CN115160415A

CN115160415A - Bmor protein mutant specifically responding to n-butanol and application thereof

Info

Publication number: CN115160415A
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: 2022-10-11
Anticipated expiration: 2041-04-02
Also published as: CN115160415B

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 wild type Bmor protein shown in a sequence table SEQ ID NO. 1. The I183T, D273N and I183T/D273N mutants are sensitive to N-butanol only and do not respond to isobutanol, so that the problem that wild Bmor protein cannot distinguish high-yield strains of N-butanol and isobutanol is solved; meanwhile, the detection range of the I183T/D273N to the N-butanol reaches 0-100mM, the response saturation of the Bmor protein to the N-butanol is improved, and the method can be used for screening and applying strains with higher yield.

Description

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

The technical field is as follows:

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.

Background art:

the microbial synthesis of n-butanol is an important transportation fuel, and the synthesis of n-butanol through metabolic engineering is realized in a plurality of microbial hosts, and the modification of host strains and the screening of high-yield hosts are the basis and key for realizing the industrial production of alcohol. Biosensors can specifically respond to a target compound to output a protein signal that is convenient to detect, and thus have been widely used for high-throughput screening.

The biosensor consists of a molecular recognition element and a signal converter. When the molecular recognition element is combined with the object to be measured, the generated signal can be converted into an optical signal or an electrical signal by the converter, and the object to be measured can be detected and analyzed. As an emerging tool in synthetic biology, biosensors can be designed and constructed to dynamically respond to changes in signal molecule concentration. Meanwhile, biosensors are designed to facilitate the optimization of microbial cell factories and the production of a range of natural products widely used in industry, such as itaconic acid, fatty acids, 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 development of the biosensor in the biological world is hindered by the disadvantages that the low dynamic range of the fluorescent protein biosensor, the difficulty of performing RNA nucleic acid switch in vitro and the capability of performing G protein coupled receptor only in the extracellular space are overcome.

Transcription Factor (TF) -based biosensors are most widely used. The most commonly used transcription factors are bacterial transcription factors, including the Ligand Binding Domain (LBD) or the Metabolic Binding Domain (MBD) and the DNA Binding Domain (DBD). Bmor is a transcription factor of the Pseudomonas normal paraffin metabolic pathway, is a member of bEBP, and is used for regulating sigma of alkane monooxygenase ⁵⁴ Dependent promoter P _bmo The signal molecule is a C2-C5 linear or branched alcohol. However, the wild-type transcription factor Bmor has poor response specificity, can respond to both substrates of n-butyl alcohol and isobutyl alcohol and cannot respond to one substrate of the n-butyl alcohol and the isobutyl alcoholThe seed alcohol performs specific response, thereby meeting the industrial requirement; and the detection range is narrow (0-40 mM), and the like, and the method cannot be widely applied to biosensors. Therefore, the realization of specific response to alcohol and the improvement of detection range become problems to be solved urgently.

The Bmor protein mutant obtained by modifying the protein can realize the specific response to n-butanol, improves the upper limit of substrate detection and response strength, and provides a solution for efficiently detecting n-butanol and quickly screening high-yield strains.

The invention content is as follows:

the invention aims to provide a Bmor protein capable of distinguishing normal butanol from isobutanol, a biosensor and application. And (3) constructing a random mutation library by using an error-prone PCR (polymerase chain reaction) technology, and screening and analyzing the mutation library by adding n-butyl alcohol and isobutanol from an external source to finally obtain the Bmor mutant protein only responding to the n-butyl alcohol.

Further, the BmoR mutant protein only responding to N-butanol is obtained by carrying out I183T and/or D273N mutation on the basis of the wild-type BmoR protein shown in SEQ ID No.1 of the sequence table, and is hereinafter referred to as I183T mutant, D273N mutant and I183T/D273N mutant (simultaneously carrying out I183T and D273N mutations), and the mutant protein is specifically:

(1) An amino acid sequence shown in SEQ ID NO.3, 5 or 7 of the sequence Listing; or

(2) An amino acid sequence having more than 75% homology with SEQ ID No.3, 5 or 7; or

(3) Amino acid sequences with the same functions as SEQ ID No.3, 5 or 7 obtained after one or more amino acid substitutions, and/or deletions, and/or additions are carried out on the basis of SEQ ID No.3, 5 or 7.

Further, the present invention also provides genes encoding the I183T mutant, the D273N mutant, and the I183T/D273N mutant;

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

It is another object of the present invention to provide the use of the I183T mutant, the D273N mutant or the I183T/D273N mutant, particularly in the detection of a sample containing N-butanol or in the screening of N-butanol producing strains, more particularly in the construction of a biosensor 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 sensor comprises a gene encoding the I183T mutant, the D273N mutant or the I183T/D273N mutant and a promoter thereof, namely a promoter P _bmo And an expression element of a reporter gene; the promoter starts the expression of the bmoR gene, the BmoR protein is combined with an alcohol molecule to form a hexamer, and then the downstream promoter P is started _bmo Thereby expressing the reporter gene and generating signals such as fluorescence; the biosensor can realize specific response and screening of n-butanol under the concentration of 0-100mM, and is further applied to industrial production to realize specific screening of a sample containing n-butanol and a n-butanol production strain;

further, the promoter of the gene encoding the mutant includes, but is not limited to, P _bmoR 、P _tac 、P _T7 、 P _LlacO1 Etc.;

further, the reporter gene includes, but is not limited to, gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp and other genes;

preferably, the biosensor is a gene encoding I183T mutant, D273N mutant or I183T/D273N mutant and its promoter P _bmoR Promoter P _bmo And a gfp reporter gene; further, expression vectors that may be used for the recombinant plasmid include, but are not limited to, those commonly used in the art, such as pET, pUC19, pMAL, and the like;

more preferably, the biosensor is obtained by replacing the wild-type Bmor protein-encoding gene on plasmid pYH1 with the gene encoding the I183T mutant, D273N mutant or I183T/D273N mutant, i.e., will be derived from P _bmoR The promoter I183T mutant, D273N mutant or I183T/D273N mutant encodes a gene linked to the colE1 replication initiation site, amp ^r And P _bmo Driven gfp gene;

further, the n-butanol producing strains include, but are not limited to, escherichia coli, saccharomyces cerevisiae, bacillus subtilis, and the like;

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

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

furthermore, 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 n-butanol detection, in particular to the application in the environment, food, medical and biological samples containing n-butanol or the screening of n-butanol producing strains, and the plasmid is introduced into producing strains, such as escherichia coli, saccharomyces cerevisiae, bacillus subtilis and the like, or introduced into a separate host (such as escherichia coli XL10-Gold and the like) and then added into a detection system to detect the production of n-butanol.

Has the advantages that:

1. the biosensor based on the Bmor can be used for screening strains with high yield of N-butanol or isobutanol, but wild Bmor has response to both N-butanol and isobutanol, cannot be distinguished, and has poor specificity.

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

Description of the drawings:

FIG. 1 is a schematic flow chart;

firstly, carrying out random mutation on the first 1000bp of the N end of wild type bmoR through error-prone PCR to obtain a random mutation library of the BmoR; GFP fluorescent protein is added at the downstream of the bmoR gene, and the response condition of the mutant BmoR to the n-butanol can be reflected by detecting the fluorescence intensity. The response of the mutant BmoR to the alcohol molecule was examined by adding n-butanol at different concentrations.

FIG. 2 shows the response of Bmor mutant and wild type to 10mM n-butanol and isobutanol;

FIG. 3 shows the response of Bmor mutant 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 implementation mode is as follows:

the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made 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 the sensor comprises an I183T mutant, a D273N mutant or an I183T/D273N mutant encoding gene and a promoter thereof, and a promoter P _bmo And an expression element of a reporter gene; the promoter starts the expression of the bmoR gene, the BmoR protein is combined with an alcohol molecule to form a hexamer, and then the downstream promoter P is started _bmo Thereby expressing the reporter gene and generating signals such as fluorescence. The person skilled in the art can select promoters to drive the expression of the Bmor mutant gene in the prior art according to the actual situation, for example, by using P _bmoR 、P _tac 、P _T7 、P _LlacO1 And the like. The reporter gene can be selected from various choices, and protein molecules which are commonly used in the field and can generate visual detection signals or small molecular substances for detection, such as fluorescent protein, chromoprotein and the like, can realize the response of the biosensor,preferably, such as gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp, etc. The sensor also comprises the necessary elements for realizing expression, such as a replication initiation site, preferably, a colE1 replication initiation site and the like. The sensor may also comprise a marker such as a resistance gene, e.g., amp ^r And the like, so that the screening is convenient. The skilled person can also add other elements to the sensor according to actual needs, for example, construct the elements on 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 following examples are commercially available unless otherwise specified.

The invention will be further explained below by means of specific examples.

Example 1 screening of Bmor mutants I183T, D273N and I183T/D273N

Construction of random mutation library of transcription factor Bmor

(1) The mutant gene of the wild type Bmor (shown in SEQ ID NO. 2) is obtained by taking a plasmid pYH1 (see DOI: https:// doi.org/10.1016/j.ymben.2019.08.015; https:// doi.org/10.1186/s 12934-019-1084-2) as a template and performing error-prone PCR amplification (by adding Mn into a PCR system ²⁺ Increasing Mg in PCR System ²⁺ Concentration and adjustment of dNTP ratio to introduce random mutations. A10 Xunbalanced dNTPs mixture was prepared in which the concentration of dCTP, dTTP was four times that of dATP, dGTP. The PCR program was set up as follows: pre-denaturation at 94 ℃ for 2min,30 amplification cycles comprising: denaturation at 95 ℃ for 1min, annealing at 55-68 ℃ for 1min, and determining the appropriate extension time at 72 ℃ according to the amplification rate of 1kb per minute. The storage temperature was set at 16 ℃. ) (ii) a Confirming the PCR product by gel electrophoresis, and recovering and purifying; the purified product was placed in a 37 ℃ water bath and digested with DpnI (1. Mu.L/50. Mu.L purified product) for 1-2h. mu.L of the bmoR mutant fragment (with the bmoR wild-type fragment as a control), 3. Mu.L of pYH1 backbone (alternatively constructed with P) _bmoR (or other promoters), P _bmo Plasmid of gfp fluorescent protein gene (or other reporter gene) as a backbone, equivalent to pYH1 backbone) and 5mu.L of Gibson Assembly Mix, mixed and placed in a 50 ℃ water bath for ligation for 1h. Transferring 5-10 μ L of the ligation product into 50 μ L of Escherichia coli XL 10-Gold-transformed competent cells, and culturing overnight at 37 ℃ to obtain a Bmor-1000bp mutation library.

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

(3) Fluorescence intensity GFP and OD Using microplate ₆₀₀ And (3) detection: blowing and beating the mixed bacteria liquid, sucking 200 mu L of the mixed bacteria liquid, putting the mixed bacteria liquid into an enzyme-labeling instrument, and quantitatively detecting the mixed bacteria liquid at 30 ℃ with the set parameters as follows: an excitation wavelength of 470nm, an emission wavelength of 510nm, and a gain value of 50; the obtained GFP and OD ₆₀₀ Values the background control values were first subtracted and the GFP/OD was calculated for each well based thereon ₆₀₀ As a relative fluorescence intensity value.

(4) After analyzing the preliminary screening results, the plasmids of the effective mutant bacteria were sequenced.

Determining mutants which only respond to the n-butanol, specifically: an I183T mutant (the amino acid sequence is shown as SEQ ID NO.3 in a sequence table, and the nucleotide sequence is shown as SEQ ID NO. 4) with the 183 th amino acid mutated from Ile to Thr, a D273N mutant (the amino acid sequence is shown as SEQ ID NO.5 in the sequence table, and the nucleotide sequence is shown as SEQ ID NO. 6) with the 273 th amino acid mutated from Asp to Asn, an I183T/D273N mutant (the amino acid sequence is shown as SEQ ID NO.7 in the sequence table, and the nucleotide sequence is shown as SEQ ID NO. 8) with the 183 th amino acid mutated from Ile to Thr and the 273 nd amino acid mutated from Asp to Asn, GFP/OD of the mutants, and the like ₆₀₀ The preliminary screening results are shown in table 1 below and fig. 2, and it can be seen that the I183T, D273N and I183T/D273N mutants respond only to N-butanol, relative to wild-type BmoR.

TABLE 1 GFP/OD ₆₀₀

Bmor protein	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 Change in detection Limit for wild type and mutant

The wild Bmor-based biosensor can realize the response to 0-40mM n-butyl alcohol or isobutyl alcohol and has response to both n-butyl alcohol and isobutyl alcohol; when the substrate concentration is higher than 40mM, the response value tends to be saturated, and the response cannot be carried out on alcohol molecules with higher concentration. The specific response of the Bmor biosensor based on the 1183T/D273N mutant to 0-100mM N-butanol/isobutanol was verified experimentally below.

Respectively carrying out concentration gradient N-butanol on strains containing I183T/D273N mutant genes and wild Bmor genes on the basis of primary screening resultsOr exogenous addition experiment of isobutanol, measuring response curve and calculating K _m Response intensity value, etc.

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

Exogenous addition experiments were performed in sterilized 2mL 96 deep well plates. 950. Mu.L of fresh LB (100. Mu.g/mL Amp) medium was added to each well, then n-butanol or isobutanol were added to the medium to give final concentrations of 0, 1, 10, 20, 40, 60, 80, 100mM, respectively, and finally 50. Mu.L of seed solution was inoculated to each well, and after sealing the sealing film, the deep well plate was incubated at 30 ℃ for 16h on a shaker at 220 rpm.

Fluorescence intensity GFP and OD Using microplate ₆₀₀ And (3) detection: blowing and beating the mixed bacteria liquid, sucking 200 mu L of the mixed bacteria liquid, putting the mixed bacteria liquid into an enzyme-labeling instrument, and quantitatively detecting the mixed bacteria liquid at 30 ℃ with the set parameters as follows: 470nm excitation wavelength and 510nm emission wavelength, gain value is 50; the obtained GFP and OD ₆₀₀ Values the background control values were first subtracted from the values, and the GFP/OD of each well was calculated based thereon ₆₀₀ As a relative fluorescence intensity value.

At GFP/OD ₆₀₀ Using the final concentrations of the n-butanol and the isobutanol as the ordinate and the final concentrations of the n-butanol and the isobutanol as the abscissa respectively, using originPro 8.5 or GraphPad Prism 8 software for mapping, carrying out Mie's fitting on the data, and calculating the K of the Bmor mutant for the n-butanol and the isobutanol according to the fitting result _m Maximum response strength, etc. (fig. 3).

Screening a random mutation library at the concentration of 10mM substrate to obtain a mutant I183T/D273N which specifically responds to N-butanol and does not respond to isobutanol; further verification is carried out under the condition of 0-100mM gradient concentration, and the result shows that the mutant still keeps specific response to the n-butanol and hardly responds to the isobutanol under the condition of different n-butanol and isobutanol concentrations. Mapping by originPro 8.5 wild type Bmor at substrate concentrations 0-100mM, K for n-butanol _m 3.78K for isobutanol _m Is 4.24; the I183T/D273N mutant has K in N-butanol at a substrate concentration of 0-100mM _m 15.7K for isobutanol _m Is 33.2；K _m As characterization of the affinity, K _m The larger the value, the smaller the affinity; k is _m The smaller the value, the greater the affinity.

The wild type Bmor can respond to 0-40mM of n-butanol or isobutanol, and when the concentration is higher than 40mM, the response is saturated, so that the wild type Bmor cannot distinguish the n-butanol or the isobutanol of more than 40 mM; the calculation result shows that the mutant has K compared with the wild type _m The value is obviously improved, and the Bmor mutant can realize the specific response (0-100 mM) of n-butanol with higher concentration; meanwhile, N-butanol K of I183T/D273N mutant _m Value less than isobutanol K _m Values further indicating that the mutant has a greater affinity for n-butanol than for isobutanol. In agreement with the experimental results.

Example 3 model analysis

Sequencing the I183T/D273N mutant, analyzing the change of amino acid at the mutation site, modeling the Bmor mutant by using software such as AUTODOCK, chimeraX and the like, butting the mutant with small molecular substrates such as N-butanol and isobutanol respectively, and analyzing the formation of the binding site and hydrogen bond of the mutant and the two alcohols.

The three-dimensional structure of the wild Bmor protein is used as a template to carry out homologous modeling on the I183T/D273N mutant, and the homology rate is 99.6%. And further carrying out molecular docking on the mutant structure and a substrate molecule (n-butyl alcohol or isobutyl alcohol). The results show that the mutant has 2 hydrogen bond interactions (Leu 260, arg 211) with n-butanol in the complex, and does not form any interaction force with isobutanol, indicating that n-butanol can tightly bind to the mutant and K _m The results of the numerical analysis remained consistent, i.e., specifically responded to n-butanol (FIG. 4).

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.

SEQUENCE LISTING

<110> Beijing university of science and technology

<120> Bmor protein mutant specifically responding to n-butanol and application thereof

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<170> PatentIn version 3.5

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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 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 carrying out mutation containing I183T and/or D273N on the basis of a wild Bmor protein shown in a sequence table SEQ ID NO. 1.

2. The Bmor mutant protein according to claim 1, wherein the mutant protein is in particular:

3. Use of a BmoR mutant according to claim 1 for detecting a sample containing n-butanol, or for screening n-butanol producing strains.

4. Use of the BmoR mutant of claim 1 to construct a biosensor for detecting n-butanol.

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

6. The coding gene of claim 5, which is represented by SEQ ID No.4, 6 or 8 of the sequence Listing.

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

8. A biosensor comprising a promoter P activated by a Bmor mutant and comprising a gene encoding the mutant of claim 5 _bmo From P _bmo A driven reporter gene and an expression element for expressing a promoter for the BmoR mutant.

9. The biosensor of claim 8, wherein the reporter genes include, but are not limited to, gfp, rfp, cfp, sfgfp, egfp, yfp, ecfp genes.

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

11. The biosensor of claim 8, wherein the sensor is to be represented by P _bmoR The initiated mutant-encoding gene is linked to the colE1 replication initiation site, amp ^r And P _bmo The gfp gene was initiated.

12. Biosensor according to claim 8, wherein the promoter P is _bmo The nucleotide sequence of (A) is shown in a sequence table SEQ ID NO. 10.

13. Use of the biosensor of claim 8 for detecting n-butanol-containing environmental, food, medical, biological samples, and screening n-butanol-producing strains.