CN115181167B

CN115181167B - Screening method of ethanol production strain based on BmoR mutant

Info

Publication number: CN115181167B
Application number: CN202110360692.0A
Authority: CN
Inventors: 霍毅欣; 毋彤; 陈振娅; 张翠英
Original assignee: Beijing Institute of Technology BIT; Tianjin University of Science and Technology
Current assignee: Beijing Institute of Technology BIT; Tianjin University of Science and Technology
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2023-08-08
Anticipated expiration: 2041-04-02
Also published as: CN115181167A

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a BmoR protein mutant specifically responding to isobutanol and application thereof in isobutanol detection or a biosensor. The BmoR protein mutant is obtained by mutating M94V and/or F272L on the basis of a wild BmoR protein shown in a sequence table SEQ ID NO. 1. The M94V/F272L mutant is only sensitive to isobutanol, is not responsive to n-butanol and is highly insensitive to ethanol, so that the problem that wild BmoR protein cannot distinguish n-butanol, isobutanol and ethanol is solved; meanwhile, the detection range of the isobutanol reaches 0-100mM, the response saturation of BmoR protein to the isobutanol is improved, and the method can be used for screening and applying strains with higher yield.

Description

Screening method of ethanol production strain based on BmoR mutant

Technical field:

the invention belongs to the technical field of bioengineering, and particularly relates to a BmoR protein mutant specifically responding to isobutanol and application thereof in isobutanol detection or a biosensor.

The background technology is as follows:

the fusel oil is an important flavor substance in the alcoholic beverage, mainly comprises n-propanol, isobutanol, isoamyl alcohol, phenethyl alcohol and the like, and can endow wine with special fragrance, but too high content of the fusel oil can damage the taste of the wine and also harm the health of human bodies; therefore, the breeding of a proper amount of bacterial strain for producing fusel oil has important significance for improving the quality of products.

In the brewing process of alcoholic beverages, fusel oil is mainly produced by fermentation of Saccharomyces cerevisiae. At present, relevant reports of breeding a proper amount of yeast strains producing fusel oil through mutation breeding exist. Heng by adding a certain amount of isoamyl chloroacetate into a plate for culturing yeast, the yeast strain with low fusel oil yield can be rapidly screened, and the bred yeast strain with low fusel oil yield can reduce the fusel oil content in the wine by 10% -15% (Chinese patent application CN 103627646A). The beer yeast after ultraviolet mutagenesis is coated on a methylpyrazole-dithiolan-ethanol resistant plate under Hong Mei and the like, and the yield of fusel oil is reduced by 45% -55% under wort fermentation conditions of the mutant strain obtained by screening (Chinese patent application CN 106834148B). However, in these studies, high performance liquid chromatography is often used to detect the fusel oil content, which greatly limits the throughput of the detection. In recent years, the yield of fusel oil is detected by a colorimetric method, but the yield is still limited by the problems of large reaction system, low detection speed and the like, and the high-throughput screening of the fusel oil cannot be realized. Therefore, development of a high-throughput screening method with high accuracy and high sensitivity is needed, so that large-scale screening of strains is realized, and the method has great application significance for breeding of food-use bacteria.

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 optical signal or the electric signal 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 biosensor, the difficulty of RNA nucleic acid switch in vitro, the G protein coupled receptor can only be performed outside the cell, etc. have hindered the development of biosensor in biology.

The BmoR-based biosensor can be applied to screening of alcohols high-yield strains such as n-butanol, isobutanol and the like. Since the wild BmoR has response to C2-C6 alcohol, the sensor cannot be directly applied to host strains such as Saccharomyces cerevisiae and the like which naturally have ethanol production capacity, so that the application of the sensor is greatly limited. For example: during alcoholic beverage fermentation, the amount of ethanol synthesized by saccharomyces cerevisiae is far higher than that of other alcohols, and a wild BmoR sensor cannot be used for screening mutant strains with different fusel oil yields. Based on the problem, the BmoR is subjected to point mutation, the mutant insensitive to ethanol is obtained by screening, the response characteristic of the mutant to isobutanol is maintained, and a high-flux screening system is constructed by utilizing the mutant, so that the high-flux screening of brewing microorganisms is realized, and the method has extremely high innovation.

The invention comprises the following steps:

the invention aims to provide a method for screening ethanol production strains, which modifies the N end of a response element BmR protein by introducing protein engineering to construct a biosensor based on BmR mutant. The invention utilizes error-prone PCR technology to construct random mutation library, and carries out screening analysis on the mutation library through exogenous addition of ethanol, n-butanol or isobutanol and fermentation experiment; finally, bmoR protein mutants and biosensors which are highly insensitive to ethanol or n-butanol in response to isobutanol specificity are obtained so as to realize high-throughput screening of a proper amount of ethanol production strains for producing fusel oil.

Further, the BmoR protein mutant is obtained by generating M94V and/or F272L mutation on the basis of the wild BmoR protein shown in a sequence table SEQ ID NO.1, hereinafter referred to as M94V mutant, F272L mutant and M94V/F272L mutant (M94V and F272L mutation are generated simultaneously), and the mutant protein specifically comprises:

(1) Amino acid sequences shown in SEQ ID NO.3, 5 or 7 of the sequence list; or (b)

(2) Amino acid sequence with homology of more than 75% of SEQ ID NO.3, 5 or 7; or (b)

(3) One or more amino acid substitutions, and/or deletions, and/or additions are made on the basis of SEQ ID NO.3, 5 or 7 to obtain an amino acid sequence with the same function as SEQ ID NO.3, 5 or 7.

Further, the invention also provides M94V mutant, F272L mutant and M94V/F272L mutant coding genes;

further, the coding gene is shown in sequence table SEQ ID NO.4, 6 or 8.

It is a further object of the present invention to provide the use of the M94V mutant, F272L mutant or M94V/F272L mutant, in particular in the detection of isobutanol containing samples or in the screening of ethanol producing strains that produce an appropriate amount of fusel oil, more particularly in the construction of biosensors for the detection of isobutanol;

further, the biosensor is based on M94V mutant, F272L mutant or M94V/F272L mutant, and the sensor comprises M94V mutant, F272L mutant or M94V/F272L mutant coding gene and 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 specific response and screening of isobutanol at the concentration of 0-100mM, is further applied to industrial production, and can realize screening of isobutanol-containing samples or ethanol production strains with proper amount of fusel oil;

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 biosensor comprises M94V mutant, F272L mutant or M94V/F272L mutant coding gene and 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, such as pET, pUC19, pMAL, etc.;

more preferably, the biosensor is obtained by replacing the wild BmoR protein encoding gene on plasmid pYH1 with the M94V mutant, F272L mutant or M94V/F272L mutant encoding gene, i.e., will be composed of P _bmoR The M94V mutant, F272L mutant or M94V/F272L mutant coding gene is connected to colE1 replication initiation site and amp ^r And P _bmo A driven gfp gene;

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

further, the promoter P _bmoR The nucleotide sequence of (2) is shown as SEQ ID NO.9 of the sequence table;

further, the promoter P _bmo The nucleotide sequence of (2) is shown as SEQ ID NO.10 of the sequence table;

further, the nucleotide sequence of the gfp reporter gene is shown in a sequence table SEQ ID NO. 11.

The invention also provides the application of the biosensor in isobutanol detection, especially in the screening of isobutanol-containing environment, food, medical and biological samples or ethanol production strains with proper amount of fusel oil, and the application is used for detecting the production of isobutanol or fusel oil by introducing the plasmid into production strains such as escherichia coli, saccharomyces cerevisiae, bacillus subtilis and the like or introducing the plasmid into a separate host (such as escherichia coli XL10-Gold and the like) and then adding the plasmid into a detection system.

The beneficial effects are that:

1. the BmoR-based biosensor can be used for screening high-yield n-butanol or isobutanol strains, but wild BmoR has response to n-butanol, isobutanol and ethanol, can not be distinguished, has poor specificity, and provides an M94V mutant, an F272L mutant or an M94V/F272L mutant which are only sensitive to isobutanol and not responsive to n-butanol, and an M94V/F272L which is highly insensitive to ethanol, so that the problem that wild BmoR protein can not distinguish n-butanol, isobutanol and ethanol is solved.

2. The detection range of the wild type BmoR is too narrow, the detection range of isobutanol is 0-40mM, and the response is saturated when the substrate concentration reaches 40mM, so that the BmoR can not be used for identifying strains with the isobutanol yield higher than 40 mM. The detection range of the M94V/F272L mutant provided by the invention for isobutanol reaches 0-100mM, the response saturation of BmoR protein for isobutanol is improved, and the method can be used for screening and applying isobutanol high-level production strains.

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 an N end random mutation library of the Bmo R; the GFP fluorescent protein is added downstream of the bmOR gene to construct a plasmid, i.e. a biosensor, which is introduced into an ethanol production strain, and when n-butanol/isobutanol/ethanol is combined with the Bmor protein, the GFP fluorescent protein generates different degrees of response to reflect the generation of n-butanol/isobutanol/ethanol.

FIG. 2 is a response of BmoR mutant/wild type to 10mM n-butanol and isobutanol;

FIG. 3 is a response of BmoR mutant/wild type to 0-100mM n-butanol and isobutanol;

FIG. 4 is a response of BmoR mutant/wild type to 0-800mM ethanol;

FIG. 5 shows the response of BmoR mutants in fermentation broths with different levels of higher alcohols;

FIG. 6 shows the docking of M94V/F272L mutants with n-butanol, isobutanol and ethanol molecules.

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 M94V mutant, F272L mutant or M94V/F272L mutant, and comprises M94V mutant, F272L mutant or M94V/F272L mutant coding gene and 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 person skilled in the art can choose a promoter to promote the expression of BmR mutant gene in the prior art according to the actual situation, e.g.by using 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 visual detection signal, or small molecule substances available for detection, to effect the response of the biosensor of the present invention, preferably gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc. 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 mutants M94V, F272L and M94V/F272L

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 (while taking the bmoR wild-type fragment as a control) and 3. Mu.L of the pYH1 backbone (P-constructed can also be used _bmoR (or other promoters), P _bmo The plasmids of gfp fluorescent protein genes (or other reporter genes) are taken as a skeleton and are equivalent to the effect of the pYH1 skeleton) and 5 mu L Gibson Assemble Mix, and the plasmids are mixed and then 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, inoculated into 5mL LB (100. Mu.g/mL amp.) liquid medium, and cultured at 37℃for 8h 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, isobutanol or ethanol 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 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, 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 to determine mutants in which only isobutanol was responsive, specifically: M94V mutant (amino acid sequence shown in sequence table SEQ ID NO.3, nucleotide sequence shown in SEQ ID NO. 4) with amino acid at 94 th position mutated from Met to Val, F272L mutant (amino acid sequence shown in sequence table SEQ ID NO.5, nucleotide sequence shown in SEQ ID NO. 6) with amino acid at 272 position mutated from Phe to Leu, M94V/F272L mutant (amino acid sequence shown in sequence table SEQ ID NO.7, nucleotide sequence shown in SEQ ID NO. 8) with amino acid at 94 th position mutated from Met to Val and amino acid at 272 position mutated from Phe to Leu ₆₀₀ The primary screening results are shown in Table 1 and FIG. 2 below, and it is seen that the M94V/F272L mutant was responsive to isobutanol only, not to n-butanol and ethanol, and the M94V mutant and F272L mutant were responsive to isobutanol only, not to n-butanol, relative to the wild-type Bmor.

TABLE 1 GFP/OD ₆₀₀

	N-butanol	Isobutanol	Ethanol
				WT	983	868	68.4
M94V/F272L	0.00	99.0	0.00
				M94V	0.00	60.6	——
F272L	0.00	40.7	——

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 0-40mM n-butanol or isobutanol, and has response to both n-butanol and isobutanol; at substrate concentrations above 40mM, the response values tend to saturate and fail to respond to higher concentrations of alcohol molecules. The specific response of BmoR biosensors based on the M94V/F272L mutant to 0-100mM n-butanol/isobutanol was verified experimentally as follows.

Based on the primary screening result, exogenous addition experiments of ethanol, n-butanol or isobutanol with concentration gradients are respectively carried out on strains containing M94V/F272L mutant genes and wild BmoR genes, response curves are measured, and K is calculated _m Response intensity value, etc.

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.

And (3) exogenously adding n-butanol or isobutanol for experiment to determine fluorescence intensity. Exogenous addition experiments were performed in sterilized 2mL 96-deep well plates. 950. Mu.L of fresh LB (100. Mu.g/mL Amp) medium is added to each well, n-butanol or isobutanol is added to the medium to make the final concentration of each of the n-butanol or isobutanol be 0, 1, 10, 20, 40, 60, 80 and 100mM, 50. Mu.L of seed solution is finally inoculated to each well, the sealing film is sealed, and the deep-well plate is placed at 30 ℃ and cultured for 16 hours by a shaking table at 220 rpm. 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.

And (5) testing the fluorescence intensity by using an exogenous ethanol adding experiment. Exogenous addition experiments were performed using 96 deep well plates. Adding 950 mu L of fresh LB (100 mu g/mL Amp) culture medium into each well by a pipette, adding absolute ethyl alcohol into the culture medium to ensure that the final concentration of the culture medium is 0, 50, 100, 200, 300, 400, 500, 600, 700 or 800mM respectively, inoculating 50 mu L of seed solution of different mutants into each well, sealing a sealing film, placing a deep-hole plate into a shaking table at 30 ℃, and culturing at 220rpm for 16 hours; detection of fluorescence intensity and OD Using microplates ₆₀₀ : 200 mu L of bacterial liquid is sucked and put into an enzyme-labeled instrument, quantitative detection is carried out at 30 ℃, and the parameters are set as follows: 470nm excitation wavelength and 510nm emission wavelength, the gain value was 50.

With GFP/OD ₆₀₀ On the ordinate, the final concentrations of n-butanol and isobutanol are respectively plotted on the abscissa, originPro 8.5 or GraphPad Prism 8 software is used for plotting, michaelis fitting is carried out on the data, and K of Bmor mutant on n-butanol and isobutanol is calculated according to the fitting result _m Maximum response intensity, etc. (fig. 3).

Screening a random mutation library under the concentration of 10mM substrate, wherein the obtained mutant M94V/F272L has specific response to isobutanol and does not respond to n-butanol; further validation under a gradient of 0-100mM, the results show that the mutant is in the presence ofUnder the condition of different n-butanol and isobutanol concentrations, the specific response to isobutanol is still maintained, and the response to n-butanol is hardly generated. Mapping was performed by OrigingPro 8.5, wild type BmoR at substrate concentration of 0-100mM for K-butanol _m K for isobutanol of 3.78 _m 4.24; k for n-butanol at a substrate concentration of 0-100mM for M94V/F272L mutant _m 83.46, K for isobutanol _m 48.99K _m As a characterization of affinity, K _m The larger the value, the smaller the affinity; k (K) _m The smaller the value, the greater the affinity.

Wild-type BmoR is able to respond to 0-40mM n-butanol or isobutanol, and at concentrations above 40mM the response will saturate, so wild-type BmoR cannot distinguish between n-butanol or isobutanol above 40 mM; the calculation result shows that the M94V/F272L mutant phase is compared with the wild type, K _m The value is obviously improved, and the BmoR mutant can realize the specific response (0-100 mM) of isobutanol with higher concentration; at the same time, the M94V/F272L mutant of isobutanol K _m Has a value less than n-butanol K _m The values further indicate that the mutant has a greater affinity for isobutanol than for n-butanol. Is consistent with the experimental result.

Response analysis of wild type BmoR and mutant M94V/F272L was first performed at 0-200mM ethanol concentration. The results showed that the mutant had little response at 0-200mM ethanol. Mapping was performed by OriginPro 8.5, wild type BmoR at substrate concentration of 0-200mM for ethanol K _m 9.70; k for ethanol at substrate concentration of 0-200mM for M94V/F272L mutant _m 174.K (K) _m As a characterization of affinity, K _m The larger the value, the smaller the affinity; k (K) _m The smaller the value, the greater the affinity. Further increasing the ethanol concentration to 800mM, the mutant was still barely responsive to ethanol, i.e., insensitive to ethanol within 800mM (FIG. 4).

Example 3 Saccharomyces cerevisiae fusel oil assay based on M94V/F272L mutant biosensor

The main culture medium comprises:

(1) LB medium: 1g of sodium chloride, 1g of peptone and 0.5g of yeast extract powder are dissolved in distilled water and sterilized for 20min at 115 ℃ to 100 mL.

(2) Corn hydrolysate seed medium: 500g of corn grit is weighed, 1.5L of water (60-70 ℃) is added, the mixture is stirred and then kept stand for 20min, and 300 mu L of high temperature resistant alpha-amylase (2X 10) is added ⁴ U/mL), placing in a water bath kettle at 85-90 ℃ for liquefying for 1.5h, stirring continuously during the liquefying, cooling to 60 ℃, adding 1mL of saccharifying enzyme, and saccharifying for 20h. Finally, the corn hydrolysate is filtered after cooling. According to the sugar degree requirement, adding a proper amount of water to dilute to a proper sugar degree for fermentation experiments.

The sugar degree of the primary seed culture medium is 8 DEG Brix; the sugar degree of the secondary seed culture medium is 12 DEG Brix, and the primary seed culture medium and the secondary seed culture medium are added with 0.5% (w/v) yeast extract powder, and the secondary seed culture medium is sterilized at 105 ℃ for 20min. The primary seed liquid is packaged in a test tube for sterilization, the secondary seed liquid is packaged in a 250mL triangular flask for sterilization, and the sterilized seed culture medium needs to be used in time and is not suitable for being stored for too long, so that bacteria infection is prevented.

(3) Corn thick mash fermentation medium: injecting 130mL of hot water at 60-70 ℃ into 60g of weighed corn residues, uniformly stirring, and standing for 20min; thereto was added 30. Mu.L of a thermostable. Alpha. -amylase (2X 10) ⁴ U/mL), placing in a water bath kettle at 85-90 ℃ for liquefying for 1.5h, and continuously stirring during the liquefying process; cooled to 60℃and 90. Mu.L of saccharifying enzyme (10X 10) ⁴ U/mL) is stirred uniformly and is put into a water bath kettle with the temperature of 55-60 ℃ for saccharification for 20min; 60mg of acid protease (2X 10) ⁴ U/mL) and uniformly stirring, and standing for 20min; after cooling to 30 ℃, the secondary seed solution and 1mL of nutrient salt solution are added. The medium does not need to be sterilized again.

(4) Nutrient salt solution: 15g of anhydrous magnesium sulfate, 7.5g of monopotassium phosphate, 8.1g of urea and sterile deionized water are prepared, the volume is fixed to 100mL, and the mixture is placed at room temperature and is ready for use.

Corn thick mash fermentation experiment:

fermentation process route diagram: corn residue, gelatinization, saccharification, liquefaction, nutrient salt addition, inoculation, fermentation and index measurement.

Fermentation flow: (1) Inoculating one-ring yeast into a test tube (6 mL) containing a primary seed culture medium, standing and culturing in a 30 ℃ incubator for 24 hours; (2) Transferring the primary seed liquid into a 250mL triangular flask filled with 54mL of secondary seed culture medium, and performing stationary culture at 30 ℃ for 16-17h; (3) Inoculating 10% of inoculum size (15 mL of secondary seed liquid) into a thick mash fermentation medium of corn, standing in a 30 ℃ incubator for fermentation, shaking the bottle once every 12h and recording the weight at the moment, taking the fermentation as the end when the weight loss in 12h is less than 0.2g, distilling the fermentation liquid after the end, and measuring the content of ethanol and fusel oil.

Metabolite detection: wine steaming conditions: 100mL of the fermentation broth was added with 100mL of water, and 100mL of wine was distilled off.

(1) Alcohol content determination: in the experiment, the content of higher alcohol in distilled liquor is measured by adopting a gas chromatography method, the content of ethanol in fermentation liquor is measured by adopting a high performance liquid chromatography method, and a standard curve is drawn in the gas (liquid) chromatography in advance according to a standard solution of the higher alcohol (ethanol) before measurement, so that the standard curve is used as a standard for measuring the data afterwards.

(2) High performance gas chromatography detection conditions: the detector is a FID, LZP-930 type capillary chromatographic column, the specification is 50m multiplied by 320 mu m multiplied by 1.0 mu m, the carrier gas is nitrogen with the purity of 99.99 percent, and the split ratio is 1:10. The temperature of the sample inlet is 200 ℃, the temperature of the detector is 200 ℃, and the sample inlet amount is 1 mu L. Heating to 50deg.C for 8min, heating to 150deg.C at 5deg.C/min, holding for 15min, and measuring for 45min.

(3) High performance liquid chromatography detection conditions: the chromatographic column is Bio-Rad HPX-87H, 300X 7.8mm; the detector is a differential refraction detector (RID); the mobile phase is 5mmol/L sulfuric acid with the flow rate of 0.6 mL/min; the detector temperature was 45℃and the column temperature was 65℃and each sample was measured for 25min.

Five strains of Saccharomyces cerevisiae strains (M1, M2, M3, M4 and M5) with different fusel oil production capacities are inoculated into a fermentation culture medium according to the method, and after the culture is carried out for 5 days at 30 ℃, the weight loss is less than 0.2g, and the fermentation is regarded as the end. Dividing the fermentation liquor into two parts, centrifuging one part of fermentation liquor at 12000rpm for 10min, taking supernatant, filtering with 0.22 μm filter membrane, and storing at 4deg.C for detection; and adding the same volume of water into the other fermentation liquor, wherein the distillate is in a wine shape and is stored at 4 ℃ for detection. The concentrations of ethanol and higher alcohols in the fermentation broth (Table 1) were measured by liquid and gas chromatography, and the results showed that the fusel oil production of the 5 strains was different, with overall higher alcohol yields ranging from high to low, M5, M4, M3, M2 and M1, respectively. While the ethanol yields of these strains were not greatly different.

TABLE 2 high alcohol and ethanol content of Saccharomyces cerevisiae corn thick mash fermentation

To verify the effect of the application of the M94V/F272L mutant sensor, the fluorescence response of the mutant in the fermentation broth was further examined. E.coli single colony containing M94V/F272L Bmor mutant coding gene sensor obtained by screening in example 1 is selected and inoculated into 5mL LB (100 mug/mL amp) liquid culture medium, and cultured for 8 hours at 37 ℃ and 220rpm to obtain seed liquid. The broth obtained from the thick mash fermentation described above was diluted 4-fold with LB (100 μg/ml) liquid medium, then 950 μl of the diluted broth was added to 96 deep wells, and then 50 μl of seed solution was aspirated and inoculated into each well. The 96 deep-well plate was placed in a shaker at 30℃and 220rpm for 16h. Fluorescence intensity GFP and OD Using microplates ₆₀₀ Detecting; 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.

As a result, as shown in FIG. 5, the fluorescence response value of the mutant M94V/F272L increased as the concentration of isobutanol (higher alcohol) in the fermentation broth increased. Further proves that the mutant can carry out specific detection on the isobutanol under the condition of ethanol noise.

Example 4 model analysis

Sequencing the M94V/F272L 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, respectively docking the mutant with substrate small molecules such as n-butanol, isobutanol and ethanol, and analyzing the combination site and hydrogen bond formation condition of the mutant and the three molecules.

The three-dimensional structure of the wild BmoR protein is used as a template for M94V/F2The 72L mutant was subjected to homology modeling with a homology of 99.6%. The mutant structure is further molecularly docked with a substrate molecule (n-butanol, isobutanol or ethanol). The results showed that in the complex, the mutant had 1 hydrophobic interaction with isobutanol (Phe 276) and did not form any interaction with both ethanol and n-butanol, indicating that isobutanol can bind tightly to the mutant, and K _m The results of the value analysis remained consistent, i.e., specific response to isobutanol (figure 6).

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.

SEQUENCE LISTING

<110> university of Beijing technology

Tianjin University of Science and Technology

<120> a screening method of Bmor mutant-based ethanol production strain

<130> 1

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 669

<212> PRT

<213> Pseudomonas (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 (Pseudomonas butanovora)

<400> 2

atgtctaaaa tgcaggaatt cgctcgtctg gaaaccgttg cttctatgcg tcgtgctgtt 60

tgggacggta acgaatgcca gccgggtaaa gttgctgacg ttgttctgcg ttcttggacc 120

cgttgccgtg ctgaaggtgt tgttccgaac gctcgtcagg aattcgaccc gatcccgcgt 180

accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240

gttgacgctc tgatggaaca gatgaacgac gctccgcgta tgatcatcct gaacgacgaa 300

cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360

gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420

gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480

accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540

ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600

gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660

cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720

cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780

gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840

gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900

gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960

ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020

ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080

gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140

aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200

ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260

gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320

ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380

gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440

atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500

ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560

tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620

cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680

ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740

gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800

ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860

caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920

cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980

cactggtctc agtctcgtat cggtacctaa 2010

<210> 3

<211> 669

<212> PRT

<213> artificial sequence

<400> 3

Met Ser Lys Met 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 Val 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 aattcgaccc gatcccgcgt 180

accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240

gttgacgctc tgatggaaca gatgaacgac gctccgcgtg tgatcatcct gaacgacgaa 300

cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360

gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420

gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480

accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540

ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600

gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660

cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720

cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780

gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840

gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900

gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960

ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020

ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080

gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140

aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200

ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260

gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320

ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380

gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440

atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500

ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560

tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620

cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680

ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740

gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800

ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860

caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920

cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980

cactggtctc agtctcgtat cggtacctaa 2010

<210> 5

<211> 669

<212> PRT

<213> artificial sequence

<400> 5

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 Leu

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> 6

<211> 2010

<212> DNA

<213> artificial sequence

<400> 6

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 gttctcgacc tgaactttgg cgcgttcctg 840

gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900

gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960

ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020

ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080

gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140

aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200

ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260

gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320

ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380

gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440

atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500

ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560

tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620

cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680

ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740

gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800

ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860

caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920

cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980

cactggtctc agtctcgtat cggtacctaa 2010

<210> 7

<211> 669

<212> PRT

<213> artificial sequence

<400> 7

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 Val 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 Leu

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> 8

<211> 2010

<212> DNA

<213> artificial sequence

<400> 8

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 gctccgcgtg 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 gttctcgacc tgaactttgg cgcgttcctg 840

gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900

gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960

ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020

ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080

gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140

aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200

ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260

gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320

ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380

gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440

atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500

ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560

tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620

cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680

ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740

gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800

ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860

caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920

cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980

cactggtctc agtctcgtat cggtacctaa 2010

<210> 9

<211> 138

<212> DNA

<213> Pseudomonas (Pseudomonas butanovora)

<400> 9

gaccttgagg tgaccttgag cgggcagata ccaccaaaat ttcccacgtg ctattatggt 60

tttgctaaag ctctcgacag cgaggagaga ctcgcgaaga taagcaattc gcccgacaga 120

ggtgaatgag gagacggt 138

<210> 10

<211> 524

<212> DNA

<213> Pseudomonas (Pseudomonas butanovora)

<400> 10

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> 11

<211> 717

<212> DNA

<213> artificial sequence

<400> 11

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 is characterized in that the BmoR mutant protein is obtained by carrying out M94V and F272L mutation on the basis of a wild BmoR protein shown in a sequence table SEQ ID NO. 1; the amino acid sequence of the mutant protein is shown as SEQ ID NO. 7.

2. Use of the BmoR mutant of claim 1 for detecting a sample containing isobutanol.

3. Use of a BmoR mutant according to claim 1 for screening an ethanol producing strain, wherein the ethanol producing strain is tested for its isobutanol producing ability and the strain is saccharomyces cerevisiae.

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

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.8 of the sequence Listing.

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

8. A biosensor comprising the mutant-encoding gene of claim 5, wherein the promoter is activated by Bmor mutantP _bmo By (1)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 togfp、rfp、cfp、sfgfp、egfp、yfp、ecfpAnd (3) a gene.

10. The biosensor of claim 8, wherein the sensor is to be formed fromP _bmoR The initiated mutant encoding gene is linked tocolE1A replication initiation site,amp ^r AndP _bmo actuated by a motorgfpObtaining a gene; the saidP _bmoR The nucleotide sequence of the promoter is shown as SEQ ID NO. 9.

11. The biosensor of claim 8, wherein the promoterP _bmo Nucleotide sequences of (A) such asThe sequence table is shown as SEQ ID NO. 10.

12. Use of the biosensor of claim 8 for detecting an isobutanol containing environmental, food, medical, biological sample.

13. Use of the biosensor according to claim 8 for screening ethanol producing strains, wherein the ethanol producing strains are tested for their isobutanol producing ability, said strains being saccharomyces cerevisiae.