A highly sensitive biosensor based on the BmoR mutant

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a BmoR protein mutant capable of sensitively detecting n-butanol or isobutanol, and its application in n-butanol or isobutanol detection or biosensors. The BmoR protein mutant is obtained by mutating the wild-type BmoR protein shown in SEQ ID NO.1 to the E54G mutation. The E54G mutant, possessing the amino acid sequence shown in SEQ ID NO.3, is more sensitive to n-butanol (2.64 × 10⁻⁶). ‑6 mM) and isobutanol (2.16×10 ‑6 The method uses mM) detection to solve the problem of low sensitivity of wild-type BmoR protein, which cannot screen for extremely low concentrations of n-butanol or isobutanol. It can be used for screening and application of strains that produce low levels of higher alcohols.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a BmoR mutant that can sensitively detect extremely low concentrations of higher alcohols, and its application in higher alcohol detection or biosensors. Background Technology

[0002] Higher alcohols synthesized by microorganisms are important transport fuels. The synthesis of higher alcohols through metabolic engineering has been achieved in many microbial hosts. Modifying host strains and screening for high- or low-yield production is fundamental and crucial for the industrial production of higher alcohols. Biosensors, capable of specifically responding to target compounds and outputting easily detectable protein signals, have been widely used in high-throughput screening. However, the wild-type transcription factor BmoR, due to its poor response specificity, narrow detection range, and low sensitivity, cannot be widely applied in biosensors and industrial production. This invention, through protein modification, yields the BmoR protein, achieving sensitive detection of higher alcohols, thus meeting industrial requirements.

[0003] As a new generation of biofuels, higher alcohols are used in many fields and have been biosynthesized in multiple microbial hosts. While screening strains that produce high levels of higher alcohols, research has not focused on the detection of extremely low concentrations of higher alcohols. The wild-type transcription factor BmoR has a minimum response concentration of 0.001 mM to the substrate higher alcohols (n-butanol or isobutanol), making it impossible to detect alcohol molecules at even lower concentrations. Therefore, achieving sensitive detection of alcohols remains a major challenge.

[0004] Biosensors consist of molecular recognition elements and signal transducers. When the molecular recognition element binds to the analyte, the resulting signal can be converted into an optical or electrical signal by the transducer, enabling the detection and analysis of the analyte. As an emerging tool in synthetic biology, biosensors can be designed and constructed to dynamically respond to changes in the concentration of signal molecules. Simultaneously, biosensors are designed to facilitate the optimization of microbial cell factories and the production of a range 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 in vitro RNA nucleic acid switching, and the extracellular limitation of G protein-coupled receptors have hindered the development of biosensors in the biological world.

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

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

[0007] Furthermore, the BmoR mutant protein capable of detecting 0-1 mM higher alcohols is obtained by undergoing an E54G mutation on the wild-type BmoR protein shown in SEQ ID NO.1, hereinafter referred to as the E54G mutant. The mutant protein specifically comprises:

[0008] (1) The amino acid sequence shown in SEQ ID NO.3 of the sequence listing; or

[0009] (2) An amino acid sequence with more than 75% homology to SEQ ID NO.3; or

[0010] (3) An amino acid sequence with the same function as SEQ ID NO.3 obtained by replacing one or more amino acids, and / or deleting, and / or adding them based on SEQ ID NO.3.

[0011] Furthermore, the present invention also provides the coding gene for the E54G mutant;

[0012] Furthermore, the encoding gene is shown in the sequence listing SEQ ID NO.4.

[0013] Another object of the present invention is to provide applications of the E54G mutant, particularly in sensors for the sensitive detection of samples containing higher alcohols or for screening strains that produce higher alcohols, and more particularly in the construction of biosensors for higher alcohols.

[0014] Furthermore, the biosensor is based on the E54G mutant, and the sensor contains the E54G mutant coding gene and its promoter, and the 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 expression of reporter genes and the generation of fluorescent signals. The biosensor can respond to and screen for higher alcohols at 0-1 mM, and can be further applied in industrial production to screen strains that produce higher alcohols.

[0015] Furthermore, the promoter of the mutant encoding gene includes P bmoR 、P tac 、P T7 、P LlacO1 wait;

[0016] Furthermore, the reporter gene includes gfp , rfp , CFP , sfgfp , egfp , yfp , ecfp Isogenes;

[0017] Preferably, the expression element comprises the E54G mutant coding gene and its promoter. P bmoR promoter P bmo and gfp A recombinant plasmid for a reporter gene; further, the expression vector that can be used for the recombinant plasmid includes, but is not limited to, expression vectors commonly used in the art;

[0018] More preferably, the biosensor is obtained by replacing the wild-type BmoR protein-coding gene on plasmid pYH1 with the E54G mutant gene, that is, replacing the wild-type BmoR protein-coding gene on plasmid pYH1 with the E54G mutant gene. P bmoR The E54G mutant gene that was initiated was linked to colE1 replication origin site, amp r andP bmo Driven gfp Genetic;

[0019] Furthermore, the strains include, but are not limited to, Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc.

[0020] Furthermore, the promoter P bmoR The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing;

[0021] Furthermore, the promoter P bmo The nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing;

[0022] Furthermore, the aforementioned gfp The nucleotide sequence of the reporter gene is shown in SEQ ID NO.7 of the sequence listing.

[0023] The present invention also provides the application of the above-mentioned biosensor in the detection of higher alcohols, especially in the screening of isobutanol producing strains. By introducing the above-mentioned plasmid into producing strains, such as Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, etc., the production of higher alcohols can be detected. Beneficial effects

[0024] The wild-type BmoR strain has excessively low detection sensitivity, with a limit of detection (LOD) of only 0.001 mM for higher alcohols. Furthermore, at this concentration, its response to higher alcohols is extremely low, showing almost no response to concentrations below 0.001 mM, making further detection impossible. Therefore, it cannot be used to identify strains with higher alcohol yields below 0.001 mM. In contrast, the E54G mutant provided by this invention achieves a limit of detection (LOD) of 2.64 × 10⁻⁶ for n-butanol. -6 The mM concentration was 500 times higher than that of the wild type; the limit of detection for isobutanol reached 2.16 × 10⁻⁶ mM. -6 The mM concentration was increased by 1000 times compared to the wild type, improving the sensitivity of BmoR protein for the detection of higher alcohols. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the principle.

[0026] First, wild-type was identified through error-prone PCR. bmoR Random mutations were performed on the first 1000 bp of the N-terminus to obtain a random mutant library of BmoR; bmoR Adding GFP fluorescent protein downstream of the gene allows for the detection of fluorescence intensity to reflect the response of the mutant BmoR to higher alcohols. The response of the mutant BmoR to higher alcohols was also detected by adding different concentrations of n-butanol or isobutanol.

[0027] Figure 2 The response of BmoR mutant and wild type to 10 mM n-butanol or isobutanol;

[0028] Figure 3 The response of BmoR mutants and wild-type to 0-1 mM n-butanol or isobutanol;

[0029] Figure 4 The molecular docking of the E54G mutant with n-butanol and isobutanol; Detailed Implementation

[0030] 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.

[0031] The biosensor provided by this invention is based on the E54G mutant. The sensor comprises the E54G 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 、P tac 、P T7 、P LlacO1 The promoter can be varied. The reporter gene can also be any protein molecule commonly used in the art that can generate a visual detection signal 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, ecfpThe aforementioned sensors also include necessary elements for expression, such as a replication origin site, preferably, such as the colE1 replication origin site. The sensors may also include markers such as resistance genes, like ampr, for easy screening. Those skilled in the art can add other elements to the sensors according to actual needs, such as constructing these elements onto existing expression vectors, like pET, pUC19, and pMAL, to obtain recombinant plasmids that can be used as sensors.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] The present invention will be further explained and illustrated below through specific embodiments.

[0034] Example 1: Screening of the BmoR mutant E54G

[0035] Constructing a random mutant library of transcription factor BmoR

[0036] (1) Using plasmid pYH1 (construction details can be found at DOI: https: / / doi.org / 10.1016 / j.ymben.2019.08.015; https: / / doi.org / 10.1186 / s12934-019-1084-2) carrying the wild-type BmoR encoding gene (SEQ ID NO.2) as a template, error-prone PCR amplification was performed to obtain bmoR Mutant gene (by adding Mn to the PCR system) 2+ Increase the amount of Mg in the PCR system 2+ The concentration and ratio of dNTPs were adjusted to introduce random mutations. A 10× unbalanced dNTPs mixture was prepared, in which the concentrations of dCTP and dTTP were four times that of dATP and dGTP. The PCR program was set as follows: 94ºC pre-denaturation for 2 min, 30 amplification cycles including: 95ºC denaturation for 1 min, 55-68ºC annealing for 1 min, and an appropriate extension time determined according to an amplification rate of 1 kb / min, with an extension temperature of 72ºC. The storage temperature was set to 16ºC. 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. Take 2 μL bmoR mutant fragment, 3 μL pYH1 backbone (or a pYH1-based backbone can be used) bmoR P bmo , gfpThe plasmid of the fluorescent protein gene (equivalent to the pYH1 backbone) and 5 μL of Gibson Assemble Mix were mixed and placed in a 50℃ water bath for 1 h 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℃ to obtain the BmoR-1000bp mutant library.

[0037] (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 ℃ 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; and 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 ℃ and 220 rpm for 16 h.

[0038] (3) Using microplates to measure the fluorescence intensity of GFP and OD 600 Detection: Mix the bacterial culture by pipetting, and transfer 200 μL into a microplate reader. Quantitative detection is performed at 30 °C. 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 value for each well is calculated. 600 As a relative fluorescence intensity value.

[0039] (4) After analyzing the initial screening results, the plasmids of the effective mutant bacteria were sequenced.

[0040] One of the amino acid mutation sites in the BmoR protein was identified as E54G (amino acid sequence as shown in SEQ ID NO. 3, nucleotide sequence as shown in SEQ ID NO. 4). This mutant exhibits GFP / OD... 600 The initial screening results are shown in Table 1 and Figure 2 As shown, compared to the wild-type BmoR, the E54G mutant has a higher response to 10 mM n-butanol and isobutanol.

[0041] Table 1 GFP / OD 600

[0042] n-Butanol Isobutanol WT 983 868 E54G 2489 2814

[0043] Example 2: Concentration gradient experiment to determine the change in detection limit between wild type and mutant

[0044] The biosensor based on wild-type BmoR can respond to 0-1 mM n-butanol or isobutanol, showing a response to both. However, when the substrate concentration is below 0.01 mM, the response value is almost zero, and it cannot respond to lower concentrations of alcohol molecules. The following experiments verify the specific response of the BmoR biosensor based on the E54G mutant to 0-1 mM n-butanol / isobutanol.

[0045] Based on the initial screening results, the strains containing the E54G mutation were subjected to exogenous addition experiments with concentration gradients of n-butanol or isobutanol, and parameters such as response curves, minimum response concentrations, and response intensity values ​​were measured.

[0046] Select a single clone from the plate and inoculate it into 5 mL LB (100 μg / mL Amp) liquid medium. Incubate at 37 ℃ and 220 rpm for 8 h to obtain the seed culture.

[0047] The exogenous addition experiments were performed in sterile 2 mL 96-well plates. Each well was first filled with 950 μL of fresh LB medium (100 μg / mL Amp), followed by the addition of either n-butanol or isobutanol to achieve final concentrations of 0 and 1 × 10⁻⁶, respectively. -6 1×10 -5 1×10 -4 1×10 -3 1×10 -2 1×10 -1 Alternatively, use 1 mM, and finally inoculate 50 μL of seed solution into each well. After sealing with a sealing film, place the deep well plate in a shaker at 30°C and 220 rpm for 16 h.

[0048] Using microplates to measure fluorescence intensity GFP and OD 600 Detection: Mix the bacterial culture by pipetting, and transfer 200 μL into a microplate reader. Quantitative detection is performed at 30℃. 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 value for each well is calculated. 600 As a relative fluorescence intensity value.

[0049] With GFP / OD 600 Plot the data on the ordinate with the final concentrations of n-butanol and isobutanol on the x-axis, respectively, using OriginPro 8.5 or GraphPad Prism 8 software, and perform Michaelis-Menten fitting. The minimum detectable concentration of BmoR for higher alcohols is defined as the substrate concentration at which 75% of the maximum response value is achieved. Based on the fitting results, the minimum detectable concentrations and maximum response intensity of the BmoR mutant for n-butanol and isobutanol were calculated. Figure 3 ).

[0050] A random mutant library was screened at a substrate concentration of 10 mM. Compared with the wild type, the mutant E54G showed extremely high fluorescence responses to n-butanol and isobutanol, with the response value to n-butanol reaching 2.53 times that of the wild type and the response value to isobutanol reaching 3.24 times that of the wild type. Further validation was performed under 0-1 mM gradient concentration conditions, and the results showed that the E54G mutant maintained a high level of response to n-butanol and isobutanol at 0-1 mM substrate concentrations. Plotting was performed using OriginPro 8.5, and Michaelis-Menten fitting was used to calculate the minimum detection concentrations (MRCs) for higher alcohols for both the wild-type BmoR and the mutant. The MRC for n-butanol in the wild-type BmoR reached 1.36 × 10⁻⁶ mM. -3 The lowest detectable concentration of isobutanol was 2.53 × 10⁻⁶ mM. -3 The lowest detectable concentration of n-butanol for the E54G mutant was 2.64 × 10⁻⁶ mM. -6 The mM concentration was increased by 500 times compared to the wild type; the lowest detectable concentration of isobutanol reached 2.16 × 10⁻⁶. -6 mM, which is 1000 times higher than that of the wild type.

[0051] Example 3 Model Analysis

[0052] The E54G mutant was sequenced to analyze the changes in amino acids at the mutation site. Autodoc and Chimera sequencing were also used. X Software was used to model the BmoR mutant, and the mutant was docked with the substrate small molecules n-butanol and isobutanol, respectively. The binding sites and hydrogen bond formation of the mutant with these two alcohols were analyzed.

[0053] Using the three-dimensional structure of the wild-type BmoR protein as a template, homology modeling of the E54G mutant was performed, with a homology rate of 99.9%. Further molecular docking was conducted between the mutant structure and substrate molecules (n-butanol or isobutanol). The results showed that in the complex, the mutant formed three hydrogen bonds with n-butanol (Asn259, Glu261) and three hydrogen bonds with isobutanol (Asn259, Glu261), indicating that both n-butanol and isobutanol can bind tightly to this mutant. experiment The results remain consistent. Figure 4 ).

[0054] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims. sequence list <110> Beijing Institute of Technology <120> A highly sensitive biosensor based on the BmoR mutant <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 669 <212> PRT <213> Pseudomonas butanovora <400> 1 Met Ser Lys Met Gln 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 Gln 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 Gln Glu Phe Asp Pro Ile Pro Arg Thr Ala Leu Asp 50 55 60 Glu Thr Val Glu Ala Lys Arg Ala Leu Ile Leu Ala Ala Glu Pro Val 65 70 75 80 Val Asp Ala Leu Met Glu Gln Met Asn Asp Ala Pro Arg Met Ile Ile 85 90 95 Leu Asn Asp Glu Arg Gly Val Val Leu Leu Asn Gln Gly Asn Asp Thr 100 105 110 Leu Leu Glu Asp Ala Arg Arg Arg Ala Val Arg Val Gly Val Cys Trp 115 120 125 Asp Glu His Ala Arg Gly Thr Asn Ala Met Gly Thr Ala Leu Ala Glu 130 135 140 Arg Arg Pro Val Ala Ile His Gly Ala Glu His Tyr Leu Glu Ser Asn 145 150 155 160 Thr Ile Phe Thr Cys Thr Ala Ala Pro Ile Tyr Asp Pro Phe Gly Glu 165 170 175 Phe Thr Gly Ile Leu Asp Ile Ser Gly Tyr Ala Gly Asp Met Gly Pro 180 185 190 Val Pro Ile Pro Phe Val Gln Met Ala Val Gln Phe Ile Glu Asn Gln 195 200 205 Leu Phe Arg Gln Thr Phe Ala Asp Cys Ile Leu Leu His Phe His Val 210 215 220 Arg Pro Asp Phe Val Gly Thr Met Arg Glu Gly Ile Ala Val Leu Ser 225 230 235 240 Arg Glu Gly Thr Ile Val Ser Met Asn Arg Ala Gly Leu Lys Ile Ala 245 250 255 Gly Leu Asn Leu Glu Ala Val Ala Asp His Arg Phe Asp Ser Val Phe 260 265 270 Asp Leu Asn Phe Gly Ala Phe Leu Asp His Val Arg Gln Ser Ala Phe 275 280 285 Gly Leu Val Arg Val Ser Leu Tyr Gly Gly Val Gln Val Tyr Ala Arg 290 295 300 Val Glu Pro Gly Leu Arg 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 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 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 tttgaccct gttcaaacgt 1620 cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680 ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740 gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800 ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860 caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920 cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980 cactggtctc agtctcgtat cggtacctaa 2010 <210> 3 <211> 669 <212> PRT <213> Artificial sequence <400> 3 Met Ser Lys Met Gln 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 Gln 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 Gln Gly Phe Asp Pro Ile Pro Arg Thr Ala Leu Asp 50 55 60 Glu Thr Val Glu Ala Lys Arg Ala Leu Ile Leu Ala Ala Glu Pro Val 65 70 75 80 Val Asp Ala Leu Met Glu Gln Met Asn Asp Ala Pro Arg Met Ile Ile 85 90 95 Leu Asn Asp Glu Arg Gly Val Val Leu Leu Asn Gln Gly Asn Asp Thr 100 105 110 Leu Leu Glu Asp Ala Arg Arg Arg Ala Val Arg Val Gly Val Cys Trp 115 120 125 Asp Glu His Ala Arg Gly Thr Asn Ala Met Gly Thr Ala Leu Ala Glu 130 135 140 Arg Arg Pro Val Ala Ile His Gly Ala Glu His Tyr Leu Glu Ser Asn 145 150 155 160 Thr Ile Phe Thr Cys Thr Ala Ala Pro Ile Tyr Asp Pro Phe Gly Glu 165 170 175 Phe Thr Gly Ile Leu Asp Ile Ser Gly Tyr Ala Gly Asp Met Gly Pro 180 185 190 Val Pro Ile Pro Phe Val Gln Met Ala Val Gln Phe Ile Glu Asn Gln 195 200 205 Leu Phe Arg Gln Thr Phe Ala Asp Cys Ile Leu Leu His Phe His Val 210 215 220 Arg Pro Asp Phe Val Gly Thr Met Arg Glu Gly Ile Ala Val Leu Ser 225 230 235 240 Arg Glu Gly Thr Ile Val Ser Met Asn Arg Ala Gly Leu Lys Ile Ala 245 250 255 Gly Leu Asn Leu Glu Ala Val Ala Asp His Arg Phe Asp Ser Val Phe 260 265 270 Asp Leu Asn Phe Gly Ala Phe Leu Asp His Val Arg Gln Ser Ala Phe 275 280 285 Gly Leu Val Arg Val Ser Leu Tyr Gly Gly Val Gln Val Tyr Ala Arg 290 295 300 Val Glu Pro Gly Leu Arg 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 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 gattcgaccc gatcccgcgt 180 accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240 gttgacgctc tgatggaaca gatgaacgac gctccgcgta tgatcatcct gaacgacgaa 300 cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360 gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420 gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480 accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540 ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600 gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660 cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720 cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780 gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840 gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900 gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960 ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020 ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080 gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140 aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200 ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260 gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320 ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380 gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 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 cactggtctc agtctcgtat cggtacctaa <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 gcagacaca CAaggagga agtg 524 <210> 7 <211> 717 <212> DNA <213> Artificial Sequence <400> 7 atgcgtaaag gagagaact ttcactgga gttgtcccaa tctgttga attagatggt 60 gatgttaatg ggcacaaattt ttctcagt ggaggggtg aaggtgatgc aacatacgga 120 aaacttaccc ttaaatttat ttgcactact ggaaactac ctgttccatg gccacactt 180 gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca tatgaacag 240 catgacttt tcaagagtgc catgcccgaa ggttatgtac aggaagaac tatattttc 300 aaagatgacg ggaactaca caacgtgct gaagtcaagt tgaaggtga tacccttgtt 360 atagaatcg agttaaaagg tattgattt aaagagatg gaacattct tggacacaaa 420 ttggaataca actataactc acacaatgta tacatcatgg cagacaaca aaagaatgga 480 atcaagtta acttcaaat tagacaac attgaatg 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 E54G mutation based on the wild-type BmoR shown in the sequence table SEQ ID NO.

1.

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

3. Use of the BmoR mutant of claim 1 in constructing biosensor for detecting n-butanol or 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. Recombinant plasmid or 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 promoter activated by the BmoR mutant P bmo , a reporter gene driven by P bmo the promoter expressing the BmoR mutant 8. The biosensor of claim 7, wherein, The reporter gene comprises gfp , rfp , cfp , sfgfp , egfp , yfp or ecfp gene 。 9. The biosensor of claim 7, wherein, Promoters that express BmoR mutants include P bmoR 、 P tac 、P T7 or P LlacO1 .

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

11. The biosensor of claim 7, wherein the enzyme is glucose oxidase. Promoter P bmo The nucleotide sequence of the promoter is shown in SEQ ID NO. 6 of the sequence listing.

12. Use of the biosensor of claim 7 in detecting biological samples containing extremely low concentration of n-butanol or isobutanol or in screening industrial microbial strains producing n-butanol or isobutanol.

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