Creation and application of an aromatic amide biosensor
By designing the optimized AmiC-AmiR biosensor, the problem of aromatic amide detection in the prior art is solved, and efficient and accurate detection of aromatic amides is achieved, which is suitable for detection and metabolic pathway research in complex environments.
Patent Information
- Application Number
- CN202211424962.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-14
- Publication Date
- 2024-12-31
- Estimated Expiration
- 2042-11-14
AI Technical Summary
The prior art is difficult to efficiently and accurately detect aromatic amide compounds, especially nicotinamides, especially in complex environments. The traditional methods are costly and have low sensitivity and are susceptible to interference.
Design a biosensor to optimize the amino acid motif of AmiC-AmiR, a transcriptional regulator derived from Pseudomonas aeruginosa, mutates tyrosine and threonine, binds the RBS sequence, and builds a plasmid carrying fluorescent protein genes. Expressed in E. coli to achieve detection of aromatic amides.
It realizes efficient and accurate detection of aromatic amides, with a response range of 0 to 10 mM, which significantly improves detection sensitivity and linear relationship, and is suitable for the detection and metabolic pathway research of aromatic amides.
Smart Images
Figure CN115851773B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the creation and application of an aromatic amide biosensor, belonging to the technical field of bioengineering. Background Art
[0002] Amide is a kind of nitrogen-containing carboxylic acid derivative. In terms of structure, amide can be regarded as a compound formed by replacing the hydroxyl group of the carboxyl group in the carboxylic acid molecule with an amino group or a hydrocarbon amino group (-NHR or -NR2); it can also be regarded as a compound formed by replacing the hydrogen on the nitrogen atom in the ammonia or amine molecule with an acyl group. It is mainly used as an industrial solvent. In the pharmaceutical industry, it is used to produce vitamins and hormones, and is also used to make chlordimeform. Nicotinamide is a very important compound. In industry, it can be used as a nutritional additive, with anti-aging effects, improving skin resistance and other effects, and is widely used in the cosmetics industry; in medicine, it is used to prevent and treat pellagra and the treatment of stomatitis, glossitis and other diseases. Isonicotinamide is an intermediate of cephalosporin drugs and plays an important role in the pharmaceutical industry.
[0003] A biosensor is a device that can quickly respond to target substances in the environment and convert their concentrations into easily recognizable signals, such as electrical signals and colors. People can judge the presence and content of the target substance based on the received signals.
[0004] Nicotinamide is a colorless, odorless compound that is difficult to detect after being dissolved in a solvent. The most commonly used detection method is high performance liquid chromatography, but this method is relatively expensive and time-consuming. In addition, nicotinamide can also be detected by colorimetry, but this method is easily interfered by other substances, has a relatively low sensitivity, and has serious errors. At the same time, other aromatic amide compounds have the same problems as nicotinamide, which are difficult to detect and require high concentrations for detection. Therefore, there is currently a lack of a means to efficiently and accurately detect aromatic amide compounds, especially nicotinamide, in a complex environment. Summary of the invention
[0005] In view of the existing technical difficulties and problems, the present invention provides an amino acid motif of a transcriptional regulator AmiC-AmiR derived from Pseudomonas aeruginosa and its application in detecting aromatic amides.
[0006] The first object of the present invention is to provide a biosensor of amide substances, wherein the biosensor comprises a RBS AmiC Sequences of AmiC structural units and RBS AmiRThe AmiR structural unit has an amino acid sequence as shown in SEQ ID NO.1, wherein the AmiC structural unit is a ligand binding domain that binds to amides, with the tyrosine at position 83 mutated to isoleucine and the threonine at position 233 mutated to cysteine.
[0007] In one embodiment, the genes in the biosensor are RBS in the order of gene expression. AmiC 、AmiC structural unit gene、RBS AmiR , AmiR structural unit genes.
[0008] In one embodiment, the amino acid sequence of the AmiC structural unit is shown as SEQ ID NO.2.
[0009] In one embodiment, the amino acid sequence of the AmiR structural unit is shown as SEQ ID NO.3.
[0010] In one embodiment, the RBS AmiC The sequence is shown as SEQ ID NO.4.
[0011] In one embodiment, the RBS AmiR The sequence is shown as SEQ ID NO.5.
[0012] In one embodiment, the amide substances include isobutylamide, valeramide, acrylamide, nicotinamide, pyrazinamide, naphthoyl, benzamide and cinnamamide.
[0013] The second object of the present invention is to provide a plasmid carrying the above-mentioned biosensor.
[0014] In one embodiment, the plasmid includes a pET series vector, and the pET series vector carries a marker gene.
[0015] In one embodiment, the pET series vectors include pET24a.
[0016] In one embodiment, the marker gene comprises a gene encoding a fluorescent protein.
[0017] In one embodiment, the fluorescent protein comprises green fluorescent protein GFP.
[0018] The second object of the present invention is to provide a cell carrying the above biosensor or the above plasmid.
[0019] In one embodiment, the cell comprises E. coli.
[0020] In one embodiment, the Escherichia coli includes any one of E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, and E. coli TOP10.
[0021] The fourth object of the present invention is to provide a genetically engineered bacterium, which uses Escherichia coli as a host and carries the above-mentioned biosensor or the above-mentioned plasmid.
[0022] The Escherichia coli includes any one of E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, and E. coli TOP10.
[0023] The fifth object of the present invention is to provide the application of the above-mentioned biosensor or the above-mentioned plasmid or the above-mentioned cell or the above-mentioned genetically engineered bacteria in the detection of aromatic amides or the screening of strains that metabolize aromatic amides or the study of aromatic amide production metabolic pathways.
[0024] Beneficial effects:
[0025] The present invention provides a biosensor that can respond to aromatic amides. Based on the amino acid motif of the transcriptional regulator AmiC-AmiR derived from Pseudomonas aeruginosa, the RBS of AmiC and AmiR are optimized respectively, and the tyrosine at position 83 of AmiC is mutated to isoleucine, and the threonine at position 233 is mutated to cysteine. Compared with the wild-type transcriptional regulator AmiC-AmiR, the biosensor provided by the present invention changes its responsiveness to aromatic amides from non-responsiveness to responsiveness. After the optimization of RBS, its responsiveness has a good linear relationship, and the response range is between 0 and 10 mM. The biosensor that can respond to aromatic amides provided by the present invention can be used for the detection of aromatic amides, screening of strains that metabolize aromatic amides, or laying the foundation for subsequent research on aromatic amide production metabolic pathways, and has a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a test of the specificity of the wild type and mutants for different substances.
[0027] Figure 2 Verification of the response of Y83I / T233C to different concentrations of nicotinamide. DETAILED DESCRIPTION
[0028] LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, kanamycin final concentration 50 μg / mL.
[0029] Table 1 Primer sequences
[0030]
[0031] (Note: F refers to the upstream primer, R refers to the downstream primer)
[0032] Table 2 PCR amplification system
[0033]
[0034]
[0035] Example 1: Construction of wild-type AmiC-AmiR
[0036] The amino acid motif of the transcriptional regulator AmiC-AmiR from Pseudomonas aeruginosa was downloaded from NCBI, and gene synthesis and codon optimization were performed at GENEWISE. Then, the synthesized gene was assembled with the existing plasmid pVEO in the laboratory using the Gibson Assembly method to construct the plasmid pVEO-AmiC-AmiR-WT. pVEO is a commercial plasmid integrated with green fluorescent protein GFP on pET24a (pET24a was purchased from the Miaoling plasmid platform).
[0037] Using plasmid pVEO and the synthesized AmiC-RBS-AmiR gene (the amino acid sequence of the AmiC structural unit is shown in SEQ ID NO.1, the amino acid sequence of the AmiR structural unit is shown in SEQ ID NO.3, and the amino acid sequence of RBS is shown in SEQ ID NO.6) as templates, primers AmiC-R_insert-SR / AmiC-R_insert-SF, AmiC-R_insert_R / AmiC-R_insert_F (Table 1) were used to amplify the target DNA fragment by PCR, and the amplification system was shown in Table 2. The PCR amplification reaction conditions were 95°C pre-denaturation for 3min, 98°C denaturation for 15s, 55°C annealing for 30s, and 72°C extension. The extension time was set according to the length of the target gene, for a total of 30 cycles. The PCR product was digested with DpnI digestion enzyme for 2-3h and connected using ABclonalMultifSeamlessAssembly Mix. The ligation product was transformed into E. coli DH5α, and the gene sequence of the positive transformant was sequenced and verified by Anshengda (Suzhou) Co., Ltd. The plasmid with correct sequencing was named pVEO-AmiC-AmiR-WT.
[0038] Example 2: Optimization of the RBS of wild-type AmiC-AmiR
[0039] According to the wild-type plasmid pVEO-AmiC-AmiR-WT constructed in Example 1, the RBS of AmiC and AmiR were screened by library construction. The library construction method used the wild-type plasmid pVEO-AmiC-AmiR-WT as a template, and used degenerate primers, RBSNNN-SF / RBSNNN-SR, RBSNNN-F / RBSNNN-R (Table 1) to amplify the target DNA fragments by PCR. The purpose of these two pairs of degenerate primers is to design and introduce NNN mutations in the GC-rich base region of RBS. The test steps of Example 1 were repeated, and the obtained PCR products were transformed into Escherichia coli JM109, respectively coated on LB medium (detecting background fluorescence value) and LB medium containing 10 mM different amides (detecting fluorescence value), and cultured at 37°C overnight. The fluorescence value was detected using a microplate reader, and the individuals that could not express fluorescence and had too high background fluorescence were removed. The individuals with significantly improved fluorescence values after adding amide were selected to obtain the RBS-optimized plasmid pVEO-AmiC-AmiR-WT*, wherein the gene sequence of the RBS of AmiC is shown in SEQ ID NO.4, and the gene sequence of the RBS of AmiR is shown in SEQ ID NO.5.
[0040] Example 3 Construction of mutant AmiC-AmiR-Y83I / T233C
[0041] The plasmid pVEO-AmiC-AmiR-WT* after RBS optimization in Example 2 was used as a template and primers Y83I-F and Y83I-R (Table 1) were used to amplify the target DNA fragment by the whole plasmid PCR method.
[0042] The amplification system is shown in Table 2. The PCR amplification reaction conditions are 95°C pre-denaturation for 3 min, 98°C denaturation for 15 s, 55°C annealing for 30 s, and 72°C extension for 1 min 30 s, for a total of 30 cycles.
[0043] The PCR product was digested with DpnI digestive enzyme for 2-3h and transformed into E. coli DH5α. The gene sequence of the positive transformant was sequenced and verified by Anshengda (Suzhou) Co., Ltd. The plasmid with the correct sequencing was named pVEO-AmiC-AmiR-Y83I.
[0044] Using pVEO-AmiC-AmiR-Y83I as a template, the above operation was repeated using primers Y83I / T233C-F and Y83I / T233C-R (Table 1) to construct pVEO-AmiC-AmiR-Y83I / T233C.
[0045] Example 4 Verification of the response ability of mutant AmiC-AmiR-Y83I / T233C to amide compounds
[0046] (1) Response of mutant AmiC-AmiR-Y83I / T233C to amide compounds
[0047] The mutant plasmid pVEO-AmiC-AmiR-Y83I / T233C and plasmid pVEO-AmiC-AmiR-WT* constructed in Example 3 were transformed into Escherichia coli JM109, and positive single colonies were picked to 5 mL LB medium, and cultured at 37°C and 200 rpm for 7-8 hours. The seed solution was transferred to 5 mL LB medium containing 10 mM different amides at 1% (v / v) and cultured for 24 hours. After culture, the fluorescence value was detected using an ELISA instrument.
[0048] Fluorescence detection method: Use an enzyme reader to detect OD 600 Absorbance value and fluorescence value at excitation wavelength 495 and absorption wavelength 525.
[0049] The results are as follows Figure 1 As shown: when the amino acid residues mutated, the response of AmiC-AmiR to nicotinamide, isonicotinamide, and benzamide changed from nothing to something, and the final induction ratio (the final fluorescence value of the mutant was divided by the fluorescence value of the wild type) could reach 7.4, 5.27, and 10 times, respectively; at the same time, the response of AmiC-AmiR to pyrazinamide cinnamamide was also improved compared with the wild type, indicating that the 83rd and 233rd amino acid residues may be the key domains of AmiC binding to aromatic amide ligands, and play an important role in the response of AmiC-AmiR to substrates.
[0050] (2) Response of mutant AmiC-AmiR-Y83I / T233C to different concentrations of nicotinamide
[0051] The positive transformants containing plasmid pVEO-AmiC-AmiR-Y83I / T233C were picked up and placed in LB medium, and cultured at 37°C and 200 rpm for 7-8 hours. The seed solution was transferred to 5 mL LB medium containing different concentrations of nicotinamide (0.0064 mM, 0.032 mM, 0.16 mM, 0.8 mM, 4 mM, 20 mM, and 100 mM) at 1% (v / v) and cultured for 24 hours. After culture, the fluorescence value was detected using an ELISA reader.
[0052] The results are as follows Figure 2As shown, the mutant AmiC-AmiR-Y83I / T233C is more sensitive to aromatic amides, has a good detection ability for millimolar-level nicotinamide, effectively detects between 0 and 4 mM, and has good linearity.
[0053] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A biosensor for amide substances, characterized in that: The biosensor comprises a RBS AmiC Sequences of AmiC structural units and RBS AmiR AmiR structural unit of the sequence; wherein the AmiC structural unit is based on the amino acid sequence as shown in SEQ ID NO.1, the tyrosine at position 83 is mutated to isoleucine, and the threonine at position 233 is mutated to cysteine; the genes in the biosensor are RBS in the direction of gene expression. AmiC 、AmiC structural unit gene、RBS AmiR , AmiR structural unit genes; The amino acid sequence of the AmiR structural unit is shown in SEQ ID NO.3; The RBS AmiC The sequence is shown in SEQ ID NO.4, the RBS AmiR The sequence is shown in SEQ ID NO.5; The amide substance is nicotinamide, isonicotinamide, benzamide, pyrazinamide, cinnamamide, naphthamide, nicotinic acid or nicotinonitrile.
2. A plasmid carrying the biosensor according to claim 1.
3. The plasmid according to claim 2, characterized in that The plasmid includes pET series vectors, and the pET series vectors carry marker genes.
4. The plasmid according to claim 3, characterized in that The marker gene includes a gene encoding a fluorescent protein.
5. A cell carrying the biosensor according to claim 1 or the plasmid according to any one of claims 2 to 4.
6. The cell according to claim 5, characterized in that The cells include Escherichia coli.
7. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria uses Escherichia coli as a host and carries the biosensor according to claim 1 or the plasmid according to any one of claims 2 to 4.
8. The genetically engineered bacterium according to claim 7, characterized in that: The Escherichia coli includes E. coli BL21, E. coli BL21 (DE3), E. coli JM109, E. coli DH5α, E. coli Any of the top 10.
9. Application of the biosensor according to claim 1, or the plasmid according to any one of claims 2 to 4, or the cell according to claim 5 or 6, or the genetically engineered bacteria according to claim 7 or 8 in detecting amide substances, or screening strains that metabolize amide substances, or studying metabolic pathways for the production of amide substances, characterized in that: The amide substance is nicotinamide, isonicotinamide, benzamide, pyrazinamide, cinnamamide, naphthamide, nicotinic acid or nicotinonitrile.