BmoR mutant biosensor with sensitive response to phenethyl alcohol

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a BmoR protein mutant sensitively responding to phenethyl alcohol and application of the BmoR protein mutant in phenethyl alcohol detection or a biosensor. The BmoR protein mutant is obtained by mutation containing S184T on the basis of a wild type BmoR protein as shown in SEQ ID NO. 1 in a sequence table. The S184T mutant sensitively responds to phenethyl alcohol, so that the problem that wild type BmoR protein does not respond to a phenethyl alcohol high-yield strain is solved; meanwhile, the detection range of S184T on phenethyl alcohol reaches 0-30 mM, the response saturation of BmoR protein on phenethyl alcohol is improved, and the S184T can be used for screening and application of strains with higher yield.

Description

Technical field:

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a BmoR protein mutant sensitively responding to phenylethanol (0-30 mM), and application of the mutant in phenylethanol detection or biosensor. Background technology:

[0002] During the fermentation process of alcoholic beverages, different alcohol byproducts are produced, and phenylethanol is one of them. It is an important component for forming the flavor of alcoholic beverages. Although the amount is small, it has a great influence on the taste and aroma characteristics of alcoholic beverages. As one of the alcohol byproducts, phenylethanol can make alcoholic beverages have a unique fragrance and improve their taste. When phenylethanol exists in excess, it will have an adverse effect on the product quality of alcoholic beverages. It will not only seriously affect the flavor of alcoholic beverages, making them have strange tastes, dryness and bitterness and unable to swallow, but also cause drinkers to get drunk, get high, and feel thirsty, and even cause severe headaches, which are dangerous for damaging the nerve center and endangering human health. Therefore, it is of great significance to control the content of alcohol byproducts in alcoholic beverages. Due to the similarity of alcohol byproducts and the main product ethanol in chemical properties, the difficulty and cost of separating alcohol byproducts from alcoholic beverages are greatly increased. Moreover, the accumulation of alcohol byproducts is not well controlled by controlling fermentation conditions and feeding, and it is impossible to accurately control the accumulation of alcohol byproducts without affecting the flavor of alcoholic beverages. Therefore, it is necessary to monitor the composition ratio and content level of alcohol by-products during the fermentation process of alcoholic beverages in real time.

[0003] Traditional detection methods, such as chemical analysis represented by spectrophotometry and colorimetry and instrumental analysis represented by gas chromatography, can accurately carry out qualitative or quantitative analysis of alcohol byproducts such as phenylethanol, but the stability of different detection methods varies, and it is impossible to take into account the convenience and speed of operation as well as the time and economic cost. These defects and deficiencies have caused certain adverse effects and limitations on the application of traditional detection methods in large-scale industrial and market production. Therefore, it is considered to use biosensors to realize real-time detection and control of phenylethanol alcohol byproducts in the fermentation process of alcoholic beverages. The efficient real-time detection of alcohol byproducts such as phenylethanol and n-propanol byproducts based on transcription factors has become one of the best choices under existing conditions. Since the effector of the transcription factor BmoR is highly consistent with the alcohol byproducts such as phenylethanol and n-propanol in the fermentation process of alcoholic beverages, the fermentation process of wine is improved. The biosensor consists of a molecular recognition element and a signal converter. When the molecular recognition element binds to the object to be measured, the generated signal can be converted into an optical signal or an electrical signal by the converter, and the object to be measured can be detected and analyzed. As an emerging tool in synthetic biology, biosensors can be designed and constructed to dynamically respond to changes in the concentration of signal molecules.

[0004] At the same time, biosensors are designed to promote the optimization of microbial cell factories and the production of a range of natural products widely used in industry, such as itaconic acid, fatty acids, n-isoamyl alcohol, phenylethanol and alkaloids. Biosensors mainly include RNA nucleic acid switches, transcription factor-regulated biosensors, G protein-coupled receptors and fluorescent protein biosensors. However, the low dynamic range of fluorescent protein biosensors, the difficulty of RNA nucleic acid switches in vitro, and the fact that G protein-coupled receptors can only be performed outside the cell have hindered the development of biosensors in the biological world.

[0005] Biosensors based on transcription factors (TFs) are the most widely used. The most commonly used transcription factors are bacterial transcription factors, which include a ligand binding domain (LBD) or a metabolic binding domain (MBD) and a DNA binding domain (DBD). BmoR is a transcription factor of the n-alkane metabolic pathway of Pseudomonas and a member of bEBP, which is used to regulate the σ 54 P-dependent promoter bmo , the signal molecule is a straight-chain or branched alcohol of C2-C5. However, the wild-type transcription factor BmoR has poor response specificity. It responds to a variety of higher alcohols such as n-pentanol, isopentanol, n-butanol, isobutanol, etc., but cannot specifically respond to one of the alcohols and produce a high response value to meet industrial needs; and the detection range is narrow, so it cannot be widely used in biosensors. Therefore, achieving a specific response to alcohol and improving the detection range has become an urgent problem to be solved.

[0006] The BmoR protein mutant obtained by protein modification in the present invention can achieve a sensitive response to phenylethanol, and also improves its substrate detection upper limit and response intensity, providing a solution for efficient detection of phenylethanol and rapid screening of high-yield strains. Summary of the invention:

[0007] The purpose of the present invention is to provide a BmoR protein and a biosensor and application that can achieve sensitive response to phenylethanol. A random mutation library is constructed using error-prone PCR technology, and the mutation library is screened and analyzed by exogenous addition of phenylethanol, and finally a BmoR mutant protein that is sensitive to phenylethanol is obtained.

[0008] Furthermore, the BmoR mutant protein that is sensitive to phenylethanol is obtained by subjecting the wild-type BmoR protein shown in SEQ ID NO.1 to S184T mutation, hereinafter referred to as S184T mutant, and the mutant protein is specifically:

[0009] (1) the amino acid sequence shown in SEQ ID NO.3 in the sequence listing; or

[0010] (2) an amino acid sequence with a homology of more than 75% to SEQ ID NO. 3; or

[0011] (3) An amino acid sequence having the same function as SEQ ID NO. 3 obtained by substituting, deleting, and / or adding one or more amino acids to SEQ ID NO. 3.

[0012] Furthermore, the present invention also provides a gene encoding the S184T mutant;

[0013] Furthermore, the coding gene is shown in the sequence listing SEQ ID NO.4.

[0014] Another object of the present invention is to provide an application of the S184T mutant, particularly in detecting samples containing phenylethanol or screening phenylethanol-producing strains, and more particularly in constructing a biosensor for detecting phenylethanol.

[0015] Furthermore, the biosensor is a biosensor based on the S184T mutant, and the biosensor comprises a gene encoding the S184T mutant and its promoter, a promoter P bmo and reporter gene expression elements; the promoter activates the expression of the bmoR gene, and the BmoR protein binds to the alcohol molecule to form a hexamer, which in turn activates the downstream promoter P bmo , thereby expressing the reporter gene and generating signals such as fluorescence; the biosensor can achieve high fluorescence response and screening for phenylethanol at a concentration of 0-30mM, and is further applied in industrial production to achieve high fluorescence response value screening for samples containing phenylethanol and phenylethanol-producing strains;

[0016] Furthermore, the promoter of the mutant encoding gene includes but is not limited to P bmoR , P tac , P T7 , P LlacO1 wait;

[0017] Furthermore, the reporter gene includes but is not limited to genes such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc.;

[0018] Preferably, the biosensor comprises a gene encoding the S184T mutant and its promoter P bmoR , promoter P bmo and a recombinant plasmid of a gfp reporter gene; further, the expression vector that can be used for the recombinant plasmid includes but is not limited to the expression vector commonly used in the art;

[0019] More preferably, the biosensor is obtained by replacing the wild-type BmoR protein encoding gene on the plasmid pYH1 with the S184T mutant encoding gene, that is, replacing the wild-type BmoR protein encoding gene on the plasmid pYH1 with the S184T mutant encoding gene. bmoR The S184T mutant encoding gene is linked to the colE1 replication origin, amp r and P bmo The driven gfp gene is obtained;

[0020] Furthermore, the phenylethanol producing strains include but are not limited to Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc.;

[0021] Furthermore, the promoter P bmoR The nucleotide sequence is shown in the sequence listing SEQ ID NO.5;

[0022] Furthermore, the promoter P bmo The nucleotide sequence is shown in the sequence listing SEQ ID NO.6;

[0023] Furthermore, the nucleotide sequence of the gfp reporter gene is shown in the sequence listing SEQ ID NO.7.

[0024] The present invention also provides the use of the above-mentioned biosensor in the detection of phenylethanol, especially in the use of environmental, food, medical, biological samples containing phenylethanol, or in the screening of phenylethanol-producing strains, by introducing the above-mentioned plasmid into a production strain, such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc., or a separate host, such as E. coli, etc., to detect the production of phenylethanol.

[0025] Beneficial effects:

[0026] 1. The BmoR-based biosensor can be used to screen high-yield phenylethanol strains, but the wild-type BmoR has no response to phenylethanol. The S184T mutant provided by the present invention has a higher response value to phenylethanol (0-30mM), which solves the problem that the wild-type BmoR protein cannot sensitively respond to phenylethanol.

[0027] 2. The wild-type BmoR has no response to phenylethanol and therefore cannot be used to identify strains that produce phenylethanol. The S184T mutant provided by the present invention has a detection range of 0-30 mM for phenylethanol, which makes the BmoR protein sensitive to phenylethanol and improves the response saturation of the BmoR protein to phenylethanol, and can be used for the screening and application of high-level phenylethanol production strains. Description of the drawings:

[0028] Figure 1 For technology roadmap;

[0029] First, the first 1000bp of the N-terminus of the wild-type bmoR was randomly mutated by error-prone PCR to obtain a random mutation library of BmoR. GFP fluorescent protein was added downstream of the bmoR gene, and the response of the mutant BmoR to n-butanol was reflected by detecting the fluorescence intensity. The response of the mutant BmoR to alcohol molecules was detected by adding different concentrations of n-pentanol.

[0030] Figure 2 The responses of BmoR mutant and wild type to phenylethanol;

[0031] Figure 3 Fluorescence images of the BmoRS184T mutant and wild type. Specific implementation method:

[0032] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0033] The biosensor provided by the present invention is a biosensor based on the S184T mutant, wherein the biosensor comprises a gene encoding the S184T mutant and its promoter, a promoter P bmo and reporter gene expression elements; the promoter activates the expression of the bmoR gene, and the BmoR protein binds to the alcohol molecule to form a hexamer, which in turn activates the downstream promoter P bmo , thereby expressing the reporter gene and generating fluorescence and other signals. The skilled person in the art can select a promoter to start the expression of the BmoR mutant gene in the prior art according to the actual situation, such as using P bmoR , P tac , P T7 , P LlacO1 The reporter gene can also be selected in many ways. Protein molecules commonly used in the art that can generate visual detection signals or detectable small molecules, such as fluorescent proteins, color proteins, etc., can achieve the response of the biosensor of the present invention, preferably, such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc. The above sensor also contains necessary elements for expression such as replication initiation sites, preferably, such as colE1 replication initiation sites, etc. The above sensor can also contain resistance genes and other markers, such as amp rThose skilled in the art can also add other elements to the above sensor according to actual needs, such as constructing the above elements into expression vectors in the prior art, such as pET, pUC19, pMAL, etc., to obtain a recombinant plasmid that can be used as a sensor.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] The present invention will be further explained below through specific examples.

[0036] Example 1 Design and screening process of BmoR mutant S184T

[0037] Construction of a random mutation library for the transcription factor BmoR

[0038] (1) Plasmid pYH1 (construction see DOI: https: / / doi.org / 10.1016 / j.ymben.2019.08.015; https: / / doi.org / 10.1186 / s12934-019-1084-2) containing the wild-type transcription factor BmoR encoding gene (shown in SEQ ID NO. 2) was used as a template to obtain the bmoR mutant gene by error-prone PCR amplification (by adding Mn 2+ , increase the Mg content in the PCR system 2+ The concentration was adjusted and the ratio of dNTPs was adjusted to introduce random mutations. A 10× unbalanced dNTPs mixture was prepared, in which the concentrations of dCTP and dTTP were four times that of dATP and dGTP. The PCR program was set as follows: 94°C pre-denaturation for 2 minutes, 30 amplification cycles including: 95°C denaturation for 1 minute, 55-68°C annealing for 1 minute, and the appropriate extension time was determined according to the amplification rate of 1 kb per minute, and the extension temperature was 72°C. The storage temperature was set to 16°C. ); The PCR product was confirmed by gel electrophoresis and recovered and purified; the purified product was placed in a 37°C water bath and digested with DpnI (1μL / 50μL purified product) for 1-2h. Take 2μL of the bmoR mutant fragment, 3μL of the pYH1 backbone (a construct with P bmoR , P bmo, gfp fluorescent protein gene plasmid as the backbone, equivalent to the pYH1 backbone effect) and 5μL Gibson Assemble Mix, after mixing, place in a 50℃ water bath for 1h, take 10μL of the ligation product and transfer it into 50μL E. coli XL10-Gold competent cells. After 1h incubation, inoculate it into 4mL liquid LB, incubate it at 37℃, 220rpm constant temperature shaker for 1h, add 5μL 50mg / mL carbenicillin after 1h, and culture overnight. After obtaining the seed solution, take 50μL and transfer it to 5mL fresh LB liquid culture medium (50μg / mL), add 10mM phenylethanol at the same time, cover the tube tightly, and induce it at 30℃, 220rpm constant temperature shaker for 8-10h to obtain the library induced sample S1. The library induced sample S1 was screened continuously for two rounds by flow cytometry to obtain the enriched excellent BmoR mutants.

[0039] (2) Rapid screening of BmoR mutant library based on flow cytometry.

[0040] After obtaining the induced BmoR mutant library samples, two rounds of continuous screening were performed by flow cytometry (FACS). First, by analyzing the samples induced by the wild-type BmoR sensor system and the control system, the location of the fluorescence peak (P1) and the appropriate sorting voltage were determined; based on this, the mutant library samples were sorted, and the first round of collecting the first 1% of the bacteria in the P3 area (about 1000) was collected and collected in 1mL of antibiotic-free liquid LB medium; after the collection was completed, 4mL of antibiotic-free liquid LB medium was added to the collected bacterial liquid, incubated on a shaking table at 37℃ for 1h, and 5μL of ampicillin antibiotics were added after 1h, and cultured overnight. The reason for incubating first and then adding ampicillin antibiotics is that the bacterial cells screened by flow cytometry are relatively fragile, and they should be incubated for 1h to restore their vitality to avoid premature addition of antibiotics to cause bacterial cell death. The reason for adding antibiotics is to maintain environmental pressure and avoid loss of the target plasmid. After obtaining the bacterial library after the first round of enrichment, the same induction method was adopted for the second round of induction and FACS sorting. The second round of sorting was based on the fluorescence peak offset of the first round of sorting, and the first 1% of the bacterial cells in the P3 region (about 10,000) were collected. After the collection was completed, 4 mL of anti-free liquid LB medium was added to the collected bacterial solution, and the culture was incubated at 37°C for 1 hour. After the incubation, 200 μL of bacterial solution was first applied to the plate containing antibiotics (Amp), and the plate was inverted and placed in a 37°C constant temperature box for overnight culture after the sealing film was fixed; then the corresponding amount of ampicillin antibiotics was added to the remaining liquid sample, and the plate was placed in a constant temperature shaker at 37°C for overnight culture. Ten single colonies obtained after the second round of sorting were randomly selected for colony PCR and sequencing to verify the construction effect of the mutant library, analyze the mutation rate of the library after sorting, and estimate the enrichment of excellent mutants and the effect of the second round of sorting.

[0041] (3) Validation of the BmoR mutation library based on 96-deep-well plates.

[0042] Randomly pick a suitable amount of single colonies (the specific number is determined by the number of single colonies on the plate) from the plate obtained after the second round of sorting, inoculate it into 3mL LB (100μg / mL Amp) liquid culture medium, and use a constant temperature shaker to culture for 8h at 37℃ and 220rpm as seed liquid. Take 100mL of fresh liquid LB culture medium, add 100μL of ampicillin antibiotics and 100μL of n-propanol, isopropanol, 2-phenylethanol or 3-methyl-1-butanol, respectively, and mix well. Take a sterilized 2mL 96 deep-well plate, first use a pipette to add 950μL of LB culture medium with antibiotics and alcohol byproducts to each well, and then add 50μL of seed liquid respectively. Make three replicates for each alcohol byproduct. Immediately cover the sealing film after adding, and place the deep-well plate in a shaker at 30℃ and 220rpm for 16h. Pipette 200 μL of bacterial solution into a black 96-well plate and measure the fluorescence intensity and OD using an ELISA reader. 600 The quantitative detection of green fluorescent protein GFP uses an excitation wavelength of 470nm and an emission wavelength of 510nm, with a gain value of 50. Each well is measured three times to obtain the average value. 600 The value of the control was first subtracted, and the GFP / OD of each well was calculated based on this. 600 As the relative fluorescence intensity value. Each alcohol was measured as GFP / OD 600 The ordinate is the concentration and the abscissa is the concentration. The graph was drawn using GraphPad Prism 8.0.2 software. 600 For strains with a higher expression than the wild type, colony PCR was performed and sequencing was performed to check whether there were mutation sites in BmoR.

[0043] (4) Verification of the BmoR mutation library based on 96-deep-well plates.

[0044] The other single colonies on the plate were screened and verified at 10mM. The specific operation is as follows: Randomly pick an appropriate number of single colonies from the plate obtained after the second round of sorting (the specific number is determined by the number of single colonies on the plate), inoculate them into 3mL LB (100μg / mL Amp) liquid culture medium, and use a constant temperature shaker to culture at 37°C, 220rpm for 8h as seed liquid. Take 100mL of fresh liquid LB culture medium, add 100μL of ampicillin antibiotics and 100μL of 2-phenylethanol respectively, and mix well. Take a sterilized 2mL 96 deep-well plate, first use a pipette to add 950μL of LB culture medium with antibiotics and alcohol byproducts to each well, and then add 50μL of seed liquid respectively, and make three replicate groups. Immediately cover the sealing film after the addition is completed, and place the deep-well plate in a shaker at 30°C, 220rpm for 16h. Pipette 200μL of bacterial solution into a black 96-well plate, and use an ELISA reader to measure the fluorescence intensity and OD respectively 600 The quantitative detection of green fluorescent protein GFP uses an excitation wavelength of 470nm and an emission wavelength of 510nm, with a gain value of 50. Each well is measured three times to obtain the average value. 600 The value of the control was first subtracted, and the GFP / OD of each well was calculated based on this. 600 As the relative fluorescence intensity value. 600 The ordinate is the concentration and the abscissa is the concentration. The graph was drawn using GraphPad Prism 8.0.2 software. 600 For strains with a higher expression than the wild type, colony PCR was performed and sequencing was performed to check whether there were mutation sites in BmoR.

[0045] Example 2 Concentration gradient experiment to determine the change in detection limit of wild type and mutant

[0046] The wild-type BmoR-based biosensor does not respond to phenylethanol, cannot respond to phenylethanol molecules in the substrate, and cannot respond to higher concentrations of alcohol molecules. The following experiments verify that the BmoR biosensor based on the S184T mutant has a sensitive response to 0-30mM phenylethanol.

[0047] The strain containing the S184T mutation was subjected to an exogenous addition experiment of phenylethanol at a concentration gradient, the response curve was determined, and K was calculated. m , response intensity value and other parameters.

[0048] A single clone on the plate was picked and inoculated into 5 mL LB (100 μg / mL Amp) liquid medium, and cultured at 37°C and 220 rpm for 8 h as seed solution.

[0049] The exogenous addition experiment was conducted in a sterilized 2 mL 96-well plate. Each well was first added with 950 μL of fresh LB (100 μg / mL Amp) medium, and then phenylethanol was added to the medium to make their final concentrations 0, 1, 10, 20, and 30 mM, respectively. Finally, 50 μL of seed solution was inoculated into each well, and after sealing the sealing film, the deep-well plate was placed at 30°C and 220 rpm in a shaker for 16 h.

[0050] Use microplate to measure fluorescence intensity GFP and OD 600 Detection: Mix the bacterial solution by blowing, draw 200 μL into the microplate reader, and perform quantitative detection at 30°C. Set the parameters as follows: 470 nm excitation wavelength and 510 nm emission wavelength, with a gain value of 50. The obtained GFP and OD 600 The background control value was first subtracted from the value, and the GFP / OD value of each well was calculated based on this. 600 As relative fluorescence intensity values.

[0051] GFP / OD 600 The data were fitted with Michaelis-Menten fitting, and the K of BmoR mutants to phenylethanol was calculated based on the fitting results. m , maximum response intensity and other parameters ( Figure 2 ).

[0052] Under the condition of 0-30mM gradient concentration, the mutant still maintained the response to phenylethanol under different gradient concentrations of phenylethanol. The K of wild-type BmoR to phenylethanol was plotted using GraphPad 9.0. m The K of mutant S184T to phenylethanol was 0 mM. m K m As a representation of affinity, K m The larger the value, the smaller the affinity; K m The smaller the value, the greater the affinity.

[0053] The wild-type BmoR cannot respond to toxic phenylethanol, so the wild-type BmoR cannot detect phenylethanol in the substrate; the calculation results show that the mutant has a significantly increased Km value for phenylethanol compared to the wild type, proving that the BmoR mutant can achieve a specific response to higher concentrations of phenylethanol (0-30mM), which is consistent with the experimental results.

[0054] Example 3: Bright field and dark field microscopes further verify the experimental results

[0055] In order to further verify the high response to phenylethanol, whether there is a difference in the response of the wild type and S184T mutant to phenylethanol under different light and dark field conditions. Fluorescence photos of the wild type, control and mutant with and without exogenous addition of alcohol molecules were taken using a fluorescence microscope; there was almost no fluorescence when no alcohol byproducts were added, and there was still no fluorescence after adding phenylethanol, indicating that the biosensor system regulated by the wild-type BmoR did not respond to phenylethanol. The constructed background control showed no fluorescence under the conditions of adding or not adding phenylethanol, indicating that it can be used as a background control during the expression of the biosensor system regulated by the wild-type BmoR. In contrast to the wild type, which had no fluorescence response to phenylethanol, obvious fluorescence was observed in the mutant after adding phenylethanol, indicating that the screened mutant can effectively respond to phenylethanol. ( Figure 3 ).

[0056] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A BmoR mutant protein, characterized in that: The mutant is obtained by causing a S184T mutation on the wild-type BmoR protein shown in SEQ ID NO.

1.

2. The BmoR mutant protein according to claim 1, characterized in that The mutant protein is specifically: (1) the amino acid sequence shown in SEQ ID NO.3 in the sequence listing; or (2) an amino acid sequence with a homology of more than 75% to SEQ ID NO. 3; or (3) An amino acid sequence having the same function as SEQ ID NO. 3 obtained by substituting, deleting, and / or adding one or more amino acids to SEQ ID NO.

3.

3. Use of the BmoR mutant according to claim 1 in detecting samples containing phenylethanol or in screening phenylethanol-producing strains.

4. Use of the BmoR mutant according to claim 1 in constructing a biosensor for detecting phenylethanol.

5. The gene encoding the BmoR mutant of claim 1.

6. The coding gene according to claim 5, characterized in that As shown in the sequence listing SEQ ID NO.

4.

7. A recombinant plasmid or recombinant strain comprising the coding gene according to claim 5.

8. A biosensor, characterized in that: The sensor comprises a gene encoding the mutant of claim 5 and a promoter P activated by the BmoR mutant. bmo , by P bmo Expression elements of the promoter driving the reporter gene and expressing the BmoR mutant.

9. The biosensor according to claim 8, wherein The reporter genes include but are not limited to gfp, rfp, cfp, sfgfp, egfp, yfp, and ecfp genes.

10. The biosensor according to claim 8, wherein The promoters expressing BmoR mutants include, but are not limited to, P bmoR , P tac , P T7 , P LlacO1 .

11. The biosensor according to claim 8, wherein The sensor is to be bmoR The mutant encoding gene was linked to the colE1 replication origin, amp r and P bmo The gfp gene was activated.

12. The biosensor according to claim 8, wherein Promoter P bmo The nucleotide sequence is shown in the sequence listing SEQ ID NO.

6.

13. Use of the biosensor according to claim 8 in detecting environmental, food, medical and biological samples containing phenylethanol, and in screening strains producing phenylethanol.

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