Transcription factor ncgl0581 mutant and use thereof in l-serine detection

Abstract

Provided are a transcription factor NCgl0581 mutant and the use thereof in L-serine detection, belonging to the technical field of biological detection. The present application comprises performing molecular docking to obtain a key binding site of a transcription factor NCgl0581 and L-serine, and further performing model analysis and virtual screening, so as to obtain an L-serine biosensor mutant NCgl0581E136P. Experiments prove that a biosensor constructed on the basis of the mutant NCgl0581 E136P can respond to serine having a concentration of 25 mM, the response concentration being reduced by one time than that before mutation. The mutation lowers the detection lower limit of biosensors, thus laying a good foundation for detection of low-concentration L-serine. Therefore, the present application has good application value and prospects.

Description

A transcription factor NCgl0581 mutant and its application in L-serine detection Technical Field

[0001] The invention relates to a transcription factor NCgl0581 mutant and application thereof in L-serine detection, belonging to the technical field of biological detection. Background Art

[0002] L-serine participates in numerous important intracellular activities and holds broad application prospects in fields such as medicine, food, and cosmetics. Recent studies have demonstrated that L-serine stimulates the proliferation of various cell types. Its metabolic pathways are closely linked to tumor cell formation and proliferation, making it a promising tumor marker. Developing efficient and convenient methods for detecting L-serine is crucial. Biosensors, which enable specific responses to target compounds, have been widely used to measure metabolite concentrations. However, L-serine biosensors based on the wild-type transcription factor NCgl0581 suffer from high detection limits, making them difficult to apply to the analysis of low L-serine concentrations.

[0003] Biosensors primarily consist of two key components: a biosensitive membrane and a transducer. When the analyte enters the biosensitive membrane through a process such as permeation or flow, it specifically binds to the membrane via molecular recognition. The resulting physicochemical or biological signal can be converted by the transducer into optical or electrical signals, enabling analysis and detection of the analyte. Biosensors primarily include FRET sensors, riboswitch sensors, and transcription factor (TF) sensors. In recent years, biosensors have been widely used in the optimization of biosynthetic pathways and the real-time monitoring of metabolites, and they hold great potential for the construction and screening of high-yield strains and efficient enzymes.

[0004] Among them, biosensors based on transcription factors are the most widely used. When the target metabolite reaches a certain concentration, the transcription factor changes its own protein conformation to achieve response characteristics and converts the concentration signal of the target metabolite into a signal output such as fluorescence. NCgl0581 is an endogenous transcriptional regulatory factor in Corynebacterium glutamicum, belonging to the LysR type transcriptional regulatory factor family. The signal molecule is L-serine, which is used to regulate the promoter P SerE. Biosensors based on the transcription factor NCgl0581 can be used to detect L-serine concentrations and screen L-serine-producing strains. However, the detection limit of the wild-type L-serine biosensor is relatively high, making it difficult to apply to the analysis and detection of low-concentration L-serine. The performance of biosensors can usually be modified by adjusting traditional strategies such as promoter strength and RBS. However, traditional static modification methods have problems such as small data volume and poor versatility. In combination with computer-aided design, the performance of biosensors can be predictably modified with the help of rational design methods such as mathematical models to achieve intelligent regulation. Therefore, the present invention is proposed.

[0005] Summary of the Invention

[0006] To solve the above problems, the present invention takes the wild-type transcription factor NCgl0581 of L-serine biosensor as the research object, obtains its key binding site with the signal molecule L-serine by analyzing and docking, further performs model analysis and virtual screening, and obtains the L-serine biosensor mutant NCgl0581. E136P , which lowered the detection limit of the biosensor, improved the sensitivity, and laid the foundation for the detection of low-concentration L-serine.

[0007] The first object of the present invention is to provide a transcription factor NCgl0581 mutant, which is obtained by mutating the 136th glutamic acid in the amino acid sequence as shown in SEQ ID NO.1 to proline (NCgl0581 E136P ).

[0008] The second object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned transcription factor NCgl0581 mutant.

[0009] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.4.

[0010] The third object of the present invention is to provide an expression vector carrying the above nucleic acid molecule.

[0011] The fourth object of the present invention is to provide a host cell expressing the above-mentioned transcription factor NCgl0581 mutant.

[0012] Furthermore, the host cell is a bacterium, a fungus, a plant cell or an animal cell.

[0013] The fifth object of the present invention is to provide the use of the above-mentioned transcription factor NCgl0581 mutant, nucleic acid molecule, expression vector or host cell in the preparation of an L-serine detection product.

[0014] The sixth object of the present invention is to provide an L-serine detection product, which contains the above-mentioned transcription factor NCgl0581 mutant, nucleic acid molecule, expression vector or host cell.

[0015] Furthermore, the detection product is a biosensor, which includes: a transcription factor NCgl0581 mutant encoding gene, a promoter for initiating expression of the transcription factor NCgl0581 mutant encoding gene, a promoter P SerE and a reporter gene expressed by the promoter, the promoter P SerE Located downstream of the gene encoding the transcription factor NCgl0581 mutant.

[0016] Taking L-serine as the detection target, the detection principle of the biosensor constructed in the present invention is as follows: the promoter activates the expression of NCgl0581 gene, and the NCgl0581 protein binds to the L-serine molecule to change its own protein conformation, thereby activating the downstream promoter P SerE , thereby expressing the reporter gene, outputting signals such as fluorescence, and realizing quantitative detection of L-serine molecules according to the strength of the fluorescence signal. Of course, the biosensor constructed by the present invention can respond to 25mM L-serine, laying the foundation for the detection of low-concentration L-serine samples, which is the advantage of the present invention.

[0017] Furthermore, the promoter for initiating the expression of the gene encoding the transcription factor NCgl0581 mutant may be an inducible promoter or a constitutive promoter, including but not limited to P NCgl0581 、P B6 、P e 、P a16 wait.

[0018] Furthermore, the reporter gene can be any identifiable signal, such as a fluorescent gene, a resistance gene, and the like.

[0019] Furthermore, the above elements are located on the same plasmid vector and introduced into host cells for detection of the target. In one embodiment of the present invention, the vector used is pDXW-11.

[0020] The seventh object of the present invention is to provide a kind of detection method of L-serine, comprising the following steps:

[0021] Qualitative detection: The sample to be tested is incubated with host cells containing the above-mentioned biosensor. After the incubation is completed, the reporter gene signal is detected. The presence or absence of the signal is used to determine whether the sample to be tested contains the target compound;

[0022] Quantitative detection:

[0023] S1. Incubate samples containing different concentrations of the target with host cells containing the above-mentioned biosensor, and detect the strength of the reporter gene signal after the incubation is completed;

[0024] S2. Establishing a relationship curve between reporter gene signal and target concentration;

[0025] S3. Repeat step S1 for the actual sample to be tested, and substitute it into the relationship curve in S2 to calculate the content of the target substance.

[0026] Beneficial effects of the present invention:

[0027] The present invention performs molecular docking on the transcription factor NCgl0581 and L-serine, and mutates the obtained binding key sites to obtain NCgl0581. E136P A biosensor was constructed based on the E136P mutant of the transcription factor NCgl0581, which contained the E136P mutant encoding gene and its promoter P NCgl0581 , promoter P SerE Among them, the transcription factor NCgl0581 can be induced by L-serine, changing its own structure to bind to L-serine, and then activate the downstream promoter P SerE After the above biosensor was transformed into Escherichia coli, the response concentration of NCgl0581 mutant to L-serine was verified by exogenously adding different concentration gradients of L-serine. It was found that the NCgl0581-based E136P The detection limit of the biosensor constructed by the mutant was reduced by 1-fold, and its L-serine response concentration was as low as 25 mM, indicating that the mutant NCgl0581 E136P Its superiority in detecting low-concentration L-serine lays the foundation for the analytical detection of low-concentration L-serine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a schematic diagram of the construction of the biosensor of the present invention.

[0029] FIG2 shows the responses of the wild-type NCgl0581 biosensor and mutants to L-serine. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0031] The scheme that the present invention relates to is as follows:

[0032] The difficulty in using mutation technology to change the detection range of L-serine lies in the selection of mutation sites. This method constructs a three-dimensional structure of the wild-type transcriptional regulatory factor NCgl0581 protein and L-serine, simulates molecular docking using MOE-Dock software, and determines the binding affinity of L-serine and the NCgl0581 tetramer. Based on the changes in binding affinity, key binding sites are identified and virtual mutations are performed to determine the mutation sites and directions. Based on the simulation results, mutants such as E136P are designed. Then, a dose-response curve of the biosensor is generated by exogenously adding L-serine concentration gradients. Finally, the changes in the detection limit of the mutant biosensor are verified.

[0033] The culture medium and various assay methods involved in the following examples are as follows:

[0034] LB medium (g / L): peptone 10; yeast extract 5; NaCl 10 (solid medium, agar powder 20); sterilization conditions: 121°C, 20 min.

[0035] Determination of bacterial fluorescence intensity and biomass: E. coli in the culture medium was centrifuged at 4°C, 4000g for 5 min to collect the bacteria, washed 2 to 3 times with pre-cooled PBS buffer, resuspended and diluted in PBS buffer (OD 600 =0.2-0.8). 200 μL of the diluted bacterial solution was pipetted into a 96-well plate, and fluorescence intensity and bacterial concentration were measured using a multifunctional microplate reader. For fluorescence intensity, the reader was set to a yellow fluorescent protein excitation wavelength of 488 nm and an emission wavelength of 590 / 9 nm; for bacterial concentration, the reader was set to λ = 600.

[0036] E. coli transformation method: Using mutant E136P as an example, thaw competent E. coli cells on ice. In a clean bench, mix 100 μg-10 ng of the plasmid containing mutant E136P with the competent cells and incubate on ice for 30 minutes. Heat shock the cells in a 42°C water bath for 2 minutes, then incubate on ice for 2 minutes. Add the cells to 900 μL of fresh LB liquid medium and incubate at 37°C, 220 rpm, for 1 hour. Collect the cells by centrifugation, resuspend, and plate on a solid LB plate containing Kan. Incubate the cells in an inverted position overnight at 37°C.

[0037] Example 1: Model analysis and virtual screening

[0038] Homology modeling of the wild-type transcriptional regulator NCgl0581 protein was performed using MOE molecular modeling software. The intermediate model with the best score, based on the GB / VI scoring function, was selected as the final model. The model was described and analyzed using PyMOL and save v6.0 software. The downloaded two-dimensional structure of L-serine was then converted to a three-dimensional structure using energy minimization in MOE.

[0039] MOE-Dock was used to perform molecular docking between the constructed NCgl0581 tetramer model and the substrate L-serine. The most promising conformation was screened by comprehensively considering factors such as the interaction between protein and L-serine, the binding site, and the docking score. PyMOL was used to analyze the binding mode.

[0040] The changes in the affinity of NCgl0581 for L-serine were determined by residue scanning under the LowMode module, the conformational space of the mutants was determined, and the mutant conformations that met the energetic criteria were output. Based on the changes in binding affinity, the mutations were simulated to construct a virtual mutant library, and the E136P mutant was designed according to the mutation sites and mutation directions guided by the simulation calculation results.

[0041] Example 2: Point mutation of transcription factor NCgl0581 to E136P

[0042] PCR was used to perform site-directed mutagenesis at position E136 of the transcription factor NCgl0581 (amino acid sequence shown in SEQ ID NO. 1, nucleotide sequence shown in SEQ ID NO. 2), mutating the glutamic acid at position 136 to proline. Using the bacterial strain genome as a template, the 500 bp preceding and 500 bp following the point mutation were selected for amplification. Primers ncgl0581-F and ncgl0581-R were used to amplify the homology arm gene fragment containing the point mutation. This fragment was then ligated with the plasmid pK18mobsacB digested with EcoRI and XbaI to construct a revertant plasmid. This plasmid was transformed into competent E. coli cells and cultured overnight at 37°C. Transformants were subjected to colony PCR and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Strains with correct results were considered successful mutants. The amino acid sequence of the E136P mutant is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 4.

[0043] The primers used for E136P mutation are shown in Table 1.

[0044] Table 1 Site-directed mutagenesis primers

[0045] Example 3: Exogenous gradient experiment to verify the change of the detection range of L-serine in the mutant biosensor

[0046] Construction of biosensor: The construction diagram is shown in Figure 1. One part of the biosensor is used for the expression of the transcriptional regulatory factor NCgl0581, and the other part is connected to the promoter P regulated by NCgl0581. SerE , and then connected to eyfp, used to characterize the biosensor binding to L-serine to regulate the transcription level of eyfp. SerE The nucleotide sequence of the reporter gene eyfp is shown in SEQ ID NO.5, and the nucleotide sequence of the reporter gene eyfp is shown in SEQ ID NO.6.

[0047] The detection limit of L-serine of the NCgl0581-based L-serine biosensor (pDSer) is 50 mM. When the concentration of L-serine is lower than 50 mM, the fluorescence value remains basically unchanged and cannot respond to lower concentrations of L-serine. The following exogenous addition experiment verifies the NCgl0581-based E136P The response of the mutant biosensor to L-serine concentration was reduced to 25 mM.

[0048] After site-directed mutagenesis, single JM109 colonies containing wild-type NCgl0581 and mutant E136P were selected and inoculated into 10 mL of LB (100 μg / mL Kan) liquid medium and cultured overnight at 37°C, 220 rpm. In a sterilized 24-well plate, 950 μL of fresh LB medium was added to each well. L-serine was then added at varying concentrations, controlling the final concentration to 0, 25, 50, 75, or 100 mM. Finally, 50 μL of the overnight culture was added to each well, and the plate was incubated at 37°C, 220 rpm, on a shaker for 2 h.

[0049] The E. coli in the culture medium was centrifuged for 5 min to collect the cells, washed 2-3 times with PBS buffer, resuspended and diluted in PBS buffer (OD 600 =0.2-0.8). Pipette 200 μL of the diluted bacterial solution into a 96-well plate and measure the bacterial concentration OD using a multifunctional microplate reader. 600 and fluorescence intensity, calculated fluorescence intensity / OD 600 It is the fluorescence intensity value per unit cell.

[0050] Example 4: Analysis of the Effect of Mutation on L-Serine Detection Range

[0051] Fluorescence intensity / OD 600The biosensor's dose-response curve for L-serine was plotted using OriginPro 8.5, with the concentration of L-serine as the vertical axis and the concentration of L-serine as the horizontal axis. The detection limit of the biosensor was defined as the L-serine concentration corresponding to the marked upward trend in the dose-response curve. The detection ranges of wild-type NCgl0581 and mutant E136P were determined from the response curves.

[0052] The results showed that the wild-type NCgl0581 biosensor began to respond at an L-serine concentration of 50 mM, with a detection limit of 50 mM. However, the response curve of the biosensor based on the E136P mutant began to show a clear upward trend at an L-serine concentration of 25 mM, lowering the detection limit to 25 mM. Experimental verification showed that the E136P point mutation at the key L-serine binding site of the transcription factor NCgl0581 altered the biosensor's detection limit; however, the E136A and E136Y mutations at this site had no effect on the L-serine response concentration (Figure 2).

[0053] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A transcription factor NCgl0581 mutant, characterized in that: The mutant is obtained by mutating the 136th glutamic acid in the amino acid sequence shown in SEQ ID NO.1 to proline.

2. A nucleic acid encoding the transcription factor NCgl0581 mutant according to claim 1.

3. An expression vector carrying the nucleic acid of claim 2.

4. A host cell expressing the transcription factor NCgl0581 mutant according to claim 1.

5. Use of the transcription factor NCgl0581 mutant according to claim 1, the nucleic acid according to claim 2, the expression vector according to claim 3 or the host cell according to claim 4 in preparing an L-serine detection product.

6. An L-serine detection product, characterized in that: The detection product contains the transcription factor NCgl0581 mutant according to claim 1, the nucleic acid according to claim 2, the expression vector according to claim 3 or the host cell according to claim 4.

7. The L-serine detection product according to claim 6, characterized in that: The L-serine detection product comprises a biosensor.

8. The L-serine detection product according to claim 7, characterized in that, The biosensor comprises: a transcription factor NCgl0581 mutant encoding gene, a promoter for initiating the expression of the transcription factor NCgl0581 mutant encoding gene, a promoter P SerE and a reporter gene whose expression is initiated thereby.

9. A method for detecting L-serine, characterized in that: The method comprises the steps of co-incubating a host cell containing the biosensor according to claim 8 with a sample to be tested, and performing signal detection.

10. The detection method according to claim 9, characterized in that: The following steps are involved: S1, incubating samples containing different concentrations of the target with host cells containing the biosensor, and detecting the strength of the reporter gene signal after the incubation is completed; S2. Establishing the relationship curve between the reporter gene signal and the target concentration; S3, replace the sample with the sample to be tested and repeat step S1, substituting it into the relationship curve of S2 to calculate the content of the target substance.

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