A biosensor based on bmo r mutant with high sensitivity

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a BmoR protein mutant capable of sensitively detecting n-butanol or isobutanol, and its application in n-butanol or isobutanol detection or biosensors. The BmoR protein mutant is obtained by mutating the wild-type BmoR protein shown in SEQ ID NO.1 to V311A. The V311A mutant, possessing the amino acid sequence shown in SEQ ID NO.3, is more sensitive to n-butanol (2.13 × 10⁻⁶). ‑6 mM) and isobutanol (1.94×10) ‑6 The method uses mM) detection to solve the problem of low sensitivity of wild-type BmoR protein, which cannot screen for extremely low concentrations of n-butanol or isobutanol. It can be used for screening and application of strains that produce low levels of n-butanol or isobutanol.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a BmoR mutant capable of sensitively detecting extremely low concentration of n-butanol or isobutanol and application thereof in n-butanol or isobutanol detection or biosensor. BACKGROUND

[0002] Microbially synthesized n-butanol or isobutanol is an important transportation fuel, and n-butanol or isobutanol synthesized through metabolic engineering has been realized in many microbial hosts, and the modification of host strains and the screening of high-yield or low-yield hosts are the basis and key to the industrial production of n-butanol or isobutanol. Biosensors can specifically respond to target compounds to output protein signals convenient for detection, and have been widely used in high-throughput screening. However, the wild-type transcription factor BmoR cannot be widely used in biosensors and industrial production due to its poor response specificity, narrow detection range and low sensitivity. The BmoR protein obtained by protein modification in the present application realizes the sensitive detection of n-butanol or isobutanol, thereby meeting the needs of industrialization.

[0003] As a new generation of biofuels, n-butanol or isobutanol is used in many fields, and has been synthesized in many microbial hosts. While screening for high-yield n-butanol or isobutanol production strains, research has not focused on the detection of extremely low concentration of n-butanol or isobutanol. The minimum response concentration of wild-type transcription factor BmoR to substrate n-butanol or isobutanol (n-butanol or isobutanol) is 0.001 mM, which cannot detect lower concentration of alcohol molecules, so it is a big problem to realize the sensitive detection of alcohol.

[0004] Biosensors are composed of molecular recognition elements and signal transducers. When the molecular recognition element binds to the measured object, the generated signal can be converted into an optical signal or an electrical signal by the transducer, and the measured object can be detected and analyzed. As an emerging tool of synthetic biology, biosensors can be designed and constructed to dynamically respond to changes in signal molecule concentration. At the same time, biosensors are designed to facilitate the optimization of microbial cell factories and the production of a series of widely used industrial natural products, such as itaconic acid, fatty acids, isobutanol, n-butanol 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 extracellular performance of G protein-coupled receptors hinder the development of biosensors in the biological world.

[0005] Transcription factor (TF)-based biosensors are the most widely used. The most commonly used transcription factors are bacterial transcription factors, including ligand-binding domains (LBDs) or metabolic-binding domains (MBDs) and DNA-binding domains (DBDs). BmoR is a transcription factor in the Pseudomonas positive-chain alkane metabolic pathway, a member of the bEBP family, and is used to regulate the σ-chain activity of alkane monooxygenases. 54 Dependency promoter P bmo The signaling molecule is a C2-C5 straight-chain or branched alcohol. BmoR-based biosensors can be used to screen strains that produce high or low yields of n-butanol or isobutanol. However, wild-type BmoR biosensors have poor response specificity, a narrow detection range, and low sensitivity, making them unsuitable for industrial production. Therefore, modifying the biosensor provides a solution for the sensitive detection of n-butanol or isobutanol production and rapid strain screening. Summary of the Invention:

[0006] The purpose of this invention is to provide a BmoR protein and biosensor capable of sensitively detecting extremely low concentrations of n-butanol or isobutanol. A random mutant library was constructed using error-prone PCR technology. The library was then screened and analyzed by exogenously adding n-butanol or isobutanol to a final concentration of 0-1 mM. Ultimately, a BmoR mutant protein capable of sensitively detecting extremely low concentrations of n-butanol or isobutanol was obtained.

[0007] Furthermore, the BmoR mutant protein capable of detecting 0-1 mM n-butanol or isobutanol is obtained by a V311A mutation on the wild-type BmoR protein shown in SEQ ID NO.1, hereinafter referred to as the V311A mutant. The mutant protein specifically has the amino acid sequence shown in SEQ ID NO.3.

[0008] Furthermore, the present invention also provides the coding gene for the V311A mutant;

[0009] Furthermore, the encoding gene is shown in the sequence listing SEQ ID NO.4.

[0010] Another object of the present invention is to provide applications of the V311A mutant, particularly in sensors for the sensitive detection of samples containing n-butanol or isobutanol, or for screening n-butanol or isobutanol producing strains, and more particularly in the construction of biosensors for n-butanol or isobutanol.

[0011] Furthermore, the biosensor is a V311A mutant-based biosensor, comprising the V311A mutant coding gene and its promoter, promoter P. bmoand a reporter gene expression element; the promoter initiates the expression of the bmoR gene, the BmoR protein binds with alcohol molecules to form a hexamer, which in turn initiates the downstream promoter P bmo , thereby expressing the reporter gene and generating signals such as fluorescence; the biosensor can be used to respond to and screen 0-1 mM n-butanol or isobutanol, and further applied to industrial production to screen samples containing n-butanol or isobutanol and n-butanol or isobutanol producing strains.

[0012] Further, the promoter of the mutant coding gene includes but is not limited to P bmoR , P tac , P T7 , P LlacO1 , etc.

[0013] Further, the reporter gene includes but is not limited to gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc.

[0014] Preferably, the expression element is a recombinant plasmid containing the V311A mutant coding gene, its promoter P bmoR , the promoter P bmo and the gfp reporter gene; further, the expression vector that can be selected for the recombinant plasmid includes but is not limited to commonly used expression vectors in the art.

[0015] More preferably, the biosensor is obtained by replacing the wild-type BmoR protein coding gene on the plasmid pYH1 with the V311A mutant coding gene, that is, connecting the V311A mutant coding gene driven by P bmoR to the colE1 replication initiation site, amp r and the gfp gene driven by P bmo .

[0016] Further, the strain includes but is not limited to Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc.

[0017] Further, the nucleotide sequence of the promoter P bmo is shown in the sequence table SEQ ID NO. 5.

[0018] Further, the nucleotide sequence of the promoter P bmo is shown in the sequence table SEQ ID NO. 6.

[0019] Further, the nucleotide sequence of the gfp reporter gene is shown in the sequence table SEQ ID NO. 7.

[0020] The application also provides the application of the above-mentioned biosensor in the detection of n-butanol or isobutanol, especially in the screening of isobutanol-producing strains, by introducing the above-mentioned plasmid into a production strain, such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc., to detect the production of isobutanol.

[0021] Beneficial effects:

[0022] The detection sensitivity of the wild-type BmoR is too low, and the minimum detection concentration of n-butanol or isobutanol is only 0.001 mM, and at this concentration, the response value of n-butanol or isobutanol is too low, and there is almost no response to n-butanol or isobutanol below 0.001 mM, which cannot be further detected, so it cannot be used to identify strains with n-butanol or isobutanol production below 0.001 mM. The minimum detection limit of the V311A mutant provided by the application for n-butanol reaches 2.13 x 10 -6 mM, which is 500 times higher than that of the wild type; the minimum detection limit for isobutanol reaches 1.94 x 10 -6 mM, which is 1000 times higher than that of the wild type, thereby improving the detection sensitivity of the BmoR protein to n-butanol or isobutanol. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a principle flowchart;

[0024] First, the N-terminal 1000 bp of the wild-type bmoR is randomly mutated by error-prone PCR to obtain a random mutation library of BmoR; a GFP fluorescent protein is added downstream of the bmoR gene, and the response of the mutant BmoR to n-butanol or isobutanol can be reflected by detecting the fluorescence intensity. By adding n-butanol or isobutanol of different concentrations, the response of the mutant BmoR to n-butanol or isobutanol is detected.

[0025] Figure 2 It is the response of the BmoR mutant and the wild type to 10 mM n-butanol or isobutanol;

[0026] Figure 3 It is the response of the BmoR mutant and the wild type to 0-1 mM n-butanol or isobutanol;

[0027] Figure 4 It is the molecular docking of the V311A mutant with n-butanol and isobutanol; DETAILED DESCRIPTION

[0028] The present application will be described in detail below through specific embodiments. Unless otherwise specified, the technical means used in the present application are methods known to those skilled in the art. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the present application, the essence and scope of which are defined only by the claims. For those skilled in the art, various changes or modifications to the composition and amount of the materials in these embodiments without departing from the essence and scope of the present application also fall within the scope of the present application.

[0029] The biosensor provided by the present application is a biosensor based on the V311A mutant, which comprises the expression element of the V311A mutant encoding gene and its promoter, the promoter P bmo and the reporter gene; the promoter initiates the expression of the bmoR gene, the BmoR protein binds with alcohol molecules to form a hexamer, which in turn initiates the downstream promoter P bmo , thereby expressing the reporter gene and producing signals such as fluorescence. Those skilled in the art can select promoters to initiate the expression of the BmoR mutant gene in the prior art according to actual conditions, such as P bmoR , P tac , P T7 , P LlacO1 , etc. The reporter gene can also have a variety of choices. Commonly used protein molecules that can produce detection signals in the art, such as fluorescent proteins, color proteins, etc. can all achieve the response of the biosensor described in the present application, preferably, such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc. The above-mentioned sensor also comprises a replication origin and other necessary elements for expression, preferably, such as colE1 replication origin, etc. The above-mentioned sensor can also comprise a marker such as amp r , etc. for easy screening. Those skilled in the art can also add other elements to the above-mentioned sensor according to actual needs, such as constructing the above-mentioned elements to the expression vectors in the prior art, such as pET, pUC19, pMAL, etc. to obtain recombinant plasmids that can be used as sensors.

[0030] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0031] The present application will be further explained and described below through specific embodiments.

[0032] Example 1 Screening of BmoR mutant V311A

[0033] Construction of random mutation library of transcription factor BmoR

[0034] (1) Using plasmid pYH1 (construction details: DOI: https: / / doi.org / 10.1016 / j.ymben.2019.08.015; https: / / doi.org / 10.1186 / s12934-019-1084-2) carrying the wild-type BmoR coding gene (SEQ ID NO.2) as a template, the bmoR mutant gene was amplified by error-prone PCR (by adding Mn to the PCR system). 2+ Increase the amount of Mg in the PCR system 2+ The concentration and ratio of dNTPs were 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℃ pre-denaturation for 2 min, 30 amplification cycles including: 95℃ denaturation for 1 min, 55-68℃ annealing for 1 min, and an appropriate extension time determined according to an amplification rate of 1 kb per minute, with an extension temperature of 72℃. The storage temperature was set to 16℃. The PCR product was confirmed by gel electrophoresis and purified. The purified product was placed in a 37℃ water bath and digested with DpnI (1 μL / 50 μL of purified product) for 1-2 h. 2 μL of the bmoR mutant fragment and 3 μL of the pYH1 backbone (or a pYH1 backbone constructed with P) were added. bmoR P bmo Using a plasmid of the GFP fluorescent protein gene as a backbone (equivalent to the pYH1 backbone) and 5 μL of Gibson Assemble Mix, the mixture was placed in a 50°C water bath for 1 hour for ligation. 5-10 μL of the ligation product was then transformed into 50 μL of E. coli XL10-Gold-transfected competent cells and cultured overnight at 37°C to obtain the BmoR-1000bp mutant library.

[0035] (2) Pick a single colony from the plate and inoculate it into 5 mL of LB (100 μg / mL Amp) liquid medium. Incubate at 37°C and 220 rpm for 8 h to obtain the seed culture. Use a 96-well plate for initial screening. Add 950 μL of fresh LB (100 μg / mL Amp) medium to each well; add n-butanol or isobutanol to each well to a final concentration of 10 mM; finally, inoculate 50 μL of the seed culture into each well. After sealing with a sealing film, place the plate in a shaker at 30°C and 220 rpm for 16 h.

[0036] (3) Using microplates to measure fluorescence intensity GFP and OD 600 Detection: Mix the bacterial culture by pipetting, take 200 μL and place it in a microplate reader. Quantitative detection is performed at 30℃. The parameters are set as follows: excitation wavelength 470 nm, emission wavelength 510 nm, and gain value 50. The obtained GFP and OD... 600The values were first subtracted by the background control values, and then the GFP / OD of each well was calculated 600 as the relative fluorescence intensity value.

[0037] (4) After analyzing the primary screening results, the plasmids of the effective mutant bacteria were sequenced.

[0038] It was determined that one of the amino acid mutation sites of the BmoR protein was V311A (amino acid sequence as SEQ ID NO. 3, nucleotide sequence as SEQ ID NO. 4), and the GFP / OD of the mutant was 600 The primary screening results are shown in Table 1 and Figure 2 It can be seen that, compared with the wild-type BmoR, the V311A mutant has a higher response to 10 mM n-butanol and isobutanol.

[0039] Table 1 GFP / OD 600

[0040] n-butanol isobutanol WT 983 868 V311A 2613 3048

[0041] Example 2 Concentration gradient experiment to determine the detection limit change of wild-type and mutant

[0042] The wild-type BmoR biosensor can respond to 0-1 mM n-butanol or isobutanol, and responds to both n-butanol and isobutanol; when the substrate concentration is lower than 0.001 mM, the response value is almost zero, and it cannot respond to lower concentration of alcohol molecules. The following experiments verify that the BmoR biosensor based on the V311A mutant has a high sensitivity response to 0-1 mM n-butanol or isobutanol.

[0043] Based on the primary screening results, the strain containing the V311A mutation was subjected to exogenous addition of n-butanol or isobutanol with a concentration gradient, and the response curve, the minimum response concentration and the response intensity value were determined.

[0044] Single colonies on the plate were picked and inoculated into 5 mL of LB (100 μg / mL Amp) liquid medium, and incubated at 37°C, 220 rpm for 8 h as seed liquid.

[0045] The exogenous addition experiment was carried out in sterilized 2 mL 96 deep-well plates. 950 μL of fresh LB (100 μg / mL Amp) medium was added to each well, and then n-butanol or isobutanol was added to the medium to make their final concentrations 0, 1 × 10 -6 , 1 × 10 -5 , 1 × 10 -4 , 1 × 10 -3 , 1 × 10 -2 , 1 × 10 -1Or 1 mM, and finally 50 μL of the seed liquid was inoculated into each well. After the cover film was sealed, the deep-well plate was placed in a 30 °C shaker at 220 rpm for 16 h.

[0046] The fluorescence intensity GFP and OD of the micro-well plate were measured 600 The bacterial liquid was mixed by blowing, 200 μL was taken and placed in an enzyme marker, and quantitative detection was performed at 30 °C. The parameters were set as follows: 470 nm excitation wavelength and 510 nm emission wavelength, and the gain value was 50. The obtained GFP and OD 600 values were first subtracted by the background control values, and then the GFP / OD 600 of each well was calculated as the relative fluorescence intensity value.

[0047] The GFP / OD 600 was taken as the vertical coordinate, and the final concentration of n-butanol and isobutanol was taken as the horizontal coordinate, and the data were plotted by OriginPro 8.5 or GraphPad Prism 8 software. The minimum detection concentration of BmoR to n-butanol or isobutanol was defined as the substrate concentration when the maximum fluorescence response value reached 75%. According to the fitting results, the minimum detection concentration of wild-type BmoR and the mutant to n-butanol and isobutanol, the maximum response intensity and other parameters were calculated. Figure 3

[0048] At a substrate concentration of 10 mM, the mutant library was screened, and compared with the wild type, the obtained mutant V311A had a very high fluorescence response to n-butanol and isobutanol, and the response value to n-butanol reached 2.67 times that of the wild type; the response value to isobutanol reached 3.51 times that of the wild type; further verification was performed at a gradient concentration of 0-1 mM, and the results showed that the response of the V311A mutant to n-butanol and isobutanol remained at a high level at a substrate concentration of 0-1 mM. The data were plotted by OriginPro 8.5, and the minimum detection concentration of wild-type BmoR and the mutant to n-butanol or isobutanol was calculated by Miichaelis fitting. The results showed that the minimum detection concentration of wild-type BmoR to n-butanol was 1.36 x 10 -3 mM, and the minimum detection concentration to isobutanol was 2.53 x 10 -3 mM; the minimum detection concentration of the V311A mutant to n-butanol reached 2.13 x 10 -6 mM, which was 500 times higher than that of the wild type; the minimum detection concentration to isobutanol reached 1.94 x 10 -6 mM, which was 1000 times higher than that of the wild type.

[0049] Example 3 Model Analysis

[0050] ​The V311A mutant was sequenced to analyze the change of amino acids at the mutation site, and AUTODOCK and ChimeraX software were used to model the BmoR mutant, and the mutant was docked with the substrate small molecules n-butanol and isobutanol, respectively, to analyze the binding site of the mutant and the formation of hydrogen bonds with the two alcohols.

[0051] The V311A mutant was modeled using the three-dimensional structure of the wild-type BmoR protein as a template, with a homology rate of 99.9%. Further molecular docking of the mutant structure with the substrate molecules (n-butanol or isobutanol) was performed. The results showed that in the complex, the mutant formed 2 hydrogen bond interactions with n-butanol (Arg211, Gln212); and 2 hydrogen bond interactions with isobutanol (Asn259, Leu260), indicating that n-butanol and isobutanol can be tightly bound to the mutant, which is consistent with the experimental results. Figure 4 ).

[0052] The above-described embodiments only express several embodiments of the present application, which are described in detail and in detail, but should not be construed as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, the above-mentioned embodiments can be made in several ways, combinations and improvements without departing from the concept of the present patent, which are within the scope of protection of the present patent. Therefore, the scope of protection of the present patent should be subject to the claims. SEQUENCE LISTING <110> Beijing University of Technology <120> A high-sensitivity biosensor based on BmoR mutant <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 669 <212> PRT <213> Pseudomonas butanovora <400> 1 Met Ser Lys Met Gin Glu Phe Ala Arg Leu Glu Thr Val Ala Ser Met 1 5 10 15 Arg Arg Ala Val Trp Asp Gly Asn Glu Cys Gin Pro Gly Lys Val Ala 20 25 30 Asp Val Val Leu Arg Ser Trp Thr Arg Cys Arg Ala Glu Gly Val Val 35 40 45 Pro Asn Ala Arg Gin Glu Phe Asp Pro lie Pro Arg Thr Ala Leu Asp 50 55 60 Glu Thr Val Glu Ala Lys Arg Ala Leu lie Leu Ala Ala Glu Pro Val 65 70 75 80 Val Asp Ala Leu Met Glu Gin Met Asn Asp Ala Pro Arg Met lie lie 85 90 95 Leu Asn Asp Glu Arg Gly Val Val Leu Leu Asn Gin Gly Asn Asp Thr 100 105 110 Leu Leu Glu Asp Ala Arg Arg Arg Ala Val Arg Val Gly Val Cys Trp 115 120 125 Asp Glu His Ala Arg Gly Thr Asn Ala Met Gly Thr Ala Leu Ala Glu 130 135 140 Arg Arg Pro Val Ala lie His Gly Ala Glu His Tyr Leu Glu Ser Asn 145 150 155 160 Thr lie Phe Thr Cys Thr Ala Ala Pro lie Tyr Asp Pro Phe Gly Glu 165 170 175 Phe Thr Gly lie Leu Asp lie Ser Gly Tyr Ala Gly Asp Met Gly Pro 180 185 190 ​​​​​​​​​​Val Pro lie Pro Phe Val Gin Met Ala Val Gin Phe lie Gin Asn Gin 195 200 205 Leu Phe Arg Gin Thr Phe Ala Asp Cys lie Leu Leu His Phe His Val 210 215 220 Arg Pro Asp Phe Val Gly Thr Met Arg Glu Gly lie Ala Val Leu Ser 225 230 235 240 Arg Gin Gly Thr lie Val Ser Met Asn Arg Ala Gly Leu Gin lie Ala 245 250 255 Gly Leu Asn Leu Gin Ala Val Ala Asp His Arg Phe Asp Ser Val Phe 260 265 270 Asp Leu Asn Phe Gly Ala Phe Leu Asp His Val Arg Gin Ser Ala Phe 275 280 285 Gly Leu Val Arg Val Ser Leu Tyr Gly Gly Val Gin Val Tyr Ala Arg 290 295 300 Val Gin Pro Gly Leu Arg Val Pro Pro Arg Pro Ala Ala His Ala Arg 305 310 315 320 Pro Pro Arg Pro Ala Pro Arg Pro Leu Asp Ser Leu Asp Thr Gly Asp 325 330 335 Ala Ala Val Arg Leu Ala lie Asp Arg Ala Arg Arg Ala lie Gly Arg 340 345 350 Asn Leu Ser lie lie Leu Gin Gly Glu Thr Gly Ala Gly Lys Glu Val 355 360 365 Phe Ala Lys His Leu His Ala Glu Ser Pro Arg Ser Lys Gly Pro Phe 370 375 380 Val Ala Val Asn Cys Ala Ala lie Pro Glu Gly Leu lie Glu Ser Glu 385 390 395 400 Leu Phe Gly Tyr Glu Glu Gly Ala Phe Thr Gly Gly Arg Arg Lys Gly 405 410 415 Asn lie Gly Lys Val Ala Gin Ala His Gly Gly Thr Leu Phe Leu Asp 420 425 430 Glu lie Gly Asp Met Ala Pro Gly Leu Gin Thr Arg Leu Leu Arg Val 435 440 445 Leu Gin Asp Arg Ala Val Met Pro Leu Gly Gly Arg Glu Pro Met Pro 450 455 460 Val Asp lie Ala Leu Val Cys Ala Thr His Arg Asn Leu Arg Ser Leu 465 470 475 480 lie Ala Gin Gly Gin Phe Arg Glu Asp Leu Tyr Tyr Arg Leu Asn Gly 485 490 495 Leu Ala lie Ser Leu Pro Pro Leu Arg Gin Arg Ser Asp Leu Ala Ala 500 505 510 Leu Val Asn His Ile Leu Phe Gin Cys Cys Gly Gly Glu Pro His Tyr 515 520 525 Ser Val Ser Pro Glu Val Met Thr Leu Phe Lys Arg His Ala Trp Pro 530 535 540 Gly Asn Leu Arg Gin Leu His Asn Val Leu Asp Ala Ala Leu Ala Met 545 550 555 560 Leu Asp Asp Gly His Val He Glu Pro His His Leu Pro Glu Asp Phe 565 570 575 Val Met Glu Val Asp Ser Gly Leu Arg Pro He Glu Glu Asp Gly Ser 580 585 590 Thr Ala Ala His Arg Ala Arg Gin Pro Ala Ser Gly Ser Gly Pro Ala 595 600 605 Lys Lys Leu Gin Asp Leu Ala Leu Asp Ala He Glu Gin Ala He Glu 610 615 620 Gln Asn Glu Gly Asn He Ser Val Ala Ala Arg Gin Leu Gly Val Ser 625 630 635 640 Arg Thr Thr He Tyr Arg Lys Leu Arg Gin Leu Ser Pro Thr Gly Cys 645 650 655 His Arg Pro Ala His Trp Ser Gin Ser Arg He Gly Thr 660 665 <210> 2 <211> 2010 <212> DNA <213> Pseudomonas butanovora <400> 2 atgtctaaaa tgcaggaatt cgctcgtctg gaaaccgttg cttctatgcg tcgtgctgtt 60 tgggacggta acgaatgcca gccgggtaaa gttgctgacg ttgttctgcg ttcttggacc 120 cgttgccgtg ctgaaggtgt tgttccgaac gctcgtcagg aattcgaccc gatcccgcgt 180 accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240 gttgacgctc tgatggaaca gatgaacgac gctccgcgta tgatcatcct gaacgacgaa 300 cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360 gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420 gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480 accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540 ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600 gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660 cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720 cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780 gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840 gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900 gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960 ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020 ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080 gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140 aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200 ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260 gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320 ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380 gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440 atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500 ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560 tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620 cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680 ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740 gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800 ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860 caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920 cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980 cactggtctc agtctcgtat cggtacctaa 2010 <210> 3 <211> 669 <212> PRT <213> Artificial sequence <400> 3 Met Ser Lys Met Gin Glu Phe Ala Arg Leu Glu Thr Val Ala Ser Met 1 5 10 15 Arg Arg Ala Val Trp Asp Gly Asn Glu Cys Gin Pro Gly Lys Val Ala 20 25 30 Asp Val Val Leu Arg Ser Trp Thr Arg Cys Arg Ala Glu Gly Val Val 35 40 45 Pro Asn Ala Arg Gin Glu Phe Asp Pro lie Pro Arg Thr Ala Leu Asp 50 55 60 Glu Thr Val Glu Ala Lys Arg Ala Leu lie Leu Ala Ala Glu Pro Val 65 70 75 80 Val Asp Ala Leu Met Glu Gin Met Asn Asp Ala Pro Arg Met lie lie 85 90 95 Leu Asn Asp Glu Arg Gly Val Val Leu Leu Asn Gin Gly Asn Asp Thr 100 105 110 Leu Leu Glu Asp Ala Arg Arg Arg Ala Val Arg Val Gly Val Cys Trp 115 120 125 Asp Glu His Ala Arg Gly Thr Asn Ala Met Gly Thr Ala Leu Ala Glu 130 135 140 Arg Arg Pro Val Ala Ile His Gly Ala Glu His Tyr Leu Glu Ser Asn 145 150 155 160 Thr Ile Phe Thr Cys Thr Ala Ala Pro Ile Tyr Asp Pro Phe Gly Glu 165 170 175 Phe Thr Gly Ile Leu Asp Ile Ser Gly Tyr Ala Gly Asp Met Gly Pro 180 185 190 Val Pro Ile Pro Phe Val Gln Met Ala Val Gln Phe Ile Glu Asn Gln 195 200 205 Leu Phe Arg Gln Thr Phe Ala Asp Cys Ile Leu Leu His Phe His Val 210 215 220 Arg Pro Asp Phe Val Gly Thr Met Arg Glu Gly Ile Ala Val Leu Ser 225 230 235 240 Arg Glu Gly Thr Ile Val Ser Met Asn Arg Ala Gly Leu Lys Ile Ala 245 250 255 Gly Leu Asn Leu Glu Ala Val Ala Asp His Arg Phe Asp Ser Val Phe 260 265 270 Asp Leu Asn Phe Gly Ala Phe Leu Asp His Val Arg Gln Ser Ala Phe 275 280 285 Gly Leu Val Arg Val Ser Leu Tyr Gly Gly Val Gln Val Tyr Ala Arg 290 295 300 Val Glu Pro Gly Leu Arg Ala Pro Pro Arg Pro Ala Ala His Ala Arg 305 310 315 320 Pro Pro Arg Pro Ala Pro Arg Pro Leu Asp Ser Leu Asp Thr Gly Asp 325 330 335 Ala Ala Val Arg Leu Ala Ile Asp Arg Ala Arg Arg Ala Ile Gly Arg 340 345 350 Asn Leu Ser Ile Leu Ile Gln Gly Glu Thr Gly Ala Gly Lys Glu Val 355 360 365 Phe Ala Lys His Leu His Ala Glu Ser Pro Arg Ser Lys Gly Pro Phe 370 375 380 Val Ala Val Asn Cys Ala Ala Ile Pro Glu Gly Leu Ile Glu Ser Glu 385 390 395 400 Leu Phe Gly Tyr Glu Glu Gly Ala Phe Thr Gly Gly Arg Arg Lys Gly 405 410 415 Asn Ile Gly Lys Val Ala Gln Ala His Gly Gly Thr Leu Phe Leu Asp 420 425 430 Glu Ile Gly Asp Met Ala Pro Gly Leu Gln Thr Arg Leu Leu Arg Val 435 440 445 Leu Gln Asp Arg Ala Val Met Pro Leu Gly Gly Arg Glu Pro Met Pro 450 455 460 Val Asp Ile Ala Leu Val Cys Ala Thr His Arg Asn Leu Arg Ser Leu 465 470 475 480 Ile Ala Gln Gly Gln Phe Arg Glu Asp Leu Tyr Tyr Arg Leu Asn Gly 485 490 495 Leu Ala Ile Ser Leu Pro Pro Leu Arg Gln Arg Ser Asp Leu Ala Ala 500 505 510 Leu Val Asn His Ile Leu Phe Gln Cys Cys Gly Gly Glu Pro His Tyr 515 520 525 Ser Val Ser Pro Glu Val Met Thr Leu Phe Lys Arg His Ala Trp Pro 530 535 540 Gly Asn Leu Arg Gln Leu His Asn Val Leu Asp Ala Ala Leu Ala Met 545 550 555 560 Leu Asp Asp Gly His Val Ile Glu Pro His His Leu Pro Glu Asp Phe 565 570 575 Val Met Glu Val Asp Ser Gly Leu Arg Pro Ile Glu Glu Asp Gly Ser 580 585 590 Thr Ala Ala His Arg Ala Arg Gln Pro Ala Ser Gly Ser Gly Pro Ala 595 600 605 Lys Lys Leu Gln Asp Leu Ala Leu Asp Ala Ile Glu Cys Ala Leu Glu 610 615 620 Gln Asn Glu Gly Asn Ile Ser Val Ala Ala Arg Gin Leu Gly Val Ser 625 630 635 640 Arg Thr Thr Ile Tyr Arg Lys Leu Arg Gin Leu Ser Pro Thr Gly Cys 645 650 655 His Arg Pro Ala His Trp Ser Gin Ser Arg Ile Gly Thr 660 665 <210> 4 <211> 2010 <212> DNA <213> Artificial sequence <400> 4 atgtctaaaa tgcaggaatt cgctcgtctg gaaaccgttg cttctatgcg tcgtgctgtt 60 tgggacggta acgaatgcca gccgggtaaa gttgctgacg ttgttctgcg ttcttggacc 120 cgttgccgtg ctgaaggtgt tgttccgaac gctcgtcagg aattcgaccc gatcccgcgt 180 accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240 gttgacgctc tgatggaaca gatgaacgac gctccgcgta tgatcatcct gaacgacgaa 300 cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360 gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420 gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480 accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540 ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600 gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660 cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720 cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780 gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840 gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900 gtttacgctc gtgttgaacc gggtctgcgt gctccgccgc gtccggctgc tcacgctcgt 960 ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020 ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080 gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa ctgttcggtt acgaagagg tgctttcacc ggtggtcgtc gtaaggtaa catcggtaaa gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320 ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380 gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560 tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt gactctggtc tgcgtccgat cgaagagac ggttctaccg ctgctcaccg tgctcgtcag ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860 caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920 cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980 cactggtctc agtctcgtat cggtacctaa 2010 <210> 5 <211> 138 <212> DNA <213> Pseudomonas butanovora <400> 5 gaccttgagg tgaccttgag cgggcagata ccaccaaaat ttcccacgtg ctattatggt 60 tttgctaaag ctctcgacag cgaggagaga ctcgcgaaga taagcaattc gcccgacaga 120 ggtgaatgag gagacggt 138 <210> 6 <211> 524 <212> DNA <213> Pseudomonas butanovora <400> 6 ccccccaacg acgtccgtca gagcccggtt cgagtggctt ctatatgccg atcatcggtg 60 gctctattgt ggcggtcagt gacaccggtc gccttcaccc ccacagatag taggtgctgc 120 ggctgctcat gctcctgtcg cggtagcgcg ctgttacgcg accgcccccg gacctcggcg 180 gacagcgcgg aagattggaa acagcccgag cgtgcgtgcc tcgggctgca tccttgccac 240 acccaaccgg attcgtcgga ccgctcgaca ttcgcgttcg ctcccgcggc gccgcgggtg 300 taccgttgcg ttacagatgt acccttcttt aacgtgtaac acacgcctgg agcggccaag 360 agccccgcac cttgcggcgc gtcttcccca ggggcccacc ggttgcggcc tttgctgcg 420 accgtccatg ctggcacgac acttgctgaa agcgttagag cggaatcggt ccgatggagc 480 attcgaagcc gctaccgaca gcagaacaca caaaggagga agtg 524 <210> 7 <211> 717 <212> DNA <213> Artificial sequence <400> 7 atgcgtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180 gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca tatgaaacag 240 catgacttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatattttc 300 aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360 aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420 ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480 atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600 ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660 cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataa 717

Claims

1. A BmoR mutant protein, characterized in that, The mutant is obtained by mutating V311A based on the wild-type BmoR protein shown in SEQ ID NO.

1.

2. Use of the BmoR mutant of claim 1 in detecting samples containing extremely low concentration of n-butanol or isobutanol, or in screening n-butanol or isobutanol producing strains.

3. Use of the BmoR mutant of claim 1 in constructing biosensors for detecting n-butanol or isobutanol.

4. A coding gene of the BmoR mutant of claim 1.

5. The genetic code of claim 4, wherein, As shown in SEQ ID NO.

4.

6. A recombinant plasmid or a recombinant strain comprising the coding gene of claim 4.

7. A biosensor characterized by The sensor is an expression element comprising the mutant-encoding gene of claim 4, a reporter gene driven by the promoter P bmo bmo activated by BmoR mutant, and a promoter expressing BmoR mutant.​ 8. The biosensor of claim 7, wherein, The reporter gene includes but is not limited to gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp genes.

9. The biosensor of claim 7, wherein, Promoters that express BmoR mutants include, but are not limited to, P bmoR .

10. The biosensor of claim 7, wherein, The sensor is a gene encoding a protein P bmoR The mutant gene under the control of the promoter P r and the gfp gene under the control of the promoter P bmo The nucleotide sequence of the promoter P bmo is shown in the sequence listing SEQ ID NO.

5.

11. Use of the biosensor of claim 7 in detecting environmental, food, medical, biological samples containing extremely low concentration of n-butanol or isobutanol, and in screening n-butanol or isobutanol producing industrial microbial strains.

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