An N-acylhomoserine lactonase AIO6 recombinant strain without a resistance screening marker and its application

Abstract

The present invention discloses a recombinant strain of N-acyl homoserine lactonase AIO6 without a resistance screening marker and its application. The present invention relates to a recombinant strain of N-acyl homoserine lactonase AIO6 without a resistance screening marker and its application in the technical field of microorganisms and enzymes. The method for constructing a recombinant Escherichia coli without a resistance screening marker includes introducing the coding gene of N-acyl homoserine lactonase AIO6 into the recipient Escherichia coli, and then performing screening without an antibiotic resistance marker to obtain the recombinant Escherichia coli. The construction of the recombinant strain of N-acyl homoserine lactonase AIO6 without a resistance screening marker greatly improves the application of AIO6 in production, and compared with the reported AIO6 strains, the N-acyl homoserine lactonase activity of this strain is further enhanced.

Description

Technical Field

[0001] The present invention relates to a recombinant strain of N-acylhomoserine lactonase AIO6 without a resistance screening marker and its application in the technical field of microorganisms and enzymes. Background Art

[0002] As the most important screening marker, resistance genes play an important role in quickly obtaining transformants. However, with the development of biotechnology and the commercialization of genetically modified products, biosafety has received extensive attention from the public, especially the safety issues of the screening marker genes introduced into species. These screening marker genes may spread among populations through some channels or transfer to other populations, posing potential hazards to the ecological environment and species diversity. In addition, people are worried that genetically modified products containing screening markers and their products may cause harm to human or animal health. Therefore, the genetic transformation technology using a non-resistance screening marker to obtain the products required by people is a current research hotspot.

[0003] Since the 1950s, antibiotics have been widely used as feed additives in the aquaculture industry due to their functions of promoting growth and preventing animal diseases. However, the long-term abuse of antibiotics will lead to the enhancement of bacterial drug resistance, damage to animal health, and destruction of the ecological environment, ultimately threatening human health. Therefore, restricting and banning antibiotics has become an inevitable choice for the green development of the aquaculture industry and ensuring food safety.

[0004] Quorum sensing (QS) is a phenomenon in which microorganisms communicate with each other by synthesizing, secreting, and responding to diffusible small-molecule signal molecules, and it is one of the key points for regulating the virulence of pathogenic bacteria. Based on the role of microbial QS in the pathogenic mechanism of bacteria, the quorum quenching (QQ) method has emerged, that is, interfering with quorum sensing by quenching the signal molecules of pathogenic bacteria and preventing the effective and large accumulation of signal molecules to achieve the effect of preventing diseases. The purpose of the quorum quenching strategy is not to kill pathogens or limit cell growth, but to turn off the expression of pathogenic genes. Therefore, it will not cause drug resistance in pathogenic bacteria and has no side effects on the outside world, and has a series of advantages such as simple and convenient operation, low cost, environmental friendliness, high efficiency, and short cycle. Quorum quenching has been proven by more and more studies to be an effective new strategy for disease prevention and control, and has become the forefront and hotspot of related research in microbial diseases and their prevention and control technologies.

[0005] N-acyl-homoserine lactones (AHLs) are a class of quorum-sensing family signal molecules widely present in Gram-negative bacteria. They are involved in regulating the pathogenic processes of various pathogenic bacteria and are therefore often used as potential targets for reducing or controlling bacterial diseases. N-acyl-homoserine lactonase can degrade the signal molecule AHL, block the bacterial quorum-sensing signal pathway, prevent bacteria from initiating the expression of virulence factors, and lead to a decrease or loss of bacterial pathogenicity and virulence. It has been successfully used in the prevention and control of bacterial diseases. Research shows that the use of N-acyl-homoserine lactonase can not only reduce the virulence of pathogenic bacteria, inhibit the formation of biofilms, but also significantly improve the intestinal microbial composition and enhance the host's disease resistance. In aquaculture, feeding N-acyl-homoserine lactonase derived from Bacillus can significantly reduce the adhesion of pathogenic bacteria and improve the disease resistance of zebrafish, koi carp, and goldfish. The N-acyl-homoserine lactonase AiiA derived from Bacillus licheniformis can inhibit the formation of Vibrio biofilms, reduce its colonization in the shrimp intestine, and improve the immunity of shrimp. In poultry farming, N-acyl-homoserine lactonase AIO6 can improve the feed conversion rate and daily weight gain of broilers. The daily weight gain of broilers in the experimental group increased by 3.37%. The feed-to-meat ratio of the group fed with AIO6 was significantly reduced by 0.04 compared with the control group, and the feed conversion rate of the broiler experimental group increased by 2.30%. Adding quorum-sensing quenching enzymes, glucose oxidase, and lactic acid bacteria to the basal diet significantly increased the growth performance of broilers in the early growth stage (1-21 days old), significantly increased the serum IL-6 content, improved the activities of serum CAT and GSH-Px, and the activity of liver SOD, and reduced the uric acid content in feces. However, recombinant strains of N-acyl-homoserine lactonase are usually obtained by the method of resistance screening markers, which has certain limitations in production applications and may pose a great threat to the public environment and health. Therefore, it is crucial to obtain recombinant Escherichia coli strains of N-acyl-homoserine lactonase without resistance screening markers. Summary of the Invention

[0006] The problem to be solved by the present invention is that there is currently no recombinant Escherichia coli strain of AIO6 without resistance screening markers.

[0007] To solve the above problems, the present invention provides a recombinant Escherichia coli strain of AIO6 without resistance screening markers and a method for constructing a recombinant Escherichia coli strain without resistance screening markers.

[0008] The present invention obtains a recombinant Escherichia coli strain without resistance screening markers, including introducing the coding gene of N-acyl-homoserine lactonase AIO6 into the recipient Escherichia coli, and then performing screening without antibiotic resistance markers to obtain the recombinant Escherichia coli. The introduction includes the following steps:

[0009] 1) Introduce the vector containing N-acyl homoserine lactonase AIO6 and the vector containing the protein-coding genes of transposases TnsA, TnsB, and TnsC into the recipient Escherichia coli, and culture to obtain transformant A;

[0010] 2) Introduce the multi-target gene editing plasmid into transformant A obtained in 1) above, and culture to obtain transformant B;

[0011] 3) Conduct a primary screening on transformant B obtained in step 2), and select strain C with high transposition efficiency and / or high enzyme activity of N-acyl homoserine lactonase AIO6;

[0012] 4) Introduce the pCutamp vector into strain C to stop the transposition process, and perform screening using an antibiotic plate. The strain that can grow on the sucrose medium but cannot grow on the antibiotic plate is the recombinant Escherichia coli without a resistance screening marker.

[0013] In the above method, the amino acid sequence of N-acyl homoserine lactonase AIO6 is SEQ ID No: 1, and the nucleotide of the coding gene is SEQ ID No: 2.

[0014] In the above method, the vector containing N-acyl homoserine lactonase AIO6 can specifically be the pRE57I-AIO6 vector. The structure of the pRE57I-AIO6 vector is described as follows: A DNA fragment with the sequence of SEQ ID No: 2 is inserted between the two restriction enzyme cleavage sites of NaeI and XhoI of the starting vector pRE57I, and other sequences of the pRE57I vector are kept unchanged to obtain the recombinant vector. The pRE57I-AIO6 vector can express N-acyl homoserine lactonase AIO6, and its amino acid sequence is SEQ ID No: 1.

[0015] In the above method, the vector containing the TnsABC protein-coding genes can be the vector pTnsABC.

[0016] The amino acid sequence of TnsA is SEQ ID No: 3, and the nucleotide of the coding gene is SEQ ID No: 4; the amino acid sequence of TnsB is SEQ ID No: 5, and the nucleotide of the coding gene is SEQ ID No: 6; the amino acid sequence of TnsC is SEQ ID No: 7, and the nucleotide of the coding gene is SEQ ID No: 8.

[0017] In the above method, the multi-target gene editing plasmid is pTetQcas-8 + IS186.

[0018] The target sites are located at positions 16413 - 16444, 150770 - 150801, 426765 - 426796, 569521 - 569552, 1794404 - 1794435, 2688153 - 2688184, 2415243 - 2415274, 745115 - 745146, 2987107 - 2987138, 1269372 - 1269403, 3551707 - 3551738, 1847019 - 1847050, 4107738 - 4107769 of the Escherichia coli genome sequence (Genbank: GCF_000833145.1, updated on Feb 5, 2015).

[0019] The resistance screening marker can be: ampicillin resistance marker, kanamycin resistance marker, and / or streptomycin resistance marker.

[0020] In the screening using antibiotic plates in step 4) above, the used antibiotic plates contain ampicillin, kanamycin, and streptomycin respectively.

[0021] In this article, the transposition efficiency can be evaluated by detecting the number of target genes inserted into the transposition sites by PCR.

[0022] The enzyme activity definition of the N - acyl - homoserine lactonase AIO6 is: under the conditions of 30 °C and pH 6.5, the amount of enzyme required to hydrolyze 1 nmoL of N - acyl - homoserine lactone from 50 μg / mL of N - acyl - homoserine lactone per minute is 1 unit of N - acyl - homoserine lactonase activity, represented by U.

[0023] The present invention also provides the following applications of the method described above:

[0024] P1. Producing N - acyl - homoserine lactonase;

[0025] P2. Increasing the yield of N - acyl - homoserine lactonase;

[0026] P3. Preparing a recombinant bacterium producing N - acyl - homoserine lactonase without a resistance screening marker.

[0027] The recombinant Escherichia coli constructed by the method described above also belongs to the scope of protection of the present invention. The recombinant Escherichia coli can express N - acyl - homoserine lactonase AIO6.

[0028] The present invention also provides the following applications of the Escherichia coli described above:

[0029] P1. Producing N - acyl - homoserine lactonase;

[0030] P2. Improve the production of N-acyl homoserine lactonase.

[0031] The present invention also provides a method for producing N-acyl homoserine lactonase, which includes culturing the recombinant Escherichia coli described above to obtain a fermentation product, and obtaining N-acyl homoserine lactonase from the fermentation product.

[0032] N-acyl homoserine lactonase recombinant strains usually contain resistance screening marker genes, which have certain limitations in production applications and seriously reduce their economic value. The present invention has obtained an N-acyl homoserine lactonase AIO6 recombinant strain without a resistance screening marker. The construction of the non-resistant strain has greatly improved the application of AIO6 in production, and compared with the reported AIO6 strain, its activity has also been further enhanced. Description of the Drawings

[0033] Figure 1 PCR identification results of the transposition efficiency of AIO6 transposons at sites 1 to 4. Among them, a is the insertion situation of AIO6 at site 1; b is the insertion situation of AIO6 at site 2; c is the insertion situation of AIO6 at site 3; d is the insertion situation of AIO6 at site 4; M: DNA Marker. Lanes 1-24 correspond to transposon strains No. 1-24.

[0034] Figure 2 PCR identification results of the transposition efficiency of AIO6 transposons at sites 5 to 8. a is the insertion situation of AIO6 at site 5; b is the insertion situation of AIO6 at site 6; c is the insertion situation of AIO6 at site 7; d is the insertion situation of AIO6 at site 8; M: DNA Marker. Lanes 1-24 correspond to transposon strains No. 1-24.

[0035] Figure 3 PCR identification results of the transposition efficiency of AIO6 transposons at sites IS186-1 to IS186-5. a is the insertion situation of AIO6 at site IS186-1; b is the insertion situation of AIO6 at site IS186-2; c is the insertion situation of AIO6 at site IS186-3; d is the insertion situation of AIO6 at site IS186-4; e is the insertion situation of AIO6 at site IS186-5; M: DNA Marker. Lanes 1-24 correspond to transposon strains No. 1-24.

[0036] Figure 4SDS-PAGE results of the induced expression of the target protein in the AIO6 transposon strain. Among them, a is the whole-cell protein of the AIO6 transposon strain after induction; b is the insoluble protein of the AIO6 transposon strain treated with BugBuster; c is the soluble protein of the AIO6 transposon strain treated with BugBuster. M: Protein Marker. AIO6 is the wild-type AIO6 strain, and 1, 2, 11, 12, 13, 19, 22, 23, 24 are the corresponding strain numbers.

[0037] Figure 5 For the elimination of the transposon plasmid in the AIO6 transposon strain. Among them, a is the growth of the 12th transposon strain on LB media containing ampicillin (Amp), kanamycin (Km), streptomycin (Strep), and 15% sucrose respectively; b is the growth of the 13th transposon strain on LB media containing ampicillin (Amp), kanamycin (Km), streptomycin (Strep), and 15% sucrose respectively.

[0038] Figure 6 For the elimination of the pCutamp plasmid in the AIO6 transposon strain. Among them, a is the growth of the transposon strain 12 on LB medium containing apramycin (Apra+LB) and LB medium (LB); b is the growth of the transposon strain 13 on LB medium containing apramycin (Apra+LB) and LB medium (LB).

[0039] Figure 7 SDS-PAGE results of the induced expression of the target protein in the AIO6 plasmid-free multi-copy integration strain. Lane 1 is the total protein of the AIO6 wild type, 2 is the soluble protein of the AIO6 wild type, 3 is the total protein of the 12-1 strain, 4 is the soluble protein of the 12-1 strain, 5 is the total protein of the 12-3 strain, 6 is the soluble protein of the 12-3 strain, 7 is the total protein of the 13-2 strain, 8 is the soluble protein of the 13-2 strain, 9 is the total protein of the 13-36 strain, 10 is the soluble protein of the 13-36 strain. Specific Embodiments

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0041] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0042] Unless otherwise specified, in the following quantitative tests, three repeated experiments were set, and the results were averaged.

[0043] pRE57I-GNac in the following examples was purchased from GenSmart Cat.NO.: MC_0101251.

[0044] pTet-tns in the following examples was purchased from GenSmart Cat.NO.:MC_0101242.

[0045] pTetQcas-8+IS186 in the following examples was purchased from GenSmart Cat.NO.:MC_0101250.

[0046] pCutamp in the following examples was purchased from GenSmart Cat.NO.:MC_0101104.

[0047] pET28a in the following examples was purchased from Beijing Zoman Biotechnology: ZK159.

[0048] The data in the following examples were processed using SPSS11.5 statistical software. The experimental results were expressed as mean ± standard deviation, and One-way ANOVA test was used. P < 0.05 (*) indicated significant difference, P < 0.01 (**) indicated extremely significant difference, and P < 0.001 (***) indicated extremely significant difference.

[0049] Example 1. Transposition test of Escherichia coli

[0050] I. Construction method of recombinant vector

[0051] 1. Construction of recombinant vector pRE57I-AIO6

[0052] The target fragment AIO6 (nucleotide sequence is SEQ ID No:2) was obtained using the primer pair AIO6-F: 5’-AGAAGGAGAGCTAGCgccggcATGAAATCCCATGAAATCGAGACCAGTCACGGT-3’ and AIO6-R: 5’-TTAACTCGAGctcgagTCAATGATGATGATGATGATGGGCCGTGCAG-3’, and cloned into the pRE57I vector using the double digestion sites of NaeI and XhoI.

[0053] The structure of the pRE57I-AIO6 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No:2 between the NaeI and XhoI restriction enzyme cleavage sites of the parental vector pRE57I-GNac, while keeping other sequences of the pRE57I vector unchanged. The pRE57I-AIO6 vector can express N-acyl homoserine lactonase AIO6, and its amino acid sequence is SEQ ID No:1.

[0054] II. Transposition experiment

[0055] 1. Co-transform pRE57I-AIO6 and pTet-tns into Escherichia coli competent cells (Novoprotein, C502-03), and screen for transformant A using an LB agar plate containing 100 μg / mL ampicillin (Yuanye, S17018-25g) and 50 μg / mL kanamycin (Lablead, 0408-10G);

[0056] 2. Prepare the positive clone transformant A screened in step 1 into competent cells, and transfer pTetQcas-8 + IS186 into the competent cells. After recovery, inoculate the bacterial suspension on an LB agar plate containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 50 μg / mL streptomycin (ThermoFisher Scientific, 15140122); After culturing at 37 °C for 16 hours, obtain the positive clone transformant B;

[0057] 3. Scrape hundreds of positive clone transformants B obtained in step 2 from the plate and resuspend them in fresh LB medium, then inoculate them on an LB agar plate containing triple antibiotics (100 μg / mL ampicillin, 50 μg / mL kanamycin, and 50 μg / mL streptomycin respectively) and 0.1 μg / mL anhydrotetracycline (Zhuangmeng Biotech, ZS815), and culture at 37 °C for 16 hours to induce the expression of transposition-related proteins;

[0058] 4. After the biofilm of the bacteria obtained in step 3 is formed, scrape it off and resuspend it in LB medium, re-dilute the bacteria and inoculate it again on an LB agar plate containing triple antibiotics and 1 μg / mL anhydrotetracycline, and culture overnight at 37 °C;

[0059] 5. Name the colonies obtained in step 4 as transposons 1. Select some transposons 1 and streak them on an LB agar plate containing triple antibiotics and 1 μg / mL anhydrotetracycline, and culture overnight at 37 °C to obtain transposons 2. Continuously transfer to generate transposons 6, and randomly select strains for colony PCR to identify the transposition efficiency ( Figure 1 in a-d, Figure 2 in a-d, Figure 3Among a - e and Table 2). When AIO6 is not inserted into the corresponding site, the band is between 500 bp and 1000 bp; when AIO6 is inserted into the corresponding site, the band is between 2000 bp and 3000 bp. The transposition efficiency is determined by the total number of insertions in Table 2. The more insertions, the higher the transposition efficiency. The primers are shown in Table 1.

[0060]

[0061] 6. By comparing the PCR identification results, it is confirmed that AIO6 inserts into different sites during the transposition process. The detailed insertion site conditions are shown in Table 2.

[0062]

[0063] Note: 1 indicates successful insertion; 1 / 0 indicates that there are strains in the colony that have not been inserted.

[0064] III. Induction and Expression of Transposon Bacteria

[0065] 1. Inoculate the transposon strain with the target gene inserted at the transposition site in step 2 into 20 mL of LB containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 50 μg / mL streptomycin, and culture overnight at 37°C, using the strain containing pET28a - AIO6 (kanamycin) as a control; the strain containing pET28a - AIO6 (kanamycin) is a recombinant strain obtained by introducing the recombinant vector pET28a - AIO6 into Escherichia coli BL21.

[0066] The structural description of the recombinant vector pET28a - AIO6 is as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No:2 between the NaeI and XhoI restriction enzyme sites of the starting vector pET28a, while keeping other sequences of the pET28a starting vector unchanged.

[0067] 2. Transfer 500 μL of the strain culture solution in step 1 to 50 mL of LB containing the corresponding antibiotics, and culture at 37°C until OD 600 = 0.6 - 0.8;

[0068] 3. Add 24 μL of IPTG (final concentration 0.4 mM) and induce at 16°C for 16 - 18 hours;

[0069] 4. Take 3 tubes of 1 mL of the induced samples in step 3, and use 1 tube to measure OD 600(200 μL; 4 replicates); Centrifuge one tube at 12,000 rpm at 4°C, discard the supernatant, collect the pellet C (total protein) and store it at -20°C; For one tube, perform soluble and insoluble experiments. Centrifuge at 12,000 rpm at 4°C, discard the supernatant, add 0.5 mL of BugBuster to the pellet, pipette evenly, and place it on a shaker at 4°C for 30 min. Then centrifuge at 12,000 rpm at 4°C for 10 min. The pellet is the insoluble protein and the supernatant is the soluble protein;

[0070] 5. Add 2 volumes of acetone to the supernatant SP in step 4 and precipitate at -20°C for 1 hour. Centrifuge at 12,000 rpm at 4°C for 10 min, discard the supernatant, and the precipitate is the soluble protein;

[0071] 6. Add appropriate amounts of PBS and SDS to the total protein, insoluble protein, and soluble protein (adjust the biomass to 6 according to the OD 600 value), boil in a water bath for 10 min, and verify protein expression by SDS-PAGE.

[0072] The results show that: Figure 4 As can be seen from a-c in [reference], the size of the target protein AIO6 is approximately 32 kDa. Select strains with good solubility and high expression levels (strains 1, 2, 11, 12, 13, 19, 22, 23, and 24) for subsequent experiments.

[0073] IV. Enzyme Activity Detection of Transposon Strains

[0074] 1. Pick the strains verified for protein expression (strains 1, 2, 11, 12, 13, 19, 22, 23, and 24) and inoculate them into 20 mL of LB containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, and 50 μg / mL streptomycin. Culture overnight at 37°C, and use the strain containing pET28a-AIO6 (kanamycin) as a control;

[0075] 2. Transfer 500 μL of the culture solution in step 1 to 50 mL of LB containing the corresponding antibiotics and culture at 37°C until OD 600 = 0.6 - 0.8;

[0076] 3. Add 24 μL of IPTG (final concentration 0.4 mM) and induce at 16°C for 16 - 18 hours;

[0077] 4. Take 50 mL of the bacterial solution and centrifuge it at 4000 rpm for 10 min to collect the precipitate. Add 10 mL of PBS to wash the bacteria, centrifuge at 4000 rpm for 10 min, discard the supernatant, add 8 mL of PBS to resuspend the bacteria, and place it in a metal ice box for ultrasonic disruption for 10 min (ultrasonic for 2 s, cool for 3 s).

[0078] 5. After ultrasonic treatment, centrifuge at 12000 rpm at 4 °C for 10 min, and take the supernatant to filter through a 0.22 um aqueous filter membrane.

[0079] 6. HPLC enzyme activity detection

[0080] a. Prepare the substrate: Dilute the substrate stock solution (3-oxo-C8-HSL (Sigma-Aldrich, O1764-100), 5 mg / mL) to 0.5 mg / mL with PBS.

[0081] b. Protein dilution: Dilute the protein appropriately with PBS.

[0082] c. Reaction system: The treatment group has a 400 μL system = 100 μL substrate + 100 μL protein + 200 μL PBS; the control group has a 400 μL system = 100 μL substrate + 200 μL PBS + 100 μL protein (the corresponding protein is added to the control group after terminating the reaction with methanol).

[0083] d. Reaction conditions: React in a 30 °C water bath for 15 min.

[0084] e. Terminate the reaction: Add 600 μL of methanol to terminate the enzyme activity reaction.

[0085] After terminating the reaction, mix well and filter through a 0.22 um organic filter membrane, and then perform HPLC detection. The detection conditions are: injection volume 20 μL, wavelength 254 nm, aqueous phase (800 mL of water plus 300 μL of triethylamine): organic phase (acetonitrile) = 0.64:0.36, flow rate 1 mL / min

[0086] Calculate the enzyme activity of the corresponding strain according to the HPLC detection data. The definition of enzyme activity is: at 30 °C and pH 6.5, the amount of enzyme required to hydrolyze 1 nmoL of N-acyl homoserine lactone from 50 μg / mL of N-acyl homoserine lactone per minute is 1 unit of N-acyl homoserine lactonase activity, denoted as U.

[0087] Enzyme activity calculation formula:

[0088]

[0089] X: Activity of N-acyl homoserine lactonase in the enzyme dilution solution, U / g;

[0090] ΔC : Change in the content of the substrate, μg / mL;

[0091] V : Dissolution volume of the sample, mL;

[0092] n : Dilution factor of the sample;

[0093] 0.1: Volume of the enzyme solution added to the total reaction system, mL;

[0094] M : Molar mass of the substrate, g / mol, M = 241.8;

[0095] t : Reaction time of the enzyme, min;

[0096] 1000: Conversion factor.

[0097] The measurement results of two parallel samples are expressed as the arithmetic mean.

[0098] The enzyme activity detection results are shown in Table 3: The enzyme activities of most AIO6 transposon strains are significantly higher than those of the control group pET28a-AIO6. Among them, the enzyme activity of strain 12 is 4560.9 U / mL / OD 600 and that of strain 13 is 4427.4 U / mL / OD 600 , with relatively high enzyme activities, and they are intended to be used as candidate strains for subsequent screening.

[0099]

[0100] Example 2. pCutamp stops transposition

[0101] 1. Obtaining of AIO6 plasmid-free multi-copy integration strains

[0102] According to the enzyme activity detection results and the number of transposon insertion sites in Example 1, the strains 12 and 13 with the most transpositions were selected. According to the method for preparing competent Escherichia coli, the transposon strains 12 and 13 containing three plasmids (pRE57I-AIO6, pTet-tns, and pTetQcas-8+IS186) were prepared into competent cells. The pCutamp vector was transformed into this competent state by electroporation (1.85 kV, 200 Ω, 25 μF), and it was resuscitated in 1 mL of SOC medium (hopebio (HaiBo, HBDC002)) at 37°C for 1 hour; the cells were collected by centrifugation and transferred to 4 mL of LB medium containing 50 μg / mL apramycin (Macklin, A899829-1g) and 10 mM rhamnose (Innochem, A65280), and shaken at 37°C for 10 h. 100 μL of the culture was inoculated on an LB agar plate containing 50 μg / mL apramycin and 10 mM rhamnose, and cultured overnight at 37°C.

[0103] The colonies growing on the above LB plates were respectively streaked on 4 kinds of LB agar plates containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 50 μg / mL streptomycin, and 15% sucrose. If they could not grow on the plates containing ampicillin, kanamycin, and streptomycin, the colonies were considered to have eliminated pRE57I-AIO6, pTnsABC, and pQCascade-IS1.

[0104] The results are as Figure 5 shown in a and b in: all the transposon plasmids in the colonies of strain 12 were eliminated, and the transposon plasmids in three colonies (2, 6, 36) of strain 13 were eliminated.

[0105] Subsequently, the corresponding colonies that only grew on the sucrose plate were respectively re-inoculated on an LB agar plate without any antibiotics and an LB plate containing apramycin to verify the curing of pCutamp. The colonies that could not grow on the plate containing apramycin were plasmid-free multi-copy integration strains.

[0106] The results are as Figure 6 shown in a and b in: clones 1 and 3 of strain 12 (i.e., 12-1 and 12-3), clones 2 and 36 of strain 13 (i.e., 13-2 and 13-36) did not grow on the plate containing apramycin, and finally, the AIO6 plasmid-free multi-copy integration strains 12-1, 12-3, 13-2, and 13-36 were obtained.

[0107] 2. Induction and expression of plasmid-free multi-copy recombinant strains

[0108] (1) Pick up the plasmid-free multi-copy recombinant strains 12-1, 12-3, 13-2, and 13-36 and inoculate them into 20 mL of LB, culture overnight at 37 °C, and use pET28a-AIO6 (kanamycin) as a control;

[0109] (2) Transfer 500 μL of the culture solution in step (1) to 50 mL of LB and culture at 37 °C until OD 600 = 0.6 - 0.8;

[0110] (3) Add 24 μL of IPTG (final concentration 0.4 mM) and induce at 16 °C for 16 - 18 hours;

[0111] (4) Take 3 tubes of 1 mL of the induced sample in step (3). One tube is used to measure OD600 (200 μl; 4 replicates); one tube is centrifuged at 12000 rpm at 4 °C, discard the supernatant, collect the precipitate C (total protein) and store it at -20 °C; one tube is used for the soluble and insoluble experiments. Centrifuge at 12000 rpm at 4 °C, discard the supernatant, add 0.5 mL of BugBuster to the precipitate and pipette evenly, then place it on a shaker at 4 °C for 30 min, and then centrifuge at 12000 rpm at 4 °C for 10 min. The precipitate is IP (insoluble protein), and the supernatant is SP (soluble protein);

[0112] (5) Add 2 volumes of acetone to the supernatant SP in step (4) and precipitate at -20 °C for 1 hour. Centrifuge at 12000 rpm at 4 °C for 10 min, discard the supernatant, and the precipitate is SP (soluble protein);

[0113] (6) Add PBS and SDS to the total protein, insoluble protein, and soluble protein (adjust the biomass to 6 according to the OD 600 value), boil in a water bath for 10 min, and verify protein expression. The SDS-PAGE results show that the selected colonies all normally express the target protein N-acylhomoserine lactonase AIO6 ( Figure 7 ).

[0114] 3. Enzyme activity detection of plasmid-free multi-copy recombinant strains

[0115] Refer to the method in step four of Example 1 to detect the enzyme activity of the plasmid-free multi-copy recombinant strains 12-1, 12-3, 13-2, and 13-36, and use Escherichia coli containing the pET28a-AIO6 (kanamycin) vector as a control strain.

[0116] The enzyme activity results are shown in Table 4: The enzyme activities of the plasmid-free multi-copy recombinant strains 12-1, 12-3, 13-2, and 13-36 obtained in the present invention without antibiotic selection markers are all higher than those of the pET28a-AIO6 strain screened by antibiotics.

[0117]

[0118] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art.

Claims

1. A method for constructing a recombinant Escherichia coli without a resistance selection marker, characterized in that: The method comprises introducing a gene encoding N-acyl homoserine lactonase AIO6 into a recipient Escherichia coli, followed by screening for antibiotic resistance markers to obtain recombinant Escherichia coli, wherein the introduction comprises the following steps: 1) Introducing the vector containing N-acyl homoserine lactonase AIO6 and the vector containing the protein encoding genes of TnsA, TnsB and TnsC into the recipient Escherichia coli, and culturing to obtain transformant A; 2) Introducing the multi-target gene editing plasmid pTetQcas-8+IS186 into the transformant A described in 1) above, and culturing to obtain transformant B; 3) Preliminary screening of the transformant B in step 2) was performed to select strain C with high transposition efficiency and / or high enzyme activity of N-acyl homoserine lactonase AIO6; 4) introducing the pCutamp vector into the strain C to stop the transposition process, and screening using an antibiotic plate to obtain a strain that grows on a rhamnose medium but cannot grow on an antibiotic plate, namely, the recombinant Escherichia coli without a resistance screening marker; The amino acid sequence of the N-acyl homoserine lactonase AIO6 is SEQ ID No: 1, and the nucleotide sequence of the encoding gene is SEQ ID No:

2.

2. The method according to claim 1 is used in any of the following applications: P1, production of N-acyl homoserine lactonase; P2, increase the production of N-acyl homoserine lactonase; P3. Prepare recombinant bacteria that produce N-acyl homoserine lactonase without resistance selection marker.

3. The recombinant Escherichia coli constructed by the method of claim 1.

4. Use of the Escherichia coli according to claim 3 in any of the following: P1, production of N-acyl homoserine lactonase; P2. Increase the production of N-acyl homoserine lactonase.

5. A method for producing N-acyl homoserine lactonase, comprising culturing the recombinant Escherichia coli according to claim 3 to obtain a fermentation product, and obtaining N-acyl homoserine lactonase from the fermentation product.

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