Bmor mutants specifically responsive to isobutanol and uses thereof

Abstract

The application belongs to the technical field of bioengineering and particularly relates to a BmoR protein mutant with specific response to isobutanol and application of the BmoR protein mutant in isobutanol detection or biosensor. The BmoR protein mutant is obtained by S240P mutation on the basis of wild-type BmoR protein shown in the sequence table SEQ ID NO. 1 and has the amino acid sequence S240P mutant shown in SEQ ID NO. 3. The S240P mutant is only sensitive to isobutanol and has no response to n-butanol, thereby solving the problem that the wild-type BmoR protein cannot distinguish n-butanol and isobutanol high-yield strains. Meanwhile, the detection range of the S240P mutant to isobutanol reaches 0-100 mM, the saturation of the BmoR protein to isobutanol is improved, and the S240P mutant can be used for screening and application of higher-yield strains.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a BmoR mutant specifically responding to isobutanol and application of the BmoR mutant in isobutanol detection or biosensor. BACKGROUND

[0002] Isobutanol synthesized by microorganisms is an important transportation fuel, and the synthesis of isobutanol by metabolic engineering is realized in many microbial hosts, and the modification of host strains and the screening of high-yield hosts are the basis and key to the industrial production of alcohol. Biosensors can specifically respond to target compounds to output protein signals that are easy to detect, and have been widely used in high-throughput screening. However, the wild-type transcription factor BmoR cannot be widely used in biosensors due to its poor response specificity and narrow detection range. The BmoR protein obtained by protein modification in the application can not only achieve specific response to isobutanol, but also improve the upper limit of substrate detection and response intensity, and most importantly, can be used for screening high-yield strains to meet the industrial demand.

[0003] As a new generation of biofuels, isobutanol is used in many fields, and has been synthesized in many microbial hosts, but specific screening of isobutanol is still a tedious engineering. The wild-type transcription factor BmoR responds to both n-butanol and isobutanol, and cannot specifically respond to one of the alcohols to meet the industrial demand. Therefore, achieving specific response to alcohol has become a big problem.

[0004] Biosensors are composed of molecular recognition elements and signal transducers. When the molecular recognition element binds to the measured object, the signal generated can be converted into an optical signal or an electrical signal by the transducer, and the measured object can be detected and analyzed. As a new 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 promote 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 domain (LBD) or metabolic binding domain (MBD) and DNA binding domain (DBD). BmoR is a transcription factor of Pseudomonas alkane metabolic pathway and a member of bEBP. It is used to regulate the expression of alkane monooxygenase. 54 Dependent promoter P bmo The signal molecule is a C2-C5 straight-chain or branched-chain alcohol. The BmoR-based biosensor can be used for screening high-yield n-butanol or isobutanol strains, but the response specificity of the wild-type BmoR biosensor is too poor and the detection range is too narrow (0-40 mM), which cannot be used in industrial production. Therefore, the improvement of the biosensor provides a solution for efficient detection of isobutanol production and rapid screening of high-yield strains. SUMMARY

[0006] The purpose of the present application is to provide a BmoR protein and biosensor that can distinguish between n-butanol and isobutanol, construct a random mutation library using error-prone PCR technology; by adding n-butanol and isobutanol exogenously, the final concentration is 0-100 mM, and the mutant library is screened and analyzed. Finally, a BmoR mutant protein that only responds to isobutanol is obtained.

[0007] Further, the BmoR mutant protein that only responds to isobutanol is obtained by S240P mutation based on the wild-type BmoR protein shown in SEQ ID NO. 1, hereinafter referred to as S240P mutant, and the mutant protein is specifically the amino acid sequence shown in SEQ ID NO. 3.

[0008] Further, the present application also provides a coding gene of S240P mutant.

[0009] Further, the coding gene is shown in SEQ ID NO. 4.

[0010] Another purpose of the present application is to provide the application of S240P mutant, especially in the sensor for detecting isobutanol-containing samples or screening isobutanol-producing strains, and more particularly, in the construction of biosensors for detecting isobutanol.

[0011] Further, the biosensor is a biosensor based on S240P mutant, and the sensor comprises the coding gene of S240P mutant and its promoter, the expression element of promoter P bmo and reporter gene; the promoter promotes the expression of bmoR gene, BmoR protein binds with alcohol molecules to form a hexamer, which further promotes the downstream promoter P bmo, so as to express the reporter gene, produce fluorescence and other signals; the biosensor can realize specific response and screening to isobutanol at a concentration of 0-100 mM, and is further applied to industrial production, so as to realize specific screening of isobutanol-containing samples and isobutanol-producing strains.

[0012] Further, the promoter of the mutant-encoding 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 biosensor is a recombinant plasmid containing the S240P mutant-encoding gene, the promoter P bmoR , the promoter P bmo and the gfp reporter gene; further, the expression vector that can be selected by the recombinant plasmid includes but is not limited to the commonly used expression vector in the art.

[0015] More preferably, the biosensor is obtained by replacing the wild-type BmoR protein-encoding gene on the plasmid pYH1 with the S240P mutant-encoding gene, that is, connecting the S240P mutant-encoding 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 bmoR 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 biosensor in isobutanol detection, in particular, in isobutanol-producing strain screening, by introducing the above plasmid into a production strain such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc., to detect the production of isobutanol.

[0021] Beneficial effects:

[0022] 1. The BmoR-based biosensor can be used for screening high butanol or isobutanol-producing strains, but wild-type BmoR responds to both n-butanol and isobutanol, cannot be distinguished, and has poor specificity. The S240P mutant provided in the application is only sensitive to isobutanol and has no response to n-butanol, solving the problem that wild-type BmoR protein cannot distinguish between n-butanol and isobutanol.

[0023] 2. The detection range of wild-type BmoR is too narrow, and the detection range of n-butanol is all 0-40 mM. When the substrate concentration reaches 40 mM, the response has reached saturation, so it cannot be used to identify strains producing isobutanol with a yield higher than 40 mM. The S240P mutant provided in the application has a detection range of 0-100 mM for isobutanol, improving the response saturation of BmoR protein to isobutanol, and can be used for screening and application of high-level isobutanol-producing strains. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a principle flow chart;

[0025] First, the N-terminal 1000 bp of 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 mutant BmoR to isobutanol can be reflected by detecting the fluorescence intensity. By adding different concentrations of isobutanol, the response of mutant BmoR to alcohol molecules is detected.

[0026] Figure 2 is the response of BmoR mutant and wild-type to 10 mM n-butanol and isobutanol;

[0027] Figure 3 is the response of BmoR mutant and wild-type to 0-100 mM n-butanol and isobutanol;

[0028] Figure 4 is the molecular docking of S240P mutant with n-butanol and isobutanol. DETAILED DESCRIPTION

[0029] The application will be described in detail below through specific embodiments. Unless otherwise specified, the technical means used in the application 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 application, and the essence and scope of the application are only limited by the claims. For those skilled in the art, various changes or modifications to the composition and amount of materials in these embodiments without departing from the essence and scope of the application also fall within the protection scope of the application.

[0030] The biosensor provided by the application is a biosensor based on S240P mutant, and the sensor comprises an S240P mutant coding gene and a promoter thereof, an expression element of a reporter gene; the promoter starts the expression of the bmoR gene, the BmoR protein combines with alcohol molecules to form a hexamer, and then starts the downstream promoter P bmo , so as to express the reporter gene and produce signals such as fluorescence. Those skilled in the art can select promoters to start the expression of the BmoR mutant gene in the prior art according to actual conditions, such as P bmo , P bmoR , P tac , P T7 , P LlacO1 and the like. The reporter gene can also be selected in multiple ways. Protein molecules commonly used in the art, which can produce visual detection signals or small molecules for detection, such as fluorescent proteins, color proteins and the like, can all realize the response of the biosensor described in the application. Preferably, such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp and the like. The above-mentioned sensor also comprises a replication origin and the like necessary element to realize expression, preferably such as a colE1 replication origin and the like. The above-mentioned sensor can also comprise a resistance gene and the like marker, such as amp r and the like, which is convenient for 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 vector in the prior art, such as pET, pUC19, pMAL and the like, to obtain a recombinant plasmid which can be used as a sensor.

[0031] The materials, reagents and the like used in the following examples can be obtained from commercial channels if there is no special description.

[0032] The application will be further explained and described below through specific examples.

[0033] Example 1 Screening of BmoR mutant S240P

[0034] Construction of a random mutation library of transcription factor BmoR

[0035] (1) Taking the plasmid pYH1 (construction is shown in DOI: https: / / doi.org / 10.1016 / j.ymben.2019.08.015; https: / / doi.org / 10.1186 / s12934-019-1084-2) with wild-type BmoR coding gene (shown in SEQ ID NO. 2) as a template, a bmoR mutant gene was obtained by error-prone PCR amplification (by adding Mn 2+ in the PCR system to improve Mg 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.

[0036] (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.

[0037] (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... 600 The background control value is first subtracted from the value, and then the GFP / OD ratio for each well is calculated. 600 As a relative fluorescence intensity value.

[0038] (4) After analyzing the initial screening results, the plasmids of the effective mutant bacteria were sequenced.

[0039] One of the amino acid mutation sites in the BmoR protein was identified as S240P (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4). This mutant has a GFP / OD...600 The initial screening results are shown in Table 1 below. Figure 2 As shown, the S240P mutant, compared to the wild-type BmoR, only responds to isobutanol.

[0040] Table 1 GFP / OD 600

[0041] n-butanol isobutanol WT 983 868 S240P 0.00 29.7

[0042] Example 2: Concentration gradient experiment to determine the change in detection limit between wild type and mutant

[0043] The wild-type BmoR-based biosensor can respond to 0-40 mM n-butanol or isobutanol, showing a response to both. However, the response tends to saturate above 40 mM, failing to respond to higher concentrations of alcohol molecules. The following experiments verify the specific response of the S240P mutant-based BmoR biosensor to 0-100 mM n-butanol / isobutanol.

[0044] Based on the initial screening results, exogenous addition experiments with concentration gradients of n-butanol or isobutanol were conducted on strains containing the S240P mutation. Response curves were measured, and K was calculated. m Parameters such as response intensity value.

[0045] Select a single clone 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.

[0046] The exogenous addition experiment was performed in sterile 2 mL 96-well plates. 950 μL of fresh LB (100 μg / mL Amp) medium was added to each well, followed by the addition of n-butanol or isobutanol to the medium at final concentrations of 0, 1, 10, 20, 40, 60, 80, and 100 mM, respectively. Finally, 50 μL of seed culture was inoculated into each well, and the plates were sealed with a sealing film and then incubated at 30°C and 220 rpm for 16 h on a shaker.

[0047] 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 and emission wavelength 510 nm, gain value 50; the obtained GFP and OD... 600 The background control value is first subtracted from the value, and then the GFP / OD ratio for each well is calculated. 600 As a relative fluorescence intensity value.

[0048] With GFP / OD 600Plot the data using OriginPro 8.5 or GraphPad Prism 8 software, with the final concentrations of n-butanol and isobutanol on the x-axis and the y-axis on the y-axis respectively. Perform Michaelis-Menten fitting on the data and calculate the Ki of the BmoR mutant for n-butanol and isobutanol based on the fitting results. m Parameters such as maximum response intensity Figure 3 ).

[0049] A random mutant library was screened at a substrate concentration of 10 mM. The resulting mutant S240P showed a specific response to isobutanol but no response to n-butanol. Further validation was performed at gradient concentrations from 0 to 100 mM. The results showed that this mutant maintained a specific response to isobutanol under different concentrations of n-butanol and isobutanol, but showed almost no response to n-butanol. Plotting with OriginPro 8.5 showed the Kt of wild-type BmoR to n-butanol at substrate concentrations from 0 to 100 mM. m The K value for isobutanol is 3.78 mM. m The K+ level of the S240P mutant at substrate concentrations of 0-100 mM was 4.24 mM; m The K value for isobutanol is 100 mM. m 43.6 mM; K m As a representation of affinity, K m The higher the value, the lower the affinity; K m The smaller the value, the greater the affinity.

[0050] Wild-type BmoR responds to n-butanol or isobutanol at concentrations from 0 to 40 mM. However, the response saturates above 40 mM, therefore wild-type BmoR cannot distinguish between n-butanol and isobutanol at concentrations above 40 mM. Calculations show that, compared to the wild-type, the mutant has a higher K... m The value showed a significant increase, demonstrating that the BmoR mutant can achieve a specific response to higher concentrations of isobutanol (0-100mM); simultaneously, the S240P mutant showed an increase in isobutanol K. m The value is less than that of n-butanol K. m The value further indicates that the mutant has a greater affinity for isobutanol than for n-butanol. This is consistent with experimental results.

[0051] Example 3 Model Analysis

[0052] The S240P mutant was sequenced to analyze the changes in amino acids at the mutation sites. The BmoR mutant was modeled using software such as Autodoc and ChimeraX. The mutant was then docked with the substrate small molecules n-butanol and isobutanol to analyze the binding sites and hydrogen bond formation between the mutant and these two alcohols.

[0053] The wild-type BmoR protein three-dimensional structure was used as a template to model the S240P mutant, and the homology rate was 99.9%. Further molecular docking was performed between the mutant structure and the substrate molecules (n-butanol or isobutanol). The results showed that in the complex, the mutant had one hydrogen bond interaction with isobutanol (Glu261), and no interaction force was formed with n-butanol, indicating that isobutanol could be tightly combined with the mutant, and K m The value analysis results remained consistent, that is, the BmoR mutant was specifically responsive to isobutanol Figure 4

[0054] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood 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 into several modifications, combinations and improvements without departing from the concept of the present patent, and these all belong to the protection scope of the present patent. Therefore, the protection scope of the present patent should be subject to the claims. SEQUENCE LISTING <110> Beijing University of Technology <120> BmoR mutant specifically responsive to isobutanol and application thereof <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 Glu Asn Gin 195 200 205 Leu Phe Arg Gin 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 Gin 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 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 Glu 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 Ile Asp Arg Ala Arg Arg Ala Ile Gly Arg 340 345 350 Asn Leu Ser Ile Leu Ile 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 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 Gln Ala Ile Glu 610 615 620 Gln Asn Glu Gly Asn Ile Ser Val Ala Ala Arg Gln Leu Gly Val Ser 625 630 635 640 Arg Thr Thr Ile Tyr Arg Lys Leu Arg Gln Leu Ser Pro Thr Gly Cys 645 650 655 His Arg Pro Ala His Trp Ser Gln Ser Arg Ile 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 GAACC GATGC CGTT GACAT AGCG CTGGT CTGCG CAACC CACC GTAACCT GCGTT CTCTG 1440 ATCGCTCAGG TTCAGTTCCG TGAAGACCTG TACTACCCTC TGAACGGTCT GGCTATCTCT 1500 CTGCCGCCGT GCCTCAGCGT TCTGACCTGG CTGCTCTGGT TAACCACATC CTGTTCCAG 1560 TGCTGCGGTG GTGAACCACA TTACTCTGTA AGCCCGGAAG TTATGACCTG TTCAACGTG 1620 CACGCTTGGC CGGGTAACCT GCCTCAGCTG CACAACGTTC TGGACGCTGC TCTGGCTATG 1680 CTGGACGACG GTCACGTTAT CGAACCACAC CTGCCGGAAG ACTTCGTATG GAAGTT 1740 GACTCTGGTC TGCCTCCGAT CGAAGAAGAC GCTTCTACC GCCTCTACCG TGCTCGTCA 1800 CCGGCTTCTG GTTCTGGTCC GGCTAAAAAA CTGCAGGACC TGGCTCTGGA CGCTATCGAA 1860 CAGGCTATCG AACAGAACGA GGCTACATC TCTGTTGCTG CGTTCAGCTG GTGTAAGC 1920 CGTACCACCA TCTACCCTAA ACTGCCTCAG CTGTCCTGAC CGGTTGCCAC CGTCCGGCT 1980 CCTTGCTCAG TCTCGTATCG GTACCTAA 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 lie 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 Pro 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 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 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 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 lie 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 lie Glu Pro His His Leu Pro Glu Asp Phe 565 570 575 Val Met Glu Val Asp Ser Gly Leu Arg Pro lie 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 lie Glu Gin Ala lie Glu 610 615 620 Gln Asn Glu Gly Asn lie 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 gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480accatcttca 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 tgttctgcct 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 CTGCTGCTGC GTTTCTGCAA GACCCTGCT GTTATGCCGC TGGGTGGTGT 120 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GGTGGTGTGT GGTGGTGGTG GTGGTGGTGG TGGTGGTGGT GGTGGTGGTG 1320 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 GTTGCTCAGG CTCACGGTGG TACCCTGTTC CTGGACGAAA TCGGTGACAT 1260 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 ttttgctgcg 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 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc 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 S240P mutation based on the wild-type BmoR protein shown in the sequence table SEQ ID NO.

1.

2. The use of the BmoR mutant of claim 1 in detecting samples containing isobutanol or in screening isobutanol-producing strains.

3. The use of the BmoR mutant of claim 1 in constructing a biosensor for detecting isobutanol.

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

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

4.

6. A recombinant plasmid or a recombinant strain comprising the encoding 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 gene.

9. The biosensor of claim 7, wherein, Promoters that express BmoR mutants include, but are not limited to, P bmoR , P tac , P T7 , P LlacO1 .

10. The biosensor of claim 7, wherein, The sensor is a plasmid p bmoR The mutant coding gene is linked to the colE1 replication origin, amp r and P bmo The gfp gene is obtained by the start.

11. The biosensor of claim 7, wherein, The nucleotide sequence of the promoter P bmo is shown in SEQ ID NO. 5 of the sequence listing.

12. The use of the biosensor of claim 7 in detecting environmental, food, medical, biological samples containing isobutanol, and screening isobutanol-producing industrial microbial strains.

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