-22b(+); The expression strain EcN△P is composed of knockout plasmids pMUT1 and pMUT2. Escherichia coli Nissle 1917.
2. The recombinant Escherichia coli Nissle 1917 as described in claim 1, characterized in that, The nucleotide sequence encoding the lysostaphin gene is SEQ ID NO.
3.
3. The method for constructing the recombinant Escherichia coli Nissle1917 producing lysostaphin according to claim 1, characterized in that, Includes the following steps: (1) The gene encoding lysostaphin lys Linked to plasmid pET-22b(+) Nde I and Xho I site, plasmid pET22b- lys ; (2) Plasmid pSC101- lacUV5-T7RNAP And the plasmid pET22b- obtained in step (1) lys The strain was transformed into the expression strain EcN△P, and after screening, recombinant strain EcN△P / pSC101- was obtained. lacUV5-T7 RNAP / pET22b- lys .
4. The construction method as described in claim 3, characterized in that, The gene sequence encoding the lysostaphin enzyme is SEQ ID NO.3; Preferably, the lysostaphin gene sequence is amplified using primers lys-F as shown in SEQ ID NO.13 and lys-R as shown in SEQ ID NO.14 to obtain the lysostaphin gene.
5. The construction method as described in claim 3, characterized in that, In step (2), the pRE112 plasmid is used to... Escherichia coli The plasmids pMUT1 and pMUT2 in Nissle 1917 were knocked out to obtain the expression strain EcN△P.
6. The use of the recombinant Escherichia coli Nissle 1917 according to any one of claims 1-2 or the recombinant Escherichia coli Nissle 1917 constructed by the method according to any one of claims 3-5 in the preparation of lysostaphin.
7. A method for preparing lysostaphin from recombinant Escherichia coli Nissle 1917 as described in any one of claims 1-2 or recombinant Escherichia coli Nissle 1917 constructed by the method described in any one of claims 3-5, comprising the following steps: Recombinant bacteria EcN△P / pSC101- lacUV5-T7RNAP / pET22b- lys Fermentation was carried out to prepare lysostaphin enzyme.
8. An Escherichia coli Nissle 1917 expression system, characterized in that, Including the following: expression strain EcN△P, plasmid pSC101- lacUV5-T7RNAP, Expression vector pET-22b(+); The plasmid pSC101- lacUV5-T7RNAP The nucleotide sequence is SEQ ID NO.1; The expression strain EcN△P is composed of knockout plasmids pMUT1 and pMUT2. Escherichia coli Nissle 1917.
9. The method for constructing the Escherichia coli Nissle 1917 expression system according to claim 8, characterized in that, Including the following: Plasmid pSC101- lacUV5-T7RNAP The expression vector pET-22b(+) was transformed into the expression strain EcN△P; Preferably, the pRE112 plasmid is used to... Escherichia coli The plasmids pMUT1 and pMUT2 in Nissle 1917 were knocked out to obtain the expression strain EcN△P.
10. The use of the Escherichia coli Nissle 1917 expression system of claim 8 in the preparation of organic acids, proteins, amino acids or polysaccharides.