A wide-range biosensor based on bmo r mutants

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a BmoR protein mutant with a wider detection range (0-200 mM) for n-butanol or isobutanol and application of the BmoR protein mutant in detection of n-butanol or isobutanol or biosensors. The BmoR protein mutant is obtained by T12N mutation on the basis of a wild-type BmoR protein shown in the sequence table SEQ ID NO. 1, and has an amino acid sequence T12N mutant shown in SEQ ID NO. 3. The T12N mutant has a wider detection range (0-200 mM) for n-butanol or isobutanol, solves the problem that the wild-type BmoR protein cannot screen n-butanol or isobutanol high-yield strains, and can be used for screening and application of higher-yield strains.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a BmoR mutant with a wider detection range (0-200 mM) for n-butanol or isobutanol and its application 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 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 n-butanol or isobutanol. Biosensors can specifically respond to target compounds to output protein signals that are 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 and narrow detection range. The BmoR protein obtained by protein modification in the present application realizes higher detection concentration upper limit and response intensity for n-butanol or isobutanol, and most importantly, can be used for screening high-yield strains of n-butanol or isobutanol, thereby meeting the industrial demand.

[0003] As a new generation of biofuels, n-butanol or isobutanol is used in many fields, and has been biologically synthesized in many microbial hosts, but the screening of higher-yield n-butanol or isobutanol is still a tedious engineering. The highest response concentration of the wild-type transcription factor BmoR to the substrate n-butanol or isobutanol is only 40 mM, which cannot detect alcohol molecules with higher concentration, thereby meeting the industrial demand. Therefore, it is a big problem to realize a wider detection range for 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 domain (LBD) or metabolic binding domain (MBD) and DNA binding domain (DBD). BmoR is a transcription factor of Pseudomonas alkane metabolic pathway, a member of bEBP, used to regulate the sigma factor 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 modification of the biosensor provides a solution for efficient detection of n-butanol or 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 detect higher concentrations of n-butanol or isobutanol, a random mutation library is constructed using error-prone PCR technology; by adding n-butanol and isobutanol exogenously, the final concentration is 0-200 mM, and the mutant library is screened and analyzed. Finally, a BmoR mutant protein capable of detecting 0-200 mM n-butanol or isobutanol is obtained.

[0007] Further, the BmoR mutant protein capable of detecting 0-200 mM n-butanol or isobutanol is obtained by T12N mutation based on the wild-type BmoR protein shown in SEQ ID NO. 1, hereinafter referred to as T12N 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 the T12N 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 the T12N mutant, particularly in the sensor for detecting samples containing n-butanol or isobutanol, or screening n-butanol or isobutanol production strains, more particularly, in the biosensor for detecting n-butanol or isobutanol;

[0011] Further, the biosensor is a T12N mutant-based biosensor, and the sensor comprises a T12N mutant coding gene and its promoter, a promoter P bmo and a reporter gene expression element; the promoter starts bmoRGene expression, BmoR protein binds with alcohol molecules to form hexamers, and then initiates downstream promoters P bmo , so as to express the reporter gene, produce signals such as fluorescence; the biosensor can realize the response and screening of 0-200 mM n-butanol or isobutanol, and is further applied to industrial production, realizes the screening of samples containing n-butanol or isobutanol, and realizes the screening of high-yield strains of n-butanol or isobutanol;

[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 T12N mutant coding gene and its promoter P bmoR , promoter P bmo , and gfp reporter gene; further, the expression vector that can be selected by the recombinant plasmid includes but is not limited to the general expression vector 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 T12N mutant coding gene, that is, the T12N mutant coding gene driven by P bmoR is connected to colE1 the replication initiation site, amp r , and P bmo driven by gfp gene;

[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 SEQ ID NO. 5.

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

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

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

[0021] The detection range of wild-type BmoR is too narrow, and the detection range of n-butanol or isobutanol is only 0-40 mM. When the substrate concentration reaches 40 mM, the response will reach saturation, so it cannot be used to identify strains with n-butanol or isobutanol concentration higher than 40 mM. The detection range of the T12N mutant provided by the application for n-butanol or isobutanol reaches 0-200 mM, which improves the response saturation of BmoR protein to n-butanol or isobutanol, and can be used for screening and application of high-level n-butanol or isobutanol production strains. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a schematic flowchart;

[0023] First, the N-terminal first 1000 bp of wild-type bmoR is randomly mutated by error-prone PCR to obtain a random mutation library of BmoR; downstream of the bmoR gene, a GFP fluorescent protein is added, and the response of mutant BmoR to n-butanol or isobutanol can be reflected by detecting the fluorescence intensity. By adding different concentrations of n-butanol or isobutanol, the response of mutant BmoR to alcohol molecules is detected.

[0024] Figure 2 is the response of BmoR mutant and wild-type to 0-200 mM n-butanol and isobutanol;

[0025] Figure 3 is the response of BmoR mutant and wild-type to isobutanol in isobutanol production strains;

[0026] Figure 4 is the response of BmoR mutant and wild-type to isobutanol in isobutanol production strains;

[0027] Figure 5This shows the molecular docking of the T12N mutant with n-butanol and isobutanol. Detailed Implementation

[0028] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined 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 invention also fall within the protection scope of this invention.

[0029] The biosensor provided by this invention is a T12N mutant-based biosensor, comprising the T12N mutant coding gene and its promoter, and a promoter... P bmo and the expression elements of reporter genes; promoter activation bmoR Gene expression occurs when the BmoR protein binds to an alcohol molecule to form a hexamer, which in turn initiates the downstream promoter. P bmo This allows the reporter gene to be expressed, producing signals such as fluorescence. Those skilled in the art can select a promoter from existing technologies to initiate the expression of the BmoR mutant gene, such as using... P bmoR Promoter. The reporter gene can also be selected from various sources. Commonly used protein molecules in the art that can generate visual detection signals or detectable small molecules, such as fluorescent proteins and color proteins, can all achieve the response of the biosensor described in this invention. Preferably, such as... gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp The aforementioned sensors also include essential elements for expression, such as a replication origin site, preferably, a colE1 replication origin site. The aforementioned sensors may also include markers such as resistance genes, such as amp... r This facilitates screening. Those skilled in the art can also add other components to the above-mentioned sensors according to actual needs, such as constructing the above-mentioned components onto expression vectors in the prior art, such as pET, pUC19, pMAL, etc., to obtain recombinant plasmids that can be used as sensors.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] The present invention will be further explained and illustrated below through specific embodiments.

[0032] Example 1: Screening of the BmoR mutant T12N

[0033] Constructing a random mutant library of transcription factor BmoR

[0034] (1) Using plasmid pYH1 (constructed 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-encoding gene (SEQ ID NO. 2) as template, mutant genes were amplified by error-prone PCR bmoR Mutant genes were introduced by adding Mn 2+ in PCR system, increasing Mg 2+ concentration in PCR system and adjusting the ratio of dNTPs. Prepare 10x unbalanced dNTPs mixture, in which the concentration of dCTP and dTTP is four times that of dATP and dGTP. The PCR program is set as follows: 94ºC pre-denaturation 2 min, 30 amplification cycles including: 95ºC denaturation 1 min, 55-68ºC annealing 1 min, determine the appropriate extension time according to the amplification speed of 1 kb per minute, and the extension temperature is 72ºC. The holding temperature is set to 16ºC.); The PCR products were confirmed by gel electrophoresis and purified by recovery; the purified product was placed in a 37ºC water bath, and DpnI (1 μL / 50 μL purified product) was digested for 1-2 h. Take 2 μL bmoR mutant fragments, 3 μL pYH1 backbone (plasmids constructed with P bmoR , P bmo , gfp fluorescent protein genes can also be used as backbone, which is equivalent to pYH1 backbone) and 5 μL Gibson Assemble Mix, mix and place in a 50ºC water bath for 1 h. Take 5-10 μL of ligation product into 50 μL E. coli XL10-Gold transformation competent cells, incubate at 37ºC overnight to obtain BmoR-1000 bp mutant library.

[0035] (2) Pick single colonies from the plate and inoculate into 5 mL LB (100 μg / mL Amp) liquid medium, incubate at 37ºC, 220 rpm for 8 h as seed liquid. Use 96-deep-well plates for primary screening. First add 950 μL fresh LB (100 μg / mL Amp) medium to each well; add n-butanol or isobutanol to each well to make the final concentration 0-200 mM; finally, inoculate 50 μL seed liquid into each well. After sealing the sealing film, place the deep-well plate in a 30ºC, 220 rpm shaker for 16 h.

[0036] (3) Use microplate to measure fluorescence intensity GFP and OD 600Detection: shake the mixed bacteria liquid, take 200 μL into the microplate reader, set the parameters as follows: 470 nm excitation wavelength, 510 nm emission wavelength, and gain value 50; the obtained GFP and OD 600 values were first subtracted from the background control values, and then the GFP / OD 600 of each well was calculated 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 S240P (amino acid sequence as SEQ ID NO. 3, nucleotide sequence as SEQ ID NO. 4), and the GFP / OD 600 of the mutant was 0. 1, which was higher than that of the wild type BmoR. Figure 2 The primary screening results are shown in Table 1.

[0039] Example 2: Shake flask fermentation experiment for determining the change of detection limit of isobutanol by wild type and mutant

[0040] The biosensor based on the wild type BmoR can respond to 0-40 mM n-butanol or isobutanol, and responds to both n-butanol and isobutanol; when the substrate concentration is higher than 40 mM, the response value tends to be saturated, and it cannot respond to higher concentration of alcohol molecules. The following experiments verify the response of the BmoR biosensor based on the T12N mutant to 0-200 mM n-butanol or isobutanol.

[0041] On the basis of further verifying the characteristics of the mutant through the exogenous gradient experiment, isobutanol shake flask fermentation experiment was carried out on the strain containing the T12N mutant to determine the yield of isobutanol and the response value of the mutant to different yields of isobutanol.

[0042] The isobutanol synthesis pathway (IOL operon) kivd , adhA , alsS , ilvC , ilvD ) was introduced into the laboratory-preserved E. coli strain JCL260, which was made into a competent cell, and then the plasmid with wild type BmoR and the plasmid with mutant T12N were respectively introduced into the competent cell, i.e., the wild type BmoR and the mutant biosensor system were introduced into the isobutanol production pathway to detect and respond to the yield of isobutanol in real time.

[0043] Single colonies of JCL260 containing wild-type BmoR or mutant T12N were picked and inoculated into 5 mL LB (100 μg / mL Amp, 25 μg / mL Cm and 50 μg / mL Kana) liquid medium, 37 °C, 220 rpm for 8 h as seed liquid. 200 μL seed liquid was inoculated into 200 mL M9 (40 % glucose, 4 g / L yeast extract and 0.1 mM IPTG) medium, 30 °C, 220 rpm for cultivation, and 1 mL was sampled at 12, 24, 36, 48, 60, 72 and 84 h, respectively.

[0044] Sample processing: The fluorescence intensity of GFP and OD 600 were detected in microplate. 200 μL of 1 mL bacterial solution was put into the microplate, and the parameters were set as follows: 470 nm excitation wavelength and 510 nm emission wavelength, and 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.

[0045] The remaining bacterial solution was centrifuged at 12000 rpm for 10 min, 100 μL supernatant was added to the gas chromatography vial, and 1 g / L n-pentanol prepared as an internal standard for gas chromatography detection was added. The gas chromatography vial was tightly capped and shaken well.

[0046] Gas chromatography detection procedure: The A91 gas chromatograph (GC) of Changzhou Pannuo Instrument Co., Ltd. and DB-FFAP capillary column (30 m x 0.32 mm x 0.25 μm; Agilent Technologies) were used for quantitative determination of isobutanol. The GC oven temperature was initially maintained at 80 °C for 3 min, then increased to 230 °C at a gradient of 115 °C per min, and finally maintained at 230 °C for 1 min. The carrier gas was nitrogen, and the temperatures of the injector and detector were maintained at 250 °C and 280 °C, respectively. The injection volume of the injector was 0.2 μL, and the split ratio was 30:1. N-pentanol was used as an internal standard. The fermentation samples were detected by the same procedure, and the isobutanol content of each sample was calculated by the internal standard method using the software batch processing function with reference to the standard.

[0047] GFP / OD 600 as the left ordinate, isobutanol yield as the right ordinate, and sampling time as the abscissa, the data were processed and analyzed by OriginPro 8.5 or GraphPad Prism 8 software, and the isobutanol yield at different sampling times and the response of wild-type BmoR and mutant T12N to isobutanol at this yield were compared. Figure 3 ).

[0048] The response value of mutant T12N at 150 mM was significantly different from that at 200 mM under 0-200 mM substrate concentration. Plotting was performed by OriginPro 8.5. The response value of wild-type BmoR to n-butanol was 3.78 mM under 0-100 mM substrate concentration. K m The response value of wild-type BmoR to iso-butanol was 4.24 mM. K m The response value of mutant T12N to n-butanol was 36.4 mM. K m The response value of mutant T12N to iso-butanol was 62.6 mM, which was increased by 9.63 times and 14.8 times compared with wild-type, respectively, indicating that the upper limit of detection of T12N mutant to n-butanol or iso-butanol was increased to 200 mM, which was increased by 5 times compared with wild-type. K m The response value of mutant T12N to iso-butanol was 62.6 mM, which was increased by 9.63 times and 14.8 times compared with wild-type, respectively, indicating that the upper limit of detection of T12N mutant to n-butanol or iso-butanol was increased to 200 mM, which was increased by 5 times compared with wild-type.

[0049] To further verify the response of T12N mutant to n-butanol or iso-butanol, the mutant was introduced into the iso-butanol synthesis pathway for shake flask fermentation experiment. The fluorescence response of T12N mutant at 72 h and 84 h was significantly different, and reached 835 at 84 h, at which time the iso-butanol yield was 215 mM. The response value of wild-type to iso-butanol was in the rising level before 36 h of fermentation, and reached 681 at 36 h, at which time the iso-butanol yield was 67 mM. Between 36 h and 84 h, the iso-butanol yield continued to increase, but the fluorescence response of wild-type BmoR did not change significantly. It was proved that in the iso-butanol production strain, the upper limit of detection of wild-type BmoR to iso-butanol was about 70 mM, and the upper limit of detection of T12N mutant to iso-butanol was about 200 mM, which was consistent with the experimental results of exogenous addition experiment, as shown in FIG. 2. Figure 4

[0050] Example 3 Model analysis

[0051] The T12N mutant was sequenced to analyze the change of amino acid at the mutation site, and AUTODOCK and Chimera X software were used to model the BmoR mutant, and the mutant was docked with the small molecule substrates n-butanol and iso-butanol, respectively, to analyze the binding sites of the mutant and the two alcohols and the formation of hydrogen bonds.

[0052] ​The wild-type BmoR protein three-dimensional structure was used as a template to model the T12N 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 show that in the complex, the mutant forms two hydrogen bond interactions with isobutanol (Asn259, Glu261), and forms one hydrogen bond with n-butanol (Arg211), indicating that n-butanol and isobutanol can be tightly combined with the mutant, and the mutant can be used as a wide-range biosensor. K m The value analysis results remain consistent Figure 5 ).

[0053] 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> A wide-range 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 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 GTAAC CTGCGT 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 CCTTGGTCTC 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 Asn 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 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 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 gaaaacgttg 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 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 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 caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 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 ggctgctcat gctcctgtcg cggtagcgcg ctgttacgcg accgcccccg gacctcggcg 180taccgttgcg 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 was obtained by a T12N mutation based on the wild-type BmoR protein shown in SEQ ID NO.1, and the amino acid sequence of the mutant is shown in SEQ ID NO.

3.

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

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

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

5. The encoding gene as described in claim 4, characterized in that, As shown in the sequence list SEQ ID NO.

4.

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

7. A biosensor, characterized in that, The sensor described herein comprises the mutant-encoding gene as described in claim 4, and a promoter activated by the BmoR mutant. P bmo， Depend on P bmo The expression elements of the promoter that drive the reporter gene and express the BmoR mutant.

8. The biosensor as described in claim 7, characterized in that, The reporter genes include gfp , rfp , CFP , sfgfp , egfp , yfp , ecfp Gene 。 9. The biosensor as described in claim 7, characterized in that, The promoters expressing the BmoR mutant include P bmoR。 10. The biosensor as described in claim 7, characterized in that, The sensor is made of P bmoR The mutant gene that was initiated was linked to colE1 replication origin site, amp r and P bmo Started gfp Gene-derived, characterized by a promoter P bmo The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing.

11. The application of the biosensor of claim 7 in detecting environmental, food, and biological samples containing n-butanol or isobutanol, and in screening industrial microbial strains that produce n-butanol or isobutanol.

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