CN106636168B - 一种调控丙酮丁醇梭菌胞外聚合物合成的方法 - Google Patents
一种调控丙酮丁醇梭菌胞外聚合物合成的方法 Download PDFInfo
- Publication number
- CN106636168B CN106636168B CN201610973505.5A CN201610973505A CN106636168B CN 106636168 B CN106636168 B CN 106636168B CN 201610973505 A CN201610973505 A CN 201610973505A CN 106636168 B CN106636168 B CN 106636168B
- Authority
- CN
- China
- Prior art keywords
- gene
- intron
- clostridium acetobutylicum
- eps
- biofilm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000193401 Clostridium acetobutylicum Species 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 19
- 230000015572 biosynthetic process Effects 0.000 title abstract description 14
- 238000003786 synthesis reaction Methods 0.000 title abstract description 12
- 230000001276 controlling effect Effects 0.000 title abstract description 11
- 230000001105 regulatory effect Effects 0.000 title abstract description 11
- 229920000642 polymer Polymers 0.000 title abstract description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 39
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 238000003209 gene knockout Methods 0.000 claims abstract description 8
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- 239000013612 plasmid Substances 0.000 claims description 23
- 238000003780 insertion Methods 0.000 claims description 11
- 230000037431 insertion Effects 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- DNZWLJIKNWYXJP-UHFFFAOYSA-N butan-1-ol;propan-2-one Chemical compound CC(C)=O.CCCCO DNZWLJIKNWYXJP-UHFFFAOYSA-N 0.000 claims description 8
- 230000001131 transforming effect Effects 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 2
- 230000003197 catalytic effect Effects 0.000 claims description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 5
- 239000008103 glucose Substances 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 239000011521 glass Substances 0.000 abstract description 4
- 238000012136 culture method Methods 0.000 abstract description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 2
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 229910052799 carbon Inorganic materials 0.000 abstract description 2
- 238000000855 fermentation Methods 0.000 description 14
- 230000004151 fermentation Effects 0.000 description 14
- 150000004676 glycans Chemical class 0.000 description 13
- 229920001282 polysaccharide Polymers 0.000 description 12
- 239000005017 polysaccharide Substances 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 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
- 239000002609 medium Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 230000002779 inactivation Effects 0.000 description 6
- 229910001220 stainless steel Inorganic materials 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 229960005091 chloramphenicol Drugs 0.000 description 4
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 229960003276 erythromycin Drugs 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- 239000004098 Tetracycline Substances 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
- 235000019257 ammonium acetate Nutrition 0.000 description 3
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000011987 methylation Effects 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000010935 stainless steel Substances 0.000 description 3
- 229960002180 tetracycline Drugs 0.000 description 3
- 229930101283 tetracycline Natural products 0.000 description 3
- 235000019364 tetracycline Nutrition 0.000 description 3
- 150000003522 tetracyclines Chemical class 0.000 description 3
- 229960003053 thiamphenicol Drugs 0.000 description 3
- OTVAEFIXJLOWRX-NXEZZACHSA-N thiamphenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 OTVAEFIXJLOWRX-NXEZZACHSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 210000001822 immobilized cell Anatomy 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000028070 sporulation Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000423302 Clostridium acetobutylicum ATCC 824 Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000055027 Protein Methyltransferases Human genes 0.000 description 1
- 108700040121 Protein Methyltransferases Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- BJDYCCHRZIFCGN-UHFFFAOYSA-N pyridin-1-ium;iodide Chemical compound I.C1=CC=NC=C1 BJDYCCHRZIFCGN-UHFFFAOYSA-N 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002522 swelling effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
-
- 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
- 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
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种调控丙酮丁醇梭菌胞外聚合物合成的方法，其特征在于，该方法是利用二型内含子基因敲除技术在丙酮丁醇梭菌的Spo0A基因中插入内含子序列。本发明提供了一种简单、高效的调控EPS合成的方法，借助此方法得到的重组菌株对EPS具有较高的调控作用，当以葡萄糖为碳源，在100ml厌氧瓶中利用载玻片生物膜培养法培养丙丁菌生物膜时，生物膜厚度为5.81μm，而同等条件培养的出发菌株生物膜厚度达到24.6μm。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种调控丙酮丁醇梭菌胞外聚合物(EPS)合成的方法。
背景技术
生物被膜又称生物膜(biofilm)是在生物或非生物介质表面由微生物及其胞外基质(Extracellular polymeric substance,EPS)形成的致密、复杂的生物聚集体。EPS作为生物膜的主要组成物质，对保持生物膜的结构和功能的完整性、代谢物质传质通道顺畅具有重要作用。EPS是细胞在固体介质诱导下自主合成的富含大分子多糖的胞外结构，具有良好的持水性、溶胀性、透过性，能够稳定细胞表面的水化层、及时泵出细胞的反应物、为细胞创造有益的微环境，可认为是生物相容性最佳的固定介质。同时，EPS为细胞隔离外界毒性物质提供了一道天然屏障，因而提高了EPS中微生物细胞的抗逆能力。因此，细胞在EPS中能够大量增殖形成密集的菌群进而展示集群效应。集群效应可使群体成员实现生理协同作用，借助个体间的互相刺激而使个体的行为、生理、形态发生变化，使多个生物个体在行为上相互协作，从而提高整个种群的代谢及适存能力。
在临床医学领域，超过80％的慢性感染疾病都与体内细菌形成EPS有关，而细菌形成EPS后对抗生素等杀菌剂和宿主免疫系统的敏感性显著降低，因此，大部分有关EPS机理的研究都集中在如何清除人体器官及外源性生物植入材料表面上的EPS。然而，作为EPS的另一种存在形式——固定化发酵过程中细胞形成的EPS却至今鲜有研究，尤其是固定化细胞形成EPS后表现出的较高底物耐受性和较快的发酵效率的机制机理到现在还不是很清楚。
发明内容
本发明所要解决的技术问题是提供一种调控丙酮丁醇梭菌胞外聚合物(EPS)合成的方法。
本发明还要解决的技术问题是提供利用调控丙酮丁醇梭菌胞外聚合物(EPS)合成的方法构建得到的丙酮丁醇基因工程菌。
本发明最后要解决的技术问题是提供利用上述基因工程菌在EPS催化体系中的应用。
为解决上述技术问题，本发明采用的技术方案如下：
一种调控丙酮丁醇梭菌胞外聚合物合成的方法，该方法是利用二型内含子基因敲除技术在丙酮丁醇梭菌的Spo0A基因中插入内含子序列。在Spo0A基因中插入内含子序列，最终使Spo0A基因不能正常表达，从而引起丙酮丁醇梭菌胞外聚合物(EPS)的合成。
其中，所述的丙酮丁醇梭菌保藏编号为CGMCC No.5234，该菌株的具体信息在申请号为201210075094.X的中国专利中详细公开。
其中，所述的Spo0A基因为CA_C2071，其核苷酸序列如SEQ ID No.：1所示。Spo0A基因编码一种孢子生成蛋白，在发育早期通过多组分磷酸化途径而被激活，是双组份应答性调节蛋白大家族的成员之一，调控并起始孢子形成作用所需基因的转录。
其中，利用二型内含子基因敲除技术将SEQ ID No.：2所示的内含子序列插入Spo0A基因的第242bp和第243bp之间
其中，所述二型内含子基因敲除技术的操作方法如下：
(1)分析SEQ ID No：1所示的序列，得到内含子的核苷酸序列和基因的插入位点；
(2)内含子序列构建到二型内含子基因敲除质粒中，得到重组质粒；
(3)将步骤(2)得到的重组质粒转化丙酮丁醇梭菌，筛选得到Spo0A基因失活的菌株
其中，所述的所述二型内含子基因敲除质粒为PMTL007C-E2质粒，该质粒的核苷酸序列如SEQ ID No：5所示。
上述调控丙酮丁醇梭菌胞外聚合物合成的方法构建得到的丙酮丁醇基因工程菌。
上述丙酮丁醇基因工程菌在发酵生产丙酮丁醇中的应用。
有益效果：
(1)本发明将丙酮丁醇梭菌B3中Spo0A基因进行插入失活后，使得此基因不能正常表达，获得CA_C2071基因插入失活的重组菌株。
(2)本发明提供了一种简单、高效的调控EPS合成的方法，借助此方法得到的重组菌株对EPS具有较高的调控作用，当以葡萄糖为碳源，在100ml厌氧瓶中利用载玻片生物膜培养法培养丙丁菌生物膜时，生物膜厚度为5.81μm，而同等条件培养的出发菌株生物膜厚度达到24.6μm。
附图说明
图1为本发明二型内含子基因敲除质粒PMTL007C-E2的质粒图谱。
图2为本发明使用二型内含子插入失活的机理图。
图3为转化子菌落PCR电泳图，泳道1：出发菌基因组验证PCR产物(846bp)；泳道2-23为挑取的红霉素抗性单菌落的菌液作为模板验证PCR产物(2755bp)；泳道M：DNA MarkerDL2000。
图4为自制反应器中细胞固定化材料的填装方法图。
图5为出发菌与敲除菌EPS宏观形态图。(在不锈钢反应器中，固定化材料与不锈钢丝网交替缠绕呈圆柱状，我们定义固定化材料靠近罐壁的一面为正面，另一面为反面反面。)
图6为CLSM观察出发菌与敲除菌EPS形成过程图。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：丙酮丁醇梭菌CA_C2071基因失活突变株的构建方法。
1、设计内含子：
根据NCBI数据库收录的丙酮丁醇梭菌的CA_C2071基因序列(如SEQ ID No：1所示)，借助软件设计合适的插入基因位点(http://www.clostron.com)，选择插入在第242-243个碱基之间，并生成内含子序列，合成内含子序列S-242其序列如SEQ ID NO：2所示。
2、CA_C2071插入失活载体的构建：
用XhoⅠ和BsrGⅠ双酶切载体PMTL007C-E2，其序列如SEQ ID NO：5所示。酶切产物经纯化试剂盒(Takara)化后，与内含子序列S-242通过一步克隆(ClonExpress)连接。将一步克隆连接的重组质粒转化到大肠杆菌E.coli DH5a(本实验室)，涂布到含有25ug/ml氯霉素抗性LB平板，37℃培养12-16h，挑取转化子，接到液体含有25ug/ml氯霉素LB培养基中，37℃、200rpm培养12h，提取重组质粒(AXYGEN)，测序验证,获得CA_C2071插入失活载体PMTL007C-E2-2071。
3、载体PMTL007C-E2-2071甲基化：
由于已经证明丙酮丁醇梭菌(Clostridium acetobutlicum)ATCC 824中含有限制性内切酶Cac824I，可以切割未甲基化的外源DNA，因此在对丙酮丁醇梭菌进行转化前对重组失活载体PMTL007C-E2-2071进行甲基化修饰。具体操作步骤为制备E.coli Top 10/pAN2(本实验室)的化学感受态，将测序成功的失活载体转化到大肠杆菌E.coli Top 10，由于pAN2质粒具有四环素抗性，故涂布到含有25ug/ml氯霉素和10ug/ml四环素双抗性LB平板，37℃培养12-16h，挑取转化子，接到液体含有25ug/ml氯霉素和10ug/ml四环素LB培养基中，37℃、200rpm培养12h，提取甲基化缺失载体(pAN2质粒含有一个枯草芽孢杆菌噬菌体基因，能编码甲基转移酶，能实现外源质粒在大肠杆菌中的甲基化)。
4、电转化甲基化的敲除质粒至丙酮丁醇梭菌：
厌氧条件下，取2xYTG培养基培养的生长至对数中期的C.acetobutylicumB3培养液60ml，4℃、4000rpm离心10min弃去上清，加入足量预冷的电转缓冲液EPB(270mM蔗糖，5mMNaH2PO4，pH7.4)，洗涤两次，并用2.3mlEPB重悬。然后取570ul加入0.4cm电转杯放置在冰浴中冷却，并加入20ul甲基化质粒，冰浴放置2min。2.0kV电压，25uF电容进行电转化。随后将电转液加入到1ml37℃的2xYTG培养基中复苏培养4h，离心收集细胞100ul并将细胞涂布于含有20ug/ml甲砜霉素的P2平板中。厌氧培养24-36h后。
5、筛选CA_C2071插入失活突变株：
挑取转化子，使用引物CA_C2071-T-F(其序列如SEQ ID NO：3所示)和CA_C2071-T-R(其序列如SEQ ID NO：4所示)，对转化子进行菌落PCR验证，筛选出内含子插入基因组的突变株(插入后，PCR扩增出基因条带电泳图上比野生型大约1Kbp)。将正确插入的突变株传代三次，同时涂布在含有红霉素抗性和红霉素、甲砜霉素抗性的P2固体培养基上，筛选出敲除质粒丢失的突变株(在同时含红霉素和甲砜霉素抗性平板上不能生长的突变株)。
实施例2：敲除菌与出发菌分别在2L发酵罐中固定化发酵合成胞外聚合物(EPS)的工艺。
平板培养基：酵母粉3g/L，蛋白胨5g/L，葡萄糖10g/L，乙酸铵2g/L，氯化钠3g/L，七水合硫酸镁3g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，七水合硫酸亚铁0.1g/L，琼脂15g/L，其余为水。
种子培养基：酵母粉3g/L，蛋白胨5g/L，葡萄糖10g/L，乙酸铵2g/L，氯化钠3g/L，七水合硫酸镁3g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，七水合硫酸亚铁0.1g/L，其余为水。
发酵培养基：葡萄糖60g/L，乙酸铵2.2g/L，磷酸二氢钾0.5g/L，磷酸氢二钾0.5g/L，氯化钠0.01g/L，七水合硫酸镁0.2g/L，七水合硫酸亚铁0.01g/L，一水合硫酸锰0.01g/L，其余为水。
将实施例1中构建的CA_C2071基因插入失活的丙酮丁醇梭菌重组菌和出发菌株CGMCC No.5234接种至平板培养基厌氧培养，培养温度37℃，培养时间12h。将平板培养的重组菌接种到种子培养基(不加琼脂，其他组分同P2平板培养基)中37℃静止培养12h。
固定化发酵：吸附固定化细胞发酵放大工艺在自制的不锈钢反应器中进行。反应器柱状，有效容积约2L，内径10cm，高30cm，外壁有夹套，可通过硅胶管与外置恒温水浴锅相连，进而实现发酵保温。反应器上下两端和侧面在适当位置装有用于取样、液体循环、探头检测的管道和阀门。材料填装时，固定化材料CTF与不锈钢丝网(孔径约2mm，厚度不足1mm)交替缠绕，松紧适度，如图4所示，然后置于2L不锈钢反应器中。发酵液静止反应12h，维持发酵温度37℃，通过蠕动泵对反应器中的发酵液进行循环，循环流速约20-35mL/min，每12h换一次液，发酵培养120h。
由图5可知，出发菌EPS宏观形态为白色粘液状团聚体，其中含有大量孔穴和通道；敲除菌肉眼基本观察不到EPS。
实施例3：利用激光共聚焦显微镜观察敲除菌与出发菌生物膜的结构与厚度。
利用载玻片生物膜培养法培养丙丁菌生物膜，用异硫氰酸荧光素(FITC)标记的刀豆蛋白A(FITC-ConA)和碘化吡啶(PI)双重免疫荧光技术染色，用激光共聚焦显微镜(CLSM)观察生物膜结构和其中胞外多糖的分布,并对生物膜进行逐层水平扫描,根据三维坐标系Z轴距离计算生物膜厚度。
具体操作如下：
(1)生物膜标本的制备
在100mL肖特瓶内放入25mm×75mm载玻片，装入50mL发酵液厌氧静置培养。在培养6、24和72h后取出载玻片，用0.1M，pH＝7.4PBS缓冲液反复冲洗,除掉表面浮游菌,无菌滤纸吸干多余液体,用2.5％戊二醛4℃固定玻片1.5h后，悬浮于0.1M，pH＝7.4的PBS缓冲溶液中，待用。
(2)生物膜标本的染色
多糖染色：FITC-ConA与生物膜中的多糖结合发出绿色荧光。将FITC-ConA配成1mg/mL溶液，呈黄色，样品4℃避光染色15-30min后，用PBS缓冲液清洗去除残留染色剂。FITC-ConA激发/发射波长：488/505nm。
细胞染色：PI与菌体内的DNA结合发出红色荧光，但无膜通透性，不能透过活细胞膜，只能染死细胞。样品4℃避光染色15-30min后，用PBS缓冲液清洗去除残留染色剂。PI激发/发射波长为：536/617nm。
(3)CLSM观察生物膜及厚度测量
生物膜观察:采用LSM 510型激光共聚焦扫描显微镜,37℃恒温载物台,物镜倒置。扫描模式:HeNe/Ar激光,Ex/Em为490/525nm和536/617nm。
生物膜厚度确定:建立三维坐标体系，调节聚焦平面位置，对生物膜标本由外(生物膜游离面)向内(生物膜与玻片相贴面)逐层水平扫描,扫描30层，并进行3D图像重建，则第一张与最后一张能观察到生物膜的扫描图片间的Z轴距离为生物膜厚度。
由图6可知，在初始黏附期，出发菌株生物膜中多糖含量较多，而细菌含量相比较少，多糖形成多处较大的积聚区域，但相互之间的联系并不紧密，细菌形成较小团块，形如细菌镶嵌于多糖基质中；而敲除菌中细菌大部分成散落分布，大部分区域细菌和多糖关系并不密切。在对数生长期，出发菌株生物膜中细菌大量繁殖，细菌和多糖的关联开始紧密，已开始形成多糖和细菌相互包裹的网络状结构；而敲除菌中菌落间的细菌分布厚薄不均，形成沟壑间隙。在成熟稳定期，出发菌株多糖染色明显多于细菌染色，细菌镶嵌在由大量多糖形成的基质中，结构紧密，形成菌囊样的结构，生物膜厚度为24.6μm；而敲除菌中细菌和多糖的关联依旧并不紧密，生物膜厚度为5.81μm。
SEQUENCE LISTING
<110> 南京工业大学
<120> 一种调控丙酮丁醇梭菌胞外聚合物合成的方法
<130> SG20181008001
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 846
<212> DNA
<213> CA_C2071基因核苷酸序列
<400> 1
atggaaagta gaaaaataag tgttttaatt gcagatgata ataaggaatt ttgtaatatt 60
cttaatgatt acttgcttaa ccaaagcgat atgatagttg ttggaatagc aaaggatgga 120
gtggaagctt taaagcttat agagaataaa aagcctgacc ttgttgttct cgatataata 180
atgccaagat tagatggact aggagtatta gaaaaactta acaataaaga tgcagaaaac 240
cttccaagaa taatagtttt atccgctgtt ggacaggata aaataacaca aagagctata 300
acattagggg cggactacta tgtagttaaa ccttttgata tggatgtatt cactaataga 360
ataagagaaa tgtttaataa tactatatca aattcagagc agaaaaggtc atatcaagtt 420
gaagagaaag aggcaagctt tgcaggtaca attgcaaatg atgtttactc tgataatata 480
ggtaataaag cggttgattt agaaagcgaa attacatcaa taatacatca aataggtgtt 540
ccagcacata taaaaggtta tatgtattta agggaagcta taacaatggt agttaataat 600
atggagcttt tatcagcagt tactaaggaa ttatatcctt ctattgcaaa aaaatacaat 660
actacagcta gcagagtaga aagagcaata agacatgcta ttgaagttgc atggtcacgt 720
ggacaggttg aaactataaa taagttattt ggatatacta tcaacaatgg caaaggtaaa 780
cctactaata gtgaatttat tgctatgatt gctgacaagc taagacttaa aaataaagtt 840
agctaa 846
<210> 2
<211> 309
<212> DNA
<213> 内含子核苷酸序列
<400> 2
ttatccttaa ttatccttgg agtgcgccca gatagggtgt taagtcaagt agtttaaggt 60
actactctgt aagataacac agaaaacagc caacctaacc gaaaagcgaa agctgatacg 120
ggaacagagc acggttggaa agcgatgagt tacctaaaga caatcgggta cgactgagtc 180
gcaatgttaa tcagatataa ggtataagtt gtgtttactg aacgcaagtt tctaatttcg 240
gttataatcc gatagaggaa agtgtctgaa acctctagta caaagaaagg taagttactt 300
ccaaggact 309
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CA_C2071-T-F
<400> 3
ggaaagtaga aaaataagtg 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> CA_C2071-T-R
<400> 4
cagcaatcat agcaataaat 20
<210> 5
<211> 8925
<212> DNA
<213> PMTL007C-E2质粒核苷酸序列
<400> 5
cctgcagggt gtagtagcct gtgaaataag taaggaaaaa aaagaagtaa gtgttatata 60
tgatgattat tttgtagatg tagataggat aatagaatcc atagaaaata taggttatac 120
agttatataa aaattacttt aaaaattaat aaaaacatgg taaaatataa atcgtataaa 180
gttgtgtaat ttttaagctt gagctcataa caatttcaca caggaaacag ctatgaccat 240
gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 300
ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 360
ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gctaataaag 420
atcttgtaca atctgtagga gaacctatgg gaacgaaacg aaagcgatgc cgagaatctg 480
aatttaccaa gacttaacac taactgggga taccctaaac aagaatgcct aatagaaagg 540
aggaaaaagg ctatagcact agagcttgaa aatcttgcaa gggtacggag tactcgtagt 600
agtctgagaa gggtaacgcc ctttacatgg caaaggggta cagttattgt gtactaaaat 660
taaaaattga ttagggagga aaacctcaaa atgaaaccaa caatggcaat tttagaaaga 720
atcagtaaaa attcacaaga aaatatagac gaagttttta caagacttta tcgttatctt 780
ttacgtccag atatttatta cgtggcgacg cgtgaagttc ctatactttc tagagaatag 840
gaacttcgcg actcatagaa ttatttcctc ccgttaaata atagataact attaaaaata 900
gacaatactt gctcataagt aacggtactt aaattgttta ctttggcgtg tttcattgct 960
tgatgaaact gatttttagt aaacagttga cgatattctc gattgaccca ttttgaaaca 1020
aagtacgtat atagcttcca atatttatct ggaacatctg tggtatggcg ggtaagtttt 1080
attaagacac tgtttacttt tggtttagga tgaaagcatt ccgctggcag cttaagcaat 1140
tgctgaatcg agacttgagt gtgcaagagc aaccctagtg ttcggtgaat atccaaggta 1200
cgcttgtaga atccttcttc aacaatcaga tagatgtcag acgcatggct ttcaaaaacc 1260
acttttttaa taatttgtgt gcttaaatgg taaggaatac tcccaacaat tttatacctc 1320
tgtttgttag ggaattgaaa ctgtagaata tcttggtgaa ttaaagtgac acgagtattc 1380
agttttaatt tttctgacga taagttgaat agatgactgt ctaattcaat agacgttacc 1440
tgtttactta ttttagccag tttcgtcgtt aaatgccctt tacctgttcc aatttcgtaa 1500
acggtatcgg tttcttttaa attcaattgt tttattattt ggttgagtac tttttcactc 1560
gttaaaaagt tttgagaata ttttatattt ttgttcatac cagcaccaga agcaccagca 1620
tctcttgggt taattgaggc ctgagtataa ggtgacttat acttgtaatc tatctaaacg 1680
gggaacctct ctagtagaca atcccgtgct aaattgtagg actgcccttt aataaatact 1740
tctatattta aagaggtatt tatgaaaagc ggaatttatc agattaaaaa tactttctct 1800
agagaaaatt tcgtctggat tagttactta tcgtgtaaaa tctgataaat ggaattggtt 1860
ctacataaat gcctaacgac tatccctttg gggagtaggg tcaagtgact cgaaacgata 1920
gacaacttgc tttaacaagt tggagatata gtctgctctg catggtgaca tgcagctgga 1980
tataattccg gggtaagatt aacgacctta tctgaacata atgccatatg aatccctcct 2040
aatttatacg ttttctctaa caacttaatt atacccacta ttattatttt tatcaatata 2100
gaagttccta tactttctag agaataggaa cttcacgcgt tgggaaatgg caatgatagc 2160
gaaacaacgt aaaactcttg ttgtatgctt tcattgtcat cgtcacgtga ttcataaaca 2220
caagtgaatg tcgacagtga atttttacga acgaacaata acagagccgt atactccgag 2280
aggggtacgt acggttcccg aagagggtgg tgcaaaccag tcacagtaat gtgaacaagg 2340
cggtacctcc ctacttcacc atatcatttt ctgcagcccc ctagaaataa ttttgtttaa 2400
ctttaagaag gagatataca tatatggcta gatcgtccat tccgacagca tcgccagtca 2460
ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt ctatgcactc gtagtagtct 2520
gagaagggta acgcccttta catggcaaag gggtacagtt attgtgtact aaaattaaaa 2580
attgattagg gaggaaaacc tcaaaatgaa accaacaatg gcaattttag aaagaatcag 2640
taaaaattca caagaaaata tagacgaagt ttttacaaga ctttatcgtt atcttttacg 2700
tccagatatt tattacgtgg cgtatcaaaa tttatattcc aataaaggag cttccacaaa 2760
aggaatatta gatgatacag cggatggctt tagtgaagaa aaaataaaaa agattattca 2820
atctttaaaa gacggaactt actatcctca acctgtacga agaatgtata ttgcaaaaaa 2880
gaattctaaa aagatgagac ctttaggaat tccaactttc acagataaat tgatccaaga 2940
agctgtgaga ataattcttg aatctatcta tgaaccggta ttcgaagatg tgtctcacgg 3000
ttttagacct caacgaagct gtcacacagc tttgaaaaca atcaaaagag agtttggcgg 3060
cgcaagatgg tttgtggagg gagatataaa aggctgcttc gataatatag accacgttac 3120
actcattgga ctcatcaatc ttaaaatcaa agatatgaaa atgagccaat tgatttataa 3180
atttctaaaa gcaggttatc tggaaaactg gcagtatcac aaaacttaca gcggaacacc 3240
tcaaggtgga attctatctc ctcttttggc caacatctat cttcatgaat tggataagtt 3300
tgttttacaa ctcaaaatga agtttgaccg agaaagtcca gaaagaataa cacctgaata 3360
tcgggagctc cacaatgaga taaaaagaat ttctcaccgt ctcaagaagt tggagggtga 3420
agaaaaagct aaagttcttt tagaatatca agaaaaacgt aaaagattac ccacactccc 3480
ctgtacctca cagacaaata aagtattgaa atacgtccgg tatgcggacg acttcattat 3540
ctctgttaaa ggaagcaaag aggactgtca atggataaaa gaacaattaa aactttttat 3600
tcataacaag ctaaaaatgg aattgagtga agaaaaaaca ctcatcacac atagcagtca 3660
acccgctcgt tttctgggat atgatatacg agtaaggaga tctggaacga taaaacgatc 3720
tggtaaagtc aaaaagagaa cactcaatgg gagtgtagaa ctccttattc ctcttcaaga 3780
caaaattcgt caatttattt ttgacaagaa aatagctatc caaaagaaag atagctcatg 3840
gtttccagtt cacaggaaat atcttattcg ttcaacagac ttagaaatca tcacaattta 3900
taattctgaa ctccgcggga tttgtaatta ctacggtcta gcaagtaatt ttaaccagct 3960
caattatttt gcttatctta tggaatacag ctgtctaaaa acgatagcct ccaaacataa 4020
gggaacactt tcaaaaacca tttccatgtt taaagatgga agtggttcgt gggggatccc 4080
gtatgagata aagcaaggta agcagcgccg ttattttgca aattttagtg aatgtaaatc 4140
cccttatcaa tttacggatg agataagtca agctcctgta ttgtatggct atgcccggaa 4200
tactcttgaa aacaggttaa aagctaaatg ttgtgaatta tgtgggacgt ctgatgaaaa 4260
tacttcctat gaaattcacc atgtcaataa ggtcaaaaat cttaaaggca aagaaaaatg 4320
ggaaatggca atgatagcga aacaacgtaa aactcttgtt gtatgctttc attgtcatcg 4380
tcacgtgatt cataaacaca agtgaatgtc gagcacccgt tctcggagca ctgtccgacc 4440
gctttggccg ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga 4500
tcatggcgac cacacccgtc ctgtggatcg ccaagccgcc gatggtagtg tggggtctcc 4560
ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact 4620
gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc 4680
cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc 4740
cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt 4800
ttctacaaac tcttcctgtc gtcatatcta caagccatcc ccccacagat acgggcgcgc 4860
cgccattatt tttttgaaca attgacaatt catttcttat tttttattaa gtgatagtca 4920
aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa agaaaattat agaatttagt 4980
atgattaatt atactcattt atgaatgttt aattgaatac aaaaaaaaat acttgttatg 5040
tattcaatta cgggttaaaa tatagacaag ttgaaaaatt taataaaaaa ataagtcctc 5100
agctcttata tattaagcta ccaacttagt atataagcca aaacttaaat gtgctaccaa 5160
cacatcaagc cgttagagaa ctctatctat agcaatattt caaatgtacc gacatacaag 5220
agaaacatta actatatata ttcaatttat gagattatct taacagatat aaatgtaaat 5280
tgcaataagt aagatttaga agtttatagc ctttgtgtat tggaagcagt acgcaaaggc 5340
ttttttattt gataaaaatt agaagtatat ttattttttc ataattaatt tatgaaaatg 5400
aaagggggtg agcaaagtga cagaggaaag cagtatctta tcaaataaca aggtattagc 5460
aatatcatta ttgactttag cagtaaacat tatgactttt atagtgcttg tagctaagta 5520
gtacgaaagg gggagcttta aaaagctcct tggaatacat agaattcata aattaattta 5580
tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa agagtttatt aaagatactg 5640
aaatatgcaa aatacattcg ttgatgattc atgataaaac agtagcaacc tattgcagta 5700
aatacaatga gtcaagatgt ttacataaag ggaaagtcca atgtattaat tgttcaaaga 5760
tgaaccgata tggatggtgt gccataaaaa tgagatgttt tacagaggaa gaacagaaaa 5820
aagaacgtac atgcattaaa tattatgcaa ggagctttaa aaaagctcat gtaaagaaga 5880
gtaaaaagaa aaaataattt atttattaat ttaatattga gagtgccgac acagtatgca 5940
ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag gatagtcact cgcattttca 6000
taatacatct tatgttatga ttatgtgtcg gtgggacttc acgacgaaaa cccacaataa 6060
aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac taaacaatca agctaggata 6120
tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt aatacatacg ctattaagat 6180
gtaaaaatac ggataccaat gaagggaaaa gtataatttt tggatgtagt ttgtttgttc 6240
atctatgggc aaactacgtc caaagccgtt tccaaatctg ctaaaaagta tatcctttct 6300
aaaatcaaag tcaagtatga aatcataaat aaagtttaat tttgaagtta ttatgatatt 6360
atgtttttct attaaaataa attaagtata tagaatagtt taataatagt atatacttaa 6420
tgtgataagt gtctgacagt gtcacagaaa ggatgattgt tatggattat aagcggccgg 6480
ccagtgggca agttgaaaaa ttcacaaaaa tgtggtataa tatctttgtt cattagagcg 6540
ataaacttga atttgagagg gaacttagat ggtatttgaa aaaattgata aaaatagttg 6600
gaacagaaaa gagtattttg accactactt tgcaagtgta ccttgtacct acagcatgac 6660
cgttaaagtg gatatcacac aaataaagga aaagggaatg aaactatatc ctgcaatgct 6720
ttattatatt gcaatgattg taaaccgcca ttcagagttt aggacggcaa tcaatcaaga 6780
tggtgaattg gggatatatg atgagatgat accaagctat acaatatttc acaatgatac 6840
tgaaacattt tccagccttt ggactgagtg taagtctgac tttaaatcat ttttagcaga 6900
ttatgaaagt gatacgcaac ggtatggaaa caatcataga atggaaggaa agccaaatgc 6960
tccggaaaac atttttaatg tatctatgat accgtggtca accttcgatg gctttaatct 7020
gaatttgcag aaaggatatg attatttgat tcctattttt actatgggga aatattataa 7080
agaagataac aaaattatac ttcctttggc aattcaagtt catcacgcag tatgtgacgg 7140
atttcacatt tgccgttttg taaacgaatt gcaggaattg ataaatagtt aacttcaggt 7200
ttgtctgtaa ctaaaaacaa gtatttaagc aaaaacatcg tagaaatacg gtgttttttg 7260
ttaccctaag tttaaactcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7320
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7380
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7440
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7500
gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7560
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 7620
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 7680
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 7740
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 7800
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 7860
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 7920
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 7980
acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 8040
ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 8100
gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcagggcccc 8160
ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac 8220
aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg 8280
caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc 8340
gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccggcta ccgccggcgt 8400
aacagatgag ggcaagcgga tggctgatga aaccaagcca accaggaagg gcagcccacc 8460
tatcaaggtg tactgccttc cagacgaacg aagagcgatt gaggaaaagg cggcggcggc 8520
cggcatgagc ctgtcggcct acctgctggc cgtcggccag ggctacaaaa tcacgggcgt 8580
cgtggactat gagcacgtcc gcgagctggc ccgcatcaat ggcgacctgg gccgcctggg 8640
cggcctgctg aaactctggc tcaccgacga cccgcgcacg gcgcggttcg gtgatgccac 8700
gatcctcgcc ctgctggcga agatcgaaga gaagcaggac gagcttggca aggtcatgat 8760
gggcgtggtc cgcccgaggg cagagccatg acttttttag ccgctaaaac ggccgggggg 8820
tgcgcgtgat tgccaagcac gtccccatgc gctccatcaa gaagagcgac ttcgcggagc 8880
tggtgaagta catcaccgac gagcaaggca agaccgatcg ggccc 8925
Claims (3)
1.丙酮丁醇基因工程菌在EPS催化体系中的应用，其特征在于，所述的丙酮丁醇基因工程菌是利用二型内含子基因敲除技术在丙酮丁醇梭菌的Spo0A基因中插入内含子序列得到的；
所述的丙酮丁醇梭菌保藏编号为CGMCC No.5234；
所述的Spo0A基因为CA_C2071，其核苷酸序列如SEQ ID No.：1所示；
利用二型内含子基因敲除技术将SEQ ID No.：2所示的内含子序列插入Spo0A基因的第242bp和第243bp之间。
2.根据权利要求1所述的应用，其特征在于，所述二型内含子基因敲除技术的操作方法如下：
(1) 分析SEQ ID No：1所示的序列，得到内含子的核苷酸序列和基因的插入位点；
(2) 内含子序列构建到二型内含子基因敲除质粒中，得到重组质粒；
(3) 将步骤(2)得到的重组质粒转化丙酮丁醇梭菌，筛选得到Spo0A基因失活的菌株。
3.根据权利要求2所述的应用，其特征在于，所述的所述二型内含子基因敲除质粒为PMTL007C-E2质粒，该质粒的核苷酸序列如SEQ ID No：5所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610973505.5A CN106636168B (zh) | 2016-11-04 | 2016-11-04 | 一种调控丙酮丁醇梭菌胞外聚合物合成的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610973505.5A CN106636168B (zh) | 2016-11-04 | 2016-11-04 | 一种调控丙酮丁醇梭菌胞外聚合物合成的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106636168A CN106636168A (zh) | 2017-05-10 |
| CN106636168B true CN106636168B (zh) | 2020-06-05 |
Family
ID=58821821
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610973505.5A Active CN106636168B (zh) | 2016-11-04 | 2016-11-04 | 一种调控丙酮丁醇梭菌胞外聚合物合成的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106636168B (zh) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201716845D0 (en) | 2017-10-13 | 2017-11-29 | Green Biologics Ltd | Process |
| CN109371052B (zh) * | 2018-12-04 | 2022-02-22 | 安徽农业大学 | 提高高gc含量噬铜菌属细菌电转化效率的方法 |
| CN109884019B (zh) * | 2019-03-25 | 2022-05-27 | 大连大学 | 一种生物膜适用的三维曲面重构方法 |
| CN111088267B (zh) * | 2019-12-18 | 2022-05-03 | 南京工业大学 | 一种提高产溶剂梭菌液体发酵细胞密度的方法 |
| CN111088201B (zh) * | 2019-12-18 | 2021-08-31 | 南京工业大学 | 一株重组丙酮丁醇梭菌及其构建方法与应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101328486A (zh) * | 2007-06-18 | 2008-12-24 | 中国科学院上海生命科学研究院 | 一种可用于丙酮丁醇梭菌基因中断的重组质粒 |
| CN103320335A (zh) * | 2012-03-20 | 2013-09-25 | 南京工业大学 | 一种丙酮丁醇梭菌及其应用 |
| CN106008738A (zh) * | 2016-07-21 | 2016-10-12 | 南京工业大学 | 一种丙酮丁醇梭菌胞外多糖的提取分离方法 |
-
2016
- 2016-11-04 CN CN201610973505.5A patent/CN106636168B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101328486A (zh) * | 2007-06-18 | 2008-12-24 | 中国科学院上海生命科学研究院 | 一种可用于丙酮丁醇梭菌基因中断的重组质粒 |
| CN103320335A (zh) * | 2012-03-20 | 2013-09-25 | 南京工业大学 | 一种丙酮丁醇梭菌及其应用 |
| CN106008738A (zh) * | 2016-07-21 | 2016-10-12 | 南京工业大学 | 一种丙酮丁醇梭菌胞外多糖的提取分离方法 |
Non-Patent Citations (7)
| Title |
|---|
| A master regulator for biofilm formation by Bacillus subtilis;Daniel B. Kearns et al;《Molecular Microbiology》;20041202;第55卷(第3期);第739-749页 * |
| ACCESSION NO.HQ263410， Group II intron-based directed mutagenesis plasmid pMTL007C-E2, complete sequence;Heap,J.T. et al;《GenBank》;20101218;FEATURES,ORIGIN * |
| ACCESSION NO.NC_003030, Clostridium acetobutylicum ATCC 824 chromosome, complete genome;Nolling，J. et al;《GenBank》;20160803;FEATURES,ORIGIN * |
| Characterization of Clostridium difficile Biofilm Formation，a Role of Spo0A;Lis F.Dawson et al;《Plos One》;20121207;第7卷(第12期);第e50527（1）页摘要，第e50527（2）页 表1-2，左栏中间段，第e50527（7）页 右栏最后一段 * |
| Comparative transcriptomic analysis of Clostridium acetobutylicum biofilm and planktonic cells;Dong Liu et al;《Journal of Biotechnology》;20151124;第218卷;第1-12页 * |
| Targeted gene disruption by use of a group II intron （targetron） vector in Clostridium acetobutylicum;Lijun Shao et al;《Cell Research》;20071106;第17卷;第963–965页 * |
| The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis;Mélanie A. Hamon et al;《Molecular Microbiology》;20020103;第42卷(第5期);第1199-1209页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106636168A (zh) | 2017-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106636168B (zh) | 一种调控丙酮丁醇梭菌胞外聚合物合成的方法 | |
| Serra et al. | Cellulose as an architectural element in spatially structured Escherichia coli biofilms | |
| Lacroix et al. | Fermentation technologies for the production of probiotics with high viability and functionality | |
| KR101511639B1 (ko) | 재조합 미생물 및 이의 사용 방법 | |
| KR101640325B1 (ko) | 재조합 미생물에 의한 일산화탄소로부터 부탄올의 제조 | |
| JP2015180227A (ja) | トランスジェニック光合成微生物およびフォトバイオリアクター | |
| CN103635571A (zh) | 调控毒素和抗毒素基因用于生物防范 | |
| JP6518955B2 (ja) | 亜リン酸デヒドロゲナーゼ遺伝子をマーカーとした微生物の選択的培養方法 | |
| US20200109395A1 (en) | Genetic systems that defend against foreign dna and uses thereof | |
| CN106967659A (zh) | 一种表达谷氨酸脱羧酶的无抗生素抗性重组枯草芽孢杆菌的构建及发酵方法 | |
| CN110846268B (zh) | 一种食品级植物乳杆菌表达系统及其在酸奶制备中的应用 | |
| Ishøy et al. | An improved method for single cell isolation of prokaryotes from meso-, thermo-and hyperthermophilic environments using micromanipulation | |
| JP2021509582A (ja) | ブドウ球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ）属細菌の栄養要求株 | |
| CN114651060A (zh) | 修饰蓝细菌、修饰蓝细菌的制造方法以及蛋白的制造方法 | |
| Zhao et al. | Engineering peptidoglycan degradation related genes of Bacillus subtilis for better fermentation processes | |
| CN110195075B (zh) | 一种链球菌基因诱导表达系统及其应用 | |
| CN103108954B (zh) | 诱导型启动子的调控 | |
| CN113943690B (zh) | 魏氏柠檬酸杆菌tpiA基因敲除突变株及其应用 | |
| CN110951760B (zh) | 一种蛋白延时表达开关及其在葡萄糖二酸生产中应用 | |
| US20160251610A1 (en) | Microbes with controlled adhesive properties | |
| CN114891806A (zh) | 一种魏氏柠檬酸杆菌yqjH基因敲除突变株及其应用 | |
| Bogdan et al. | A one-step method for genetic transformation of non-piliated Neisseria meningitidis | |
| CN110257375A (zh) | 一种表达系统及其制备方法和应用 | |
| Condori et al. | Construction and phenotypic characterization of M68, an RruI quorum sensing knockout mutant of the photosynthetic alphaproteobacterium Rhodospirillum rubrum | |
| Paik et al. | Heterologous expression of α-amylase gene of Bifidobacterium adolescentis Int57 in Bacillus polyfermenticus SCD |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |