Mutant for vitamin detection, biosensor containing mutant as well as construction method and application of biosensor

Abstract

The invention relates to the field of gene engineering, in particular to a mutant for vitamin detection, a biosensor containing the mutant and a construction method and application of the biosensor. The invention provides a mutant CaiF protein for vitamin detection, the mutant CaiF protein is obtained by substituting an original protein CaiF with alanine, the substitution is selected from one of R131A, R91A, Q119A, S31A, E67A and E84A, the invention provides a biosensor containing the CaiF protein mutant, the detection concentration range of the sensor on vitamin BT is obviously expanded through site-specific mutagenesis, and the biosensor can be used for detecting vitamin BT. The maximum detection range is 10 <-3 >-10 mM, and the biosensor can be used for screening strains with high yield of L-carnitine in a relatively rapid and easy manner, and has important application prospects in the aspect of detecting and screening microorganisms with high yield of L-carnitine.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a mutant for vitamin detection and a biosensor containing the mutant, and a construction method and application thereof. Background Art

[0002] Vitamin B T Also known as L-carnitine, it is an amino acid and is an isomer of D-carnitine. T Widely present in the human body and a variety of foods, such as red meat, dairy products, etc. Vitamin B T It plays a key role in the intermediate metabolism of eukaryotic organisms, participating in the β-oxidation process of medium- and long-chain fatty acids and the exchange of acyl and acetyl groups with coenzyme A (CoA) in mitochondria, which is essential for energy production and fatty acid metabolism. Therefore, L-carnitine has been widely used in medicine, health care and animal husbandry.

[0003] The production processes of L-carnitine include natural animal extraction, biosynthesis and chemical synthesis. In industrial production, it mainly relies on chemical synthesis methods, but the chemical synthesis process usually requires the use of harmful chemicals. These substances may pollute the environment during the production process, and may also irritate the workers' respiratory system, skin and eyes, and even cause more serious health problems.

[0004] Compared with chemical synthesis methods, biological production of L-carnitine is a more environmentally friendly and sustainable method. The raw materials used in the biological production process are safer, less waste is generated, and the impact on the environment is smaller. As an efficient and green method, it has great potential in industrial production, especially in the field of biosynthesis.

[0005] In the biosynthesis of L-carnitine, biosensors have significant advantages under high-throughput large-scale screening conditions, especially in the screening of high-yield strains and the modification of key rate-limiting enzymes. Through biosensors, microbial strains that can efficiently produce L-carnitine can be quickly and accurately identified and selected, thereby optimizing the production process. The construction of L-carnitine biosensors depends on the transcription factor CaiF, so the identification of the key binding sites of CaiF not only helps to understand its regulatory mechanism, but also provides a molecular basis for the design of more efficient biosensors.

[0006] In addition, the development of an L-carnitine biosensor with an expanded response range has important application value for industrial production. Such a sensor can improve screening efficiency and identify strains that produce L-carnitine at higher concentrations, thereby accelerating the production process and reducing costs. This modification can not only enhance the application of biosensors in basic research, but also promote their commercialization in the fields of biopharmaceuticals, food industry and environmental monitoring.

[0007] In summary, at present, there is an urgent need to develop a biosensor for vitamin detection, improve its response intensity and broaden its detection range to meet the needs of high-throughput and rapid detection of L-carnitine, which will help further promote the biosynthesis technology of L-carnitine and enable it to be more widely used in multiple fields. Summary of the invention

[0008] The object of the present invention is to provide a mutant for vitamin detection;

[0009] Another object of the present invention is to provide a biosensor containing mutants for vitamin detection and a construction method and application thereof.

[0010] CaiF gene as vitamin B T Although the CaiF gene is not directly involved in vitamin B T detection process, but it can affect the vitamin B in the fermentation broth T The concentration and molar conversion rate of vitamin B T Screening and optimizing the expression of CaiF gene not only helps to increase vitamin B T It can not only improve production efficiency, but also accelerate the screening process of high-yield strains, thereby improving the efficiency and accuracy of the entire detection and screening process.

[0011] The technical problem to be solved by the present invention is to construct a L-carnitine biosensor with improved response intensity and wider detection range to meet the demand for high-throughput and rapid detection of L-carnitine.

[0012] The present application provides a mutant for vitamin detection, wherein the mutant for vitamin detection is a CaiF protein mutant, wherein the gene of the CaiF protein mutant is obtained by site-directed mutagenesis of the original protein CaiF and an alanine substitution, wherein the substitution is selected from one of R131A, R91A, Q119A, S31A, E67A, and E84A, and a mutant library is constructed;

[0013] The amino acid sequence of the original protein CaiF is SEQ ID NO.1, and SEQ ID NO.1 is:

[0014] MCEGYVEKPLYLLIAEWMMAENRWVIAREISIHFDIEHSKAVNTLTYILSE VTEISCEVKMIPNKLEGRGCQCQRLVKVVDIDEQIYARLRNNSREKLVGVRKT PRIPAVPLTELNREQKWQMMLSKSMRR.

[0015] In the present application, a biosensor for vitamin detection is provided, wherein the biosensor comprises the CaiF protein mutant gene binding site as described above;

[0016] Specifically, the biosensor contains a promoter P fix ,caiC gene, CaiF protein mutant gene binding site and gene encoding marker for vitamin detection;

[0017] Furthermore, the P fix The sequence of the gene binding site for regulating the CaiF protein mutant is integrated and the expression of the gene encoding the marker is regulated; the amino acid sequence encoding the protein CaiF is shown in SEQ ID NO.1, and SEQ ID NO.1 is:

[0018] MCEGYVEKPLYLLIAEWMMAENRWVIAREISIHFDIEHSKAVNTLTYILSE VTEISCEVKMIPNKLEGRGCQCQRLVKVVDIDEQIYARLRNNSREKLVGVRKT PRIPAVPLTELNREQKWQMMLSKSMRR;

[0019] Furthermore, the CaiF protein mutant and the gene encoding the marker are located on a plasmid vector, that is, the biosensor is a plasmid;

[0020] Furthermore, the plasmid vector includes but is not limited to pBBR series plasmids;

[0021] Further, the plasmid vector is pBBR-caicM;

[0022] Furthermore, the coding marker is a fluorescent protein coding gene;

[0023] Furthermore, the coding marker is a green fluorescent protein coding gene;

[0024] Furthermore, the coding marker is a green fluorescent protein coding gene sfGFP, and the amino acid sequence of the green fluorescent protein coding gene sfGFP is SEQ ID NO.2, and SEQ ID NO.2 is: SEQ ID NO.2 is:

[0025] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.

[0026] On this basis, the present application further provides a method for constructing a biosensor, which specifically includes the following steps:

[0027] (1) The CaiF protein mutants were subjected to circular PCR and cloned into the vector pBBR-caicM to construct a plasmid mutation library;

[0028] The CaiF protein mutants include those obtained by substituting an alanine in the original protein CaiF, wherein the substitutions are selected from R131A, R91A, Q119A, S31A, E67A, and E84A, and constructing the plasmid mutation library;

[0029] (2) Introducing the mutant plasmid into Escherichia coli DH5α, plating, and culturing on solid medium;

[0030] (3) Verify the single colony on the solid culture medium, and after verification, perform plasmid extraction;

[0031] (4) The mutant plasmid of the biosensor was introduced into the competent BW25113 (DE3) ΔcaiA. After verification, a single colony was picked and placed in a 5 mL LB test tube containing apramycin, and cultured overnight at 37°C, 200 rpm; (5) The cells were transferred to a 96-well plate containing 0.8 mL M9 culture medium at a ratio of 1%, and the fluorescence intensity (AFU) and absorbance (OD) at 600 nm were measured for 20 h; the M9 culture medium contained apramycin and different concentrations of L-carnitine.

[0032] The present application also includes a genetically engineered strain containing the above-mentioned biosensor;

[0033] The present application also includes the use of the above-mentioned biosensor in screening vitamin strains.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The CaiF protein was modified through site-directed mutagenesis to construct a biosensor carrying the CaiF protein mutant. The biosensor includes a rich plasmid mutation library, which significantly broadens the detection boundary of the biosensor, enabling it to quickly and conveniently screen out high-L-carnitine-producing strains in the microbial population. It has made significant progress in the rapid identification and utilization of efficient production strains of specific metabolites: it not only provides a powerful tool for the industrial production of L-carnitine, but also opens up a new way for the microbial screening of other bioactive substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the plasmid map of the biosensor pBBR-caicM in the present invention;

[0037] Figure 2 This is a graph showing the response of the biosensor pBBR-caicM plasmid of the present invention to the addition of different concentrations of L-carnitine outside the cell;

[0038] Figure 3 The graph is a response diagram of the biosensor containing different mutants in the present invention to the extracellular addition of L-carnitine. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0041] Example 1

[0042] Using the E.coli MG1655 genome as a template, primers pfix-F and pfix-R were used to amplify the fix fragment with a linker. The sfGFP gene fragment with a linker amplified by sfGFP-F and sfGFP-R primers was combined and inserted into the pBBR-PQS-fre vector to produce the recombinant plasmid pBBR-PQS-fix-sfGFP. Subsequently, the caiF gene with a linker was amplified from the E. coli genome using primers caiF-R and caiF-F; the caiC fragment with a linker was amplified using primers caic-F and caic-R and cloned into the recombinant plasmid to obtain pBBR-PQS-fix-sfGFP-caiC-caiF. Subsequently, circular PCR was performed using the recombinant plasmid pBBR-PQS-caiC-caiF as a template and phom-F and phom-R as primers, and then transformed into E.coli DH5α to obtain the biosensor plasmid pBBR-caicM. The plasmid map is shown in the figure. Figure 1 shown.

[0043] The primers used in this section are as follows:

[0044] Table 1 Primers used to construct L-car sensor plasmid

[0045]

[0046]

[0047] The strains and plasmids used in this section are as follows:

[0048] Table 2 Strains and plasmids used to construct L-car sensor plasmids

[0049]

[0050] Example 2: Testing of biosensors

[0051] The sensor plasmid pBBR-caicM was transformed into BW25113(DE3)ΔcaiA competent cells to construct strain BW25113(DE3)ΔcaiA-pBBR-caicM. A single colony was picked and placed in 5 mL of LB medium containing apramycin. The culture was incubated overnight at 37°C and 200 rpm to form a seed solution. 10 μL of the seed solution was transferred to fresh 0.8 mL of M9 medium containing apramycin and different concentrations of L-carnitine (10 -6 mM, 10 -5 mM, 10 -4 mM, 10 -3 mM, 10 -2 mM, 10 -1mM, 1mM, 10mM), 37°C, 800rpm, and then 200μL of the sample was taken to measure the fluorescence intensity (AFU) and absorbance (OD) at 600nm. Figure 2 As shown in Figure 2, the response range of the biosensor to L-carnitine is 10 -4 -10 -2 mM, and the fluorescence intensity was between 100-2000 a.u.

[0052] Table 3 Relationship between L-carnitine production and fluorescence intensity

[0053]

[0054]

[0055] Example 3: Alanine scanning of CaiF protein to construct library

[0056] Using the biosensor plasmid pBBR-caicM as a template, the following mutant plasmids were constructed:

[0057] E3A, Y5A, E7A, K8A, Y11A, E16A, E21A, N22A, R23A, R28A, E29A, S31A, H33A, D35A, E37A, H38A, S39A, K40A, N 43A, T44A, T46A, Y47A, S50A, E51A, T53A, E54A, S56A, E58A, K60A, N64A, K65A, E67A, R69A, Q72A, Q74A, R75 A. K78A, D81A, D83A, E84A, Q85A, Y87A, R89A, R91A, N92A, N93A, S94A, R95A, E96A, K97A, R102A, K103A, T10 4A, R106A, T113A, E114A, N116A, R117A, E118A, Q119A, K120A, Q122A, S126A, K127A, S128A, R130A, R131A.

[0058] The sensor mutant plasmid was transformed into BW25113(DE3)ΔcaiA competent cells to construct the mutant strain BW25113(DE3)ΔcaiA-Mn.

[0059] Single colonies were picked and placed in 0.8mL 96-well plate in LB medium containing apramycin, and cultured overnight at 37°C, 200rpm to form seed solution. 10 μL of seed solution was transferred to fresh 0.8mL M9 medium containing apramycin and different concentrations of L-carnitine (10 -3 mM, 10 -1mM, 10mM), 37°C, 800rpm, and then 200μL of the sample was taken to measure the fluorescence intensity (AFU) and absorbance at 600nm (OD). The results are shown in Figure 3 As shown, the normalized fluorescence signals of R75A, N43A, Y11A, Y47A, and T46A decreased to varying degrees; while the fluorescence response ranges of R131A, R91A, Q119A, S31A, E67A, and E84A were significantly expanded compared with the other mutants, and the response ranges of the other sites did not expand significantly after mutation to alanine.

[0060] As shown in Table 4, after R75 was mutated to alanine, it did not show a dose-dependent fluorescence response to L-carnitine, and the fluorescence signals of mutants N43A, Y11A, Y47A, and T46A also decreased to varying degrees. According to the response value, after structural simulation of CaiF, R75A is at the key site of DNA binding, and N43A, Y11A, Y47A, and T46A indirectly affect DNA binding; as shown in Table 5, the response range of the wild-type biosensor is 10 -1 mM reached saturation, which was consistent with our previous test results on the response range of the sensor. The mutants R131A, R91A, Q119A, S31A, E67A, and E84A were able to detect fluorescence signals at a concentration of 10 mM, and the response range to L-carnitine was expanded to 10 -3 mM-10mM, with improved response intensity and wider detection range, can meet the needs of high-throughput and rapid detection of L-carnitine and has further application value.

[0061] Table 4 Relationship between mutation site and fluorescence intensity

[0062]

[0063] Table 5 Relationship between mutation sites and normalized fluorescence signals

[0064]

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant for vitamin detection, characterized in that: The protein mutant used for vitamin detection is a CaiF protein mutant, which is obtained by substituting an alanine in the original protein CaiF, wherein the substitution is selected from one of R131A, R91A, Q119A, S31A, E67A, and E84A.

2. A mutant for vitamin detection as claimed in claim 1, characterized in that: The amino acid sequence of the original protein CaiF is SEQ ID NO.

1.

3. A biosensor, characterized in that: The biosensor comprises a promoter P fix , caiC gene, a plasmid containing a CaiF protein mutant gene binding site and a gene encoding a marker for vitamin detection as claimed in claim 1.

4. A biosensor according to claim 3, characterized in that: The plasmid includes but is not limited to pBBR series plasmids.

5. A biosensor as claimed in claim 3, characterized in that: The coding marker is the green fluorescent protein coding gene sfGFP, and the amino acid sequence of the green fluorescent protein coding gene sfGFP is SEQ ID NO.

2.

6. A method for constructing a biosensor, characterized in that: The following steps are involved: (1) performing circular PCR on the CaiF protein mutant for vitamin detection as claimed in claim 1, cloning it into the vector pBBR-caicM, and constructing a plasmid mutation library; (2) Introduce the mutant plasmid into Escherichia coli DH5α and plate it; (3) verifying the single colony on the solid culture medium and extracting the mutant plasmid after verification; (4) The extracted mutant plasmid was introduced into the competent BW25113 (DE3) ΔcaiA; after verification, a single colony was picked and placed in a 5 mL LB tube containing apramycin, and cultured overnight at 37°C and 200 rpm; (5) The cells were transferred to a 96-well plate containing 0.8 mL of M9 medium at a ratio of 1% and cultured for 20 h to measure the fluorescence intensity and absorbance at 600 nm.

7. The method for constructing a biosensor according to claim 6, wherein: The M9 culture medium in step (5) contains apramycin and L-carnitine.

8. A genetically engineered bacterium containing the biosensor according to any one of claims 3 to 5.

9. Use of the biosensor according to any one of claims 3 to 5 in screening vitamin-producing strains.

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