CN103911334A - Clostridium beijerinckii with high stress resistance and application thereof - Google Patents
Clostridium beijerinckii with high stress resistance and application thereof Download PDFInfo
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- CN103911334A CN103911334A CN201410153818.7A CN201410153818A CN103911334A CN 103911334 A CN103911334 A CN 103911334A CN 201410153818 A CN201410153818 A CN 201410153818A CN 103911334 A CN103911334 A CN 103911334A
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- cbei
- clostridium beijerinckii
- stress resistance
- bai shi
- shi clostridium
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- 241000193454 Clostridium beijerinckii Species 0.000 title abstract description 10
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000000855 fermentation Methods 0.000 claims abstract description 22
- 230000004151 fermentation Effects 0.000 claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 229920002522 Wood fibre Polymers 0.000 claims abstract description 7
- 230000004853 protein function Effects 0.000 claims abstract description 4
- 241000193403 Clostridium Species 0.000 claims description 41
- 201000010099 disease Diseases 0.000 claims description 23
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 23
- 230000002779 inactivation Effects 0.000 claims description 23
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 17
- 238000001784 detoxification Methods 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 abstract description 8
- 235000000346 sugar Nutrition 0.000 abstract description 5
- YQUVCSBJEUQKSH-UHFFFAOYSA-N protochatechuic acid Natural products OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 abstract description 4
- WKOLLVMJNQIZCI-UHFFFAOYSA-N vanillic acid Chemical compound COC1=CC(C(O)=O)=CC=C1O WKOLLVMJNQIZCI-UHFFFAOYSA-N 0.000 abstract description 4
- TUUBOHWZSQXCSW-UHFFFAOYSA-N vanillic acid Natural products COC1=CC(O)=CC(C(O)=O)=C1 TUUBOHWZSQXCSW-UHFFFAOYSA-N 0.000 abstract description 4
- 231100000614 poison Toxicity 0.000 abstract 3
- 239000003440 toxic substance Substances 0.000 abstract 3
- 239000002025 wood fiber Substances 0.000 abstract 3
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 abstract 1
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 abstract 1
- 229940114124 ferulic acid Drugs 0.000 abstract 1
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 abstract 1
- 235000001785 ferulic acid Nutrition 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 20
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 16
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- 241000609240 Ambelania acida Species 0.000 description 10
- 239000010905 bagasse Substances 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 150000002989 phenols Chemical class 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 238000003780 insertion Methods 0.000 description 8
- 230000037431 insertion Effects 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- 238000011218 seed culture Methods 0.000 description 8
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 7
- 239000005695 Ammonium acetate Substances 0.000 description 7
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 7
- 229940043376 ammonium acetate Drugs 0.000 description 7
- 235000019257 ammonium acetate Nutrition 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 6
- 240000008042 Zea mays Species 0.000 description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 6
- 235000005822 corn Nutrition 0.000 description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 6
- 235000003891 ferrous sulphate Nutrition 0.000 description 6
- 239000011790 ferrous sulphate Substances 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 6
- 235000019341 magnesium sulphate Nutrition 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- 229910052700 potassium Inorganic materials 0.000 description 6
- 239000011591 potassium Substances 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000013598 vector Substances 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 229960003276 erythromycin Drugs 0.000 description 4
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- NWXMGUDVXFXRIG-WESIUVDSSA-N (4s,4as,5as,6s,12ar)-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O NWXMGUDVXFXRIG-WESIUVDSSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000000394 mitotic effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 241001110912 Clostridium beijerinckii NCIMB 8052 Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001035 methylating effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 229960004793 sucrose Drugs 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000193401 Clostridium acetobutylicum Species 0.000 description 1
- 241000732800 Cymbidium Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000979 retarding effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/205—Bacterial isolates
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
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- C12R2001/145—Clostridium
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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Abstract
The invention discloses clostridium beijerinckii with high stress resistance and an application of the clostridium beijerinckii. A gene Cbei_3304 of the clostridium beijerinckii strain with high stress resistance cannot be expressed normally, and a normal Cbei_3304 protein function is deleted, so that the clostridium beijerinckii has high stress resistance to toxic substances such as ferulic acid and vanillic acid, also has high stress resistance to toxic substances in virus-carrying wood fiber acidolysis sugar liquor, and can be used for producing butyl alcohol through fermentation very well. The invention provides a simple and efficient method for improving stress resistance of the clostridium beijerinckii. The clostridium beijerinckii which has high stress resistance to the toxic substances in the virus-carrying wood fiber acidolysis sugar liquor and can be used for efficiently producing butyl alcohol can be obtained by only enabling the clostridium beijerinckii to delete the normal Cbei_3304 protein function, so as to be helpful in promoting the efficient utilization of wood fiber resources.
Description
Technical field
The present invention relates to a kind of high resistance to cold and diseases Bai Shi clostridium and application thereof, belong to genetically engineered and fermentation engineering field.
Background technology
Day by day exhausted along with fossil energy, utilizes reproducible wood fibre raw material (as corn cob, bagasse etc.) by Production by Microorganism Fermentation bioenergy and bio-based chemical, becomes one of the emphasis of research and focus.Lignocellulose raw material must pass through pre-treatment, obtains the fermentable sugar of microorganism, could well be utilized by microorganism.But lignocellulose raw material after pretreatment, can produce the inhibitions such as organic acid, furfural, phenols, these inhibitions to remove cost higher and microorganism growth is had to certain restraining effect.Therefore, the resistance of raising microorganism is even more important.
Butanols have energy density large, can be with gasoline arbitrarily than mixing, can being directly used in the advantages such as oil engine, butanols, as novel reproducible liquid energy, is paid attention to widely.Utilize lignocellulose raw material fermentation to prepare fuel butanols, become one of focus of research.Thaddeus Ezeji etc. (Bioresource Technol. 2008,99:5915-5922) utilize Bai Shi clostridium mutant strain BA101, and taking the corncob acid hydrolysis of XAD-4 resin detoxification and enzymolysis liquid glucose as fermenting substrate, total solvent output is 9.30 g/L; But mutant strain BA101 can not utilize the acidolysis liquid glucose fermentation of not detoxification to produce butanols.Chinese patent ZL 201110020102.6 reports: the Bai Shi clostridium that obtains by particle beam selection by mutation (
clostridium beijerinckii) IB4 has higher resistance to phenolic compound, it can be using the corncob acid hydrolysis liquid glucose of not detoxification as carbon source, and in 2L fermentor tank, total solvent output and butanols output have reached respectively 10.3g/L and 7.1 g/L.But Guo Ting etc. (J and Microbiol Biotechnol., 2012,39 (3), 401-407) study discovery, when the concentration of the phenolic compound in corn cob and bagasse acidolysis liquid glucose rises to 1.5 g/L when above, Bai Shi clostridium (
clostridium beijerinckii) IB4 do not grow substantially.(Appl Environ Microbiol, 2003,69 (8): 4,951 4965) in clostridium acetobutylicum, cross and express coding heat shock protein such as Tomas
groeSL gene, makes butanols reduce by 85% to the restraining effect of somatic cells, and finally makes production concentration improve 33%.(the Biotechnol Bioeng 2007,97 (6): 1460-1469) research finds that organic acid, furfural etc. do not affect the fermentation of butanols, and phenols inhibition has obvious retarding effect to butylic fermentation such as Thaddeus Ezeji.But, the inhibition mechanism more complicated of phenolic compound to microorganism growth and fermentation, it is also unintelligible that it suppresses mechanism.
Based on this, growth and the leavening property of the pretreated toxin inhibition of wood fibre (as forulic acid, chinese cymbidium shortcake etc.) severe inhibition Bai Shi clostridium; Improving the Bai Shi clostridium resistance to toxin such as phenolic compounds, is that lignocellulose raw material is prepared one of key issue that the industrialization of fuel butanols must solve.
Summary of the invention
In order to address the above problem, the invention provides a kind of high resistance to cold and diseases Bai Shi clostridium of easy acquisition, it has high resistance to cold and diseases to toxin such as phenolic compounds.
The object of the present invention is to provide a kind of high resistance to cold and diseases Bai Shi clostridium.
Another object of the present invention is to provide the construction process of a kind of high resistance to cold and diseases Bai Shi clostridium.
A further object of the present invention is to provide the application of a kind of high resistance to cold and diseases Bai Shi clostridium.
The technical solution used in the present invention is:
A kind of high resistance to cold and diseases Bai Shi clostridium, it lacks normal Cbei_3304 protein function.
Further, above-mentioned high resistance to cold and diseases Bai Shi clostridium, its gene C bei_3304 can not normal expression.
Further, above-mentioned high resistance to cold and diseases Bai Shi clostridium, its gene C bei_3304 is inserted into inactivation.
Above-mentioned a kind of high resistance to cold and diseases Bai Shi clostridium is prepared the application in butanols in fermentation.
Further, above-mentioned application is specially: high resistance to cold and diseases Bai Shi clostridium is prepared butanols taking the wood fibre acidolysis liquid glucose of not detoxification as raw material direct fermentation.
The invention has the beneficial effects as follows:
The present invention inserts the Cbei_3304 gene of the membranin of encoding in Bai Shi clostridium after inactivation, makes this gene can not normal expression, obtains the recombinant bacterial strain of Cbei_3304 gene disruption.The invention provides a kind of method that improves simply, efficiently Bai Shi clostridium resistance, the recombinant bacterial strain contratoxin material (forulic acid, vanillic acid etc.) obtaining by this method has compared with high resistance to cold and diseases, when the bagasse acidolysis liquid glucose taking not detoxification is during as carbon source, in 2L fermentor tank, total solvent output and butanols output have reached respectively 7.0 g/L and 5.1 g/L, and the starting strain that equal conditions is cultivated is not grown substantially.The recombinant bacterial strain that utilizes the inventive method to build, its strong stress resistance, butanols output are high, are a kind of excellent species that is applicable to the lignocellulose raw material fermentative production butanols such as bagasse.
Brief description of the drawings
Fig. 1 is the plasmid map that the present invention inserts inactivation carrier pWJ;
Fig. 2 is the mechanism figure that the present invention uses two type Intron insertion inactivations;
Fig. 3 is transformant bacterium colony PCR electrophorogram.
Embodiment
According to following embodiment, the present invention may be better understood.But, those skilled in the art will readily understand, the described concrete material proportion of embodiment, processing condition and result thereof be only for the present invention is described, and should also can not limit the present invention described in detail in claims.
embodiment 1
one, the structure of Bai Shi clostridium Cbei_3304 gene disruption mutant strain
Build the principle of Bai Shi clostridium Cbei_3304 gene disruption mutant strain as shown in Figure 2, concrete building process comprises the following steps:
(1) Cbei_3304 inserts the structure of inactivation carrier
1) design intron
The Cbei_3304 gene order (as shown in SEQ ID No:1) of the Bai Shi clostridium of including according to ncbi database, by the suitable insertion gene locus of software design (
http:// www.clostron.com), select to be inserted between 101-102 base, and generate intron sequences, synthetic its sequence of intron sequences S-101(is as shown in SEQ ID NO:2), and the following primer of design.
Clone's primer:
PWJ-OSC-101-S:5 '-ggagtgtcgaggatc
ctcgagataattatccttacacttcgcc-3 ', its sequence is as shown in SEQ ID NO:3;
PWJ-OSC-101-A:5 '-ggttctcctacagat
tgtacaaatgtggtgataacagataag-3 ', its sequence is as shown in SEQ ID NO:4.
Checking primer:
Cbei-3304-T-S:5 '-taaattacctacagcaaaactgtg-3 ', sequence is as shown in SEQ ID NO:5;
Cbei-3304-T-A:5 '-ggaattaagaaccttgaatctatc-3 ', sequence is as shown in SEQ ID NO:6.
Primer pWJ-OSC-101-S introduces Xho I restriction enzyme site (underscore part), and primer pWJ-OSC-101-A introduces BsrG I restriction enzyme site (underscore part).
2) Cbei_3304-pWJ-101 construction of recombinant vector
With Xho I and BsrG I double digestion carrier pWJ, as shown in Figure 1, its sequence is as shown in SEQ ID NO:7 for pWJ plasmid map.Enzyme is cut after the purified test kit of product (Takara) purifying, is connected by one-step cloning (ClonExpress) with intron sequences S-101.The recombinant plasmid transformed that one-step cloning is connected is to intestinal bacteria E.
colidH5a, be applied to and contain 50 μ g/ml ammonia benzyl mycin resistance LB flat boards, cultivate 12～16h for 37 DEG C, picking transformant, receive liquid and contain in 50 μ g/ml ammonia benzyl mycin LB substratum, 37 DEG C, 200rpm are cultivated 12h, extract recombinant plasmid (AXYGEN), sequence verification, obtains Cbei_3304-pWJ-101 recombinant vectors.
3) methylating of Cbei_3304-pWJ-101 recombinant vectors
Preparation E.
colithe chemoreception state of Top 10/pAN2, is transformed into intestinal bacteria E. by successful order-checking Cbei_3304-pWJ-101 recombinant vectors
colitop 10, because pAN2 plasmid has tetracyclin resistance, contain 50 μ g/ml ammonia benzyl mycins and the dual anti-property of 10 μ g/ml tsiklomitsin LB flat board therefore be applied to, cultivate 12～16h for 37 DEG C, picking transformant, receiving liquid contains in 50 μ g/ml ammonia benzyl mycins and 10 μ g/ml tsiklomitsin LB substratum, 37 DEG C, 200rpm cultivates 12h, (pAN2 plasmid contains a bacillus subtilis phage gene to extract methylated Cbei_3304-pWJ-101 recombinant vectors, the methyltransgerase of encoding, can realize exogenous plasmid methylating in intestinal bacteria), be that Cbei_3304 inserts inactivation carrier.
(2) Cbei_3304 insert inactivation carrier transform Bai Shi clostridium (
clostridium beijerinckiiNCIMB8052)
1) will
clostridium beijerinckiinCIMB 8052 is seeded to CGM substratum (yeast powder 3 g/L, peptone 5 g/L, Zulkovsky starch 10 g/L, ammonium acetate 2 g/L, NaCl 2 g/L, MgSO
47H
2o 3 g/L, KH
2pO
41 g/L, K
2hPO
41 g/L, FeSO
47H
2o 0.1 g/L) 37 DEG C of incubated overnight, be inoculated into CGM substratum with 5% ratio next day, cultivates 6-8h for 37 DEG C, is inoculated into 37 DEG C of 2 × YTG substratum (yeast powder 16 g/L, peptone 10 g/L, glucose 5 g/L, sodium-chlor 5 g/L) cultivates 3h, OD with 10%
600nm=1;
2) get 50ml Bai Shi clostridium bacterium liquid, 5000rpm, 4 DEG C of centrifugal 10 min, abandon supernatant.With ETM damping fluid (270mM sucrose, 0.6mM Na
2hPO
4, 4.4mM Na
2hPO
4, 10mM MgCl
2) resuspended; The same centrifugal, remove supernatant, again resuspended with ETM damping fluid, the same centrifugal, thoroughly get supernatant;
3) with 1ml ET damping fluid (270mM sucrose, 0.6mM Na
2hPO
4, 4.4mM NaH
2pO
4resuspended, get 200 μ l, add 1 μ g Cbei_3304 to insert inactivation carrier, add the electric revolving cup of 0.2cm precooling, mix gently;
4) use MicroPulserTM electroporation electricity to turn, condition is voltage 1.8kV, resistance 200 Ω, and electric capacity 2.5 μ F, add 1mL 2 × YTG substratum at once after electric shock, transfer to the 2～3h that recovers in aseptic centrifuge tube;
5) get the above-mentioned bacterium liquid of 200 μ l, be applied to the CGM solid medium that contains 10 μ g/ml erythromycin, cultivate 2～3 days.
(3) Cbei_3304 inserts the screening of inactivation mutant strain
Picking above-mentioned steps 5) the middle transformant of cultivating 2～3 days, use primer Cbei-3304-T-S and Cbei-3304-T-A to carry out bacterium colony PCR checking to transformant, filter out the genomic mutant strain of Intron insertion (after insertion, pcr amplification goes out on gene band electrophorogram than the about 1Kbp of wild-type), as shown in Figure 3, the mutant strain correctly inserting is gone down to posterity three times, be coated on the CGM solid medium that contains erythromycin resistance and there is no erythromycin resistance simultaneously, filter out the mutant strain (mutant strain that can not grow) that knocks out plasmid loss in erythromycin resistant panel.
two, Cbei_3304 inserts the mitotic stability of inactivation mutant strain
The Bai Shi clostridium mutant strain of the Cbei_3304 gene disruption of above-mentioned structure is transferred 7 times continuously on solid medium, find that obtained strains is identical on morphological specificity and cultural characteristic with starting strain, growth conditions is good.In the fermention medium taking glucose as carbon source, detect the mitotic stability of recombinant bacterium, insert the inactivation mutant bacteria fermentation test result that goes down to posterity as shown in table 1.
The Cbei_3304 of the different passage numbers of table 1 inserts the fermentation situation of inactivation mutant bacteria
From experimental result, through 7 continuous passages, total solvent output and the butanols output of two plant mutant strains are more stable, have good mitotic stability, can be used as the production bacterial strain of further research and development.
three, the high resistance to cold and diseases of the Bai Shi clostridium of Cbei_3304 insertion inactivation to pattern phenolic compound forulic acid
Culture medium prescription (% is mass percent):
Plate culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar 1.5%, all the other are water, pH 6.
Seed culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, all the other are water, pH 6.
Fermention medium: glucose 3%, ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1%, forulic acid 0.06%, all the other are water, pH 6.6.
The Bai Shi clostridium mutant strain that above-mentioned Cbei_3304 is inserted to inactivation is seeded to the cultivation of plate culture medium anaerobism, 35 DEG C of culture temperature, incubation time 12 h.The mutant strain that flat board is cultivated is inoculated in seed culture medium, 35 DEG C of culture temperature, bottled liquid measure 30 mL of 50 mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 DEG C of culture temperature, incubation time 12 h; Seed is inoculated in fermention medium, inoculum size 10% (v/v), 35 DEG C of leavening temperatures, bottled liquid measure 50 mL of 100 mL Xiao Te anaerobism, inflated with nitrogen 3min, after fermentation culture 72 h, detect butanols output and reached 6.1 g/L, and the starting strain NCIMB 8052 that equal conditions is cultivated does not grow substantially.
four, the high resistance to cold and diseases of the Bai Shi clostridium of Cbei_3304 insertion inactivation to pattern phenolic compound vanillic acid
Culture medium prescription (% is mass percent):
Plate culture medium: with above-mentioned " three ".
Seed culture medium: with above-mentioned " three ".
Fermention medium: glucose 3%, ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1%, vanillic acid 0.05%, all the other are water, pH 6.6.
The Bai Shi clostridium mutant strain that above-mentioned Cbei_3304 is inserted to inactivation is seeded to the cultivation of plate culture medium anaerobism, 35 DEG C of culture temperature, incubation time 12 h.The mutant strain that flat board is cultivated is inoculated in seed culture medium, 35 DEG C of culture temperature, bottled liquid measure 30 mL of 50 mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 DEG C of culture temperature, incubation time 12 h; Seed is inoculated in fermention medium, inoculum size 10% (v/v), 35 DEG C of leavening temperatures, bottled liquid measure 50 mL of 100 mL Xiao Te anaerobism, inflated with nitrogen 3min, after fermentation culture 72 h, detects butanols output and has reached 4.6 g/L, and the starting strain NCIMB 8052 that equal conditions is cultivated can only be faint growth, but substantially do not produce butanols.
five, the high resistance to cold and diseases of the Bai Shi clostridium of Cbei_3304 insertion inactivation to bagasse acidolysis liquid glucose
The Bai Shi clostridium that this part description of contents Cbei_3304 inserts inactivation utilizes bagasse acidolysis liquid glucose (solubility total phenol content is 2.4 g/L), produces the technique of butanols in 1L fermentation cylinder for fermentation.
Culture medium prescription (% is mass percent):
Plate culture medium: with above-mentioned " three ".
Seed culture medium: with above-mentioned " three ".
Fermention medium: ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1%, with bagasse acidolysis liquid glucose (total reducing sugars the is 4 %) configuration of not detoxification, all the other are water, pH 6.6, and in substratum, solubility total phenol content is 2.4 g/L.
The Bai Shi clostridium mutant strain that above-mentioned Cbei_3304 is inserted to inactivation is seeded to the cultivation of plate culture medium anaerobism, 35 DEG C of culture temperature, incubation time 12 h.The mutant strain that flat board is cultivated is inoculated in seed culture medium, bottled liquid measure 150 mL of 250 mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 DEG C of culture temperature, incubation time 12 h; Seed is inoculated in the 2L fermentor tank that 1 L fermention medium is housed, inoculum size 10%(v/v), 35 DEG C of leavening temperatures, pass into continuously nitrogen, flow velocity is 0.3 L/min, after fermentation culture 90 h, detect total solvent output and butanols output and reached respectively 3.1 g/L and 2.2 g/L, and the starting strain NCIMB 8052 that equal conditions is cultivated does not grow substantially.
six, the high resistance to cold and diseases of the Bai Shi clostridium of Cbei_3304 insertion inactivation to bagasse acidolysis liquid glucose
The Bai Shi clostridium that this part description of contents Cbei_3304 inserts inactivation utilizes bagasse acidolysis liquid glucose (solubility total phenol content is 2.0 g/L), produces the technique of butanols in 2 L fermentation cylinder for fermentation.
Culture medium prescription (% is mass percent):
Plate culture medium: with above-mentioned " three ".
Seed culture medium: with above-mentioned " three ".
Fermention medium: ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1%, with bagasse acidolysis liquid glucose (total reducing sugars the is 3.6%) configuration of not detoxification, all the other are water, pH 6.6, and in substratum, solubility total phenol content is 2.0 g/L.
The Bai Shi clostridium mutant strain that above-mentioned Cbei_3304 is inserted to inactivation is seeded to the cultivation of plate culture medium anaerobism, 35 DEG C of culture temperature, incubation time 12 h.The mutant strain that flat board is cultivated is inoculated in seed culture medium, bottled liquid measure 150 mL of 250 mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 DEG C of culture temperature, incubation time 12 h; Seed is inoculated in the 2L fermentor tank that 1 L fermention medium is housed, inoculum size 10%(v/v), 35 DEG C of leavening temperatures, pass into continuously nitrogen, flow velocity is 0.3 L/min, after fermentation culture 90 h, detect total solvent output and butanols output and reached respectively 7.0 g/L and 5.1g/L, and the starting strain NCIMB 8052 that equal conditions is cultivated does not grow substantially.
<110> Guangzhou Inst of Cane Sugar
<120> high resistance to cold and diseases Bai Shi clostridium and application thereof
<130>
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 723
<212> DNA
<213> Bai Shi clostridium
<400> 1
atgacaaagg ttaataaatt acctacagca aaactgtgga atccgaaatc atttattata 60
ttttccgtat ttttctcatt tctgccagct ggaatcatgt gtgctttgaa ttatggacga 120
tctggaagtc aaaaaaagaa gtggatattt cttttagcaa gcatcttagt gtttatagca 180
cttatagcac ttctgcctat attatcaatt aatacttcta tcatattttt cagtattaat 240
atagctttag gcataattct tatgtttaca cagttaaagc tgtataatga acatattcaa 300
aatggcgggc aaagtgcttc gtatttattt cctataatca tagggctttt gattttttca 360
ttgtcagctg cttcaattct atactcaatt tacgtgccaa aaaatgctct tgattatggc 420
gaaaatcact tattttatac taataagata acagaatccc aagcgaaaaa acttggagat 480
tatttaaatt cagaaggata cttcactcct agttcaaaag tggacgttaa gattgataaa 540
caagatacac tctatatttt atctctcgtt gtagaaggtg actataaaag tgatacaagc 600
tatgtagaac ctatgaaagc aatatcaaaa gagatttcaa aaaatgtttt tgaaaataat 660
aaagttagaa tagatttatg caatgataga ttcaaggttc ttaattccat caatgttgat 720
taa 723
<210> 2
<211> 344
<212> DNA
<213> artificial synthesized sequence
<400> 2
ctcgagataa ttatccttac acttcgcccg cgtgcgccca 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 gttaagtgtc gatagaggaa agtgtctgaa acctctagta caaagaaagg 300
taagttatgg cgggcgactt atctgttatc accacatttg taca 344
<210> 3
<211> 43
<212> DNA
The artificial primer of <213>
<400> 3
ggagtgtcga ggatcctcga gataattatc cttacacttc gcc 43
<210> 4
<211> 42
<212> DNA
The artificial primer of <213>
<400> 4
ggttctccta cagattgtac aaatgtggtg ataacagata ag 42
<210> 5
<211> 24
<212> DNA
The artificial primer of <213>
<400> 5
taaattacct acagcaaaac tgtg 24
<210> 6
<211> 24
<212> DNA
The artificial primer of <213>
<400> 6
ggaattaaga accttgaatc tatc 24
<210> 7
<211> 8601
<212> DNA
<213> pWJ plasmid
<400> 7
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 (5)
1. a high resistance to cold and diseases Bai Shi clostridium, is characterized in that: it lacks normal Cbei_3304 protein function.
2. a kind of high resistance to cold and diseases Bai Shi clostridium according to claim 1, is characterized in that: its gene C bei_3304 can not normal expression.
3. a kind of high resistance to cold and diseases Bai Shi clostridium according to claim 2, is characterized in that: its gene C bei_3304 is inserted into inactivation.
4. in claim 1～3, arbitrary described a kind of high resistance to cold and diseases Bai Shi clostridium is prepared the application in butanols in fermentation.
5. application according to claim 4, is characterized in that: high resistance to cold and diseases Bai Shi clostridium claimed in claim 1 is prepared butanols taking the wood fibre acidolysis liquid glucose of not detoxification as raw material direct fermentation.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410153818.7A CN103911334B (en) | 2014-04-16 | 2014-04-16 | A kind of high resistance to cold and diseases Bai Shi clostridium and application thereof |
| PCT/CN2014/085468 WO2015158094A1 (en) | 2014-04-16 | 2014-08-29 | Clostridium beijerinckii with high stress resistance and uses thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410153818.7A CN103911334B (en) | 2014-04-16 | 2014-04-16 | A kind of high resistance to cold and diseases Bai Shi clostridium and application thereof |
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| CN103911334A true CN103911334A (en) | 2014-07-09 |
| CN103911334B CN103911334B (en) | 2016-04-20 |
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| CN (1) | CN103911334B (en) |
| WO (1) | WO2015158094A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894048A (en) * | 2015-06-12 | 2015-09-09 | 南京工业大学 | Method for improving ferulic acid stress resistance of clostridium beijerinckii |
| WO2015158094A1 (en) * | 2014-04-16 | 2015-10-22 | 广州甘蔗糖业研究所 | Clostridium beijerinckii with high stress resistance and uses thereof |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174433A (en) * | 2011-01-18 | 2011-09-07 | 南京工业大学 | Clostridium beijerinckii with high stress resistance and application thereof |
| CN103667147A (en) * | 2013-12-11 | 2014-03-26 | 南京工业大学 | High ferulic acid stress resistance clostridium beijerinckii and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8685729B2 (en) * | 2007-02-26 | 2014-04-01 | The Regents Of The University Of California | Heterologous expression of extremophile heat shock proteins and chaperones in microorganisms to increase tolerance to toxic compounds |
| WO2009143455A2 (en) * | 2008-05-22 | 2009-11-26 | Microbia, Inc. | Engineering resistance to aliphatic alcohols |
| GB201205795D0 (en) * | 2012-03-30 | 2012-05-16 | Univ Nottingham | Vector |
| CN103911334B (en) * | 2014-04-16 | 2016-04-20 | 广州甘蔗糖业研究所 | A kind of high resistance to cold and diseases Bai Shi clostridium and application thereof |
-
2014
- 2014-04-16 CN CN201410153818.7A patent/CN103911334B/en not_active Expired - Fee Related
- 2014-08-29 WO PCT/CN2014/085468 patent/WO2015158094A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174433A (en) * | 2011-01-18 | 2011-09-07 | 南京工业大学 | Clostridium beijerinckii with high stress resistance and application thereof |
| CN103667147A (en) * | 2013-12-11 | 2014-03-26 | 南京工业大学 | High ferulic acid stress resistance clostridium beijerinckii and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 董红军 等: "丙酮丁醇梭菌的遗传操作系统", 《生物工程学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015158094A1 (en) * | 2014-04-16 | 2015-10-22 | 广州甘蔗糖业研究所 | Clostridium beijerinckii with high stress resistance and uses thereof |
| CN104894048A (en) * | 2015-06-12 | 2015-09-09 | 南京工业大学 | Method for improving ferulic acid stress resistance of clostridium beijerinckii |
| CN104894048B (en) * | 2015-06-12 | 2018-03-09 | 南京工业大学 | A kind of method for improving Clostridium beijerinckii forulic acid resistance |
| 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 |
| CN105567603B (en) * | 2016-02-03 | 2019-08-09 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A method of Clostridium beijerinckii is improved to 4- hydroxycinnamic acid resistance |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2015158094A1 (en) | 2015-10-22 |
| CN103911334B (en) | 2016-04-20 |
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Address after: 510316 Haizhuqu District, Guangdong Province, No. ten, road Patentee after: Guangdong Institute of Biotechnology (Guangzhou sugar cane sugar Research Institute) Address before: 510316 Haizhuqu District, Guangdong Province, No. ten, road Patentee before: Guangzhou Sugarcane Industry Research Institute |
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