JP2014187892A - ブタノールの製造方法 - Google Patents
ブタノールの製造方法 Download PDFInfo
- Publication number
- JP2014187892A JP2014187892A JP2013063887A JP2013063887A JP2014187892A JP 2014187892 A JP2014187892 A JP 2014187892A JP 2013063887 A JP2013063887 A JP 2013063887A JP 2013063887 A JP2013063887 A JP 2013063887A JP 2014187892 A JP2014187892 A JP 2014187892A
- Authority
- JP
- Japan
- Prior art keywords
- butanol
- gene
- culture
- acetic acid
- culturing
- 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.)
- Granted
Links
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 title claims abstract description 144
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 89
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 84
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims abstract description 54
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 claims abstract description 12
- CRFNGMNYKDXRTN-CITAKDKDSA-N butyryl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CCC)O[C@H]1N1C2=NC=NC(N)=C2N=C1 CRFNGMNYKDXRTN-CITAKDKDSA-N 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 55
- 239000001257 hydrogen Substances 0.000 claims description 23
- 229910052739 hydrogen Inorganic materials 0.000 claims description 23
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 21
- 244000005700 microbiome Species 0.000 claims description 21
- 238000012258 culturing Methods 0.000 claims description 15
- 230000037361 pathway Effects 0.000 claims description 10
- 241000193403 Clostridium Species 0.000 claims description 9
- 238000011084 recovery Methods 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 7
- 238000000638 solvent extraction Methods 0.000 claims description 7
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 claims description 4
- 230000002950 deficient Effects 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 24
- 238000000855 fermentation Methods 0.000 abstract description 15
- 230000004151 fermentation Effects 0.000 abstract description 15
- 102000004190 Enzymes Human genes 0.000 abstract description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 6
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract 2
- 241000193464 Clostridium sp. Species 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 32
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 30
- 239000006227 byproduct Substances 0.000 description 29
- 108020004414 DNA Proteins 0.000 description 25
- 239000002609 medium Substances 0.000 description 19
- 239000013612 plasmid Substances 0.000 description 19
- 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 16
- 239000000243 solution Substances 0.000 description 15
- 206010059866 Drug resistance Diseases 0.000 description 12
- 101150108780 pta gene Proteins 0.000 description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 239000007789 gas Substances 0.000 description 9
- 101150048333 ptb gene Proteins 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000012217 deletion Methods 0.000 description 8
- 230000037430 deletion Effects 0.000 description 8
- 229960003276 erythromycin Drugs 0.000 description 8
- 230000006801 homologous recombination Effects 0.000 description 8
- 238000002744 homologous recombination Methods 0.000 description 8
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 230000008685 targeting Effects 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 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 description 5
- 108091029499 Group II intron Proteins 0.000 description 5
- 101150052992 PTBP1 gene Proteins 0.000 description 5
- 238000004821 distillation Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 150000007524 organic acids Chemical class 0.000 description 5
- 235000005985 organic acids Nutrition 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108700023175 Phosphate acetyltransferases Proteins 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 239000004310 lactic acid Substances 0.000 description 4
- 235000014655 lactic acid Nutrition 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000005373 pervaporation Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- ALSTYHKOOCGGFT-KTKRTIGZSA-N (9Z)-octadecen-1-ol Chemical compound CCCCCCCC\C=C/CCCCCCCCO ALSTYHKOOCGGFT-KTKRTIGZSA-N 0.000 description 3
- 108700024126 Butyrate kinases Proteins 0.000 description 3
- 241001508458 Clostridium saccharoperbutylacetonicum Species 0.000 description 3
- 239000007993 MOPS buffer Substances 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 150000001298 alcohols Chemical class 0.000 description 3
- BTANRVKWQNVYAZ-UHFFFAOYSA-N butan-2-ol Chemical compound CCC(C)O BTANRVKWQNVYAZ-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 150000002576 ketones Chemical class 0.000 description 3
- 229940055577 oleyl alcohol Drugs 0.000 description 3
- XMLQWXUVTXCDDL-UHFFFAOYSA-N oleyl alcohol Natural products CCCCCCC=CCCCCCCCCCCO XMLQWXUVTXCDDL-UHFFFAOYSA-N 0.000 description 3
- 238000010979 pH adjustment Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 238000001223 reverse osmosis Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 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 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- 108010092060 Acetate kinase Proteins 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000193401 Clostridium acetobutylicum Species 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010046276 FLP recombinase Proteins 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 108010020056 Hydrogenase Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 description 2
- 108700005078 Synthetic Genes Proteins 0.000 description 2
- 241000616244 Thetys Species 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- JSHMCUNOMIZJDJ-UHFFFAOYSA-N butanoyl dihydrogen phosphate Chemical compound CCCC(=O)OP(O)(O)=O JSHMCUNOMIZJDJ-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 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 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 101150036876 cre gene Proteins 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 150000002431 hydrogen Chemical class 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 229910001410 inorganic ion Inorganic materials 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000013605 shuttle vector Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- -1 sorbitol Chemical class 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 1
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 101000695175 Bacillus subtilis (strain 168) Probable phosphate butyryltransferase Proteins 0.000 description 1
- 101100385573 Clostridium acetobutylicum (strain ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787) ctfB gene Proteins 0.000 description 1
- 241000193454 Clostridium beijerinckii Species 0.000 description 1
- 241000193171 Clostridium butyricum Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 229920002472 Starch Polymers 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
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 108091022873 acetoacetate decarboxylase Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 101150042777 flp gene Proteins 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229910001437 manganese ion Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
【解決手段】本発明は、ブチリルCoAから酪酸が生成する経路に関与する酪酸生成酵素遺伝子の機能、およびアセチルCoAから酢酸が生成する経路に関与する酢酸生成酵素遺伝子の機能を欠損させたクロストリジウム属微生物の形質転換体を、炭素源を含む培地中で、還元力を上昇させて培養する工程を含む、ブタノールの製造方法に関する。
【選択図】図1
Description
(1)ブチリルCoAから酪酸が生成する経路に関与する酪酸生成酵素遺伝子の機能、およびアセチルCoAから酢酸が生成する経路に関与する酢酸生成酵素遺伝子の機能を欠損させたクロストリジウム属微生物の形質転換体を、炭素源を含む培地中で、pHが4.6を下回らないようにして培養する工程を含む、ブタノールの製造方法。
(2)培養工程において、還元力を上昇させて培養する、(1)記載の方法。
(3)培養工程において、培養槽内の水素分圧を上昇させて培養する、(2)記載の方法。
(4)培養工程において、培養槽内の水素分圧を0.06〜10atmに維持して培養する、(3)記載の方法。
(5)酪酸生成酵素遺伝子として、ptbおよび/またはbukの機能が欠損している、(1)〜(4)のいずれかに記載の方法。
(6)酢酸生成酵素遺伝子として、ptaおよび/またはackの機能が欠損している、(1)〜(5)のいずれかに記載の方法。
(7)培養液からブタノールを回収する工程を含む、(1)〜(6)のいずれかに記載の方法。
(8)ブタノール回収工程が、溶媒抽出を用いる工程を含む、(7)に記載の方法。
まず、Perutka et al., J. Mol. Biol. 13;336(2):421-39(2004)をもとにして、標的とする遺伝子の塩基配列を入力することにより、その遺伝子へのグループIIイントロンの挿入箇所とターゲッティング配列の改変方法が出力されるようなエクセルマクロのプログラミングを行った。次に、実際に遺伝子破壊を実施するpta遺伝子およびptb遺伝子の塩基配列をこれに入力し、ターゲッティング配列の改変箇所を出力させた。pta遺伝子の塩基配列を配列番号1に、ptb遺伝子の塩基配列を配列番号2に示す。
実施例1で作製した形質転換微生物(ΔptaΔptb株)を培養して、その性能を評価した。形質転換微生物のグリセロールストックを500μl分、TYA培地5mlに植菌し、試験管内で30℃にて培養を24時間行った。取得した前培養液を、新たなTYA、TYSまたはTYS−CaCO3培地5mlに50μl分植菌し、試験管内で30℃にて培養を行った。TYS培地の組成を以下に示す。なお、TYS−CaCO3培地とは、TYS培地にCaCO3を5g/L添加した培地のことを指す。
実施例1で作製した形質転換微生物(ΔptaΔptb株)を溶媒抽出培養し、評価した。形質転換微生物のグリセロールストックを500μl分、TYA培地5mlに植菌し、試験管内で30℃にて培養を24時間行った。取得した前培養液を、新たなTYS−CaCO3培地5mlに50μl分植菌し、さらに培養液層(水層)の上部に抽出溶媒としてオレイルアルコールを5ml重層し、試験管内で30℃にて培養を行った。抽出溶媒としてオレイルアルコールを重層することにより、ブタノールなどの生成物が溶媒層に移動し、生成物による生育阻害が防止されることが考えられたため、24時間ごとに50%グルコース溶液を一定量添加し、グルコースの消費ができなくなるまで培養を続けた。培養は96時間実施した。野生株についても上記と同様に培養評価を行った。
Claims (8)
- ブチリルCoAから酪酸が生成する経路に関与する酪酸生成酵素遺伝子の機能、およびアセチルCoAから酢酸が生成する経路に関与する酢酸生成酵素遺伝子の機能を欠損させたクロストリジウム属微生物の形質転換体を、炭素源を含む培地中でpHが4.6を下回らないようにして培養する工程を含む、ブタノールの製造方法。
- 培養工程において、還元力を上昇させて培養する、請求項1項記載の方法。
- 培養工程において、培養槽内の水素分圧を上昇させて培養する、請求項2記載の方法。
- 培養工程において、培養槽内の水素分圧を0.06〜10atmに維持して培養する、請求項3記載の方法。
- 酪酸生成酵素遺伝子として、ptbおよび/またはbukの機能が欠損している、請求項1〜4のいずれか1項記載の方法。
- 酢酸生成酵素遺伝子として、ptaおよび/またはackの機能が欠損している、請求項1〜5のいずれか1項記載の方法。
- 培養液からブタノールを回収する工程を含む、請求項1〜6のいずれか1項記載の方法。
- ブタノール回収工程が、溶媒抽出を用いる工程を含む、請求項7に記載の方法。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2013063887A JP6236209B2 (ja) | 2013-03-26 | 2013-03-26 | ブタノールの製造方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2013063887A JP6236209B2 (ja) | 2013-03-26 | 2013-03-26 | ブタノールの製造方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2014187892A true JP2014187892A (ja) | 2014-10-06 |
JP6236209B2 JP6236209B2 (ja) | 2017-11-22 |
Family
ID=51834883
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2013063887A Active JP6236209B2 (ja) | 2013-03-26 | 2013-03-26 | ブタノールの製造方法 |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP6236209B2 (ja) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010508017A (ja) * | 2006-10-31 | 2010-03-18 | メタボリック エクスプローラー | n−ブタノールを高収量で生物学的に製造する方法 |
EP2267141A1 (en) * | 2009-06-26 | 2010-12-29 | Metabolic Explorer | Process for the biological production of n-Butanol with high yield |
JP2011522543A (ja) * | 2008-06-04 | 2011-08-04 | ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー | 二相抽出発酵を用いてブタノールを生産するための方法 |
EP2481793A2 (en) * | 2009-09-22 | 2012-08-01 | Korea Advanced Institute of Science and Technology | Recombinant microorganism with increased butanol production ability, and preparation method of butanol using same |
-
2013
- 2013-03-26 JP JP2013063887A patent/JP6236209B2/ja active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010508017A (ja) * | 2006-10-31 | 2010-03-18 | メタボリック エクスプローラー | n−ブタノールを高収量で生物学的に製造する方法 |
JP2011522543A (ja) * | 2008-06-04 | 2011-08-04 | ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー | 二相抽出発酵を用いてブタノールを生産するための方法 |
EP2267141A1 (en) * | 2009-06-26 | 2010-12-29 | Metabolic Explorer | Process for the biological production of n-Butanol with high yield |
EP2481793A2 (en) * | 2009-09-22 | 2012-08-01 | Korea Advanced Institute of Science and Technology | Recombinant microorganism with increased butanol production ability, and preparation method of butanol using same |
Non-Patent Citations (2)
Title |
---|
APPL. MICROBIOL. BIOTECHNOL., 1985, VOL. 22, NO. 2, PP. 103-107, JPN6016043119, ISSN: 0003436279 * |
MBIO, 2012, VOLUME 3, ISSUE 5, E00314-12, JPN6016043118, ISSN: 0003436278 * |
Also Published As
Publication number | Publication date |
---|---|
JP6236209B2 (ja) | 2017-11-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhang et al. | Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production | |
Rogers et al. | Zymomonas mobilis for fuel ethanol and higher value products | |
RU2461627C2 (ru) | СПОСОБ БИОЛОГИЧЕСКОГО ПРОИЗВОДСТВА н-БУТАНОЛА | |
US9057071B2 (en) | Recombinant microorganisms and methods of use thereof | |
EP3800258A1 (en) | Novel promoter derived from organic acid-resistant yeast and method for expression of target gene by using same | |
JP6404575B2 (ja) | 遺伝子改変クロストリジウム・サッカロパーブチルアセトニカム | |
JP5350806B2 (ja) | 生物変換により芳香族分子を産生するためのシステム | |
JP2009529905A (ja) | 微生物によるエタノール製造の強化 | |
EP3164485A1 (en) | Microorganisms and methods for producing vanillin | |
CN105492613B (zh) | 用于使用代谢工程化丙酸杆菌产生正丙醇和丙酸的方法 | |
JP5813977B2 (ja) | Kluyveromyces属の変異体酵母及びこれを用いたエタノールの製造方法 | |
EP3102675B1 (en) | Improved microorganisms for succinic acid production | |
US9309542B2 (en) | Recombinant Caldicellulosiruptor bescii and methods of use | |
JP5649119B2 (ja) | シロ−イノシトール産生細胞および当該細胞を用いたシロ−イノシトール製造方法 | |
Kita et al. | Isolation of thermophilic acetogens and transformation of them with the pyrF and kanr genes | |
JP6236209B2 (ja) | ブタノールの製造方法 | |
KR101647143B1 (ko) | 클로스트리듐 속 미생물 유전자를 불활성화시키는 방법 | |
JP6012371B2 (ja) | 4−ヒドロキシ−2−ブタノンまたはブタノールの製造方法 | |
JP7126403B2 (ja) | 遺伝子改変クロストリジウム・サッカロパーブチルアセトニカム種微生物 | |
JP2008283917A (ja) | 乳酸の製造方法 | |
Bengelsdorf et al. | Host organisms: Clostridium acetobutylicum/Clostridium beijerinckii and related organisms | |
JP2014068555A (ja) | ブタノールの製造方法 | |
JP2014068554A (ja) | ブタノールの製造方法 | |
WO2016026954A1 (en) | Ars induced gene amplification | |
US20050026293A1 (en) | Plasmids from an extremely thermophilic microorganism and derived expression vectors |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20151204 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20161108 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20170110 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20170620 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20170920 |
|
A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20170927 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20171017 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20171030 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6236209 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |