CN107287145A - One plant of electricity production Clostridium beijerinckii and its construction method and application - Google Patents
One plant of electricity production Clostridium beijerinckii and its construction method and application Download PDFInfo
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- CN107287145A CN107287145A CN201710638736.5A CN201710638736A CN107287145A CN 107287145 A CN107287145 A CN 107287145A CN 201710638736 A CN201710638736 A CN 201710638736A CN 107287145 A CN107287145 A CN 107287145A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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- C12Y106/05—Oxidoreductases acting on NADH or NADPH (1.6) with a quinone or similar compound as acceptor (1.6.5)
- C12Y106/05003—NADH dehydrogenase (ubiquinone) (1.6.5.3)
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M8/00—Fuel cells; Manufacture thereof
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P70/00—Climate change mitigation technologies in the production process for final industrial or consumer products
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Abstract
The invention discloses one plant of electricity production Clostridium beijerinckii, it is characterised in that Cbei_2996 genes are inactivated in Clostridium beijerinckii, the gene is unable to normal expression in Clostridium beijerinckii.The invention also discloses the construction method of above-mentioned high electricity production restructuring Clostridium beijerinckii and application.The recombinant bacterial strain 2996 of the present invention is in 1g/L glucose seed liquors, and when 0.1g/L methyl viologens (MV) are electron mediator, maximum output voltage is up to 194mV；Under equal conditions, the maximum output voltage of starting strain 8052 only has 85mV；The maximum output voltage of recombinant bacterial strain 2996 is 2.3 times of starting strain.Method of the invention is easy to operate, cost is low, can effectively improve the output voltage of Clostridium beijerinckii, and recombinant bacterium 2996 is one plant and is suitable for microbiological fuel cell research and the strain excellent applied.
Description
Technical field
The invention belongs to environment and field of new energy technologies, and in particular to a kind of method of raising Clostridium beijerinckii electricity production.
Background technology
Microbiological fuel cell (Microbial Fuel Cell, MFC) is one kind using electricity-producing microorganism as anode catalyst
Chemical energy in organic matter is converted into the device of electric energy, microorganism electronics can be transferred directly to compound or
Electron acceptor is connect, and then worked in MFC (Bioresource Technol., 2009,101 (6):1533–1543).Mesh
Before, technology is increasingly mature for microbiological fuel cell (Microbial Fuel Cell, MFC), and is paid attention to.Microbial fuel
Battery in terms of alternative energy source, biology sensor and wastewater treatment have potential application value (Microb Ecol.,
2004,48(2):178-190)。
At present, it has been found that many electricity-producing microorganism such as Shewanella, ground bacillus, klebsiellas etc., electricity-producing microorganism
It is anaerobe mostly, and is mostly gramnegative bacterium, the report produced electricity using gram-positive bacterium is few.
Liu Jun et al. also studied the MFC electricity production situations of Clostridium beijerinckii, and Clostridium beijerinckii mutant strain is situated between by the use of methyl viologen as electronics
Body, glucose is used as acquisition maximum voltage 230mV, peak power output density 79.2mW/m during carbon source2。(Biotechnology
Lett.,2015,37:95-100).Researcher tentatively discloses the mechanism of microorganism electricity generation, but specific mechanism is still unclear
Clear, microorganism electricity generation ability is weaker in MFC, and one of the bottleneck problem of technology development is produced electricity as MFC.Based on microorganism electricity generation machine
The research of reason, by molecular biology and genetic engineering techniques microorganism, and then improves the efficiency of fuel cell generation of microorganism；To have
Make every effort to promote the application and popularization into MFC.
The content of the invention
The technical problem to be solved in the present invention is to provide one plant of electricity production Clostridium beijerinckii.
The technical problem of the invention also to be solved is to provide the construction method of above-mentioned electricity production Clostridium beijerinckii.
The technical problem of the invention finally to be solved is to provide above-mentioned electricity production Clostridium beijerinckii in microbiological fuel cell
Using.
In order to solve the above technical problems, the present invention is adopted the following technical scheme that：
One plant of electricity production Clostridium beijerinckii, it is the inactivation Cbei_2996 genes in Clostridium beijerinckii, makes the gene in Clostridium beijerinckii
In be unable to normal expression.The Cbei_2996 genes are to rely on NADH CoQ oxide-reductase subunit A, the method for inactivation
Selected from base mutation inactivation, base insertion inactivation, base deletion inactivation
Wherein, the nucleotide sequence of the Cbei_2996 genes such as SEQ ID NO：Shown in 1.
Wherein, the Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052.
Wherein, it is described in Clostridium beijerinckii inactivate Cbei_2996 genes, be in Cbei_2996 genes 62bp and
Intron sequences are inserted between 63bp.
Wherein, the intron sequences such as SEQ ID NO：Shown in 2.
The construction method of above-mentioned electricity production Clostridium beijerinckii, 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_2996 genes inactivation
Strain, both obtains producing electricity Clostridium beijerinckii.
Wherein, the two types introne gene knockout plasmid is pWJ plasmids.
Wherein, the intron sequences such as SEQ ID NO：Shown in 2, the insertion point of introne is Cbei_2996 genes
In between 62bp and 63bp.
The construction method of above-mentioned electricity production Clostridium beijerinckii build obtained electricity production Clostridium beijerinckii protection scope of the present invention it
It is interior.
Above-mentioned electricity production Clostridium beijerinckii applying within protection scope of the present invention in microbiological fuel cell.
Beneficial effect：
(1) present invention depends on coding in Clostridium beijerinckii NADH CoQ oxide-reductase subunit A gene Cs bei_
2996 are carried out after insertion inactivation so that this gene is unable to normal expression, obtain the recombinant bacterium of Cbei_2996 gene disruptions
Strain.
(2) the invention provides a kind of simple, efficient method for improving Clostridium beijerinckii electricity production, obtained by the method
Recombinant bacterial strain 2996 is in 1g/L glucose seed liquors, when 0.1g/L methyl viologens (MV) are electron mediator, maximum output voltage
Up to 194mV；Under equal conditions, the maximum output voltage of starting strain 8052 only has 85mV；Recombinant bacterial strain 2996 it is maximum defeated
Go out 2.3 times that voltage is starting strain.
Brief description of the drawings
Fig. 1 is plasmid pWJ plasmid map；
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, and swimming lane 1 is Marker, and swimming lane 2 is insertion Inactivating mutations strain, and swimming lane 3 is
Wild-type strain Cbei_2996 genes；
Fig. 4 is that Clostridium beijerinckii 2996 of the present invention utilizes the electricity production that 1g/L glucose is fuel with initial strains NCIMB 8052
Voltage condition.
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 construction method of Clostridium beijerinckii Cbei_2996 genes Inactivating mutations strain
(1) introne is designed：
Cbei_2996 gene orders (such as SEQ ID No for the Clostridium beijerinckii included according to ncbi database：Shown in 1), i.e.,
CoQ oxide-reductase subunit A base sequence (nuoA) is encoded, gene loci is suitably inserted by Software for Design
(http://www.clostron.com), experiment screening and checking by early stage, selection are inserted in Cbei_2996 gene sequences
Arrange between the 62nd and 63 base, and generate intron sequences, the introne of synthesis is S-62, its sequence such as SEQ ID NO：2
It is shown.
(2) Cbei_2996 inserts the structure of inactivating vectors：
With Xho I and BsrG I difference double digestion carriers pWJ, (Shanghai Sheng Ke institutes teacher Yang Sheng provides, its sequence such as SEQ ID
NO：Shown in 5, pWJ plasmid map is as shown in Figure 1) and DNA fragmentation S-62.The purified kit of digestion products (Takara) is pure
After change, connection is stayed overnight by T4 ligases (Takara).By the recombinant plasmid transformed of connection to E. coli DH5a
(this laboratory), is applied to containing 50ug/ml ammonia benzyl chloramphenicol resistance LB flat boards, 37 DEG C of culture 12-16h, picking transformant is connected to
Liquid contains in 50ug/ml ammonia benzyl mycin LB culture mediums, 37 DEG C, 200rpm culture 12h, extracts recombinant plasmid (AXYGEN plasmids
Extracts kit), sequence verification obtains two type introne gene knockout carrier pWJ-62 of Cbei_2996 genes.
(3) carrier pWJ-62 methylates：
The Competent of the E.coli Top 10 (this laboratory) containing pAN2 plasmids is prepared, sequencing is successful
Cbei_2996 insertions inactivating vectors pWJ-62 is transformed into E. coli Top 10, because pAN2 plasmids have tetracycline
Resistance, therefore be 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 culture 12-16h, choose
Transformant is taken, liquid is connected to and contains in 50ug/ml ammonia benzyl mycins and 10ug/ml tetracycline LB culture mediums, 37 DEG C, 200r cultures
12h, (pAN2 plasmids contain a bacillus subtilis phage gene to the carrier pWJ-62 that extraction methylates, and can encode methyl
Transferase, can realize exogenous plasmid methylating in Escherichia coli).
(4) the electricity knockout plasmid that methylates of conversion is to Clostridium beijerinckii (Clostridium beijerinckii NCIMB
8052)：
1) Clostridium beijerinckii NCIMB 8052 (the conventional bacterial strain that this laboratory normal phase has) are connect
Kind to YPS seed culture mediums (dusty yeast 3g/L, peptone 5g/L, soluble starch 10g/L, ammonium acetate 2g/L, NaCl 3g/L,
MgSO4·7H2O 3g/L, KH2PO41g/L, K2HPO41g/L, FeSO4·7H2O 0.1g/L) 37 DEG C of incubated overnights, next day with
5% ratio is inoculated into YPS culture mediums, and 37 DEG C of culture 6-8h are inoculated into 2 × YTG culture mediums (dusty yeast 16g/L, albumen with 10%
Peptone 10g/L, glucose 5g/L, sodium chloride 5g/L) 37 DEG C of culture 3h, OD600=1.
2) 50ml bacterium solutions are taken, 5000rpm, 4 DEG C of centrifugation 10min abandon supernatant.Again with ETM buffer solutions (270mM sucrose,
0.6mM Na2HPO4, 4.4mM Na2HPO4, 10mM MgCl2·7H2O) it is resuspended；Ibid centrifuge, remove supernatant, buffered again with ETM
Liquid is resuspended, and ibid centrifuges, thoroughly takes supernatant.
3) the pWJ-62 plasmids for taking 1 μ g to methylate are added to the electric revolving cup of 0.2cm precoolings；1mLET buffer solutions are used again
(270mM sucrose, 0.6mM Na2HPO4, 4.4mM Na2HPO4) step 2 is resuspended) bacterium mud, take 200 μ l to be added to electric revolving cup, 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, recovery 2-3h in sterile centrifugation tube is transferred to.
5) the above-mentioned bacterium solutions of 200ul are taken, the YPS solid mediums containing 10 μ g/ml erythromycin are applied to, cultivated 2-3 days.
(5) screening Cbei_2996 insertions Inactivating mutations strain：
Picking transformant, uses primer Cbei-2996-T-S (its sequence such as SEQ ID NO：Shown in 3) and Cbei-2996-
T-A (its sequence such as SEQ ID NO：Shown in 4), bacterium colony PCR checkings are carried out to transformant, Intron insertion genome is filtered out
Mutant strain (after insertion, PCR is amplified than wild type about 1Kbp on gene band electrophoretogram, as shown in Figure 3).It will be correctly inserted into
Mutant strain pass on three times, while be coated on the YPS solid mediums containing Erythromycinresistant and without Erythromycinresistant, sieve
The mutant strain (mutant strain that can not be grown on Erythromycinresistant flat board) for knocking out plasmid loss is selected, the present invention is used in two types
The mechanism figure inactivated containing sub- insertion is as shown in Figure 2.
Embodiment 2：
This example demonstrates that Clostridium beijerinckii is tested as anode catalyst using the electricity production of 1g/L glucose sugar.
(1) Microbial fuel electricity is built：
The present embodiment establishes according to existing technology and method and utilizes micro- life that Clostridium beijerinckii is anode catalyst generating
Thing fuel cell, as shown in Figure 2, including anode chamber, cathode chamber, four parts of PEM and external circuit.Anode electrode
It is PAN bases graphite soft felt (5 × 5cm) with cathode electrode, using titanium silk as external circuitses, outer meeting resistance is 1000 Ω, proton
Exchange membrane is Du Pont NafionN117, uses data acquisition unit for Keithley series.
(2) culture medium prescription：
YPS seed culture mediums：Dusty yeast 3g/L, peptone 5g/L, glucose 1g/L, ammonium acetate 2g/L, sodium chloride 3g/L,
Bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, pH=6.
Dual chamber MFC Anodic liquid：Dusty yeast 3g/L, peptone 5g/L, glucose 1g/L, ammonium acetate 2g/L, sodium chloride 3g/
L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, crystal violet
Essence (MV concentration is 0.4g/L in 3304-MFC, and MV concentration is 0.1g/L in 8052-MFC), pH=6.
Catholyte in dual chamber MFC：Two hypophosphite monohydrate sodium dihydrogen 2.772g/L, disodium hydrogen phosphate dodecahydrate 11.5g/L, chlorine
Change potassium 0.13g//l, the potassium ferricyanide 40mM, pH=7.
(3) high electricity production recombinant bacterial strain 3304 and initial strains 8052,37 DEG C of cultivation temperature are activated in YPS seed liquors, is detested
Oxygen incubation time 12h.Then 250mL dual chamber MFC technologies are carried out, anolyte 225mL, methyl viologen are accessed into anode chamber
(MV), it is passed through N23 minutes, then 25mL Clostridium beijerinckii (inoculum concentration 10% (v/v)) is accessed, it is passed through N23 minutes, discharge in solution
Oxygen, anode chamber is sealed rapidly, catholyte is added to cathode chamber, seals cathode chamber rapidly, 30 DEG C of cultivation temperature, during electricity production detection
Between 33h, final result is as shown in Figure 4.
The maximum output voltage of recombinant bacterial strain 3304 of the present invention is up to 409mV, improves 3.13 times than initial strains 8052, most
Big output power density is 144.4mW/m2, 1.71 times are improved than initial strains 8052.
SEQUENCE LISTING
<110>Nanjing Zhong Tai bio tech ltd
<120>One plant of electricity production Clostridium beijerinckii and its construction method and application
<130> SG20170725001
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 357
<212> DNA
<213>Cbei_2996 gene nucleotide series
<400> 1
atgatacaag attatttgat tattggaatt tttcttatag cttcatttat ttttggaatg 60
gtcgtcttat taacggcttc attagtaaga ccgaagaagc caaataaaga aaaattatct 120
acttatgagt gtggtgttga aactactggt tcaacgtgga ttagatttaa ggtcagttac 180
tttatgtacg gcttagtatt tctactcttt gatgttgaaa cagtattttt attgccatgg 240
gcagtgaaat ttaaatcatt aggattattc gcactttttg aaatggttat ttttataggt 300
attttaatca ttgggttatg gtatgcgtgg aaagaggggg cattagagtg gaagtaa 357
<210> 2
<211> 344
<212> DNA
<213> Artificial Sequence
<220>
<223>Introne S-62 nucleotide sequences
<400> 2
ctcgagataa ttatccttag ttaacaagac ggtgcgccca gatagggtgt taagtcaagt 60
agtttaaggt actactctgt aagataacac agaaaacagc caacctaacc gaaaagcgaa 120
agctgatacg ggaacagagc acggttggaa agcgatgagt tacctaaaga caatcgggta 180
cgactgagtc gcaatgttaa tcagatataa ggtataagtt gtgtttactg aacgcaagtt 240
tctaatttcg attttaactc gatagaggaa agtgtctgaa acctctagta caaagaaagg 300
taagttagtc gtcttgactt atctgttatc accacatttg taca 344
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Cbei-2996-T-S
<400> 3
atgatacaag attatttgat 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Cbei-2996-T-A
<400> 4
ttacttccac tctaatgccc 20
<210> 5
<211> 8601
<212> DNA
<213>PWJ plasmid nucleotide sequences
<400> 5
tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 60
aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 120
gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 180
gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 240
acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 300
taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 360
aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 420
tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 480
ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 540
tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 600
taataatagt atatacttaa tgtgctgcag tataaaatat aaataatttt ctaaaaaact 660
taacttcatg tgaaaagttt gttaaaatat aaatgagcac gttaatcatt taacatagat 720
aattaaatag taaaagggag tgtcgaggat cctcgagata attatcctta aaggacgtag 780
atgtgcgccc agatagggtg ttaagtcaag tagtttaagg tactactctg taagataaca 840
cagaaaacag ccaacctaac cgaaaagcga aagctgatac gggaacagag cacggttgga 900
aagcgatgag ttacctaaag acaatcgggt acgactgagt cgcaatgtta atcagatata 960
aggtataagt tgtgtttact gaacgcaagt ttctaatttc ggtttccttc cgatagagga 1020
aagtgtctga aacctctagt acaaagaaag gtaagttaac atctacgact tatctgttat 1080
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 (10)
1. one plant of electricity production Clostridium beijerinckii, it is characterised in that Cbei_2996 genes are inactivated in Clostridium beijerinckii, the gene is being visitd
Normal expression is unable in family name clostridium.
2. electricity production Clostridium beijerinckii according to claim 1, it is characterised in that the nucleotides sequence of the Cbei_2996 genes
Row such as SEQ ID NO：Shown in 1.
3. electricity production Clostridium beijerinckii according to claim 1, it is characterised in that the Clostridium beijerinckii is Clostridium beijerinckii NCIMB
8052。
4. electricity production Clostridium beijerinckii according to claim 1, it is characterised in that described that Cbei_ is inactivated in Clostridium beijerinckii
2996 genes, are intron sequences to be inserted between 62bp and 63bp in Cbei_2996 genes.
5. electricity production Clostridium beijerinckii according to claim 1, it is characterised in that the intron sequences such as SEQ ID NO：2
It is shown.
6. the construction method of any electricity production Clostridium beijerinckii of Claims 1 to 5, 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_2996 genes inactivation, both
Obtain producing electricity Clostridium beijerinckii.
7. the construction method of Clostridium beijerinckii is produced electricity according to claim 6, it is characterised in that the two types introne clpp gene
Except plasmid is pWJ plasmids.
8. the construction method of Clostridium beijerinckii is produced electricity according to claim 6, it is characterised in that the intron sequences such as SEQ
ID NO：Shown in 2, the insertion point of introne is in Cbei_2996 genes the between 62bp and 63bp.
9. the construction method of any electricity production Clostridium beijerinckii of claim 6~8 builds obtained electricity production Clostridium beijerinckii.
10. application of the Clostridium beijerinckii in microbiological fuel cell is produced electricity described in claim 9.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567603A (en) * | 2016-02-03 | 2016-05-11 | 广州甘蔗糖业研究所 | Method for improving stress resistance of clostridium beijerinckii to 4-hydroxycinnamic acid |
| 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 |
-
2017
- 2017-07-31 CN CN201710638736.5A patent/CN107287145B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567603A (en) * | 2016-02-03 | 2016-05-11 | 广州甘蔗糖业研究所 | Method for improving stress resistance of clostridium beijerinckii to 4-hydroxycinnamic acid |
| 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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| JULIANE NIESSEN等: "exploiting complex carbohydrates for microbial electricity generataion-a bacteriral fuel cell operating on starch", 《ELECTROCHEMISTRY COMMUNICATIONS》 * |
| 林海龙等: "《厌氧环境微生物学》", 31 October 2014 * |
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