An engineered Escherichia coli strain that secretes lactate oxidase under hypoxia and its application

Abstract

This invention discloses an engineered *E. coli* strain that secretes lactate oxidase under hypoxia-induced conditions and its applications, relating to the field of biomedical technology. The engineered *E. coli* strain provided by this invention can responsively synthesize lactate oxidase in vitro and under culture medium conditions when exposed to a hypoxic environment. Lactate oxidase decomposes localized lactic acid to produce hydrogen peroxide, thereby killing cells and local bacteria. This process, while treating local symptoms, also kills the *E. coli* vector during culture, reducing the risk of exogenous infection caused by this engineered *E. coli* strain, making it a relatively safe therapeutic strain overall.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an Escherichia coli engineering bacterium secreting lactic acid oxidase under hypoxia induction and application thereof. BACKGROUND

[0002] A plasmid is an extrachromosomal genetic factor, which can autonomously replicate and does not have an extracellular phase, and is not necessary for the life activities such as growth, metabolism and reproduction of a non-host cell, but can endow the host cell with certain special properties. The transformation process of the plasmid to the Escherichia coli is simple, such as electric stimulation, heat stimulation and high-concentration calcium chloride, which can make the Escherichia coli into a competent cell, take in the plasmid and maintain the stability of the plasmid under normal conditions. At the same time, based on the technology of DNA endonuclease, some special sequences in the plasmid can be easily cut by the DNA endonuclease, and then a specific DNA sequence can be inserted therein, that is, the plasmid can be designed controllably. This process can make the plasmid have corresponding gene sequences of many proteins. After the plasmid is transformed into bacteria, the bacteria can produce specific proteins.

[0003] A promoter is a DNA sequence recognized, combined and started to be transcribed by RNA polymerase, which contains a conserved sequence required for specific binding and transcription initiation of RNA polymerase, and most of the promoters are located upstream of the transcription initiation point of the structural gene, and the promoter itself is not transcribed. Some promoters have external field inducibility, and will induce transcription function under some environmental stimulation, such as some heat-induced promoters (for example, Tcl) and radiation-induced promoters (for example, Egrl) in nature. There are also some anoxic promoters in nature, such as PSnirB. PSnirB is an anaerobic inducible promoter derived from Salmonella enterica, and the activity of the promoter is significantly improved under anoxic conditions.

[0004] Lactic acid oxidase can oxidize lactic acid to produce pyruvic acid and hydrogen peroxide, and since no exogenous coenzyme is required as an electron acceptor in the reaction process, it has good application prospects. However, free lactic acid oxidase has poor stability and is easy to be inactivated, especially if it is in the body fluid, it is easy to be hydrolyzed and inactivated by proteases in the blood. How to effectively deliver lactic acid oxidase to the action site, or respond to the production of lactic acid oxidase at the site where the action is needed, has been a difficulty in the field of disease treatment.

[0005] Low oxygen concentration is a characteristic of many disease lesion areas, such as hypoxia caused by rapid growth and metabolism of tumors and lack of blood supply of gangrene. One of the characteristics of low oxygen concentration to tissues is the accumulation of lactic acid.

[0006] The hydrogen peroxide produced by lactic acid oxidase catalyzing lactic acid is a common reactive oxygen species (ROS), and hydrogen peroxide has a high oxidation potential and has a certain killing effect on cells and bacteria.

[0007] The plasmid is transformed into E. coli to construct an anaerobic response synthetic lactate oxidase E. coli, which in-situ synthesizes lactate oxidase in an anaerobic environment, catalyzes local lactate to produce hydrogen peroxide, and realizes the killing of cells and bacteria, which is an ideal way to effectively utilize lactate oxidase and get rid of the easy inactivation characteristics of lactate oxidase. SUMMARY

[0008] Therefore, the present application aims to provide an anaerobic induction secreted lactate oxidase E. coli engineering bacteria and its application, and to provide an anaerobic response secreted lactate oxidase engineering E. coli preparation method and a plasmid nucleotide sequence used in the engineering process.

[0009] The specific technical solutions are as follows:

[0010] The present application provides an anaerobic induction secreted lactate oxidase E. coli engineering bacteria, which is constructed by introducing a plasmid carrying an anaerobic induction promoter, a secretion peptide gene and a lactate oxidase gene into E. coli as a starting bacterium.

[0011] The anaerobic induction promoter is an anaerobic induction promoter PSnirB derived from Salmonella Choleraesuis C500, the FNR promoter family derived from Salmonella Choleraesuis C500, the secretion peptide gene is a secretion peptide gene pelB derived from Pectobacterium carotovorum, which is a common signal peptide sequence in E. coli, and the lactate oxidase gene is a lactate oxidase gene lcto derived from Streptococcus iniae, which is optimized according to the codon bias of E. coli to improve its expression efficiency in E. coli.

[0012] Preferably, the sequence of the anaerobic induction promoter is shown as SEQ ID NO. 1, the sequence of the secretion peptide gene is shown as SEQ ID NO. 3, and the sequence of the lactate oxidase gene is shown as SEQ ID NO. 2.

[0013] The present application also provides a construction method of the E. coli engineering bacteria, which comprises the following steps:

[0014] (1) constructing a prokaryotic expression plasmid containing an anaerobic induction promoter sequence, a secretion peptide gene sequence and a lactate oxidase gene sequence;

[0015] (2) transforming the prokaryotic expression plasmid containing the anaerobic induction promoter sequence, the secretion peptide gene sequence and the lactate oxidase gene sequence into E. coli to obtain the E. coli engineering bacteria.

[0016] Preferably, the transformation is by heat shock method.

[0017] Specifically, the engineered E. coli is constructed by plasmid transformation, and the successfully plasmid-transformed E. coli is obtained by ampicillin screening.

[0018] Preferably, the prokaryotic expression plasmid is pET-20b(+) plasmid.

[0019] The application further provides application of the E. coli engineering bacteria in preparation of a medicine for treating tumors or gangrene with low oxygen characteristics.

[0020] The application further provides a medicine, and an active ingredient of the medicine contains the E. coli engineering bacteria.

[0021] The application has the beneficial effects that the engineered E. coli can synthesize lactic acid oxidase in response to low oxygen environment in vitro and under culture conditions, the lactic acid oxidase can decompose local lactic acid to produce hydrogen peroxide, and then kill cells and local bacteria. This process can treat local diseases, and at the same time, the E. coli carrier is killed along with the culture, so that the risk of exogenous infection caused by the engineered E. coli is reduced, and the engineered E. coli as a whole shows a relatively safe disease treatment strain. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a gel electrophoresis photo of pET-PL-EGFP plasmid (1) in Example 3, fragments after pET-PL-EGFP treated by restriction endonuclease AhdI and XhoI (2), and KB ladder (M) for reference.

[0023] Figure 2 It is a map of the pET-PL-EGFP plasmid.

[0024] Figure 3 It is a scanning electron microscope photo of the engineered E. coli in Example 4.

[0025] Figure 4 It is a green fluorescence expression gray scale photo of the engineered E. coli induced by hypoxia in Example 4.

[0026] Figure 5 It is hydrogen peroxide production of the engineered E. coli culture solution and centrifugal supernatant mixed with lactic acid for 6 hours under different oxygen concentrations in Example 5; wherein, ① E. coli solution cultured under hypoxia for 6 hours, ② supernatant of the E. coli solution cultured under hypoxia for 6 hours after centrifugation at 8000 r / min, ③ E. coli solution cultured under normoxia for 6 hours, and ④ supernatant of the E. coli solution cultured under normoxia for 6 hours after centrifugation at 8000 r / min.

[0027] Figure 6 Graph of the activity ability of the original E. coli and the engineered E. coli.

[0028] Figure 7 Graph of the cytotoxicity of the engineered E. coli in Example 4 to 4T1 cells. DETAILED DESCRIPTION

[0029] Example 1

[0030] This example provides a design for synthesizing a PL-EGFP gene.

[0031] The hypoxia-inducible promoter PSnirB of the FNR promoter family of Salmonella Choleraesuis C500 and the lctO gene of lactic acid oxidase (LOX) derived from Streptococcus iniae were introduced. The sequences of PSnirB (the sequence is shown as SEQ ID NO. 1) and lctO (the sequence is shown as SEQ ID NO. 2) were designed. Considering that the synthesized LOX needs to be secreted to function more effectively, a secretory peptide gene pelB (the sequence is shown as SEQ ID NO. 3) was synthesized by referring to the common secretory peptide sequence of the prokaryotic promoter in Pectobacterium carotovorum, and it was placed in front of the lctO gene corresponding to LOX. In order to more intuitively observe the expression, according to the characteristics of multiple cistrons of the prokaryotic expression system, the enhanced green fluorescent protein (EGFP) gene sequence was directly fused. In addition, the sequences of LOX and EGFP were optimized according to the preference of E. coli codon. The overall gene sequence of the synthesized PL-EGFP gene is shown as SEQ ID NO. 4.

[0032] Example 2

[0033] This example provides a design for synthesizing a PL gene.

[0034] To introduce the hypoxia-inducible promoter PSnirB of FNR promoter family of Salmonella Choleraesuis C500 and lactate oxidase (LOX) gene lctO from Streptococcus iniae. The sequences of PSnirB (the sequence is shown as SEQ ID NO. 1) and lctO (the sequence is shown as SEQ ID NO. 2) were designed. Considering that the synthesized LOX needs to be secreted to play a more effective role, the secretory peptide gene pelB (the sequence is shown as SEQ ID NO. 3) was synthesized by referring to the common secretory peptide sequence of prokaryotic promoters in Pectobacterium carotovorum, and was placed in front of the lctO gene corresponding to LOX, to obtain the PL gene, and the overall gene sequence is shown as SEQ ID NO. 5

[0035] Example 3

[0036] This example provides a way to introduce the gene described in any one of the above embodiments 1-2 into a commercial pET-20b(+) plasmid to construct an engineered plasmid.

[0037] Taking the introduction of the gene in embodiment 1 into the pET-20b(+) plasmid as an example, the commercial plasmid pET-20b(+) was treated with restriction endonuclease AhdI and XhoI, and the gene sequence synthesized in step 1 and subjected to PCR amplification was introduced, and the plasmid and the synthesized gene were connected by T4 DNA ligase to construct pET-PL-EGFP, as shown in the map Figure 2 The electrophoresis photograph of the synthesized pET-PL-EGFP and the electrophoresis photograph of pET-PL-EGFP treated with AhdI and XhoI are shown in Figure 1 The results prove the successful synthesis of the PL-EGFP gene and the successful construction of the pET-PL-EGFP plasmid.

[0038] Example 4

[0039] This example provides a method for transforming the engineered plasmid based on the gene of any one of embodiments 1-2 and the commercial pET-20b(+) plasmid described in embodiment 3 into Escherichia coli. Taking the transformation of pET-PL-EGFP into Escherichia coli as an example, an engineered Escherichia coli is constructed.

[0040] 1. Constructing Escherichia coli competent cells:

[0041] Prepare LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1 L of deionized water, and adjust the pH to 7.0 with 5 mol / L sodium hydroxide. Then use a high-temperature sterilization pot to sterilize under high-pressure steam at 121℃ for 30 min.

[0042] Prepare Tris-CaCl2 solution: weigh 4.1621 g calcium chloride and 0.6057 g Tris into a beaker, add 200 mL ultrapure water to dissolve completely, then add ultrapure water to make up to 500 mL, adjust pH to 6.5 with hydrochloric acid. Filter the resulting solution with a 0.45 μm filter to obtain the Tris-CaCl2 solution.

[0043] Take 2 mL of DH5α E. coli bacterial solution and inoculate into 100 mL of LB liquid medium preheated at 37°C, and cultivate at 37°C for 3 hours. Under sterile conditions, transfer the culture solution into two sterile cold-resistant 50 mL centrifuge tubes, and cool in an ice bath for 30 minutes to 0°C. Then, centrifuge the bacterial solution at 5000 rpm for 10 minutes at 4°C using a refrigerated centrifuge. Discard the supernatant, invert the centrifuge tube to drain the residual liquid, and add 10 mL of pre-cooled Tris-CaCl2 solution to each centrifuge tube, and ice-bath for 15 minutes. Then, centrifuge at 5000 rpm for 10 minutes at 4°C, discard the supernatant, and store each tube with 2 mL of pre-cooled Tris-CaCl2 solution containing 20% glycerol. Use a sterile pipette to dispense the competent cells into sterile centrifuge tubes, 50 μL per tube, and store at -80°C for later use.

[0044] 2. Heat shock method to transform pET-PL-EGFP into E. coli:

[0045] Adjust the temperature of the water bath to 42°C, and preheat to the preset temperature. Thaw the frozen competent E. coli prepared in step 3 at -80°C in 20% glycerol on ice, and add 1 μL of 1 μg / μL of the plasmid pET-PL-EGFP synthesized in step 2 to 10 μL of E. coli bacterial solution, and mix the mixture on ice for 30 minutes. Then, heat shock the mixture in a 42°C water bath for 90 seconds, and quickly transfer to ice to cool for 5 minutes. In a sterile workbench, add 200 μL of LB culture solution (without resistance) to each tube containing the E. coli bacterial solution with the plasmid, and then transfer to a 50 mL centrifuge tube, which is sealed with a filter membrane, and is air-permeable and resistant to contamination. Cultivate and proliferate at 37°C in a shaker at 120 rpm for 3 hours.

[0046] 3. Ampicillin screening and enrichment of successfully transformed E. coli:

[0047] Commercial pET-20b(+) comes with ampicillin resistance. Add 10 mL of E. coli treated in step 2 to LB liquid medium containing 100 μg / mL ampicillin. Cultivate the mixed bacteria at 37°C at 120 rpm for 24 hours to achieve bacterial enrichment. Then, centrifuge at 3000 rpm for 5 minutes to collect the microorganisms, and observe the morphology of the engineered E. coli.Figure 3 ) As the conventional E. coli. Then resuspend the microorganism with LB medium, take 6 groups of 5mL E. coli with absorbance of 1, put them in 6 petri dishes, put three in each of the normoxic incubator with 21% oxygen concentration and the anoxic incubator with 4% oxygen concentration, take out the microorganism after 12 hours of culture, and detect whether the hypoxia inducible promoter is successfully expressed based on the inserted EGFP gene. If green fluorescence is successfully observed and the fluorescence intensity of the anoxic culture is stronger than that of the normoxic culture, it proves that the hypoxia inducible promoter PSnirB is successfully functional. The results show that: under the observation of the inverted fluorescence microscope, it is confirmed that the anoxic condition can effectively induce the engineered E. coli involved in the present application to express green fluorescence Figure 4 ).

[0048] Example 5

[0049] This example evaluates whether the engineered E. coli is successfully constructed and whether it has the ability to secrete lactate oxidase by detecting the concentration of hydrogen peroxide produced by the catalysis of lactate by the engineered E. coli or its extract described in Example 4. Take the engineered E. coli into which pET-PL-EGFP is transferred as an example.

[0050] Grouping: the initial E. coli has an OD600 absorbance of 1, take 1mL of each of ① the E. coli culture under anoxic condition for 6 hours, ② the supernatant of the E. coli culture under anoxic condition for 6 hours after centrifugation at 8000rpm, ③ the E. coli culture under normoxic condition for 6 hours, and ④ the supernatant of the E. coli culture under normoxic condition for 6 hours after centrifugation at 8000rpm, and mix them with 0.5mL of 15mM lactate solution. After 30 minutes, detect the hydrogen peroxide concentration of the four mixed solutions by using the hydrogen peroxide kit, and the results prove that the hydrogen peroxide concentration of the lactate mixed solution of ① and ② groups is significantly higher than that of ③ and ④ groups Figure 5 ). The results prove that the recombinant E. coli described in the present application can successfully synthesize and secrete lactate oxidase, and the secreted lactate oxidase can achieve the expected function.

[0051] Example 6

[0052] This example is used to evaluate the proliferation activity of the recombinant E. coli in Example 4. Take the engineered E. coli into which pET-PL-EGFP is transferred as an example.

[0053] The product of some exogenous genes may have a great impact on the survival and activity of E. coli. In order to detect whether the activity of the recombinant E. coli is normal, based on the proliferation experiment, 1mL of 10 8The original E. coli and the engineered E. coli of CFU / mL were sealed with a single filter membrane, and cultured in a shaker at 37℃ and 120 rpm. The E. coli concentration was measured at selected time, and the test results are shown in Figure 6 The transformation process did not significantly affect the activity of E. coli.

[0054] Example 7

[0055] This example is used to evaluate the in vitro hypoxia-responsive killing performance of the recombinant E. coli of Example 4 on 4T1 breast cancer cells, and specifically using the engineered E. coli into which pET-PL-EGFP is transferred as an example.

[0056] To test the hypoxia-responsive toxicity of the recombinant E. coli on tumor cells, 4T1 cells were used as a detection model. To avoid the influence of excessive bacterial proliferation on the CCK8 test, a Transwell chamber system (0.4 μm) pore size was used for cell experiments, i.e., 15000 cells were inoculated in each 24-well plate, and after 12 hours, the Transwell chamber was placed in the 24-well plate, and 200 μL of different concentrations (16×10 5 , 32×10 5 , 64×10 5 , 128×10 5 , 256×10 5 CFU / mL) of the recombinant E. coli were inoculated in each Transwell chamber. The cell plates were cultured in normal oxygen / hypoxic environment for 24 hours, and the cell activity was analyzed by CCK8. The results are shown in Figure 7 The E. coli prepared by the present application has obvious hypoxia-responsive killing on cancer cells.

Claims

1. An engineered Escherichia coli bacterium that secretes a low-oxygen induced lactate oxidase, characterized in that, The plasmid carrying a low-oxygen inducible promoter, a secretion peptide gene and a lactate oxidase gene is introduced into E. coli to obtain the E. coli engineering bacteria, the low-oxygen inducible promoter is activated under low-oxygen condition to start the expression of the lactate oxidase gene, and the protein encoded by the secretion peptide gene mediates the transport of the lactate oxidase to the extracellular interstitium; The hypoxia-inducible promoter is derived from Salmonella choleraesuis (Salmonella cholerae). Salmonella Choleraesuis The anaerobic inducible promoter PSnirB of C500 contains a secretory peptide gene derived from *Pectinobacterium*. Pectobacterium carotovorum The secretory peptide gene pelB and the lactate oxidase gene are derived from Streptococcus dolphinus ( Streptococcus iniae The lactate oxidase gene lcto; The sequence of the low-oxygen inducible promoter is shown as SEQ ID NO. 1, the sequence of the secretion peptide gene is shown as SEQ ID NO. 3, and the sequence of the lactate oxidase gene is shown as SEQ ID NO.

2.

2. The method for constructing an engineered E. coli bacterium according to claim 1, wherein, The method comprises the following steps: (1) constructing a prokaryotic expression plasmid containing a low-oxygen inducible promoter sequence, a secretion peptide gene sequence and a lactate oxidase gene sequence; (2) transforming the prokaryotic expression plasmid containing the low-oxygen inducible promoter sequence, the secretion peptide gene sequence and the lactate oxidase gene sequence into E. coli to obtain the E. coli engineering bacteria.

3. The method for constructing an engineered E. coli bacterium according to claim 2, wherein, The transformation method is heat shock method.

4. The method for constructing an engineered E. coli bacterium according to claim 2, wherein, The prokaryotic expression plasmid is pET-20b(+) plasmid.

Images