CN108795834A - The pseudomonas aeruginosa gene engineering bacteria and its construction method of a kind of attenuation and application - Google Patents
The pseudomonas aeruginosa gene engineering bacteria and its construction method of a kind of attenuation and application Download PDFInfo
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Abstract
The present invention relates to the pseudomonas aeruginosa gene engineering bacteria and its construction method of a kind of attenuation and applications.Pseudomonas aeruginosa gene engineering bacteria provided by the invention is that the phz operons that pyo synthesizes and its attenuated strain that the double deletion mutations of operon copy inactivate are controlled in pseudomonas aeruginosa.The pathogenicity island that the phz operons are made of seven genes of phzA, phzB, phzC, phzD, phzE, phzF and phzG, is named as phzA1-G1；And its operon copy is named as phzA2-G2.Attenuated pseudomonas aeruginosa genetic engineering bacterium provided by the invention is generated without pyo in metabolic process, and non-resistant genetic marker.The genetic engineering bacterium of the present invention can be used in the large-scale production of rhamnolipid and the biological prosthetic of the environmental contaminants such as polycyclic aromatic hydrocarbon, petroleum hydrocarbon contaminated.
Description
Technical field
The present invention relates to engineering strain technical field, more particularly to the pseudomonas aeruginosa gene engineering of a kind of attenuation
Bacterium and its construction method and application.
Technical background
Pseudomonas aeruginosa (Pseudomonas aeruginosa) be most common pseudomonad present in soil it
One.Pseudomonas aeruginosa is conditioned pathogen, its pathogenic a variety of virulence factors for being heavily dependent on its generation.Pheno
Piperazine substance is virulence factor important in pseudomonas aeruginosa, including：Phenazine-1-carboxylic acid (phenazine-1-
Carboxylic acid, PCA), pyo etc..Pyo is blue azophenlyene class chemical combination specific to pseudomonas aeruginosa
Object is important azophenlyene class compound and virulence factor, the lung with cystic fibrosis (cystic fibrosis, CF) patient
Portion's infection is related.Pyo is the metabolite of PCA, and phz operons are the synthetic genes of PCA.Pseudomonas aeruginosa point
The virulence factor secreted causes it to cause safety and health problem in actual application, to limit its application value.
Pseudomonas aeruginosa is the most important rhamnolipid producer.Rhamnolipid is microorganism in specified conditions culture
A kind of metabolite with surface-active of secretion.Compared with chemical surfactant, rhamnolipid biological surface activator
Have many advantages, such as it is nontoxic, be easily biodegradable, efficient foaming characteristic, good breaking, be widely used in environmental pollution improvement,
The fields such as oil exploitation, biological medicine, papermaking and cosmetics, have broad application prospects.In cosmetic field for manufacturing
With skin compatibility and moisture-keeping function liposome；In field of food, as can digest, the guarantor of antibacterial flavorant and fruits and vegetables
Fresh, preservative etc..But it is pathogenic possessed by pseudomonas aeruginosa itself, it may be in the large-scale production and application of rhamnolipid
Cause safety and health problem in the process.
Meanwhile pseudomonas aeruginosa is generally acknowledged polycyclic aromatic hydrocarbons contaminated reparation bacterium.Polycyclic aromatic hydrocarbon (polycyclic
Aromatic hydrocarbons, PAHs) be it is a kind of contain two or more phenyl ring, with linear array, curved connect or cluster
The poly- organic compound for waiting different modes arrangement form, prodigious threat is caused to ecological environment and human health.Macromolecule has
The imperfect combustion of machine fuel (oil, coal tar, fossil fuel etc.) is smoked or the artificial origins such as baked goods, is generally acknowledged
The main pollution source of environment polycyclic aromatic hydrocarbon.Due to polycyclic aromatic hydrocarbon molecular structure is complicated, thermal stability is strong, be difficult aoxidized, water
Dissolubility difference and with extremely strong " three cause " effect, control with repair have become the current important environmental problem for being badly in need of solving it
One.And pseudomonad (Pseudomonas) with its accretion rate fast, environment adapt to it is strong, can be in specificity metabolism soil it is organic
Pollutant is translated into carbon dioxide and the other carbon-based macromoleculars of cell, becomes the biological prosthetic main bacteria seed of polycyclic aromatic hydrocarbon
One of.Compared with traditional physics, chemical method, there is incomparable advantage.But pseudomonas aeruginosa is divided by itself
The virulence factor secreted, it is possible to limit its application in environmental organism reparation as the source of pollution environment again.
The toxicity for how reducing pseudomonas aeruginosa itself makes it play rhamnolipid production and each side such as biological prosthetic
The extensive advantage in face, it has also become this field researcher falls over each other the project explored.However it really is able to the attenuation verdigris constructed
Pseudomonas genetic engineering bacteria is fewer and fewer【1】【2】【3】.This important virulence factor for pyo there is no effectively to be attenuated at present
Construction method of gene engineering strain report.It is sieved simultaneously because being often used resistance gene marker in construction method of gene engineering strain
Choosing, and resistant gene is considered as a kind of novel environmental contaminants, and easily shift in the horizontal direction so that the research of this project
It is extremely difficult.
Invention content
In view of this, for overcome the deficiencies in the prior art, the present invention provides a kind of pseudomonas aeruginosa gene of attenuation
Engineering bacteria is generated without pyo in metabolic process, and non-resistant genetic marker, the engineering bacteria can be used for rhamnolipid
Large-scale production and the environmental contaminants such as polycyclic aromatic hydrocarbon, petroleum hydrocarbon contaminated it is biological prosthetic in.
A kind of pseudomonas aeruginosa gene engineering bacteria of attenuation provided by the invention, the genetic engineering bacterium are that verdigris is false single
The operon of pyo synthesis is controlled in born of the same parents bacterium and its operon copies the attenuated strain that double deletion mutations inactivate.
The operon of the control pyo synthesis is that phz operons and its operon copy.
The poison that the phz operons are made of seven genes of phzA, phzB, phzC, phzD, phzE, phzF and phzG
Power island, is named as phzA1-G1；And its operon copy is named as phzA2-G2.
The construction method of attenuated pseudomonas aeruginosa genetic engineering bacterium of the present invention, includes the following steps：
1) upstreams gene phzA1-G1 are connect with plasmid pEX18Amp respectively with downstream homology arm segment, obtains clpp gene
Except carrier pEX-phzA1-G1；
2) gene knockout carrier pEX-phzA1-G1 electricity is gone into pseudomonas aeruginosa DN1 bacterial strain competent cells, passed through
Homologous recombination is screened twice, obtains single mutation strain DN1- Δs phzA1-G1；
3) upstreams gene phzA2-G2 are connect with plasmid pEX18Amp respectively with downstream homology arm segment, obtains clpp gene
Except carrier pEX-phzA2-G2；
4) gene knockout carrier pEX-phzA2-G2 electricity is gone into the impression of pseudomonas aeruginosa DN1- Δ phzA1-G1 bacterial strains
State cell screens twice by homologous recombination, obtains double-mutant strain DN1- Δs phz.
Gene knockout the primer phzA1-G1-up and phzA1-G1-down of the gene phzA1-G1 be respectively：
SEQ ID NO.1-2,SEQ ID NO.3-4.Primer phzA1-G1 is verified after the knockout is:SEQ ID NO.9-
10。
Gene knockout the primer phzA2-G2-up and phzA2-G2-down of the gene phzA2-G2 be respectively：
SEQ ID NO.5-6,SEQ ID NO.7-8.Primer phzA2-G2 is verified after the knockout is:SEQ ID
NO.11-12。
It is described to knock out the homology arm segment that primer extension product is upstream region of gene to be knocked out and each 2kb in downstream or so size,
It is to wait knocking out intragenic a part of segment to verify primer extension product.
It is screened using resistance gene marker in usual gene knockout, and resistant gene is considered as a kind of novel environment
Pollutant, while resistant gene easily shifts in the horizontal direction.Attenuated pseudomonas aeruginosa genetic engineering bacterium provided by the present invention
It is to be not added with the gene knockout method structure of resistance gene marker, not cause environmental pollution.
The present invention also provides pseudomonas aeruginosa gene engineering bacteria the answering in industrial production rhamnolipid of the attenuation
With.
Meanwhile the present invention also provides the pseudomonas aeruginosa gene engineering bacterias of the attenuation in environment polycyclic aromatic hydrocarbon or oil
Hydrocarbon decomposes and the application in removal.
Preferably, the pseudomonas aeruginosa gene engineering bacteria of the attenuation is decomposing the application with fluoranthene in removal environment.
The construction method for the pseudomonas aeruginosa gene engineering bacteria that the present invention is attenuated, is described as follows：
The pseudomonas aeruginosa DN1 bacterial strains of the present invention are to detach to obtain from oil-polluted soils；Bacillus coli DH 5 alpha exists
It helps out, is bought from Tiangeng biochemical technology Co., Ltd in knockout carrier structure；The present invention is selected to be caused containing sacB sucrose
The plasmid pEX18Amp of dead gene carries out the knockout of target gene in pseudomonas aeruginosa, is carried by the laboratories Tung T.Hoang
For.
First, gene knockout carrier pEX-phzA1-G1 is built, pseudomonas aeruginosa DN1 bacterial strain competent cells are prepared.
It is thin that gene knockout carrier pEX-phzA1-G1 by electrotransformation is directed into pseudomonas aeruginosa DN1 bacterial strain competence
Born of the same parents screen twice by homologous recombination, PCR amplification and to be verified through electrophoresis without band be DN1- Δ phzA1-G1 bacterial strains.
Then, gene knockout carrier pEX-phzA2-G2 is built, and prepares pseudomonas aeruginosa DN1- Δ phzA1-G1 bacterium
Strain competent cell.
Finally, gene knockout carrier pEX-phzA2-G2 is directed into pseudomonas aeruginosa DN1- Δs through electrotransformation again
PhzA1-G1 bacterial strain competent cells screen twice by homologous recombination, and electrophoresis verification is DN1- Δ phz bacterium without band
Strain.
The advantageous effect of technical solution of the present invention is：
1. the attenuated pseudomonas aeruginosa genetic engineering bacterium that the present invention is built, eliminates the virulence factor of pseudomonas aeruginosa
Pyo；Being tested by mouse toxicity confirms that the genetic engineering bacterium is substantially reduced mouse toxic action.
2. the cytotoxicity experiment for the attenuated pseudomonas aeruginosa engineering bacteria fermentation liquid that the present invention is built confirms, the base
The toxicity during commercial Application can be reduced because of engineering bacteria, can be used for the large scale fermentation production of rhamnolipid.
3. the attenuated pseudomonas aeruginosa genetic engineering bacterium that the present invention is built is during being metabolized polycyclic aromatic hydrocarbon, petroleum hydrocarbon
Do not generate pyo.
4. the attenuated pseudomonas aeruginosa genetic engineering bacterium that the present invention is built, using 50 μ g/mL fluoranthene as sole carbon source
Shaking flask culture 9 days in MSM culture mediums, fluoranthene degradation rate reach 80.4%.Characteristic with efficient degradation polycyclic aromatic hydrocarbon, is applied to
Polycyclic aromatic hydrocarbons contaminated environmental improvement and soil remediation.
Description of the drawings
Fig. 1 digestion connection procedures；
It is prepared by Fig. 2 P. aeruginosa bacterium competence cells；
The genome of Fig. 3 plasmids pEX18Amp；
Wherein：CDS1 is sucrose lethal gene (sacB), and CDS2 is alpha-galactosidase, and CDS3 is beta galactosidase,
Misc_Feature_1 is the sequence containing oriT, and rRNA1 is 5S rRNAs；
The rhamnolipid yield of Fig. 4 wild type DN1 bacterial strains and mutant strain DN1- Δs phz；
The pyo rhzomorph yield of Fig. 5 wild type DN1 bacterial strains and mutant strain DN1- Δs phz；
The mouse toxicity of Fig. 6 wild type DN1 bacterial strains and mutant strain DN1- Δs phz are tested；
The cytotoxicity experiment of Fig. 7 wild type DN1 bacterial strains and mutant strain DN1- Δs phz；
The fluoranthene of Fig. 8 wild type DN1 bacterial strains and mutant strain DN1- Δs phz are degraded.
Specific implementation mode
Embodiment one：Gene knockout design of primers
The whole genome sequence that pseudomonas aeruginosa DN1 bacterial strains are searched in ncbi database, with gene phzA1-G1 and
Verification primer is as shown in table 1 after designing gene knockout primer for phzA2-G2 and knocking out.Wherein knocking out primer extension product is
The homology arm segment that upstream region of gene to be knocked out is about 2kb or so with each size in downstream, verification primer extension product is base to be knocked out
A part of segment because in.
Primer is verified after the gene knockout primer of table 1 gene phzA1-G1 and phzA2-G2 and knockout.
Embodiment two：The structure of single mutation strain DN1- Δs phzA1-G1：
1) acquisition of gene knockout carrier pEX-phzA1-G1.
Using pseudomonas aeruginosa DN1 bacterial strains as template, PCR amplification gene phzA1-G1 fragment upstreams (1895bp) and downstream
Segment (1805bp)；Upstream PCR product EcoRI and XbaI enzyme cutting, downstream PCR product XbaI and HindIII digestions, plasmid
PEX18Amp EcoRI and HindIII digestions, then connect simultaneously.Digestion connection procedure is as shown in Figure 1.Connection product is through warm
Sharp method is converted into bacillus coli DH 5 alpha competent cell, and converted product is applied to the LB tablets containing 100 μ g/mL carbenicillins
On, it selects after the monoclonal grown expands culture and extracts plasmid, gene knockout carrier pEX-phzA1-G1 is obtained after digestion verification.
2) preparation of pseudomonas aeruginosa DN1 competent cells and electrotransformation.
Pseudomonas aeruginosa DN1 competent cell preparation process：(1) pseudomonas aeruginosa DN1 bacterial strains are drawn to LB solids
On tablet, 37 DEG C are incubated overnight.In picking monoclonal to LB liquid medium, 8000rpm centrifugations 5min is received after 37 DEG C of culture 12h
Collect thalline.(2) sucrose solution of 1mL 0.3M is added in thalline obtained in the previous step, is resuspended, 8000rpm centrifuges 5min.Weight
Duplicate step is twice.(3) glycerine that 1mL 10% is added is resuspended, and 8000rpm centrifuges 5min, abandons supernatant and collects thalline.It is added one
10% quantitative glycerine is resuspended, packing, -80 DEG C of preservations.As shown in Figure 2.
Electrotransformation：(1) the DN1 bacterial strain competent cells of 60 μ L are added in 1.5mL centrifuge tubes, 2 μ L gene knockouts are added
Carrier pEX-phzA1-G1 is transferred in electric revolving cup after blowing and beating mixing, electric revolving cup is placed in Bio-Rad electrotransformation instrument and is shocked by electricity,
Voltage is set as 1.8KV.(2) cell of conversion is washed out into 2mL centrifuge tubes with 500 μ L SOC culture mediums, 200rpm, 37 DEG C
Shaking table culture 2h takes 100 μ L to be coated on the LB solid plates containing 300 μ g/mL carbenicillins, cultivates 16h-24h.(3) it chooses
The monoclonal colonies being coated with after converting on the tablet of culture are taken, are crossed on the LB solid plates containing 10% sucrose, 37 DEG C of cultures
Case culture, picking monoclonal verify primer with phzA1-G1 and do PCR verifications, and electrophoresis is then single mutation strain DN1- Δs without band
phzA1-G1。
Embodiment three：The structure of double-mutant strain DN1- Δs phz.
1) acquisition of gene knockout carrier pEX-phzA2-G2.
Using pseudomonas aeruginosa DN1 bacterial strains as template, PCR amplification gene phzA2-G2 fragment upstreams (1953bp) and downstream
Segment (1926bp)；Upstream PCR product EcoRI and XbaI enzyme cutting；Downstream PCR product XbaI and HindIII digestions, plasmid
PEX18Amp EcoRI and HindIII digestions, then connect simultaneously.Connection product is converted through heat shock method to bacillus coli DH 5 alpha
In competent cell, converted product is applied on the LB tablets containing 100 μ g/mL carbenicillins, is selected the monoclonal grown and is expanded
Plasmid is extracted after big culture, gene knockout carrier pEX-phzA2-G2 is obtained after digestion verification.
2) preparation of DN1- Δs phzA1-G1 competent cells and electrotransformation.
Using DN1- Δs phzA1-G1 as starting strain, competent cell is prepared, method is the same as embodiment two.By gene knockout
Carrier pEX-phzA2-G2 electricity is transferred to DN1- Δ phzA1-G1 bacterial strain competent cells, and method takes monoclonal to use with embodiment two
PhzA1-G1 and phzA2-G2 verification primers do PCR verifications respectively.Electrophoresis is then double-mutant strain DN1- Δs phz without band.
Of particular note is that：Pseudomonas aeruginosa gene knockout carrier pEX18Amp contains Carbenicillin resistance base
Cause and sacB sucrose lethal genes.In experimental procedure, pass through the LB solid plates containing 300 μ g/mL carbenicillins first
Screening obtains a homologous recombination bacterial strain.Secondary homologous recombination bacterium is then obtained by the LB solid plates screening containing 10% sucrose
Strain, the carrier with resistance gene marker and sacB sucrose lethal genes are set to change by homologous recombination, therefore obtain
Mutant DN1- Δ phzA1-G1 and DN1- Δs phz no longer contains resistance gene marker.The genome of plasmid pEX18Amp
See Fig. 3.CDS1 is sucrose lethal gene (sacB) in collection of illustrative plates；CDS2 is alpha-galactosidase；CDS3 is beta galactosidase；
Misc_Feature_1 is the sequence containing oriT；RRNA1 is 5S rRNAs.
Experimental example one：The rhamnolipid assay of wild-type strain and mutant strain.(see Fig. 4)
Pseudomonas aeruginosa DN1 bacterial strains and double-mutant strain DN1- Δs phz are drawn to LB solid plates, 37 DEG C of trainings overnight
It supports；In picking monoclonal to LB liquid medium, 8000rpm centrifugations 5min collects thalline after 37 DEG C of culture 12h.With sterile washing
The seed liquor of OD600=1 is washed and is diluted to, seed liquor is added using 1% inoculum concentration using palm oil as the BPLM of sole carbon source
In fluid nutrient medium, 37 DEG C, 200rpm/min shaking flasks culture 7 days.The rhamnolipid content in zymotic fluid is measured daily.
H2SO4-anthrone method measures rhamnolipid yield：
BPLM culture medium zymotic fluids are diluted 500 times, take 1mL to teat glass, while taking 1mL distilled water to another glass
Glass test tube is placed in ice bath 3min in mixture of ice and water as a contrast, by two test tubes, and sulfuric acid-anthrone reagent is added in taking-up immediately,
It is shaken up with forced oscillation test tube, to developing the color, taking-up is cooled to room temperature boiling water bath 5min, and OD values are measured at 620nm, according to standard song
The yield of line computation rhamnolipid.
It is computed and show that the rhamnolipid yield of the DN1- Δ phz bacterial strains DN1 that compares is declined, about 16g/L.It can be with
Meet the emulsification of degradation process.
Experimental example two：The pyo of wild-type strain and mutant strain detects.(see Fig. 5)
Cultural method is the same as rhamnolipid assay (see experimental example one).The pyo measured daily in zymotic fluid contains
Amount.
The detection of pyo：BPLM culture medium zymotic fluids 8000rpm centrifugation 8min obtains supernatant, take 5mL supernatants with
The chloroform of 3mL mixes, and after 8000rpm centrifuges 8min, chloroform is mutually transferred in another centrifuge tube, and 1mL 0.2mol/L salt is added
Acid, 8000rpm centrifuges 8min after mixing, and upper layer inorganic phase is taken to survey light absorption value at 520nm, what every milliliter of culture supernatant generated
The amount (milligram) of pyo is equal in OD520The extinction coefficient at place is multiplied by 17.8g/L.It is repeated 3 times, is averaged.
From fig. 5, it can be seen that in fermentation in 7 days, the pyo yield of DN1 bacterial strains is about 0.05-0.1mg/mL, and
The pyo yield of DN1- Δ phz mutant strains is 0 always.
Experimental example three：The mouse toxicity of wild type DN1 bacterial strains and double-mutant strain DN1- Δs phz detect.(see Fig. 6)
Mouse toxicity is tested：BALB/c mouse is purchased from Xi'an Jiaotong University Medical College, 6-8 week old, aseptic condition raising.With
After PBS washs wild type DN1 bacterial strains and double-mutant strain DN1- Δs phz, respectively by two kinds of bacterium according to 1 × 108The agent of CFU
Amount injection mouse peritoneal, 5 mouse (average weight 16g) of each experimental group.After injection in preceding 12 hours, every 1 hour
Observe the Survival of a mouse.
The experiment mices of wild type DN1 bacterial strains is injected it can be seen from lab diagram 6, and in the 5th hour to begin with mouse dead
It dies；In the 16th hour, the mouse for injecting wild type DN1 bacterial strains is all dead.And the experiment for injecting mutant strain DN1- Δs phz is small
Mouse still all survivals in 48 hours after injection, it is rear to start death successively, it was demonstrated that toxic actions of the mutant strain DN1- Δs phz to mouse
It is substantially reduced.
Experimental example four：The cytotoxicity of wild type DN1 bacterial strains and mutant strain DN1- Δs phz detect.(see Fig. 7)
Cytotoxicity experiment：(1) mouse of logarithmic growth phase is at fiber L929 cells, the digestion of 0.25% pancreatin, 800-
1000rpm centrifuges 3min, and after blood counting chamber counts, adjustment cell concentration is 5 × 104Cells/mL is laid on 96 orifice plate, 100 μ
L/well；(2) be added 10 μ LPBS are diluted filtered and sterilize after zymotic fluid, if three repeating holes, while setting containing only training
It supports the blank control of base, be not added with the cell controls of zymotic fluid, culture plate is placed in 37 DEG C, 5% CO2Incubator culture；(3)24
10 μ L cck-8 solution, 37 DEG C, 5% CO is added in experimental port after hour2Incubator is incubated 2 hours, and each hole is surveyed with microplate reader
OD450Value；(4) cell survival rate is calculated：Cell survival rate (%)=(dosing cell OD/ control cell OD) × l00%.
From figure 7 it can be seen that the cell of injection wild type DN1 bacterial strain fermentation liquors is all dead when being diluted at 1 times, and it is dead
Rate highest also has part cell death in the cell for diluting 50 times of zymotic fluids.And in injection mutant strain DN1- Δ phz zymotic fluids
The survival rate of cell, different extension rates is all higher.Above it is demonstrated experimentally that double deletion mutation strain DN1- Δs phz successfully subtract
Poison.
Experimental example five：The measurement of fluoranthene degradation rate.(see Fig. 8)
Pseudomonas aeruginosa DN1 bacterial strains and double-mutant strain DN1- Δs phz are drawn to LB solid plates, 37 DEG C of trainings overnight
It supports；Picking monoclonal is in the MSM culture mediums containing 50 μ g/mL fluoranthene, 30 DEG C, 200rpm/min shaking flasks culture 9 days.It surveys daily
Measure the fluoranthene content in zymotic fluid.
It takes 5mL cultivation and fermentations liquid in 50mL centrifuge tubes, isometric hexamethylene is added, whirlpool shakes 5min so that two
The liquid of phase is evenly dispersed；Supernatant is taken to do appropriate dilution simultaneously tube stand 10min, 8000rpm, 4 DEG C of centrifugation 10min
Measure OD232.Standard curve is substituted into, the residual volume of fluoranthene is calculated.
As seen from Figure 8, at 2-3 days, the rate of 2 plants of bacterium degradation fluoranthene is all very fast；Mutant strain DN1- Δs phz and open country
Raw type DN1 bacterial strains shaking flask culture 9 days in using fluoranthene as the MSM culture mediums of sole carbon source, fluoranthene degradation rate can reach respectively
80.4% and 84.1%.The two degradation rate is not much different, and illustrates that DN1- Δ phz bacterial strains can be used in the life of the PAHs such as fluoranthene pollutions
Object reparation.
The above embodiments are merely illustrative of the technical scheme of the present invention and are not intended to be limiting thereof, although with reference to above-described embodiment pair
The present invention is described in detail, those of ordinary skill in the art still can to the present invention specific implementation mode into
Row changes either equivalent replacement and these exist without departing from any modification of spirit and scope of the invention or equivalent replacement
Apply within the claim protection model of the pending present invention.
Bibliography：
【1】CN201410458141- pseudomonas aeruginosa mutative symptom method and its application
【2】CN201710106794- pseudomonas aeruginosa pyoS5 gene knockout mutant strains and construction method and application
【3】The engineered strain and its structure of mono- plant of attenuation producing rhamnolipid with high yield of CN201710172680- and application
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Claims (10)
1. a kind of pseudomonas aeruginosa gene engineering bacteria of attenuation, which is characterized in that the genetic engineering bacterium is P. aeruginosa
The operon of pyo synthesis is controlled in bacterium and its operon copies the attenuated strain that double deletion mutations inactivate.
2. genetic engineering bacterium described in accordance with the claim 1, which is characterized in that it is described control pyo synthesis operon be
Phz operons and its operon copy.
3. genetic engineering bacterium according to claim 2, which is characterized in that the phz operons be by phzA, phzB,
The pathogenicity island of seven genes of phzC, phzD, phzE, phzF and phzG composition, is named as phzA1-G1；And its operon copy life
Entitled phzA2-G2.
4. the construction method of attenuated pseudomonas aeruginosa genetic engineering bacterium described in accordance with the claim 1, includes the following steps：
1) upstreams gene phzA1-G1 are connect with plasmid pEX18Amp respectively with downstream homology arm segment, obtains gene knockout and carries
Body pEX-phzA1-G1；
2) the gene knockout carrier pEX-phzA1-G1 electricity is gone into pseudomonas aeruginosa DN1 bacterial strain competent cells, passed through
Homologous recombination is screened twice, obtains single mutation strain DN1- Δs phzA1-G1；
3) upstreams gene phzA2-G2 are connect with plasmid pEX18Amp respectively with downstream homology arm segment, obtains gene knockout and carries
Body pEX-phzA2-G2；
4) pEX-phzA2-G2 electricity described in gene knockout carrier is gone into the pseudomonas aeruginosa DN1- Δs phzA1-G1 bacterial strains
Competent cell screens twice by homologous recombination, obtains double-mutant strain DN1- Δs phz.
5. according to the construction method of genetic engineering bacterium described in claim 4, which is characterized in that the gene of the gene phzA1-G1
Knocking out primer phzA1-G1-up and phzA1-G1-down is respectively：SEQ ID NO.1-2,SEQ ID NO.3-4；The knockout
Verification primer phzA1-G1 is afterwards:SEQ ID NO.9-10.
6. according to the construction method of genetic engineering bacterium described in claim 4, which is characterized in that the gene of the gene phzA2-G2
Knocking out primer phzA2-G2-up and phzA2-G2-down is respectively：SEQ ID NO.5-6,SEQ ID NO.7-8；The knockout
Verification primer phzA2-G2 is afterwards:SEQ ID NO.11-12.
7. according to the construction method of the genetic engineering bacterium of claim 5 or 6, which is characterized in that the knockout primer amplification production
Object is the homology arm segment of upstream region of gene to be knocked out and each 2kb in downstream or so size, and verification primer extension product is base to be knocked out
A part of segment because in.
8. according to pseudomonas aeruginosa gene engineering bacteria the answering in industrial production rhamnolipid being attenuated described in claim 1
With.
9. being decomposed in environment polycyclic aromatic hydrocarbon or petroleum hydrocarbon according to the pseudomonas aeruginosa gene engineering bacteria being attenuated described in claim 1
With the application in removal.
10. being decomposed and fluoranthene in removal environment according to the pseudomonas aeruginosa gene engineering bacteria being attenuated described in claim 9
Using.
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