CN107164292A - One plant of Clostridium beijerinckii and its construction method and application to the efficient dechlorination of pentachlorophenol - Google Patents

One plant of Clostridium beijerinckii and its construction method and application to the efficient dechlorination of pentachlorophenol Download PDF

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CN107164292A
CN107164292A CN201710492586.1A CN201710492586A CN107164292A CN 107164292 A CN107164292 A CN 107164292A CN 201710492586 A CN201710492586 A CN 201710492586A CN 107164292 A CN107164292 A CN 107164292A
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clostridium beijerinckii
pentachlorophenol
dechlorination
cbei
clostridium
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CN107164292B (en
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郭亭
席梓淇
朱刚强
李永
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Nanjing Zhonghuan Biotechnology Co., Ltd
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Nanjing Zhongtai Biological Science And Technology Co Ltd
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    • A—HUMAN NECESSITIES
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    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/36—Organic compounds containing halogen

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Abstract

The invention discloses a kind of Clostridium beijerinckii recombinant bacterial strain to the efficient reduction dechlorination of pentachlorophenol, the bacterial strain is by after Cbei_3304 gene disruptions so that this albumen in Clostridium beijerinckii NCIMB 8052 cisco unity malfunction and obtain.The Clostridium beijerinckii recombinant bacterium M 3304 of the present invention, in the simulated wastewater of the pentachlorophenol containing 3.0mg/L, 0.15g/L methyl viologens (MV) and 6.44g/L ironic citrates, during 60h, the reduction dechlorination rate of pentachlorophenol reaches 90%；Under equal conditions, and starting strain Clostridium beijerinckii NCIMB 8052 only has 40% to the reduction dechlorination rate of pentachlorophenol；Clostridium beijerinckii recombinant bacterium M 3304 to the reduction dechlorination efficiency of pentachlorophenol is starting strain NCIMB 8052 2.25 times.Present invention can apply to the degraded of pentachlorophenol in water, solid refuse, solid waste and soil, the invention belongs to field of Environment Protection.

Description

One plant of Clostridium beijerinckii and its construction method and application to the efficient dechlorination of pentachlorophenol
Technical field
The invention belongs to environmental technology field, and in particular to a kind of Clostridium beijerinckii recombinant bacterium to the efficient dechlorination of pentachlorophenol Strain and its application on construction method and field of Environment Protection.
Background technology
Pentachlorophenol (pentachlorophenol, PCP), also known as pentachlorophenol；Chang Hanyi molecular crystalline water is slightly warm to have pole Strong pungent stink.Pentachlorophenol can prevent fungi growth, suppress bacterium, be used as a long time herbicide, desinsection, bactericide, Mould inhibitor etc..However, toxicity of the PCP in all phenol poisoning materials is maximum, with very strong " three cause " (carcinogenic, teratogenesis, Mutagenesis) effect.The 1970's, Environmental Protection Agency USA included priority pollutant list (Water Res., 2004, 38：663-672), in China, PCP is listed in 68 kinds of pollutant lists of water environment priority acccess control.Pentachlorophenol is difficult by oxygen Change, it is also difficult to hydrolyze, there is cumulative effect, it can largely be enriched with biology, and concentration is dense in water considerably beyond it Degree.PCP is on soil or accumulation thing, with very high adsorptivity, can be absorbed by plants, enter food chain by biological concentration (Trends in Biotehcnolgoy,20:243-248.).Migration, conversion of the PCP biohazardous in natural environment And its degraded, it is always the focus of industry research and concern.
Three kinds of the processing method of pentachlorophenol, Main physical method, chemical method and bioanalysis.Physical mainly includes absorption Method, bifurcation ray method, supercritical ultrasonics technology etc..Chemical method mainly includes electrochemistry, Fenton reagent method, zero-valent metal reducing process, dioxy Change manganese method etc..Comprising a variety of microorganisms in bioanalysis, different types of microorganism is due to the biochemical mechanism of its degradation of contaminant It is different so that PCP degradation pathway variation, but degradation process is that first dechlorination is converted into after low chlorinated compound that open loop is obtained again To degraded or mineralising.Most important conditioning step is the removal of chlorine substituent in PCP biodegradation process, and PCP biology takes off Chlorine mainly has oxidative dechlorination mechanism and anaerobic reductive dechlorination mechanism (Applied and Environmental Microbiology,75:5910-5918.).Wherein, anaerobic reductive dechlorination mechanism is most common and effective biodegrading process.
Bioanalysis has the advantages that low cost, efficiency high, not non-secondary pollution, welding, will be in PCP governance processes Have broad application prospects.During anaerobic reductive dechlorination, some Fe (III) reducing bacterias Bacillus, Geobacter, Pesudomonas and Clostridium etc. plays extremely important effect (Science of the Total Environment,473:215-223.)。
The content of the invention
The technical problems to be solved by the invention are to provide one plant of type introne gene disruption technology of process two and obtained The Clostridium beijerinckii to the efficient dechlorination of pentachlorophenol.
The of the invention technical problem also to be solved is to provide above-mentioned Clostridium beijerinckii to the application in terms of pentachlorophenol dechlorination, To improve bioanalysis degraded PCP efficiency.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is as follows：
A kind of Clostridium beijerinckii to the efficient dechlorination of pentachlorophenol, Cbei_3304 genes are inactivated in Clostridium beijerinckii, make the base Because being unable to normal expression in Clostridium beijerinckii.A kind of doubtful memebrane protein of Cbei_3304 gene codes, the function of this memebrane protein lacks Lose, it is possible to increase the exchange of some materials of intracellular and mobility, and then lift dechlorination efficiency of the Clostridium beijerinckii to pentachlorophenol.
Wherein, the nucleotide sequence of the Cbei_3304 genes such as SEQ ID NO：Shown in 1.
Wherein, the Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052.
The construction method of the above-mentioned Clostridium beijerinckii to the efficient dechlorination of pentachlorophenol, comprises the following steps：
(1) intron sequences are building up in two type introne gene knockout plasmids, obtain recombinant plasmid；
(2) the recombinant plasmid transformed Clostridium beijerinckii for obtaining step (1), screening obtains the bacterium of Cbei_3304 genes inactivation Strain, had both obtained the Clostridium beijerinckii of the efficient dechlorination of pentachlorophenol.
Preferably, the nucleotide sequence of the intron sequences such as SEQ ID NO：Shown in 2, the insertion position of intron sequences Point is SEQ ID No：Between 335bp and 336bp in 1.
Preferably, the two types introne gene knockout plasmid is pWJ plasmids.
The construction method of the above-mentioned Clostridium beijerinckii to the efficient dechlorination of pentachlorophenol, the Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052。
The construction method of the above-mentioned Clostridium beijerinckii to the efficient dechlorination of pentachlorophenol builds obtained Clostridium beijerinckii in the present invention Send out within protection domain.
Above-mentioned Clostridium beijerinckii applying within protection scope of the present invention in the efficient dechlorination of pentachlorophenol.Should Bacterial strain can be used for the removing of pentachlorophenol in soil, water body.
The beneficial effects of the invention are as follows：
The present invention carries out the Cbei_3304 genes that memebrane protein is encoded in Clostridium beijerinckii after insertion inactivation so that this gene Be unable to normal expression, obtain the recombinant bacterial strain of Cbei_3304 gene disruptions, both recombinant bacterial strain M-3304, containing In the simulated wastewater of 3.0mg/L pentachlorophenol, 0.15g/L methyl viologens (MV) and 6.44g/L citric acid armors, 60 hours Interior, the reduction dechlorination rate of pentachlorophenol reaches 90%；Under equal conditions, 8052 couple five of starting strain Clostridium beijerinckii NCIMB The reduction dechlorination rate of chlorophenol only has 40%；Clostridium beijerinckii recombinant bacterium M-3304, to the reduction dechlorination efficiency of pentachlorophenol, is 2.25 times of bacterium germination strain.The recombinant bacterium that the present invention is obtained is one plant of excellent species suitable for pentachlorophenol reduction dechlorination, can be wide The general degraded applied to pentachlorophenol in water, solid refuse, solid waste and soil, the invention belongs to field of Environment Protection.
Brief description of the drawings
Fig. 1 inserts inactivating vectors pWJ plasmid map for the present invention；
The mechanism figure that Fig. 2 is inactivated for the present invention using two type Intron insertions；
Fig. 3 is conversion daughter colony PCR electrophoretograms；Swimming lane 1, swimming lane 2, swimming lane 4 are wild-type strain NCIMB's 8052 Cbei_3304 genetic fragment sizes, swimming lane 3 is insertion Inactivating mutations strain M-3304 Cbei_3304 genetic fragment sizes, swimming lane 5 be Marker；
Fig. 4 be Clostridium beijerinckii M-3304 of the present invention and initial strains NCIMB 8052 in simulated wastewater, to pentachlorophenol Dechlorination experimental result.
Embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real Apply specific material proportion, process conditions and its result described by example and be merely to illustrate the present invention, without that will not also should limit The present invention described in detail in claims processed.
Embodiment 1：The structure of Clostridium beijerinckii Cbei_3304 gene disruption mutant strains.
The principle of Clostridium beijerinckii Cbei_3304 gene disruption mutant strains is built as shown in Fig. 2 specific building process bag Include following steps：
(1) Cbei_3304 inserts the structure of inactivating vectors
1) introne is designed
Cbei_3304 gene orders (such as SEQ ID No for the Clostridium beijerinckii included according to ncbi database：Shown in 1), borrow Help Software for Design suitably insert gene loci (http://www.clostron.com), selection is inserted in the 101st~102 Between base, and generate intron sequences, synthesis intron sequences S-304 (its sequence such as SEQ ID NO：Shown in 2), and design Following primer.
Cloning primer：
pWJ-OSC-304-S：5’-ggagtgtcgaggatcctcgagAtaattatccttacacttcgcc-3 ', its sequence Row such as SEQ ID NO：Shown in 3；
pWJ-OSC-304-A：5’-ggttctcctacagattgtacaAatgtggtgataacagataag-3 ', its sequence Such as SEQ ID NO：Shown in 4.
Verify primer：
Cbei-3304-T-S：5 '-taaattacctacagcaaaactgtg-3 ', sequence such as SEQ ID NO：Shown in 5；
Cbei-3304-T-A：5 '-ggaattaagaaccttgaatctatc-3 ', sequence such as SEQ ID NO：Shown in 6.
Primer pWJ-OSC-304-S introduces the restriction enzyme sites of Xho I (underscore part), and primer pWJ-OSC-304-A is introduced The restriction enzyme sites of BsrG I (underscore part).
2) Cbei_3304-pWJ-304 construction of recombinant vector
With Xho I and double digestion carrier pWJ, the pWJ plasmid maps of BsrG I as shown in figure 1, its sequence such as SEQ ID NO：7 institutes Show.The purified kit of digestion products (Takara) after purification, one-step cloning is passed through with intron sequences S-304 (ClonExpress) connect.The recombinant plasmid transformed that one-step cloning is connected is applied to and contained to E. coli DH5a There are 50 μ g/ml ammonia benzyl chloramphenicol resistance LB flat boards, 37 DEG C of 12~16h of culture, picking transformant is connected to liquid and contains 50 μ g/ml ammonia In benzyl mycin LB culture mediums, 37 DEG C, 200rpm culture 12h extract recombinant plasmid (AXYGEN), sequence verification obtains Cbei_ 3304-pWJ-304 recombinant vectors.
3) Cbei_3304-pWJ-304 recombinant vectors methylate
E.coli Top 10/pAN2 Competent is prepared, successful Cbei_3304-pWJ-304 will be sequenced and recombinates Carrier is transformed into E. coli Top 10, because pAN2 plasmids have tetracyclin resistance, therefore is applied to containing 50 μ g/ Ml ammonia benzyl mycins and the 10 Double LB flat boards of μ g/ml tetracyclines, 37 DEG C of 12~16h of culture, picking transformant are connected to liquid and contained In 50 μ g/ml ammonia benzyl mycins and 10 μ g/ml tetracycline LB culture mediums, 37 DEG C, 200rpm culture 12h extract the Cbei_ methylated 3304-pWJ-304 recombinant vectors (pAN2 plasmids contain a bacillus subtilis phage gene, can encode transmethylase, Exogenous plasmid methylating in Escherichia coli can be realized), i.e. Cbei_3304 insertion inactivating vectors.
(2) Cbei_3304 inserts inactivating vectors conversion Clostridium beijerinckii (Clostridium beijerinckii NCIMB 8052)
1) Clostridium beijerinckii NCIMB 8052 are seeded to YPS culture mediums (dusty yeast 3g/L, egg White peptone 5g/L, glucose 1g/L, ammonium acetate 2g/L, sodium chloride 3g/L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, phosphorus Sour hydrogen dipotassium 1g/L, green vitriol 0.1g/L, pH=6.) 37 DEG C of incubated overnights, next day is inoculated into YPS with 5% ratio Culture medium, 37 DEG C of culture 6-8h, 2 × YTG culture mediums (dusty yeast 16g/L, peptone 10g/L, glucose are inoculated into 10% 5g/L, sodium chloride 5g/L) 37 DEG C of culture 3h, OD600Nm=1；
2) 50ml Clostridium beijerinckii bacterium solutions are taken, 5000rpm, 4 DEG C of centrifugation 10min abandon supernatant.With ETM buffer solutions (270mM Sucrose, 0.6mM Na2HPO4, 4.4mM Na2HPO4, 10mM MgCl2) be resuspended；Ibid centrifuge, remove supernatant, buffered again with ETM Liquid is resuspended, and ibid centrifuges, thoroughly takes supernatant；
3) with 1ml ET buffer solutions (270mM sucrose, 0.6mM Na2HPO4, 4.4mM NaH2PO4It is resuspended, takes 200 μ l, plus Enter 1ug Cbei_3304 insertion inactivating vectors, add the electric revolving cup of 0.2cm precoolings, gently mix；
4) turned using MicroPulserTM electroporations electricity, condition is voltage 1.8kV, the Ω of resistance 200, the μ F of electric capacity 2.5, electricity 1mL 2 × YTG culture mediums are added immediately after hitting, 2~3h of recovery in sterile centrifugation tube is transferred to；
5) the 200 above-mentioned bacterium solutions of μ l are taken, the YPS solid mediums containing 10 μ g/ml erythromycin are applied to, cultivated 2~3 days.
(3) Cbei_3304 inserts the screening of Inactivating mutations strain
Picking above-mentioned steps 5) the middle transformant for cultivating 2~3 days, use primer Cbei-3304-T-S and Cbei-3304- T-A carries out bacterium colony PCR checkings to transformant, and filtering out the mutant strain of Intron insertion genome, (after insertion, PCR amplifies base Because on band electrophoretogram than wild type about 1Kbp), as shown in figure 3, by the mutant strain being correctly inserted into pass on three times, simultaneously be coated with On the YPS solid mediums containing Erythromycinresistant and without Erythromycinresistant, the mutant strain for knocking out plasmid loss is filtered out (mutant strain that can not be grown on Erythromycinresistant flat board).
Embodiment 2：Clostridium beijerinckii is tested in simulated wastewater to the dechlorination of pentachlorophenol.
(1) culture medium prescription：
YPS culture mediums：Dusty yeast 3g/L, peptone 5g/L, glucose 1g/L, ammonium acetate 2g/L, sodium chloride 3g/L, seven water Close magnesium sulfate 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, pH=6.
Simulated wastewater：Glucose 2g/L, potassium chloride 0.9g/L, ammonium chloride 1.0g/L, a hypophosphite monohydrate sodium dihydrogen 0.6g/L, One calcium chloride hydrate 0.02g/L, yeast extract 0.05g/L, electron mediator (MV) 0.15g/L, ironic citrate 6.44g/L, pentachlorobenzene Phenol 3mg/L and trace element (H3BO33g/L, ZnSO4·7H2O 0.9g/L,MnCl2·2H2O 2.4g/L,CuSO4·5H2O 0.6g/L,CoCl2·6H2O 3g/L,(NH4)6Mo7O24·2H2O 0.02g/L,NiCl2·6H2O 0.1g/L)5ml/L。
(2) Clostridium beijerinckii group bacterium M-3304 and initial strains NCIMB 8052, cultivation temperature 37 are activated in YPS culture mediums DEG C, Anaerobic culturel time 12h.Then 40mL bacterium solutions are taken, 10000rpm centrifugation 5min outwell supernatant, thalline is transferred into 20mL Simulated wastewater in, be passed through N25 minutes, discharge oxygen, rapid sealing, 30 DEG C of cultivation temperature, 150rpm shaking table cultures.Bai Shi Clostridium is to the reduction dechlorination effect of pentachlorophenol in simulated wastewater, as shown in Figure 4.
As can be seen that in 60h, Clostridium beijerinckii recombinant bacterium M-3304 reaches to the reduction dechlorination rate of pentachlorophenol 90%, under equal conditions, and starting strain NCIMB 8052 only has 40% or so to the reduction dechlorination rate of pentachlorophenol.Bai Shi shuttles Bacterium recombinant bacterium M-3304, to the reduction dechlorination efficiency of pentachlorophenol, is 2.25 times of starting strain.
SEQUENCE LISTING
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<120>One plant of Clostridium beijerinckii and its construction method and application to the efficient dechlorination of pentachlorophenol
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caccacattt gtacaatctg taggagaacc tatgggaacg aaacgaaagc gatgccgaga 1140
atctgaattt accaagactt aacactaact ggggataccc taaacaagaa tgcctaatag 1200
aaaggaggaa aaaggctata gcactagagc ttgaaaatct tgcaagggta cggagtactc 1260
gtagtagtct gagaagggta acgcccttta catggcaaag gggtacagtt attgtgtact 1320
aaaattaaaa attgattagg gaggaaaacc tcaaaatgaa accaacaatg gcaattttag 1380
aaagaatcag taaaaattca caagaaaata tagacgaagt ttttacaaga ctttatcgtt 1440
atcttttacg tccagatatt tattacgtgg cgacgcgttg ggaaatggca atgatagcga 1500
aacaacgtaa aactcttgtt gtatgctttc attgtcatcg tcacgtgatt cataaacaca 1560
agtgaatgtc gacagtgaat ttttacgaac gaacaataac agagccgtat actccgagag 1620
gggtacgtac ggttcccgaa gagggtggtg caaaccagtc acagtaatgt gaacaaggcg 1680
gtacctccct acttcaccat atcattttct gcagccccct agaaataatt ttgtttaact 1740
ttaagaagga gatatacata tatggctaga tcgtccattc cgacagcatc gccagtcact 1800
atggcgtgct gctagcgcta tatgcgttga tgcaatttct atgcactcgt agtagtctga 1860
gaagggtaac gccctttaca tggcaaaggg gtacagttat tgtgtactaa aattaaaaat 1920
tgattaggga ggaaaacctc aaaatgaaac caacaatggc aattttagaa agaatcagta 1980
aaaattcaca agaaaatata gacgaagttt ttacaagact ttatcgttat cttttacgtc 2040
cagatattta ttacgtggcg tatcaaaatt tatattccaa taaaggagct tccacaaaag 2100
gaatattaga tgatacagcg gatggcttta gtgaagaaaa aataaaaaag attattcaat 2160
ctttaaaaga cggaacttac tatcctcaac ctgtacgaag aatgtatatt gcaaaaaaga 2220
attctaaaaa gatgagacct ttaggaattc caactttcac agataaattg atccaagaag 2280
ctgtgagaat aattcttgaa tctatctatg aaccggtatt cgaagatgtg tctcacggtt 2340
ttagacctca acgaagctgt cacacagctt tgaaaacaat caaaagagag tttggcggcg 2400
caagatggtt tgtggaggga gatataaaag gctgcttcga taatatagac cacgttacac 2460
tcattggact catcaatctt aaaatcaaag atatgaaaat gagccaattg atttataaat 2520
ttctaaaagc aggttatctg gaaaactggc agtatcacaa aacttacagc ggaacacctc 2580
aaggtggaat tctatctcct cttttggcca acatctatct tcatgaattg gataagtttg 2640
ttttacaact caaaatgaag tttgaccgag aaagtccaga aagaataaca cctgaatatc 2700
gggagctcca caatgagata aaaagaattt ctcaccgtct caagaagttg gagggtgaag 2760
aaaaagctaa agttctttta gaatatcaag aaaaacgtaa aagattaccc acactcccct 2820
gtacctcaca gacaaataaa gtattgaaat acgtccggta tgcggacgac ttcattatct 2880
ctgttaaagg aagcaaagag gactgtcaat ggataaaaga acaattaaaa ctttttattc 2940
ataacaagct aaaaatggaa ttgagtgaag aaaaaacact catcacacat agcagtcaac 3000
ccgctcgttt tctgggatat gatatacgag taaggagatc tggaacgata aaacgatctg 3060
gtaaagtcaa aaagagaaca ctcaatggga gtgtagaact ccttattcct cttcaagaca 3120
aaattcgtca atttattttt gacaagaaaa tagctatcca aaagaaagat agctcatggt 3180
ttccagttca caggaaatat cttattcgtt caacagactt agaaatcatc acaatttata 3240
attctgaact ccgcgggatt tgtaattact acggtctagc aagtaatttt aaccagctca 3300
attattttgc ttatcttatg gaatacagct gtctaaaaac gatagcctcc aaacataagg 3360
gaacactttc aaaaaccatt tccatgttta aagatggaag tggttcgtgg gggatcccgt 3420
atgagataaa gcaaggtaag cagcgccgtt attttgcaaa ttttagtgaa tgtaaatccc 3480
cttatcaatt tacggatgag ataagtcaag ctcctgtatt gtatggctat gcccggaata 3540
ctcttgaaaa caggttaaaa gctaaatgtt gtgaattatg tgggacgtct gatgaaaata 3600
cttcctatga aattcaccat gtcaataagg tcaaaaatct taaaggcaaa gaaaaatggg 3660
aaatggcaat gatagcgaaa caacgtaaaa ctcttgttgt atgctttcat tgtcatcgtc 3720
acgtgattca taaacacaag tgaatgtcga gcacccgttc tcggagcact gtccgaccgc 3780
tttggccgcc gcccagtcct gctcgcttcg ctacttggag ccactatcga ctacgcgatc 3840
atggcgacca cacccgtcct gtggatcgcc aagccgccga tggtagtgtg gggtctcccc 3900
atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg 3960
gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg 4020
ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca 4080
taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt 4140
ctacaaactc ttcctgtcgt catatctaca agcatatggt gcactctcag tacaatctgc 4200
tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 4260
cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 4320
atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 4380
cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 4440
tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 4500
tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 4560
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 4620
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 4680
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 4740
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 4800
cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 4860
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 4920
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 4980
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 5040
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 5100
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 5160
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 5220
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 5280
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 5340
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 5400
tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 5460
ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 5520
accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 5580
aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 5640
ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 5700
gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 5760
ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 5820
ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 5880
ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 5940
gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 6000
cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 6060
cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 6120
cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 6180
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 6240
ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 6300
gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 6360
gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg 6420
cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag 6480
ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga 6540
attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt 6600
tggctaacac acacgccatt ccaaccaata gttttctcgg cataaagcca tgctctgacg 6660
cttaaatgca ctaatgcctt aaaaaaacat taaagtctaa cacactagac ttatttactt 6720
cgtaattaag tcgttaaacc gtgtgctcta cgaccaaaag tataaaacct ttaagaactt 6780
tcttttttct tgtaaaaaaa gaaactagat aaatctctca tatcttttat tcaataatcg 6840
catcagattg cagtataaat ttaacgatca ctcatcatgt tcatatttat cagagctcgt 6900
gctataatta tactaatttt ataaggagga aaaaataaag agggttataa tgaacgagaa 6960
aaatataaaa cacagtcaaa actttattac ttcaaaacat aatatagata aaataatgac 7020
aaatataaga ttaaatgaac atgataatat ctttgaaatc ggctcaggaa aagggcattt 7080
tacccttgaa ttagtacaga ggtgtaattt cgtaactgcc attgaaatag accataaatt 7140
atgcaaaact acagaaaata aacttgttga tcacgataat ttccaagttt taaacaagga 7200
tatattgcag tttaaatttc ctaaaaacca atcctataaa atatttggta atatacctta 7260
taacataagt acggatataa tacgcaaaat tgtttttgat agtatagctg atgagattta 7320
tttaatcgtg gaatacgggt ttgctaaaag attattaaat acaaaacgct cattggcatt 7380
atttttaatg gcagaagttg atatttctat attaagtatg gttccaagag aatattttca 7440
tcctaaacct aaagtgaata gctcacttat cagattaaat agaaaaaaat caagaatatc 7500
acacaaagat aaacagaagt ataattattt cgttatgaaa tgggttaaca aagaatacaa 7560
gaaaatattt acaaaaaatc aatttaacaa ttccttaaaa catgcaggaa ttgacgattt 7620
aaacaatatt agctttgaac aattcttatc tcttttcaat agctataaat tatttaataa 7680
gtaagttaag ggatgcataa actgcatccc ttaacttgtt tttcgtgtac ctattttttg 7740
tgaatcgata atttacagaa aagaaaatta tagaatttag tatgattaat tatactcatt 7800
tatgaatgtt taattgaata caaaaaaaaa tacttgttat gtattcaatt acgggttaaa 7860
atatagacaa gttgaaaaat ttaataaaaa aataagtcct cagctcttat atattaagct 7920
accaacttag tatataagcc aaaacttaaa tgtgctacca acacatcaag ccgttagaga 7980
actctatcta tagcaatatt tcaaatgtac cgacatacaa gagaaacatt aactatatat 8040
attcaattta tgagattatc ttaacagata taaatgtaaa ttgcaataag taagatttag 8100
aagtttatag cctttgtgta ttggaagcag tacgcaaagg cttttttatt tgataaaaat 8160
tagaagtata tttatttttt cataattaat ttatgaaaat gaaagggggt gagcaaagtg 8220
acagaggaaa gcagtatctt atcaaataac aaggtattag caatatcatt attgacttta 8280
gcagtaaaca ttatgacttt tatagtgctt gtagctaagt agtacgaaag ggggagcttt 8340
aaaaagctcc ttggaataca tagaattcat aaattaattt atgaaaagaa gggcgtatat 8400
gaaaacttgt aaaaattgca aagagtttat taaagatact gaaatatgca aaatacattc 8460
gttgatgatt catgataaaa cagtagcaac ctattgcagt aaatacaatg agtcaagatg 8520
tttacataaa gggaaagtcc aatgtattaa ttgttcaaag atgaaccgat atggatggtg 8580
tgccataaaa atgagatgtt t 8601

Claims (9)

1. a kind of Clostridium beijerinckii to the efficient dechlorination of pentachlorophenol, it is characterised in that Cbei_3304 bases are inactivated in Clostridium beijerinckii Cause, makes the gene be unable to normal expression in Clostridium beijerinckii.
2. the Clostridium beijerinckii according to claim 1 to the efficient dechlorination of pentachlorophenol, it is characterised in that the Cbei_ The nucleotide sequence of 3304 genes such as SEQ ID NO：Shown in 1.
3. the Clostridium beijerinckii according to claim 1 to the efficient dechlorination of pentachlorophenol, it is characterised in that the Clostridium beijerinckii For Clostridium beijerinckii NCIMB 8052.
4. the construction method of any Clostridium beijerinckii to the efficient dechlorination of pentachlorophenol of claims 1 to 3, it is characterised in that Comprise the following steps：
(1) intron sequences are building up in two type introne gene knockout plasmids, obtain recombinant plasmid；
(2) the recombinant plasmid transformed Clostridium beijerinckii for obtaining step (1), screening obtains the bacterial strain of Cbei_3304 genes inactivation, both Obtain the Clostridium beijerinckii of the efficient dechlorination of pentachlorophenol.
5. according to claim 4 to the construction method of the Clostridium beijerinckii of the efficient dechlorination of pentachlorophenol, it is characterised in that described The nucleotide sequence of intron sequences such as SEQ ID NO：Shown in 2, the insertion point of intron sequences is SEQ ID No：In 1 Between 335bp and 336bp.
6. according to claim 4 to the construction method of the Clostridium beijerinckii of the efficient dechlorination of pentachlorophenol, it is characterised in that described Two type introne gene knockout plasmids are pWJ plasmids.
7. according to claim 4 to the construction method of the Clostridium beijerinckii of the efficient dechlorination of pentachlorophenol, it is characterised in that described Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052.
What 8. the construction method structure of any Clostridium beijerinckii to the efficient dechlorination of pentachlorophenol of claim 4~7 was obtained visits Family name clostridium.
9. application of the Clostridium beijerinckii described in claim 8 in pentachlorophenol in removing soil, water body.
CN201710492586.1A 2017-06-26 2017-06-26 Clostridium beijerinckii for efficiently dechlorinating pentachlorophenol and construction method and application thereof Active CN107164292B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106047754A (en) * 2016-06-07 2016-10-26 广州甘蔗糖业研究所 Method for improving power generation capacity of clostridium beijerinckii and application of clostridium beijerinckii

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106047754A (en) * 2016-06-07 2016-10-26 广州甘蔗糖业研究所 Method for improving power generation capacity of clostridium beijerinckii and application of clostridium beijerinckii

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GENBANK: "CP000721", 《GENBANK》 *
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