CN115160415B

CN115160415B - BmoR protein mutant specifically responding to n-butanol and application thereof

Info

Publication number: CN115160415B
Application number: CN202110361566.7A
Authority: CN
Inventors: 霍毅欣; 陈振娅; 毋彤
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2024-02-09
Anticipated expiration: 2041-04-02
Also published as: CN115160415A

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a BmoR protein mutant specifically responding to n-butanol and application thereof in n-butanol detection or a biosensor. The BmoR protein mutant is obtained by mutating I183T and/or D273N on the basis of a wild BmoR protein shown in a sequence table SEQ ID NO. 1. The I183T, D N and I183T/D273N mutants are only sensitive to N-butanol and do not respond to isobutanol, so that the problem that the wild BmoR protein cannot distinguish N-butanol from isobutanol high-yield strains is solved; meanwhile, the detection range of I183T/D273N to N-butanol reaches 0-100mM, so that the response saturation of BmoR protein to N-butanol is improved, and the BmoR protein can be used for screening and application of strains with higher yields.

Description

BmoR protein mutant specifically responding to n-butanol and application thereof

Technical field:

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

The background technology is as follows:

the synthesis of n-butanol by microorganisms is an important transportation fuel, and the synthesis of n-butanol by metabolic engineering is realized in many microbial hosts, and the transformation of host strains and the screening of high-yield hosts are the basis and key for realizing the industrial production of alcohols. Biosensors are capable of specifically responding to a target compound to output a protein signal that is convenient for detection, and thus have been widely used for high-throughput screening.

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

Transcription Factor (TF) -based biosensors are most widely used. The most commonly used transcription factors are bacterial transcription factors, including Ligand Binding Domains (LBD) or Metabolic Binding Domains (MBD) and DNA Binding Domains (DBD). Bmor is a transcription factor of the Pseudomonas n-paraffin metabolic pathway, a member of bEBP, for modulating sigma of alkane monooxygenases ⁵⁴ Dependent promoter P _bmo The signal molecule is a C2-C5 linear or branched alcohol. However, the wild transcription factor Bmor has poor response specificity, can respond to both n-butanol and isobutanol, and cannot specifically respond to one of the alcohols, thereby meeting the industrial requirements; and narrow detection range (0-40 mM), and cannot be widely applied to biosensors. Therefore, achieving a specific response to alcohol and increasing the detection range are urgent problems to be solved.

The BmoR protein mutant obtained by protein modification not only can realize specific response to n-butanol, but also can improve the upper limit of substrate detection and response intensity of the BmoR protein mutant, and provides a solution for high-efficiency detection of n-butanol and rapid screening of high-yield strains.

The invention comprises the following steps:

the invention aims to provide BmoR protein capable of distinguishing n-butanol and isobutanol, a biosensor and application thereof. A random mutation library is constructed by utilizing an error-prone PCR technology, and the mutant library is screened and analyzed by exogenously adding n-butanol and isobutanol, so that BmoR mutant proteins responding to only n-butanol are finally obtained.

Further, the Bmo R mutant protein which only responds to N-butanol is obtained by generating I183T and/or D273N mutation based on the wild Bmo R protein shown in a sequence table SEQ ID NO.1, hereinafter referred to as I183T mutant, D273N mutant and I183T/D273N mutant (simultaneously generating I183T and D273N mutation), and the mutant proteins are specifically:

(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 coding genes of the I183T mutant, the D273N mutant and the I183T/D273N mutant;

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 I183T mutant, the D273N mutant or the I183T/D273N mutant, in particular in the detection of N-butanol containing samples or the screening of N-butanol producing strains, more particularly in the construction of biosensors for the detection of N-butanol;

further, the biosensor is based on an I183T mutant, a D273N mutant or an I183T/D273N mutant, and the biosensor is a biosensor comprising an I183T mutant, a D273N mutant or an I183T/D273N mutant encoding gene and a promoter, a promoter P thereof _bmo An expression element for a reporter gene; the promoter initiates expression of the bmoR gene, and the bmoR protein binds to the alcohol molecule to form hexamers, thereby initiating the downstream promoter P _bmo Thereby expressing the reporter gene and generating signals such as fluorescence; the biosensor can realize specific response and screening of n-butanol at the concentration of 0-100mM, and is further applied to industrial production to realize specific screening of n-butanol-containing samples and n-butanol production strains;

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

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

preferably, the biosensor comprises an I183T mutant, a D273N mutant or an I183T/D273N mutant encoding gene and a promoter P thereof _bmoR Promoter P _bmo Recombinant plasmids of gfp reporter genes; further, expression vectors optionally used for the recombinant plasmid include, but are not limited to, expression vectors commonly used in the art, 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 I183T mutant, D273N mutant or I183T/D273N mutant encoding gene, i.e. will be composed of P _bmoR The started I183T mutant, D273N mutant or I183T/D273N mutant coding gene is connected to colE1 replication initiation site and amp ^r And P _bmo A driven gfp gene;

further, the n-butanol producing strain includes, but is not limited to, escherichia coli, saccharomyces cerevisiae, bacillus subtilis, and the like;

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 application of the biosensor in n-butanol detection, especially in n-butanol-containing environment, food, medical and biological samples or n-butanol production strain screening, and the biosensor is used for detecting n-butanol production by introducing the plasmid into a production strain, 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 and isobutanol, can not be distinguished, has poor specificity, and the I183T, D273N and I183T/D273N mutants provided by the invention are only sensitive to N-butanol and have no response to isobutanol, so that the problem that the wild BmoR protein can not distinguish N-butanol and isobutanol is solved.

2. The detection range of the wild BmoR is too narrow, the detection range of the normal butanol is 0-40mM, and the response is saturated when the substrate concentration reaches 40mM, so that the BmoR can not be used for identifying the strain with the normal butanol yield higher than 40 mM. The detection range of the I183T/D273N mutant provided by the invention for N-butanol reaches 0-100mM, so that the response saturation of BmoR protein for N-butanol is improved, and the BmoR mutant can be used for screening and applying strains produced in high-level N-butanol.

Description of the drawings:

FIG. 1 is a schematic flow chart;

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

FIG. 2 is a graph showing the response of BmoR mutants and wild type to 10mM n-butanol and isobutanol;

FIG. 3 is a graph showing the response of BmoR mutants and wild type to 0-100mM n-butanol and isobutanol;

FIG. 4 shows the molecular docking of the I183T/D273N mutant with N-butanol and isobutanol.

The specific embodiment is as follows:

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

The biosensor provided by the invention is based on an I183T mutant, a D273N mutant or an I183T/D273N mutant, and comprises an I183T mutant, a D273N mutant or an I183T/D273N mutant coding gene, a promoter thereof and a promoter P _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 I183T, D273N and I183T/D273N

Construction of random mutation library of transcription factor Bmor

(1) With a gene encoding the wild Bmor (SEQ ID NO.2) plasmid pYH1 (see DOI for construction: https:// doi.org/10.1016/j.ymben.2019.08.015; https:// doi.org/10.1186/s 12934-019-1084-2) as template, and the bmOR mutant gene was amplified by error-prone PCR (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 and inoculated into 5mL LB (100. Mu.g/mL Amp) liquid medium, and cultured at 37℃for 8 hours at 220rpm as seed liquid. Preliminary screening was performed using 96 deep well plates. 950. Mu.L of fresh LB (100. Mu.g/mL Amp) medium was added to each well; n-butanol or isobutanol was added to the wells to a final concentration of 10mM, respectively; finally, 50. Mu.L of seed solution was pipetted into each well. After sealing the sealing film, the deep-hole plate was placed in a shaker at 30℃and 220rpm for 16h.

(3) Fluorescence intensity GFP 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 value is first subtracted from the background control value, on whichGFP/OD per well was calculated above ₆₀₀ As relative fluorescence intensity values.

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

Mutants were determined in which only n-butanol was responsive, specifically: I183T mutant (amino acid sequence shown in sequence table SEQ ID NO.3, nucleotide sequence shown in sequence table SEQ ID NO. 4) in which amino acid at position 183 is mutated from Ile to Thr, D273N mutant (amino acid sequence shown in sequence table SEQ ID NO.5, nucleotide sequence shown in sequence table SEQ ID NO. 6) in which amino acid at position 273 is mutated from Asp to Asn, and I183T/D273N mutant (amino acid sequence shown in sequence table SEQ ID NO.7, nucleotide sequence shown in sequence table SEQ ID NO. 8) in which amino acid at position 183 is mutated from Ile to Thr and amino acid at position 273 is mutated from Asp to Asn ₆₀₀ The primary screening results are shown in Table 1 and FIG. 2 below, and it is seen that the I183T, D N and I183T/D273N mutants had responses to only N-butanol relative to wild-type Bmor.

TABLE 1 GFP/OD ₆₀₀

Bmor proteins	N-butanol	Isobutanol
			WT	983	868
I183T/D273N	54.0	0.00
			I183T	39.9	0.00
D273N	25.0	0.00

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 1183T/D273N mutants to 0-100mM N-butanol/isobutanol was verified experimentally as follows.

Based on the primary screening result, exogenous addition experiments of N-butanol or isobutanol with concentration gradients are respectively carried out on strains containing the I183T/D273N mutant gene and the wild BmoR gene, 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 of LB (100. Mu.g/mLAMP) liquid medium, and cultured at 37℃for 8 hours at 220rpm as a seed solution.

Exogenous addition experiments were performed in sterilized 2ml 96 deep well plates. 950. Mu.L of fresh LB (100. Mu.g/mL Amp) medium 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 value is first subtracted by the background control value, on the basis of which each is calculatedGFP/OD in individual wells ₆₀₀ As relative fluorescence intensity values.

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 I183T/D273N has specific response to N-butanol and does not respond to isobutanol; further, the verification is carried out under the condition of 0-100mM gradient concentration, and the result shows that the mutant still keeps specific response to n-butanol and hardly responds to isobutanol under the condition of different n-butanol and isobutanol concentrations. 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 substrate concentration of 0-100mM for I183T/D273N mutant _m 15.7, K for isobutanol _m 33.2; 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.

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 results show that the 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 the n-butanol with higher concentration; at the same time, N-butanol K of I183T/D273N mutant _m A value less than isobutanol K _m The values further indicate that the mutant has a greater affinity for n-butanol than for isobutanol. Is consistent with the experimental result.

Example 3 model analysis

Sequencing the I183T/D273N 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 of N-butanol and isobutanol, and analyzing the combination site of the mutant and the two alcohols and the formation condition of hydrogen bonds.

The three-dimensional structure of the wild BmoR protein is used as a template, and the homology modeling is carried out on the I183T/D273N mutant, wherein the homology rate is 99.6%. The mutant structure is further molecularly docked to a substrate molecule (n-butanol or isobutanol). The results showed that the mutant had 2 hydrogen bond interactions with n-butanol (Leu 260, arg 211) in the complex, while no interaction was formed with isobutanol, indicating that n-butanol could bind tightly to the mutant, and K _m The results of the value analysis remained consistent, i.e., specific response to n-butanol (fig. 4).

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

SEQUENCE LISTING

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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 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 Thr 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 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

ctggacacct 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 Phe

260 265 270

Asn 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 gttttcaacc 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 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 Thr 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

Asn 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 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

ctggacacct 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 gttttcaacc 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 mutant is obtained by mutating I183T and D273N on the basis of a wild BmoR protein shown in a sequence table SEQ ID NO.1, and the amino acid sequence of the mutant is shown in SEQ ID NO. 7.

2. Use of the BmoR mutant of claim 1 for detecting a sample containing n-butanol, or for screening a strain producing n-butanol.

3. Use of the BmoR mutant of claim 1 for constructing a biosensor for detecting n-butanol.

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

5. The coding gene of claim 4, wherein the coding gene is shown in a sequence table SEQ ID NO. 8.

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

7. A biosensor comprising the mutant-encoding gene of claim 4, wherein the Bmor mutant-activated promoter P _bmo From P _bmo Driven reporter genes and expression elements of promoters expressing BmoR mutants.

8. The biosensor of claim 7, wherein the reporter gene comprises, but is not limited to, a gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp gene.

9. The biosensor of claim 7, wherein the promoter expressing the Bmor mutant is P _bmoR 。

10. The biosensor of claim 7, wherein the sensor is to be defined by P _bmoR The started mutant coding gene is connected to colE1 replication initiation site and amp ^r And P _bmo The gfp gene is started;

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

11. Use of the biosensor of claim 7 for detecting n-butanol-containing environmental, food, medical, biological samples, and screening strains producing n-butanol.