CN115991748A - BmoR mutant-based high-sensitivity biosensor - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a BmoR protein mutant capable of sensitively detecting higher alcohols and application thereof in higher alcohols detection or biosensors. The BmoR protein mutant is obtained by carrying out V311A mutation on the basis of wild BmoR protein shown in a sequence table SEQ ID NO.1, and has an amino acid sequence V31 shown in SEQ ID NO.3The 1A mutant was able to more sensitively react to n-butanol (2.13X10 ^‑6 mM) and isobutanol (1.94X10) ^‑6 mM), the problem that the wild BmoR protein has low sensitivity and cannot screen the higher alcohol with extremely low concentration is solved, and the method can be used for screening and application of low-yield higher alcohol strains.

Description

BmoR mutant-based high-sensitivity biosensor

Technical field:

the invention belongs to the technical field of bioengineering, and particularly relates to a BmoR mutant capable of sensitively detecting extremely low-concentration higher alcohol and application thereof in higher alcohol detection or a biosensor.

The background technology is as follows:

the microbial synthesis of higher alcohols is an important transportation fuel, and the synthesis of higher alcohols by metabolic engineering has been achieved in many microbial hosts, and the transformation of host strains and the screening of hosts for high or low yields are fundamental and critical to the industrial production of higher alcohols. Biosensors are capable of specifically responding to a target compound to output a protein signal that is convenient for detection, and thus have been widely used for high-throughput screening. However, the wild transcription factor Bmor cannot be widely applied to the biosensor and the industrial production due to the poor response specificity, narrow detection range, low sensitivity and the like. The BmoR protein obtained by protein transformation realizes the sensitive detection of higher alcohol, thereby meeting the industrial requirement.

As a new generation of biofuels, higher alcohols are used in many fields while biosynthesis has been achieved in a plurality of microbial hosts, and research has not focused on the detection of very low concentrations of higher alcohols while screening for high yield higher alcohol producing strains. The lowest response concentration of the wild-type transcription factor Bmor to the substrate higher alcohol (n-butanol or isobutanol) is 0.001mM, and detection of lower concentration of alcohol molecules cannot be performed, so that the realization of sensitivity detection of alcohol is a big problem.

The biosensor consists of a molecular recognition element and a signal transducer. When the molecular recognition element is combined with the detected object, the generated signal can be converted into an optical signal or an electric signal through a converter, and the detected object can be detected and analyzed. As a synthetic biology emerging tool, biosensors can be designed to be constructed to dynamically respond to changes in signal molecule concentration. Meanwhile, the biosensor is designed to promote optimization of a microbial cell factory and production of a series of natural products widely used in industry, such as itaconic acid, fatty acid, isobutanol, n-butanol and alkaloids. The biosensor mainly comprises an RNA nucleic acid switch, a transcription factor-regulated biosensor, a G protein coupled receptor and a fluorescent protein biosensor. The low dynamic range of fluorescent protein biosensors, the difficulty in-vitro RNA nucleic acid switching, the fact that G protein-coupled receptors can only be performed outside cells, and the like have prevented the development of biosensors in the biological world.

Transcription Factor (TF) -based biosensors are most widely used. The most commonly used transcription factors are bacterial transcription factors, including Ligand Binding Domains (LBD) or Metabolic Binding Domains (MBD) and DNA Binding Domains (DBD). Bmor is a transcription factor of the Pseudomonas n-paraffin metabolic pathway, a member of the bEBP family, for modulating sigma of alkane monooxygenases ⁵⁴ Dependent promoter P _bmo The signal molecule is a C2-C5 linear or branched alcohol. BmoR-based biosensors can be used for screening high-yield or low-yield n-butanol or isobutanol strains, but the wild BmoR-based biosensors have poor response specificity, narrow detection range and poor sensitivity and cannot be used in industrial production. The modification of the biosensor thus provides a solution for sensitive detection of higher alcohol production and rapid strain screening.

The invention comprises the following steps:

the invention aims to provide BmoR protein and a biosensor which can sensitively detect extremely low-concentration higher alcohol, and a random mutation library is constructed by utilizing an error-prone PCR technology; and (3) screening and analyzing the random mutant library by exogenously adding n-butanol or isobutanol to make the final concentration of the n-butanol or isobutanol be 0-1 mM. Finally, bmoR mutant protein capable of sensitively detecting the high alcohol with extremely low concentration is obtained.

Further, the Bmo R mutant protein capable of detecting 0-1mM higher alcohol is obtained by generating V311A mutation on the basis of a wild Bmo R protein shown in a sequence table SEQ ID NO.1, hereinafter referred to as V311A mutant, and the mutant protein specifically comprises:

(1) An amino acid sequence shown in a sequence table SEQ ID NO. 3; or (b)

(2) An amino acid sequence with more than 75% of SEQ ID NO.3 homology; or (b)

(3) One or more amino acid substitutions and/or deletions are carried out on the basis of SEQ ID NO.3, and/or the amino acid sequence with the same function as SEQ ID NO.3 is obtained after addition.

Furthermore, the invention also provides a coding gene of the V311A mutant;

further, the coding gene is shown in a sequence table SEQ ID NO. 4.

It is a further object of the present invention to provide the use of the V311A mutant, in particular in a sensor for the sensitive detection of samples containing higher alcohols, or for the screening of higher alcohol producing strains, more particularly in the construction of a biosensor for higher alcohols;

further, the biosensor is based on a V311A mutant, and the sensor comprises a V311A mutant coding gene and a promoter P thereof _bmo An expression element for a reporter gene; the promoter initiates expression of the bmoR gene, and the bmoR protein binds to the alcohol molecule to form hexamers, thereby initiating the downstream promoter P _bmo Thereby expressing the reporter gene and generating signals such as fluorescence; the biosensor can realize the response and screening of 0-1mM to higher alcohols, is further applied to industrial production, and realizes the screening of samples containing higher alcohols and higher alcohol production strains.

Further, promoters of the mutant encoding genes include, but are not limited to, P _bmoR 、P _tac 、P _T7 、P _LlacO1 Etc.;

further, the reporter genes include, but are not limited to, gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp isogenes;

preferably, the expression element comprises a V311A mutant coding gene and a promoter P thereof _bmoR Promoter P _bmo Recombinant plasmids of gfp reporter genes; further, expression vectors optionally used for the recombinant plasmid include, but are not limited to, expression vectors commonly used in the art;

more preferably, the biosensor is obtained by replacing the wild BmoR protein encoding gene on plasmid pYH1 with the V311A mutant encoding gene, i.e. will be composed of P _bmoR The V311A mutant coding gene is connected to colE1 replication initiation site and amp ^r And P _bmo A driven gfp gene;

further, the strains include, but are not limited to, E.coli, saccharomyces cerevisiae, bacillus subtilis, etc.;

further, the promoter P _bmo The nucleotide sequence of (2) is shown as SEQ ID NO.5 of the sequence table;

further, the promoter P _bmo The nucleotide sequence of (2) is shown as SEQ ID NO.6 of the sequence table;

further, the nucleotide sequence of the gfp reporter gene is shown in a sequence table SEQ ID NO. 7.

The invention also provides application of the biosensor in higher alcohol detection, especially in screening of isobutanol producing strain, and the application of the biosensor in detecting the production of isobutanol by introducing the plasmid into producing strain, such as escherichia coli, saccharomyces cerevisiae, bacillus subtilis and the like.

The beneficial effects are that:

the detection sensitivity of the wild-type BmoR is too low, the lowest detection concentration for higher alcohols is only 0.001mM, and at this concentration, the response value for higher alcohols is too low, and there is little response for higher alcohols below 0.001mM, and further detection is not possible, and thus it is not possible to identify strains having higher alcohol yields below 0.001 mM. The minimum detection limit of the V311A mutant provided by the invention on the n-butanol reaches 2.13 multiplied by 10 ^-6 mM, 500-fold increase compared to wild type; the lowest detection limit of the isobutanol reaches 1.94 multiplied by 10 ^-6 mM, 1000-fold higher than wild type, and increased the sensitivity of BmoR protein to higher alcohols.

Description of the drawings:

FIG. 1 is a schematic flow chart;

firstly, randomly mutating 1000bp before the N end of a wild Bmo R by error-prone PCR to obtain a Bmo R random mutation library; the GFP fluorescent protein is added downstream of the bmoR gene, and the response of the mutant Bmor to higher alcohols can be reflected by detecting the fluorescence intensity. The response of the mutant Bmor to higher alcohols was tested by adding different concentrations of n-butanol or isobutanol.

FIG. 2 shows the response of BmoR mutants and wild type to 10mM n-butanol or isobutanol;

FIG. 3 shows the response of BmoR mutants and wild type to 0-1mM n-butanol or isobutanol;

FIG. 4 shows the molecular docking of the V311A mutant with n-butanol and isobutanol;

the specific embodiment is as follows:

the invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

The biosensor provided by the invention is based on a V311A mutant, and comprises a V311A mutant coding gene, a promoter and a promoter P thereof _bmo An expression element for a reporter gene; the promoter initiates expression of the bmoR gene, and the bmoR protein binds to the alcohol molecule to form hexamers, thereby initiating the downstream promoter P _bmo Thereby expressing the reporter gene and generating a signal such as fluorescence. The skilled worker can choose a promoter to promote the expression of BmR mutant genes in the prior art according to the actual situation, such as P _bmoR 、P _tac 、P _T7 、P _LlacO1 Such promoters. The reporter gene may be selected from various protein molecules commonly used in the art, such as fluorescent proteins, colored proteins, etc., capable of producing a detection signal, and preferably, such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc., may be used to effect the response of the biosensor of the present invention. The sensor also comprises necessary elements for realizing expression, such as a replication initiation site, and preferably, a colE1 replication initiation site, and the like. The sensor may further comprise a marker such as a resistance gene, e.g. amp ^r And the like, and is convenient for screening. The person skilled in the art can add other elements to the above-mentioned sensor according to actual requirements, such as constructing the above-mentioned elements onto expression vectors in the prior art, such as pET, pUC19, pMAL, etc., to obtain recombinant plasmids that can be used as sensors.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The invention will be further illustrated by the following examples.

EXAMPLE 1 screening of Bmor mutant V311A

Construction of random mutation library of transcription factor Bmor

(1) The BmoR mutant gene was obtained by error-prone PCR amplification using plasmid pYH1 (construction see DOI: https:// doi.org/10.1016/j. Ymben.2019.08.015; https:// doi.org/10.1186/s 12934-019-1084-2) as template with the wild-type Bmor encoding gene (SEQ ID NO. 2) (by adding Mn to the PCR system) ²⁺ Improving Mg in PCR system ²⁺ The concentration and ratio of dNTPs were adjusted to introduce random mutations. A10 Ximbalanced dNTPs mixture was prepared, in which the concentration of dCTP, dTTP was four times that of dATP, dGTP. The PCR procedure was set as follows: the pre-denaturation at 94℃for 2min,30 amplification cycles included: denaturation at 95℃for 1min, annealing at 55-68℃for 1min, and proper extension time at 72℃was determined according to an amplification rate of 1kb per minute. The storage temperature was set at 16 ℃. ) The method comprises the steps of carrying out a first treatment on the surface of the Confirming the PCR product by gel electrophoresis, and recovering and purifying; the purified product was placed in a 37℃water bath and DpnI (1. Mu.L/50. Mu.L of purified product) was digested for 1-2h. mu.L of the bmoR mutant fragment and 3. Mu.L of the pYH1 backbone (P-constructed can also be used _bmoR 、P _bmo The gfp fluorescent protein gene plasmid is taken as a framework and is equivalent to the effect of the pYH1 framework) and 5 mu L Gibson Assemble Mix, and the mixture is placed in a water bath kettle at 50 ℃ for connection for 1h. Transferring 5-10 mu L of the ligation product into 50 mu L of E.coli XL10-Gold transformation competent cells, and culturing at 37 ℃ overnight to obtain Bmor-1000bp mutation library.

(2) Single colonies on the plates were picked and inoculated into 5mL LB (100. Mu.g/mL Amp) liquid medium, and cultured at 37℃for 8 hours at 220rpm as seed liquid. Preliminary screening was performed using 96 deep well plates. 950. Mu.L of fresh LB (100. Mu.g/mL Amp) medium was added to each well; n-butanol or isobutanol was added to the wells to a final concentration of 10mM, respectively; finally, 50. Mu.L of seed solution was pipetted into each well. After sealing the sealing film, the deep-hole plate was placed in a shaker at 30℃and 220rpm for 16h.

(3) Fluorescence intensity GFP using microplatesAnd OD (optical density) ₆₀₀ And (3) detecting: blowing and beating the mixed bacterial liquid, absorbing 200 mu L, putting into an enzyme-labeled instrument, quantitatively detecting at 30 ℃, and setting parameters as follows: 470nm excitation wavelength, 510nm emission wavelength, gain value of 50; the GFP and OD obtained ₆₀₀ The values were first subtracted from the background control value, and the GFP/OD was calculated for each well based on this ₆₀₀ As relative fluorescence intensity values.

(4) After analysis of the primary screening results, plasmids of the active mutant bacteria were sequenced.

The mutation site of one BmoR-containing protein is determined to be V311A (the amino acid sequence is shown as SEQ ID NO.3 and the nucleotide sequence is shown as SEQ ID NO. 4), and the GFP/OD of the mutant ₆₀₀ The primary screening results are shown in Table 1 and FIG. 2, which demonstrate that the V311A mutant has a higher response to 10mM n-butanol and isobutanol than the wild type Bmor.

TABLE 1 GFP/OD ₆₀₀

	N-butanol	Isobutanol
			WT	983	868
V311A	2613	3048

EXAMPLE 2 concentration gradient assay to determine the limit of detection variation of wild type and mutant

The wild BmoR-based biosensor can realize response to 0-1mM n-butanol or isobutanol, and has response to both n-butanol and isobutanol; when the substrate concentration is less than 0.001mM, the response value is almost zero, and no response can be made to alcohol molecules at lower concentrations. The highly sensitive response of BmoR biosensors based on the V311A mutant to 0-1mM n-butanol or isobutanol was verified experimentally as follows.

Based on the primary screening result, exogenous addition experiments of n-butanol or isobutanol with concentration gradient are respectively carried out on the strain containing the V311A mutation, and parameters such as a response curve, the minimum response concentration, a response intensity value and the like are measured.

The single clone on the plate was picked up and inoculated into 5mL LB (100. Mu.g/mL Amp) liquid medium, and cultured at 37℃for 8 hours at 220rpm as a seed solution.

Exogenous addition experiments were performed in sterilized 2ml 96 deep well plates. 950. Mu.L of fresh LB (100. Mu.g/mL Amp) medium was added to each well, and then n-butanol or isobutanol were added to the medium to give final concentrations of 0, 1X 10, respectively ^-6 、1×10 ^-5 、1×10 ^-4 、1×10 ^-3 、1×10 ^-2 、1×10 ^-1 Or 1mM, and finally 50. Mu.L of seed solution is inoculated into each well, after sealing the sealing film, the deep-well plate is placed at 30℃and shaking culture at 220rpm is performed for 16 hours.

Fluorescence intensity GFP and OD Using microplates ₆₀₀ And (3) detecting: blowing and beating the mixed bacterial liquid, absorbing 200 mu L, putting into an enzyme-labeled instrument, quantitatively detecting at 30 ℃, and setting parameters as follows: 470nm excitation wavelength and 510nm emission wavelength, the gain value is 50; the GFP and OD obtained ₆₀₀ The values were first subtracted from the background control value, and the GFP/OD was calculated for each well based on this ₆₀₀ As relative fluorescence intensity values.

With GFP/OD ₆₀₀ On the ordinate, the final concentrations of n-butanol and isobutanol were plotted on the abscissa, and data were subjected to Mi fitting using OriginPro 8.5 or GraphPad Prism 8 software, respectively. The lowest detected concentration of Bmor on higher alcohols is defined as the substrate concentration at which 75% of the maximum fluorescence response value is reached. Calculating the lowest value of wild BmoR and mutant to n-butanol and isobutanol according to the fitting resultConcentration, maximum response intensity, etc. were measured (fig. 3).

Screening the random mutant library under the concentration of 10mM substrate, wherein compared with the wild type, the obtained mutant V311A has extremely high fluorescence response to the n-butyl alcohol and the isobutanol, and the response value to the n-butyl alcohol reaches 2.67 times of the wild type; the response value to isobutanol reaches 3.51 times of that of the wild type isobutanol; further validation was performed at a gradient of 0-1mM, and the results indicated that the V311A mutant remained high in response to n-butanol and isobutanol at a substrate concentration of 0-1 mM. The data were subjected to a Mi fit by an OriginPro 8.5 plot and the lowest detected concentrations of wild type BmoR and mutant on higher alcohols were calculated. The results showed that the lowest detection concentration of wild type BmoR on n-butanol was 1.36×10 ^-3 mM, the lowest detection concentration of isobutanol was 2.53X10 ^-3 mM; the lowest detection concentration of the V311A mutant on the n-butanol reaches 2.13 multiplied by 10 ^-6 mM, 500-fold increase compared to wild type; the lowest detection concentration of the isobutanol reaches 1.94 multiplied by 10 ^-6 mM, 1000-fold higher compared to wild type.

Example 3 model analysis

Sequencing the V311A mutant, analyzing the change of amino acid at a mutation site, modeling the Bmor mutant by using software such as AUTODOCK, chimeraX and the like, butting the mutant with substrate micromolecule n-butanol and isobutanol respectively, and analyzing the formation condition of the mutant and the binding sites and hydrogen bonds of the two alcohols.

And (3) taking the three-dimensional structure of the wild BmoR protein as a template, and carrying out homologous modeling on the V311A mutant, wherein the homology rate is 99.9%. The mutant structure is further molecularly docked to a substrate molecule (n-butanol or isobutanol). The results showed that in the complex, the mutant formed 2 hydrogen bond interactions together with n-butanol (Arg 211, gln 212); 2 hydrogen bond interactions with isobutanol (Asn 259, leu 260) indicate that both n-butanol and isobutanol bind tightly to the mutant, consistent with experimental results (fig. 4).

The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.

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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 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> 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 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> 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 (13)

1. A BmoR mutant protein is characterized in that the mutant is obtained by generating a mutant containing V311A on the basis of a wild type BmoR protein shown in a sequence table SEQ ID NO. 1.

2. The BmoR mutant protein of claim 1, wherein the mutant protein is specifically:

(1) An amino acid sequence shown in a sequence table SEQ ID NO. 3; or (b)

(2) An amino acid sequence with more than 75% of SEQ ID NO.3 homology; or (b)

(3) One or more amino acid substitutions and/or deletions are carried out on the basis of SEQ ID NO.3, and/or the amino acid sequence with the same function as SEQ ID NO.3 is obtained after addition.

3. Use of the BmoR mutant of claim 1 for detecting samples containing very low concentrations of higher alcohols or for screening higher alcohol producing strains.

4. Use of the BmoR mutant of claim 1 for constructing a biosensor for detecting higher alcohols.

5. A gene encoding the Bmor mutant of claim 1.

6. The coding gene according to claim 5, wherein the coding gene is shown in SEQ ID NO.4 of the sequence Listing.

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

8. A biosensor comprising the mutant code of claim 5Gene and promoter P activated by Bmor mutant _bmo From P _bmo Driven reporter genes and expression elements of promoters expressing BmoR mutants.

9. The biosensor of claim 8, wherein the reporter gene comprises, but is not limited to, a gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp gene.

10. The biosensor of claim 8, wherein the promoter expressing the Bmor mutant includes, but is not limited to, P _bmoR 、P _tac 、P _T7 、P _LlacO1 。

11. The biosensor of claim 8, wherein the sensor is to be defined by P _bmoR The started mutant coding gene is connected to colE1 replication initiation site and amp ^r And P _bmo The initiated gfp gene.

12. The biosensor of claim 8, wherein promoter P _bmo The nucleotide sequence of (2) is shown in a sequence table SEQ ID NO. 5.

13. Use of the biosensor of claim 8 for detecting environmental, food, medical, biological samples containing very low concentrations of higher alcohols, and for screening industrial microbial strains producing higher alcohols.

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