WO2014063156A2 - Micro-organismes modifiés et leurs procédés d'utilisation pour produire du butadiène et un ou plusieurs parmi le 1,3-butanediol, le 1,4-butanediol et/ou le 1,3-propanediol - Google Patents
Micro-organismes modifiés et leurs procédés d'utilisation pour produire du butadiène et un ou plusieurs parmi le 1,3-butanediol, le 1,4-butanediol et/ou le 1,3-propanediol Download PDFInfo
- Publication number
- WO2014063156A2 WO2014063156A2 PCT/US2013/065957 US2013065957W WO2014063156A2 WO 2014063156 A2 WO2014063156 A2 WO 2014063156A2 US 2013065957 W US2013065957 W US 2013065957W WO 2014063156 A2 WO2014063156 A2 WO 2014063156A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- butanediol
- coa
- enzymes
- pathway
- conversion
- Prior art date
Links
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 title claims abstract description 412
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 title claims abstract description 242
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 title claims abstract description 234
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 title claims abstract description 115
- YPFDHNVEDLHUCE-UHFFFAOYSA-N 1,3-propanediol Substances OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 title claims abstract description 115
- 229920000166 polytrimethylene carbonate Polymers 0.000 title claims abstract description 115
- 244000005700 microbiome Species 0.000 title claims abstract description 90
- 238000000034 method Methods 0.000 title claims description 74
- 102000004190 Enzymes Human genes 0.000 claims abstract description 410
- 108090000790 Enzymes Proteins 0.000 claims abstract description 410
- 238000006243 chemical reaction Methods 0.000 claims abstract description 353
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 344
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 344
- 239000002157 polynucleotide Substances 0.000 claims abstract description 344
- 230000037361 pathway Effects 0.000 claims abstract description 326
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 92
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 90
- 238000004519 manufacturing process Methods 0.000 claims abstract description 75
- 235000019437 butane-1,3-diol Nutrition 0.000 claims description 114
- 229940035437 1,3-propanediol Drugs 0.000 claims description 112
- 239000000543 intermediate Substances 0.000 claims description 112
- 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 description 110
- KFWWCMJSYSSPSK-PAXLJYGASA-N crotonoyl-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=C/C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 KFWWCMJSYSSPSK-PAXLJYGASA-N 0.000 claims description 39
- 238000000855 fermentation Methods 0.000 claims description 37
- 230000004151 fermentation Effects 0.000 claims description 37
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims description 34
- POODSGUMUCVRTR-IEXPHMLFSA-N acryloyl-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=C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 POODSGUMUCVRTR-IEXPHMLFSA-N 0.000 claims description 31
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 30
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 26
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 26
- OJFDKHTZOUZBOS-CITAKDKDSA-N acetoacetyl-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)CC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 OJFDKHTZOUZBOS-CITAKDKDSA-N 0.000 claims description 26
- BERBFZCUSMQABM-IEXPHMLFSA-N 3-hydroxypropanoyl-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)CCO)O[C@H]1N1C2=NC=NC(N)=C2N=C1 BERBFZCUSMQABM-IEXPHMLFSA-N 0.000 claims description 25
- SXMOKYXNAPLNCW-GORZOVPNSA-N formyl-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)O[C@H]1N1C2=NC=NC(N)=C2N=C1 SXMOKYXNAPLNCW-GORZOVPNSA-N 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 17
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims description 15
- 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 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 241000206602 Eukaryota Species 0.000 claims description 8
- 241000233866 Fungi Species 0.000 claims description 7
- 241000588722 Escherichia Species 0.000 claims description 6
- 241000186660 Lactobacillus Species 0.000 claims description 6
- 229940039696 lactobacillus Drugs 0.000 claims description 6
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 5
- 241000193403 Clostridium Species 0.000 claims description 5
- 241000195493 Cryptophyta Species 0.000 claims description 5
- 241000235058 Komagataella pastoris Species 0.000 claims description 5
- 241000863392 Pelobacter Species 0.000 claims description 5
- 241000186429 Propionibacterium Species 0.000 claims description 5
- 241001656788 Propionispira Species 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 4
- 240000000111 Saccharum officinarum Species 0.000 claims description 4
- 235000007201 Saccharum officinarum Nutrition 0.000 claims description 4
- 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 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
- 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 claims description 2
- 229930091371 Fructose Natural products 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims 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 claims description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 claims description 2
- 239000012978 lignocellulosic material Substances 0.000 claims description 2
- 235000013379 molasses Nutrition 0.000 claims description 2
- 150000002772 monosaccharides Chemical class 0.000 claims description 2
- 229920001542 oligosaccharide Polymers 0.000 claims description 2
- 150000002482 oligosaccharides Chemical class 0.000 claims description 2
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 description 94
- 210000004027 cell Anatomy 0.000 description 90
- 102000004169 proteins and genes Human genes 0.000 description 46
- 108090000765 processed proteins & peptides Proteins 0.000 description 44
- 239000013598 vector Substances 0.000 description 40
- 150000007523 nucleic acids Chemical group 0.000 description 37
- 102000004196 processed proteins & peptides Human genes 0.000 description 36
- 229920001184 polypeptide Polymers 0.000 description 32
- VNOYUJKHFWYWIR-ITIYDSSPSA-N succinyl-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)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 VNOYUJKHFWYWIR-ITIYDSSPSA-N 0.000 description 30
- 230000014509 gene expression Effects 0.000 description 26
- KHPXUQMNIQBQEV-UHFFFAOYSA-L oxaloacetate(2-) Chemical compound [O-]C(=O)CC(=O)C([O-])=O KHPXUQMNIQBQEV-UHFFFAOYSA-L 0.000 description 26
- 229940049920 malate Drugs 0.000 description 25
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 25
- 239000000758 substrate Substances 0.000 description 23
- AINRQBNLOBQURT-UHFFFAOYSA-N 3-hydroxypent-4-enoic acid Chemical compound C=CC(O)CC(O)=O AINRQBNLOBQURT-UHFFFAOYSA-N 0.000 description 21
- BAMBWCGEVIAQBF-CITAKDKDSA-N 4-hydroxybutyryl-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)CCCO)O[C@H]1N1C2=NC=NC(N)=C2N=C1 BAMBWCGEVIAQBF-CITAKDKDSA-N 0.000 description 21
- 102000039446 nucleic acids Human genes 0.000 description 21
- 108020004707 nucleic acids Proteins 0.000 description 21
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 21
- 108090000992 Transferases Proteins 0.000 description 19
- 102000004357 Transferases Human genes 0.000 description 19
- -1 polybutylene terephthalate Polymers 0.000 description 19
- 150000001413 amino acids Chemical class 0.000 description 18
- 239000000047 product Substances 0.000 description 18
- KDOCIZRMPUBINQ-XMWLYHNJSA-N s-[2-[3-[[(2r)-4-[[[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyl] 3-hydroxypent-4-enethioate 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)CC(O)C=C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 KDOCIZRMPUBINQ-XMWLYHNJSA-N 0.000 description 18
- SJZRECIVHVDYJC-UHFFFAOYSA-M 4-hydroxybutyrate Chemical compound OCCCC([O-])=O SJZRECIVHVDYJC-UHFFFAOYSA-M 0.000 description 17
- 238000010367 cloning Methods 0.000 description 17
- 239000013604 expression vector Substances 0.000 description 17
- 230000004048 modification Effects 0.000 description 17
- 238000012986 modification Methods 0.000 description 17
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 17
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 17
- HSJKGGMUJITCBW-UHFFFAOYSA-N 3-hydroxybutanal Chemical compound CC(O)CC=O HSJKGGMUJITCBW-UHFFFAOYSA-N 0.000 description 16
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 16
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 16
- QHHKKMYHDBRONY-RMNRSTNRSA-N 3-hydroxybutanoyl-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)CC(O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 QHHKKMYHDBRONY-RMNRSTNRSA-N 0.000 description 15
- 239000007795 chemical reaction product Substances 0.000 description 14
- 238000006911 enzymatic reaction Methods 0.000 description 14
- 239000013612 plasmid Substances 0.000 description 14
- 230000008569 process Effects 0.000 description 14
- 238000001890 transfection Methods 0.000 description 14
- 230000002255 enzymatic effect Effects 0.000 description 13
- 230000010076 replication Effects 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 108091026890 Coding region Proteins 0.000 description 12
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 12
- VIWKEBOLLIEAIL-FBMOWMAESA-N lactoyl-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)O[C@H]1N1C2=NC=NC(N)=C2N=C1 VIWKEBOLLIEAIL-FBMOWMAESA-N 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 description 11
- UIUJIQZEACWQSV-UHFFFAOYSA-M 4-oxobutanoate Chemical compound [O-]C(=O)CCC=O UIUJIQZEACWQSV-UHFFFAOYSA-M 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 102000004316 Oxidoreductases Human genes 0.000 description 10
- 108090000854 Oxidoreductases Proteins 0.000 description 10
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 10
- 230000001965 increasing effect Effects 0.000 description 10
- UUFDQTFCJFWTDW-XMWLYHNJSA-N s-[2-[3-[[(2r)-4-[[[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyl] 3,5-dihydroxypentanethioate 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)CC(O)CCO)O[C@H]1N1C2=NC=NC(N)=C2N=C1 UUFDQTFCJFWTDW-XMWLYHNJSA-N 0.000 description 10
- 238000013518 transcription Methods 0.000 description 10
- 108090000489 Carboxy-Lyases Proteins 0.000 description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 9
- 230000012010 growth Effects 0.000 description 9
- 230000001939 inductive effect Effects 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- KGWOBFSKGHXRMY-ZMHDXICWSA-N s-[2-[3-[[(2r)-4-[[[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyl] 5-hydroxy-3-oxopentanethioate 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)CC(=O)CCO)O[C@H]1N1C2=NC=NC(N)=C2N=C1 KGWOBFSKGHXRMY-ZMHDXICWSA-N 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 230000035897 transcription Effects 0.000 description 9
- AKXKFZDCRYJKTF-UHFFFAOYSA-N 3-Hydroxypropionaldehyde Chemical compound OCCC=O AKXKFZDCRYJKTF-UHFFFAOYSA-N 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 8
- 101710088194 Dehydrogenase Proteins 0.000 description 8
- 108010036781 Fumarate Hydratase Proteins 0.000 description 8
- 102100036160 Fumarate hydratase, mitochondrial Human genes 0.000 description 8
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 8
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 8
- 108090000472 Phosphoenolpyruvate carboxykinase (ATP) Proteins 0.000 description 8
- 108010053763 Pyruvate Carboxylase Proteins 0.000 description 8
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 description 8
- 102000019259 Succinate Dehydrogenase Human genes 0.000 description 8
- 108010012901 Succinate Dehydrogenase Proteins 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 210000003292 kidney cell Anatomy 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 239000003623 enhancer Substances 0.000 description 7
- 230000004077 genetic alteration Effects 0.000 description 7
- 231100000118 genetic alteration Toxicity 0.000 description 7
- 210000004962 mammalian cell Anatomy 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 230000000813 microbial effect Effects 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
- 229920000642 polymer Polymers 0.000 description 7
- WCASXYBKJHWFMY-NSCUHMNNSA-N 2-Buten-1-ol Chemical compound C\C=C\CO WCASXYBKJHWFMY-NSCUHMNNSA-N 0.000 description 6
- 241000701022 Cytomegalovirus Species 0.000 description 6
- MLUCVPSAIODCQM-NSCUHMNNSA-N crotonaldehyde Chemical compound C\C=C\C=O MLUCVPSAIODCQM-NSCUHMNNSA-N 0.000 description 6
- MLUCVPSAIODCQM-UHFFFAOYSA-N crotonaldehyde Natural products CC=CC=O MLUCVPSAIODCQM-UHFFFAOYSA-N 0.000 description 6
- 230000009483 enzymatic pathway Effects 0.000 description 6
- 238000001638 lipofection Methods 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 108010037073 succinate semialdehyde reductase Proteins 0.000 description 6
- UIUJIQZEACWQSV-UHFFFAOYSA-N succinic semialdehyde Chemical compound OC(=O)CCC=O UIUJIQZEACWQSV-UHFFFAOYSA-N 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- PIAOXUVIBAKVSP-UHFFFAOYSA-N γ-hydroxybutyraldehyde Chemical compound OCCCC=O PIAOXUVIBAKVSP-UHFFFAOYSA-N 0.000 description 6
- 241000700605 Viruses Species 0.000 description 5
- 239000000356 contaminant Substances 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 230000002503 metabolic effect Effects 0.000 description 5
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 229920002857 polybutadiene Polymers 0.000 description 5
- JTSWAWHLWFZNMF-XMWLYHNJSA-N s-[2-[3-[[(2r)-4-[[[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyl] 3-hydroxy-5-oxopentanethioate 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)CC(O)CC=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 JTSWAWHLWFZNMF-XMWLYHNJSA-N 0.000 description 5
- MGKKVVWYGTWMNB-TVCSPYKZSA-N s-[2-[3-[[4-[[[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyl] 3-oxopent-4-enethioate Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCSC(=O)CC(=O)C=C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MGKKVVWYGTWMNB-TVCSPYKZSA-N 0.000 description 5
- 241000701161 unidentified adenovirus Species 0.000 description 5
- 108010011958 1,3-propanediol dehydrogenase Proteins 0.000 description 4
- 241000282693 Cercopithecidae Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 4
- 108010025885 Glycerol dehydratase Proteins 0.000 description 4
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 4
- 239000005062 Polybutadiene Substances 0.000 description 4
- 239000002174 Styrene-butadiene Substances 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 244000000188 Vaccinium ovalifolium Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 150000001336 alkenes Chemical class 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 229920001577 copolymer Polymers 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 108010032776 glycerol-1-phosphatase Proteins 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 210000005229 liver cell Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 229920001707 polybutylene terephthalate Polymers 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 230000003362 replicative effect Effects 0.000 description 4
- 229920003048 styrene butadiene rubber Polymers 0.000 description 4
- 229920001791 ((R)-3-Hydroxybutanoyl)(n-2) Polymers 0.000 description 3
- 241000699800 Cricetinae Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 3
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 description 3
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 108090000604 Hydrolases Proteins 0.000 description 3
- 102000004157 Hydrolases Human genes 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 102000002932 Thiolase Human genes 0.000 description 3
- 108060008225 Thiolase Proteins 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 239000013611 chromosomal DNA Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 229950006238 nadide Drugs 0.000 description 3
- 229920001084 poly(chloroprene) Polymers 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- 238000003151 transfection method Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 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 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
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 2
- 101710084782 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA-Delta-isomerase Proteins 0.000 description 2
- 108010037188 5-hydroxyvaleryl-CoA dehydratase Proteins 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 241000255789 Bombyx mori Species 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- 241000282552 Chlorocebus aethiops Species 0.000 description 2
- 101710168556 Chromodomain Y-like protein Proteins 0.000 description 2
- 102100031668 Chromodomain Y-like protein Human genes 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 229920001634 Copolyester Polymers 0.000 description 2
- 101710188748 Crotonyl-CoA hydratase Proteins 0.000 description 2
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 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 2
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 2
- 108090001042 Hydro-Lyases Proteins 0.000 description 2
- 102000004867 Hydro-Lyases Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 244000285963 Kluyveromyces fragilis Species 0.000 description 2
- 241001138401 Kluyveromyces lactis Species 0.000 description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 2
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 101150003085 Pdcl gene Proteins 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 241000700157 Rattus norvegicus Species 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 241000714474 Rous sarcoma virus Species 0.000 description 2
- 241000607720 Serratia Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002334 Spandex Polymers 0.000 description 2
- 241000256251 Spodoptera frugiperda Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 239000004676 acrylonitrile butadiene styrene Substances 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 238000010564 aerobic fermentation Methods 0.000 description 2
- 229920003232 aliphatic polyester Polymers 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 229950006334 apramycin Drugs 0.000 description 2
- XZNUGFQTQHRASN-XQENGBIVSA-N apramycin Chemical compound O([C@H]1O[C@@H]2[C@H](O)[C@@H]([C@H](O[C@H]2C[C@H]1N)O[C@@H]1[C@@H]([C@@H](O)[C@H](N)[C@@H](CO)O1)O)NC)[C@@H]1[C@@H](N)C[C@@H](N)[C@H](O)[C@H]1O XZNUGFQTQHRASN-XQENGBIVSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000011942 biocatalyst Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000006664 bond formation reaction Methods 0.000 description 2
- NTXGQCSETZTARF-UHFFFAOYSA-N buta-1,3-diene;prop-2-enenitrile Chemical compound C=CC=C.C=CC#N NTXGQCSETZTARF-UHFFFAOYSA-N 0.000 description 2
- MTAZNLWOLGHBHU-UHFFFAOYSA-N butadiene-styrene rubber Chemical compound C=CC=C.C=CC1=CC=CC=C1 MTAZNLWOLGHBHU-UHFFFAOYSA-N 0.000 description 2
- 108010036467 butanediol dehydrogenase Proteins 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 208000019065 cervical carcinoma Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000005547 deoxyribonucleotide Substances 0.000 description 2
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 229920001971 elastomer Polymers 0.000 description 2
- 229920006351 engineering plastic Polymers 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940023064 escherichia coli Drugs 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000004880 explosion Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 230000005714 functional activity Effects 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 108010067653 lactate dehydratase Proteins 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 125000005647 linker group Chemical group 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000005265 lung cell Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- 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 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 229920002215 polytrimethylene terephthalate Polymers 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- FGDZQCVHDSGLHJ-UHFFFAOYSA-M rubidium chloride Chemical compound [Cl-].[Rb+] FGDZQCVHDSGLHJ-UHFFFAOYSA-M 0.000 description 2
- MGKKVVWYGTWMNB-ZMHDXICWSA-N s-[2-[3-[[(2r)-4-[[[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyl] 3-oxopent-4-enethioate 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)CC(=O)C=C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MGKKVVWYGTWMNB-ZMHDXICWSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 210000000717 sertoli cell Anatomy 0.000 description 2
- 239000004759 spandex Substances 0.000 description 2
- 239000011115 styrene butadiene Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000004114 suspension culture Methods 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 241000186426 Acidipropionibacterium acidipropionici Species 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000256118 Aedes aegypti Species 0.000 description 1
- 241000256173 Aedes albopictus Species 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 241001136167 Anaerotignum propionicum Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000351920 Aspergillus nidulans Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241001367049 Autographa Species 0.000 description 1
- 241000713842 Avian sarcoma virus Species 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 241000409811 Bombyx mori nucleopolyhedrovirus Species 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000193401 Clostridium acetobutylicum Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108091006149 Electron carriers Proteins 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 230000004619 Entner-Doudoroff pathway Effects 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 241001522878 Escherichia coli B Species 0.000 description 1
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000219146 Gossypium Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 241000701109 Human adenovirus 2 Species 0.000 description 1
- 241001135569 Human adenovirus 5 Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241000481961 Lachancea thermotolerans Species 0.000 description 1
- 241000235651 Lachancea waltii Species 0.000 description 1
- 241000209499 Lemna Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930189782 Methylenomycin Natural products 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 101000969137 Mus musculus Metallothionein-1 Proteins 0.000 description 1
- 241000713883 Myeloproliferative sarcoma virus Species 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000221960 Neurospora Species 0.000 description 1
- 241000221961 Neurospora crassa Species 0.000 description 1
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 229920000459 Nitrile rubber Polymers 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- COPKIUCAOUAEPL-ZMHDXICWSA-N 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=C=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 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=C=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 COPKIUCAOUAEPL-ZMHDXICWSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 241001148572 Pelobacter propionicus Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241001505332 Polyomavirus sp. Species 0.000 description 1
- 241000186428 Propionibacterium freudenreichii Species 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 241000311088 Schwanniomyces Species 0.000 description 1
- 241001123650 Schwanniomyces occidentalis Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 241000255588 Tephritidae Species 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- NSFFHOGKXHRQEW-UHFFFAOYSA-N Thiostrepton B Natural products N1C(=O)C(C)NC(=O)C(=C)NC(=O)C(C)NC(=O)C(C(C)CC)NC(C(C2=N3)O)C=CC2=C(C(C)O)C=C3C(=O)OC(C)C(C=2SC=C(N=2)C2N=3)NC(=O)C(N=4)=CSC=4C(C(C)(O)C(C)O)NC(=O)C(N=4)CSC=4C(=CC)NC(=O)C(C(C)O)NC(=O)C(N=4)=CSC=4C21CCC=3C1=NC(C(=O)NC(=C)C(=O)NC(=C)C(N)=O)=CS1 NSFFHOGKXHRQEW-UHFFFAOYSA-N 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 241001149964 Tolypocladium Species 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 208000010094 Visna Diseases 0.000 description 1
- 241000235013 Yarrowia Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- WNLRTRBMVRJNCN-UHFFFAOYSA-L adipate(2-) Chemical compound [O-]C(=O)CCCCC([O-])=O WNLRTRBMVRJNCN-UHFFFAOYSA-L 0.000 description 1
- BTGRAWJCKBQKAO-UHFFFAOYSA-N adiponitrile Chemical compound N#CCCCCC#N BTGRAWJCKBQKAO-UHFFFAOYSA-N 0.000 description 1
- 125000003342 alkenyl group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940059260 amidate Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000002528 anti-freeze Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000010310 bacterial transformation Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000008236 biological pathway Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 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
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- LNDKRVOOYMEYTC-UHFFFAOYSA-N buta-1,3-dien-1-ol Chemical compound OC=CC=C LNDKRVOOYMEYTC-UHFFFAOYSA-N 0.000 description 1
- 229920005549 butyl rubber Polymers 0.000 description 1
- 229960005069 calcium Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229960002713 calcium chloride Drugs 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 125000006355 carbonyl methylene group Chemical group [H]C([H])([*:2])C([*:1])=O 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 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 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 230000035071 co-translational protein modification Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 125000000392 cycloalkenyl group Chemical group 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- 108020001096 dihydrofolate reductase Proteins 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000806 elastomer Substances 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- NPUKDXXFDDZOKR-LLVKDONJSA-N etomidate Chemical compound CCOC(=O)C1=CN=CN1[C@H](C)C1=CC=CC=C1 NPUKDXXFDDZOKR-LLVKDONJSA-N 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 108010008221 formate C-acetyltransferase Proteins 0.000 description 1
- MUQBUKGCVPYANP-UHFFFAOYSA-N formonitrile Chemical compound N#C.N#C MUQBUKGCVPYANP-UHFFFAOYSA-N 0.000 description 1
- 108010028610 formyl-coenzyme A transferase Proteins 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 108060003196 globin Proteins 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000029226 lipidation Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000006680 metabolic alteration Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000006241 metabolic reaction Methods 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000017066 negative regulation of growth Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 230000007030 peptide scission Effects 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920002961 polybutylene succinate Polymers 0.000 description 1
- 239000004631 polybutylene succinate Substances 0.000 description 1
- 229920003225 polyurethane elastomer Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 229930010796 primary metabolite Natural products 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000005060 rubber Substances 0.000 description 1
- 229940102127 rubidium chloride Drugs 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229920003051 synthetic elastomer Polymers 0.000 description 1
- 239000005061 synthetic rubber Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229930188070 thiostrepton Natural products 0.000 description 1
- NSFFHOGKXHRQEW-AIHSUZKVSA-N thiostrepton Chemical compound C([C@]12C=3SC=C(N=3)C(=O)N[C@H](C(=O)NC(/C=3SC[C@@H](N=3)C(=O)N[C@H](C=3SC=C(N=3)C(=O)N[C@H](C=3SC=C(N=3)[C@H]1N=1)[C@@H](C)OC(=O)C3=CC(=C4C=C[C@H]([C@@H](C4=N3)O)N[C@H](C(N[C@@H](C)C(=O)NC(=C)C(=O)N[C@@H](C)C(=O)N2)=O)[C@@H](C)CC)[C@H](C)O)[C@](C)(O)[C@@H](C)O)=C\C)[C@@H](C)O)CC=1C1=NC(C(=O)NC(=C)C(=O)NC(=C)C(N)=O)=CS1 NSFFHOGKXHRQEW-AIHSUZKVSA-N 0.000 description 1
- 229940063214 thiostrepton Drugs 0.000 description 1
- NSFFHOGKXHRQEW-OFMUQYBVSA-N thiostrepton A Natural products CC[C@H](C)[C@@H]1N[C@@H]2C=Cc3c(cc(nc3[C@H]2O)C(=O)O[C@H](C)[C@@H]4NC(=O)c5csc(n5)[C@@H](NC(=O)[C@H]6CSC(=N6)C(=CC)NC(=O)[C@@H](NC(=O)c7csc(n7)[C@]8(CCC(=N[C@@H]8c9csc4n9)c%10nc(cs%10)C(=O)NC(=C)C(=O)NC(=C)C(=O)N)NC(=O)[C@H](C)NC(=O)C(=C)NC(=O)[C@H](C)NC1=O)[C@@H](C)O)[C@](C)(O)[C@@H](C)O)[C@H](C)O NSFFHOGKXHRQEW-OFMUQYBVSA-N 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- 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
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- 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
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
Definitions
- Butadiene, 1 ,3-butanediol, 1 ,4- butanediol, and/or 1 ,3-propanediol are typically manufactured (along with other 4-carbon or 6-carbon molecules) in processes that involve harsh conditions and high temperatures (> 850°C). Other methods for their production involve toxic and/or expensive catalysts, highly flammable and/or gaseous carbon sources, and high temperatures.
- butadiene, 1 ,3-butanediol, 1 ,4- butanediol, and/or 1 ,3- propanediol in demand globally and are used in a variety of commercial applications.
- butadiene including butadiene formed from the dehydration of 1 ,3-butanediol, can be polymerized to form polybutadiene, or reacted with hydrogen cyanide (prussic acid) in the presence of a nickel catalyst to form adiponitrile, a precursor to nylon.
- butadiene is polymerized with other olefins to form copolymers such as acrylonitrile-butadiene-styrene (ABS), acrylonitrile -butadiene (ABR), or styrene-butadiene (SBR) copolymers.
- ABS acrylonitrile-butadiene-styrene
- ABR acrylonitrile -butadiene
- SBR styrene-butadiene copolymers.
- 1 ,4-butanediol is commonly used in the production of tetrahydrofuran, which is an intermediate of spandex and other performance polymers, and polybutylene terephthalate (PBT) resins for engineering plastics.
- PBT polybutylene terephthalate
- 1 ,3-propanediol is commonly used as a building block in the production of polymers such as polytrimethylene terephthalate and can be formulated into a variety of industrial products including, composites, adhesives, laminates, coatings, moldings, aliphatic polyesters, and copolyesters.
- the present disclosure generally relates to microorganisms (e.g., non-naturally occurring microorganisms) that comprise one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a carbon source (e.g., a fermentable carbon source) to i) butadiene; and one or more of ii) 1 ,3-butanediol, 1 ,4-butanediol, and/or 1 ,3-propanediol and the use of such microorganisms for the production of i) butadiene; and one or more of ii) 1,3- butanediol, 1 ,4-butanediol, and/or 1,3-propanediol.
- a carbon source e.g., a fermentable carbon source
- the present disclosure also provides a method of co-producing butadiene and one or more of 1,3-butanediol, 1 ,4-butanediol, and/or 1,3-propanediol from a fermentable carbon source, the method comprising: providing a fermentable carbon source; expressing one or more polynucleotides in a microorganism that encode one or more enzymes in a pathway that catalyze a conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more of 1,3-butanediol, 1 ,4-butanediol, and/or 1,3- propanediol, expressing one or more polynucleotides in a microorganism that encode one or more enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and one or more of 1,3-butanediol, 1 ,
- the enzymes that catalyze the conversion of the one or more intermediates to butadiene and one or more of 1,3-butanediol, 1 ,4-butanediol, and/or 1,3-propanediolare set forth in any one of Figures 1-12.
- 1,3-butanediol and butadiene are produced, 1,4- butanediol and butadiene are produced, or 1,3-propanediol and butadiene are produced.
- the microorganism is a bacteria selected from the genera consisting of: Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, and Lactobacillus.
- the microorganism is a eukaryote selected from the group consisting of yeast, filamentous fungi, protozoa, and algae.
- the yeast is Saccharomyces cerevisiae or Pichia pastoris.
- the fermentable carbon source comprises sugarcane juice, sugarcane molasses, hydrolyzed starch, hydrolyzed lignocellulosic materials, glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, or glycerol in any form or mixture thereof.
- the fermentable carbon source is a monosaccharide, oligosaccharide, or polysaccharide.
- the method further comprises recovering the produced butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3 -propanediol from the fermentation media.
- the microorganism has been genetically modified to express the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3- propanediol
- the present disclosure also provides a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3- propanediol, wherein the one or more intermediates in the pathway for the production of butadiene are selected from the group consisting of: crotonyl-CoA, acryloyl-CoA and acetyl- CoA, 3-hydroxypropionyl-CoA and
- the enzymes that catalyze a conversion of the fermentable carbon source to one or more intermediates in the pathway for the production of butadiene and one or more of 1,3- butanediol, 1,4- butanediol, and/or 1,3-propanediol are set forth in any one of Figures 1-12.
- the enzymes that catalyze a conversion of the one or more intermediates to butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1 ,3-propanediol are set forth in any one of Figures 1-12.
- the microorganism is a bacteria selected from the genera consisting of: Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, and Lactobacillus.
- the microorganism is a eukaryote selected from the group consisting of yeast, filamentous fungi, protozoa, and algae.
- the yeast is Saccharomyces cerevisiae or Pichia pastoris.
- the microorganism has been genetically modified to express the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol, and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3- propanediol.
- Figure 1 depicts an exemplary pathway for the production of 1,3-butanediol via an oxalacetate intermediate and butadiene via a crotonyl-CoA intermediate.
- Figure 2 depicts an exemplary pathway for the production of 1,3- butanediol via an oxalacetate intermediate and butadiene via acryloyl-CoA and acetyl-CoA and acetyl-CoA intermediates.
- Figure 3 depicts an exemplary pathway for the production of 1 ,3- butanediol via an oxalacetate intermediate and butadiene via 3-hydroxypropionyl-CoA and acetyl-CoA intermediates.
- Figure 4 depicts an exemplary pathway for the production of 1 ,3-butanediol via an oxalacetate intermediate and butadiene via formyl-CoA and acetoacetyl-CoA intermediates.
- Figure 5 depicts an exemplary pathway for the production of 1 ,4-butanediol via an oxalacetate intermediate and butadiene via a crotonyl-CoA intermediate.
- Figure 6 depicts an exemplary pathway for the production of 1 ,4-butanediol via an oxalacetate intermediate and butadiene via acryloyl-CoA and acetyl-CoA intermediates.
- Figure 7 depicts an exemplary pathway for the production of 1 ,4-butanediol via an oxalacetate intermediate and butadiene via 3-hydroxypropionyl-CoA and acetyl-CoA intermediates.
- Figure 8 depicts an exemplary pathway for the production of 1 ,4-butanediol via an oxalacetate intermediate and butadiene via formyl-CoA and acetoacetyl-CoA intermediates.
- Figure 9 depicts an exemplary pathway for the production of 1 ,3-propanediol via a glycerol-3 -phosphate intermediate and butadiene via a crotonyl-CoA intermediate.
- Figure 10 depicts an exemplary pathway for the production of 1 ,3-propanediol via a glycerol-3 -phosphate intermediate and butadiene via acryloyl-CoA and acetyl-CoA intermediates.
- Figure 1 1 depicts an exemplary pathway for the production of 1 ,3-propanediol via a glycerol-3 -phosphate intermediate and butadiene via 3-hydroxypropionyl-CoA and acetyl- CoA intermediates.
- Figure 12 depicts an exemplary pathway for the production of 1 ,3-propanediol via a glycerol-3-phosphate intermediate and butadiene via formyl-CoA and acetoacetyl-CoA intermediates.
- the present disclosure generally relates to microorganisms (e.g., non-naturally occurring microorganisms) that comprise one or more genetically modified pathways and uses of such microorganisms for the conversion of a fermentable carbon source to butadiene and one or more of 1 ,3-butanediol, 1 ,4- butanediol, and/or 1 ,3-propanediol (see, Figures 1-12).
- microorganisms e.g., non-naturally occurring microorganisms
- microorganisms e.g., non-naturally occurring microorganisms
- uses of such microorganisms for the conversion of a fermentable carbon source to butadiene and one or more of 1 ,3-butanediol, 1 ,4- butanediol, and/or 1 ,3-propanediol (see, Figures 1-12).
- Such microorganisms may comprise one or more polynucleotides coding for enzymes that catalyze a conversion of a fermentable carbon source to butadiene and one or more of 1 ,3-butanediol, 1 ,4- butanediol, and/or 1,3-propanediol.
- This disclosure provides, in part, the discovery of novel enzymatic pathways including, for example, novel combinations of enzymatic pathways, for the production of butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol from a carbon source (e.g., a fermentable carbon source).
- a carbon source e.g., a fermentable carbon source.
- butadiene and 1,3- butanediol are produced; butadiene and 1 ,4-butanediol are produced; or butadiene and 1,3-
- the methods provided herein provide end-results similar to those of sterilization without the high capital expenditure and continuing higher management costs that are typically required to establish and maintain sterility throughout a production process.
- most industrial-scale butadiene and 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol production processes are operated in the presence of measurable numbers of bacterial contaminants due to the aerobic nature of their processes.
- the enzymatic pathways disclosed herein are advantageous over prior known enzymatic pathways for the production of butadiene and one or more of 1,3-butanediol, 1 ,4- butanediol, and/or 1,3-propanediol in that the enzymatic pathways disclosed herein are anaerobic. While it is possible to use aerobic processes to produce butadiene and one or more of 1,3- butanediol, 1,4- butanediol, and/or 1,3-propanediol, anaerobic processes are preferred due to the risk incurred when olefins (which are by nature are explosive) are mixed with oxygen during the fermentation process.
- olefins which are by nature are explosive
- the supplementation of oxygen and nitrogen in a fermenter requires an additional investment for aerobic process and another additional investment for the purification from the nitrogen from the butadiene and one or more of 1,3-butanediol, 1 ,4- butanediol, and/or 1,3-propanediol.
- the presence of oxygen can also catalyze the polymerization of butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediolnd can promote the growth of aerobic contaminants in the fermentor broth.
- aerobic fermentation processes for the production of butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol present several drawbacks at industrial scale (where it is technically challenging to maintain aseptic conditions) such as the fact that: (i) greater biomass is obtained reducing overall yields on carbon for the desired products; (ii) the presence and oxygen favors the growth of contaminants (Weusthuis et ah, 2011, Trends in Biotechnology, 2011, Vol. 29, No.
- the present disclosure provides methods of producing butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3- propanediol from a fermentable carbon source, comprising: providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol, and one or more polynucleotides coding for enzymes in a pathway that catalyze a
- expression of the one or more polynucleotides coding for the enzymes in the pathway that catalyzes a conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the one or more intermediates to butadiene and one or more of 1,3- butanediol, 1,4- butanediol, and/or 1,3-propanediol in the microorganism to produce butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol may be performed prior to or after contacting the fermentable carbon source with a microorganism comprising one or
- Any molecule disclosed herein including, enzyme, substrate, or intermediate may be present in any of its enantiomeric forms (e.g., (S)-(+) or (R)-(-) forms).
- lactate includes R-lactate, S-lactate, or both R-lactate and S-lactate.
- biological activity when referring to a protein, polypeptide or peptide, may mean that the protein, polypeptide or peptide exhibits a functionality or property that is useful as relating to some biological process, pathway or reaction.
- Biological or functional activity can refer to, for example, an ability to interact or associate with (e.g., bind to) another polypeptide or molecule, or it can refer to an ability to catalyze or regulate the interaction of other proteins or molecules (e.g., enzymatic reactions).
- butadiene is intended to mean 1 ,3-butadiene with a general formula CH 2 CHCHCH 2 (CAS number- 106-99-0).
- 1 ,3-butanediol is intended to mean butane-l ,3-diol with a general formula CH 3 CHOH CH 2 CH 2 OH (CAS number- 107-88-0).
- 1 ,4-butanediol is intended to mean butane- 1 ,4-diol with a general formula HOCH 2 CH 2 CH 2 CH 2 OH (CAS number- 1 10-63-4).
- 1 ,3-propanediol is intended to mean propane-l ,3-diol with a general formula OHCH 2 CH 2 CH 2 OH (CAS number- 504-63-2).
- the term “culturing” may refer to growing a population of cells, e.g., microbial cells, under suitable conditions for growth, in a liquid or on solid medium.
- the term "derived from” may encompass the terms originated from, obtained from, obtainable from, isolated from, and created from, and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to the another specified material.
- exogenous polynucleotide refers to any deoxyribonucleic acid that originates outside of the microorganism.
- an expression vector may refer to a DNA construct containing a polynucleotide or nucleic acid sequence encoding a polypeptide or protein, such as a DNA coding sequence (e.g. gene sequence) that is operably linked to one or more suitable control sequence(s) capable of affecting expression of the coding sequence in a host.
- control sequences include a promoter to affect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mR A ribosome binding sites, and sequences which control termination of transcription and translation.
- the vector may be a plasmid, cosmid, phage particle, bacterial artificial chromosome, or simply a potential genomic insert.
- the vector may replicate and function independently of the host genome (e.g. , independent vector or plasmid), or may, in some instances, integrate into the genome itself (e.g. , integrated vector).
- the plasmid is the most commonly used form of expression vector. However, the disclosure is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.
- the term "expression” may refer to the process by which a polypeptide is produced based on a nucleic acid sequence encoding the polypeptides (e.g. , a gene). The process includes both transcription and translation.
- the term "gene” may refer to a DNA segment that is involved in producing a polypeptide or protein (e.g., fusion protein) and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).
- heterologous with reference to a nucleic acid, polynucleotide, protein or peptide, may refer to a nucleic acid, polynucleotide, protein or peptide that does not naturally occur in a specified cell, e.g. , a host cell. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes.
- homologous with reference to a nucleic acid, polynucleotide, protein or peptide, refers to a nucleic acid, polynucleotide, protein or peptide that occurs naturally in the cell.
- a "host cell” may refer to a cell or cell line, including a cell such as a microorganism which a recombinant expression vector may be transfected for expression of a polypeptide or protein (e.g. , fusion protein).
- Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
- a host cell may include cells transfected or transformed in vivo with an expression vector.
- the term "introduced,” in the context of inserting a nucleic acid sequence or a polynucleotide sequence into a cell, may include transfection, transformation, or transduction and refers to the incorporation of a nucleic acid sequence or polynucleotide sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence or polynucleotide sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.
- the genome of the cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
- non-naturally occurring when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
- Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species.
- Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon.
- a non-naturally occurring microbial organisms of the disclosure can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration.
- stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely.
- Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway.
- E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species.
- Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.
- operably linked may refer to a juxtaposition or arrangement of specified elements that allows them to perform in concert to bring about an effect.
- a promoter may be operably linked to a coding sequence if it controls the transcription of the coding sequence.
- a promoter may refer to a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene.
- a promoter may be an inducible promoter or a constitutive promoter.
- An inducible promoter is a promoter that is active under environmental or developmental regulatory conditions.
- a polynucleotide or “nucleic acid sequence” may refer to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs.
- Such polynucleotides or nucleic acid sequences may encode amino acids (e.g., polypeptides or proteins such as fusion proteins).
- polynucleotides which encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2'-0-Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin.
- polynucleotide also includes peptide nucleic acids (PNA).
- Polynucleotides may be naturally occurring or non-naturally occurring.
- the terms polynucleotide, nucleic acid, and oligonucleotide are used herein interchangeably.
- Polynucleotides may contain R A, DNA, or both, and/or modified forms and/or analogs thereof.
- a sequence of nucleotides may be interrupted by non-nucleotide components.
- One or more phosphodiester linkages may be replaced by alternative linking groups.
- linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S (thioate), P(S)S (dithioate), (0)NPv 2 (amidate), P(0)R, P(0)OR', COCH 2 (formacetal), in which each R or R* is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (- 0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions.
- a "protein” or “polypeptide” may refer to a composition comprised of amino acids and recognized as a protein by those of skill in the art.
- the conventional one-letter or three-letter code for amino acid residues is used herein.
- the terms protein and polypeptide are used interchangeably herein to refer to polymers of amino acids of any length, including those comprising linked (e.g., fused) peptides/polypeptides (e.g., fusion proteins).
- the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
- the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
- related proteins, polypeptides or peptides may encompass variant proteins, polypeptides or peptides.
- Variant proteins, polypeptides or peptides differ from a parent protein, polypeptide or peptide and/or from one another by a small number of amino acid residues. In some embodiments, the number of different amino acid residues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, variants differ by about 1 to about 10 amino acids. Alternatively or additionally, variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g.
- variant proteins or nucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or even 99.5% amino acid sequence identity with a reference sequence.
- the term “recovered,” “isolated,” “purified,” and “separated” may refer to a material (e.g., a protein, peptide, nucleic acid, polynucleotide or cell) that is removed from at least one component with which it is naturally associated.
- a material e.g., a protein, peptide, nucleic acid, polynucleotide or cell
- these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.
- the term “recombinant” may refer to nucleic acid sequences or polynucleotides, polypeptides or proteins, and cells based thereon, that have been manipulated by man such that they are not the same as nucleic acids, polypeptides, and cells as found in nature.
- Recombinant may also refer to genetic material (e.g., nucleic acid sequences or polynucleotides, the polypeptides or proteins they encode, and vectors and cells comprising such nucleic acid sequences or polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another coding sequence or gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at decreased or elevated levels, expressing a gene conditionally or constitutively in manners different from its natural expression profile, and the like.
- genetic material e.g., nucleic acid sequences or polynucleotides, the polypeptides or proteins they encode, and vectors and cells comprising such nucleic acid sequences or polynucleotides
- selectable marker may refer to a gene capable of expression in a host cell that allows for ease of selection of those hosts containing an introduced nucleic acid sequence, polynucleotide or vector.
- selectable markers include but are not limited to antimicrobial substances (e.g. , hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.
- nucleic acid, polynucleotide, protein or polypeptide comprises a sequence that has at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference (e.g., wild-type) nucleic acid, polynucleotide, protein or polypeptide.
- a reference e.g., wild-type
- Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters.
- BLAST Altshul et al. (1990) J. Mol. Biol. 215:403-410; Henikoff et al. (1989) Proc. Natl. Acad. Sci. 89: 10915; Karin et al. (1993) Proc. Natl. Acad. Sci. 90:5873; and Higgins et al. (1988) Gene 73:237).
- Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Person et al. (1988) Proc. Natl. Acad. Sci.
- substantially identical polypeptides differ only by one or more conservative amino acid substitutions.
- substantially identical polypeptides are immunologically cross-reactive.
- substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).
- transfection may refer to the insertion of an exogenous nucleic acid or polynucleotide into a host cell.
- the exogenous nucleic acid or polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.
- transfecting or transfection is intended to encompass all conventional techniques for introducing nucleic acid or polynucleotide into host cells. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, and microinjection.
- transformed may refer to a cell that has a non-native (e.g., heterologous) nucleic acid sequence or polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.
- non-native e.g., heterologous
- vector may refer to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types.
- Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, single and double stranded cassettes and the like.
- wild-type As used herein, the term “wild-type,” “native,” or “naturally-occurring” proteins may refer to those proteins found in nature.
- wild-type sequence refers to an amino acid or nucleic acid sequence that is found in nature or naturally occurring.
- a wild-type sequence is the starting point of a protein engineering project, for example, production of variant proteins.
- nucleic acids sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
- a microorganism may be modified (e.g. , genetically engineered) by any method known in the art to comprise and/or express one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more of 1 ,3-butanediol, 1 ,4- butanediol, and/or 1 ,3-propanediol (e.g., butadiene and 1 ,3-butanediol; butadiene and 1 ,4-butanediol; butadiene and 1 ,3-propanediol).
- a microorganism may be modified (e.g. , genetically engineered) by any method known in the art to comprise and/or express one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion
- Such enzymes may include any of those enzymes as set forth in any one of Figures 1-12.
- the microorganism may be modified to comprise one or more polynucleotides coding for enzymes that catalyze a conversion of crotonyl-CoA, acryloyl- CoA and acetyl-CoA, 3-hydroxypropionyl-CoA and acetyl-CoA, and/or formyl-CoA to butadiene.
- the microorganism may additionally comprise one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxalacetate to 1,3-butadienol, one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxalacetate to 1 ,4-butanediol, and/or one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol-3 -phosphate to 1 ,3-propanediol.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-butanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as crotonyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-butanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to malate e.g. , PEP carboxykinase or PEP carboxylase or pyruvate carboxylase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxaloacetate to malate e.g., malate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of malate to fumarate e.g., fumarase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of fumarate to succinate e.g., fumarate reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate to succinyl-CoA e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl-CoA to succinic semialdehyde e.g., succinyl-CoA reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinic semialdehyde to 4-hydroxybutyrate (e.g., succinate semialdehyde reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA e.g., 4-hydroxybutyryl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA (e.g., 4-hydroxybutyryl-CoA dehydratase/ vinylacetyl-CoA-Delta-isomerase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to 3 -hydroxybutyryl-Co A (e.g., crotonyl-CoA hydratase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3 -hydroxybutyryl-Co A to 3-hydroxybutyraldehyde (e.g., 3 -hydroxybutyryl-Co A reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutyraldehyde to 1 ,3 butanediol (e.g., 1 ,3 butanediol dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutanoyl-CoA to 1 ,3 -butanediol;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA to 3-hydroxybutanoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA to 3-hydroxybutanoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutanoyl-CoA to crotonoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonyl alcohol
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonaldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonoyl-CoA to crotonaldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl alcohol to butadiene.
- Exemplary enzymes that convert crotonyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 1 below.
- the enzyme reference identifier listed in Table 1 correlates with the enzyme numbering used in Figure 1, which schematically represents the enzymatic conversion of a fermentable carbon source to 1,3-butanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-butanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as acryloyl-CoA and acetyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-butanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to malate e.g. , PEP carboxykinase or PEP carboxylase or pyruvate carboxylase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxaloacetate to malate e.g., malate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of malate to fumarate e.g., fumarase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of fumarate to succinate e.g., fumarate reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate to succinyl-CoA e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl-CoA to succinyl semialdehyde (e.g., succinyl-CoA reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl semialdehyde to 4-hydroxybutyrate (e.g., succinate semialdehyde reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA e.g., 4-hydroxybutyryl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA (e.g., 4-hydroxybutyryl-CoA dehydratase/ vinylacetyl-CoA-Delta-isomerase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to 3 -hydroxybutyryl-Co A (e.g., crotonyl-CoA hydratase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3 -hydroxybutyryl-Co A to 3-hydroxybutyraldehyde (e.g., 3 -hydroxybutyryl-Co A reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutyraldehyde to 1 ,3 butanediol (e.g., 1 ,3 butanediol dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutanoyl-CoA to 1 ,3 -butanediol;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoyl-CoA and acetic acid;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA and Acetyl-CoA to 3-keto-4-pentenoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-keto-4-pentenoyl-CoA to R/S 3- hydroxy-4-pentenoyl-CoA
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid and/or
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene.
- Exemplary enzymes that convert cryloyl-CoA and acetyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 2 below.
- the enzyme reference identifier listed in Table 2 correlates with the enzyme numbering used in Figure 2, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,3- butanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-butanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as 3- hydroxypropionyl-CoA and acetyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3- butanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to malate e.g. , PEP carboxykinase or PEP carboxylase or pyruvate carboxylase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxaloacetate to malate e.g., malate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of malate to fumarate e.g., fumarase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of fumarate to succinate e.g., fumarate reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate to succinyl-CoA e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl-CoA to succinyl semialdehyde (e.g., succinyl-CoA reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl semialdehyde to 4-hydroxybutyrate (e.g., succinate semialdehyde reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA e.g., 4-hydroxybutyryl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA e.g. 4-hydroxybutyryl-CoA reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA and H20;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-Coa to 3-hydroxybutyryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutanoyl-CoA to 3-hydroxybutyraldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutyraldehyde to 1 ,3-butanediol
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutanoyl-CoA to 1 ,3-butanediol;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoyl-CoA and acetic acid;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA to 3-hydroxypropionyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxypropionyl-CoA and acetyl-CoA to 5-hydroxy-3-kethovaleryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-Ketovaleryl-CoA to R/S 3,5-dihydroxy-valeryl-CoA; - one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3,5-dihydroxy-valeryl-CoA to 3-hydroxy-4-pentenoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene.
- Exemplary enzymes that convert 3-hydroxypropionyl-CoA and acetyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 3 below.
- the enzyme reference identifier listed in Table 3 correlates with the enzyme numbering used in Figure 3, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,3- butanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-butanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as formyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-butanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to malate e.g. , PEP carboxykinase or PEP carboxylase or pyruvate carboxylase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxaloacetate to malate e.g., malate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of malate to fumarate e.g., fumarase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of fumarate to succinate e.g., fumarate reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate to succinyl-CoA e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl-CoA to succinyl semialdehyde (e.g., succinyl-CoA reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl semialdehyde to 4-hydroxybutyrate (e.g., succinate semialdehyde reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA e.g., 4-hydroxybutyryl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to 3-hydroxybutyryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutanoyl-CoA to 3-hydroxybutyraldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutyraldehyde to 1 ,3-butanediol; - one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutanoyl-CoA to 1 ,3-butanediol;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2 acetyl-Coa to acetoacetyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of Pyruvate to acetyl-CoA and formate;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA and formyl-CoA to 3,5-ketovaleryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3,5-ketovaleryl-CoA to R/S 3-hydroxy-5-Ketovaleryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3,5-dihydroxy-valeryl-CoA to 3-hydroxy-4-pentenoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene.
- Exemplary enzymes that convert formyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 4 below.
- the enzyme reference identifier listed in Table 4 correlates with the enzyme numbering used in Figure 4, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,3-butanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as crotonyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to malate e.g. , PEP carboxykinase or PEP carboxylase or pyruvate carboxylase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxaloacetate to malate e.g., malate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of malate to fumarate e.g., fumarase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of fumarate to succinate e.g., fumarate reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate to succinyl-CoA e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl-CoA to succinyl semialdehyde (e.g., succinyl-CoA reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl semialdehyde to 4-hydroxybutyrate (e.g., succinate semialdehyde reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA e.g., 4-hydroxybutyryl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA e.g., 4-hydroxybutyryl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to 3-hydroxybutyryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutanoyl-CoA to 4-hydroxybutyraldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of4-hydroxybutyraldehyde to 1 ,4-butanediol
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of4-hydroxybutanoyl-CoA to 1 ,4-butanediol;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA to 3-hydroxybutanoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion oO-hydroxybutanoyl-CoA to crotonoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonaldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonoyl-CoA to crotonaldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonyl alcohol
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl alcohol to butadiene.
- Exemplary enzymes that convert crotonyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 5 below.
- the enzyme reference identifier listed in Table 5 correlates with the enzyme numbering used in Figure 5, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as acryloyl-CoA and acetyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of PEP to oxaloacetate; - one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of PEP to pyruvate;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to malate e.g., PEP carboxykinase or PEP carboxylase or pyruvate carboxylase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxaloacetate to malate e.g., malate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of malate to fumarate e.g., fumarase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of fumarate to succinate e.g., fumarate reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate to succinyl-CoA e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl-CoA to succinyl semialdehyde (e.g., succinyl-CoA reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl semialdehyde to 4-hydroxybutyrate (e.g., succinate semialdehyde reductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA e.g., 4-hydroxybutyryl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutanoyl-CoA to 4-hydroxybutyraldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of4-hydroxybutyraldehyde to 1 ,4-butanediol
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of4-hydroxybutanoyl-CoA to 1 ,4-butanediol;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoyl-CoA and acetic acid;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA; - one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA and acetyl-CoA to 3-keto-4-pentenoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-keto-4-pentenoyl-CoA to R/S 3- hydroxy-4-pentenoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene.
- Exemplary enzymes that convert acryloyl-CoA and acetyl-CoA intermediates to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 6 below.
- the enzyme reference identifier listed in Table 6 correlates with the enzyme numbering used in Figure 6, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as 3- hydroxypropionyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of PEP to oxaloacetate or pyruvate to malate e.g., PEP carboxykinase or PEP carboxylase or pyruvate carboxylase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxaloacetate to malate e.g., malate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of malate to fumarate e.g., fumarase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of fumarate to succinate e.g., fumarate reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate to succinyl-CoA e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate semialdehyde to 4-hydroxybutyrate; - one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutanoyl-CoA to 4-hydroxybutyraldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyraldehyde to 1 ,4-butanediol
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutanoyl-CoA to 1 ,4-butanediol;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate and acetyl-CoA to lactoyl-CoA and acetic acid;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA to 3-hydroxypropionyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxypropionyl-CoA and acetyl-CoA to 5-hydroxy-3-kethovaleryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-Ketovaleryl-CoA to R/S 3,5-dihydroxy-valeryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3,5-dihydroxy-valeryl-CoA to 3-hydroxy-4-pentenoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene.
- Exemplary enzymes that convert 3-hydroxypropionyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 7 below.
- the enzyme reference identifier listed in Table 7 correlates with the enzyme numbering used in Figure 7, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene.
- Table 7 Coproduction of 1 ,4-butanediol via an oxalacetate intermediate and butadiene via 3- hydroxypropionyl-CoA and acetyl-CoA intermediates
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as formyl-CoA and acetoacetyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of PEP to oxaloacetate or pyruvate to malate e.g., PEP carboxykinase or PEP carboxylase or pyruvate carboxylase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of oxaloacetate to malate e.g., malate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of malate to fumarate e.g., fumarase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of fumarate to succinate e.g., fumarate reductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinate to succinyl-CoA e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of succinyl-CoA to succinate semialdehyde e.g., succinyl-CoA transferase or succinyl-CoA synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutanoyl-CoA to 4-hydroxybutyraldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutyraldehyde to 1 ,4-butanediol
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 4-hydroxybutanoyl-CoA to 1 ,4-butanediol;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 2 acetyl-CoA to acetoacetyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of Pyruvate to acetyl-CoA + formate
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA and formyl-CoA to 3,5-ketovaleryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3,5-ketovaleryl-CoA to R S 3-hydroxy-5-Ketovaleryl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3,5-dihydroxy-valeryl-CoA to 3-hydroxy-4-pentenoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene.
- Exemplary enzymes that convert oxalacetate to 1 ,4-butanediol and formyl-CoA and acetoacetyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 8 below.
- the enzyme reference identifier listed in Table 8 correlates with the enzyme numbering used in Figure 8, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,4-butanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as oxalacetate to 1 ,3-propanediol and a conversion of an intermediate such as crotonyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to glycerol-3 -phosphate (e.g., glycerol-3 -phosphate dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol-3 -phosphate to glycerol (e.g., glycerol 3-phosphatase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol to 3-hydroxypropionaldehyde e.g., glycerol dehydratase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxypropionaldehyde to 1 ,3-propanediol e.g., 1 ,3-propanediol oxidoreductase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetyl-CoA to acetoacetyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA to 3-hydroxybutanoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxybutanoyl-CoA to crotonoyl-CoA;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonaldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonoyl-CoA to crotonaldehyde;
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA to crotonyl alcohol
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl alcohol to butadiene.
- Exemplary enzymes that convert oxalacetate to 1 ,3-propanediol and crotonyl- CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 9 below.
- the enzyme reference identifier listed in Table 9 correlates with the enzyme numbering used in Figure 9, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as glycerol-3 - phosphate to 1 ,3-propanediol and a conversion of intermediates such as acryloyl-CoA and acetyl- CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to glycerol-3 -phosphate (e.g., glycerol-3 -phosphate dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol-3 -phosphate to glycerol (e.g., glycerol 3-phosphatase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol to 3-hydroxypropionaldehyde e.g., glycerol dehydratase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxypropionaldehyde to 1 ,3-propanediol (e.g., 1 ,3-propanediol oxidoreductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA (e.g., pyruvate dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate e.g. , lactate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA e.g., lactoyl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA e.g. , lactoyl-CoA dehydratase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA and acetyl-CoA to 3-keto-pent-4-enoyl-CoA e.g., keto-4-pentenoyl-CoA thiolase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-keto-pent-4-enoyl-CoA to R/S 3- hydroxy-4-pentenoyl-CoA (e.g., 3-keto-4-pentenoyl-CoA dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid e.g., 3-hydroxy-4-pentenoyl- CoA transferase or hydrolase or synthase
- enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid e.g., 3-hydroxy-4-pentenoyl- CoA transferase or hydrolase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene e.g., 3-hydroxy-4-pentenoic acid decarboxylase
- Exemplary enzymes that convert oxalacetate to 1 ,3-propanediol and acryloyl- CoA and acetyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 10 below.
- the enzyme reference identifier listed in Table 10 correlates with the enzyme numbering used in Figure 10, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as glycerol-3 - phosphate to 1 ,3-propanediol and a conversion of intermediates such as 3-hydroxypropionyl- CoA and acetyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of dihydroxyacetone-phosphate to glycerol-3 -phosphate (e.g., glycerol-3 -phosphate dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol-3 -phosphate to glycerol (e.g., glycerol 3-phosphatase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol to 3-hydroxypropionaldehyde e.g., glycerol dehydratase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxypropionaldehyde to 1 ,3-propanediol (e.g., 1 ,3-propanediol oxidoreductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA (e.g., pyruvate dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to lactate e.g. , lactate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactate to lactoyl-CoA e.g., lactoyl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of lactoyl-CoA to acryloyl-CoA e.g., lactoyl-CoA dehydratase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acryloyl-CoA to 3- hydroxypropionyl-CoA e.g., acryloyl-CoA hydratase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetyl-CoA and 3-hydroxypropionyl-CoA to 5-hydroxy-3-ketovaleryl-CoA (e.g., 5-hydroxy-3- ketovaleryl-CoA thiolase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-Ketovaleryl-CoA and NADH to R/S 3,5-dihydroxy-valeryl-CoA e.g., 5- hydroxy-3-Ketovaleryl-CoA dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3,5-dihydroxy-valeryl-CoA to R/S 3-hydroxy-4-pentenoyl-CoA (e.g., 3,5-hydroxyvaleryl- CoA dehydratase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid e.g., 3-hydroxy-4-pentenoyl- CoA hydrolase, transferase or synthase
- enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid e.g., 3-hydroxy-4-pentenoyl- CoA hydrolase, transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxy-4-pentenoic acid to butadiene e.g., 3-hydroxy-4-pentenoic acid decarboxylase
- Exemplary enzymes that convert oxalacetate to 1 ,3 -propanediol and 3- hydroxypropionyl-CoA and acetyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 1 1 below.
- the enzyme reference identifier listed in Table 1 1 correlates with the enzyme numbering used in Figure 1 1 , which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,3- propanediol and butadiene.
- the one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene include enzymes that catalyze a conversion of an intermediate such as glycerol-3 - phosphate to 1 ,3-propanediol and a conversion of intermediates such as formyl-CoA and acetoacetyl-CoA to butadiene.
- Representative polynucleotides coding for enzymes in a pathway that catalyze a conversion of a fermentable carbon source to 1 ,3-propanediol and butadiene include:
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol-3 -phosphate to glycerol (e.g., glycerol 3-phosphatase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of glycerol to 3-hydroxypropionaldehyde e.g., glycerol dehydratase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxypropionaldehyde to 1 ,3-propanediol (e.g., 1 ,3-propanediol oxidoreductase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate to acetyl-CoA (e.g., pyruvate dehydrogenase); - one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of pyruvate and CoA to acetyl-CoA and formate (e.g., pyruvate formate-lyase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of C0 2 to formate e.g., formate dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of formate to formyl-CoA e.g., formyl-CoA transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of acetoacetyl-CoA and formyl-CoA to 3,5-ketovaleryl-CoA (e.g., 3,5-ketovaleryl-CoA thiolase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3,5-ketovaleryl-CoA to 5-hydroxy-3-ketovaleryl-CoA e.g., 5-hydroxy-3-ketovaleryl-CoA dehydrogenase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3,5-ketovaleryl-CoA to R/S 5-keto-3-hydroxyvaleryl-CoA (e.g., 3-hydroxy-5-Ketovaleryl- CoA dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 5-hydroxy-3-ketovaleryl-CoA or R/S 5-keto-3-hydroxyvaleryl-CoA to R/S 3,5-dihydroxy- valeryl-CoA (e.g., 3,5-hydroxyaleryl-CoA dehydrogenase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3,5-dihydroxy-valeryl-CoA to R/S 3-hydroxy-4-pentenoyl-CoA (e.g., 3,5-hydroxyvaleryl- CoA dehydratase);
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxypent-4-enoic acid e.g., 3-hydroxy-4- pentenoyl-CoA hydrolase, transferase or synthase
- enzymes in a pathway that catalyze a conversion of R/S 3-hydroxy-4-pentenoyl-CoA to 3-hydroxypent-4-enoic acid e.g., 3-hydroxy-4- pentenoyl-CoA hydrolase, transferase or synthase
- polynucleotides coding for enzymes in a pathway that catalyze a conversion of 3-hydroxypent-4-enoic acid to butadiene (e.g., 3-hydroxy-4-pentenoic acid decarboxylase).
- Exemplary enzymes that convert oxalacetate to 1 ,3-propanediol and formyl- CoA and acetoacetyl-CoA to butadiene including, enzyme substrates, and enzyme reaction products associated with the conversions are presented in Table 12 below.
- the enzyme reference identifier listed in Table 12 correlates with the enzyme numbering used in Figure 12, which schematically represents the enzymatic conversion of a fermentable carbon source to 1 ,3- propanediol and butadiene.
- Table 12 Production of 1 ,3 -propanediol via a glycerol-3 -phosphate intermediate and butadiene via formyl-CoA and acetoacetyl-CoA intermediates
- the microorganism may be an archea, bacteria, or eukaryote.
- the bacteria is a Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus including, for example, Pelobacter propionicus, Clostridium propionicum, Clostridium acetobutylicum, Lactobacillus, Propionibacterium acidipropionici or Propionibacterium freudenreichii.
- the eukaryote is a yeast, filamentous fungi, protozoa, or algae.
- the yeast is Saccharomyces cerevisiae or Pichia pastoris.
- the microorganism is additionally modified to comprise one or more tolerance mechanisms including, for example, tolerance to a produced biofuel, and/or organic solvents.
- a microorganism modified to comprise such a tolerance mechanism may provide a means to increase titers of fermentations and/or may control contamination in an industrial scale process.
- the pathways may additionally comprise one or more polynucleotides coding for enzymes in any known pathway that catalyze a conversion of glucose to dihydroxyacetone-phosphate, PEP, or pyruvate including, for example, the Embden-Meyerhof- Parnas or Entner-Doudoroff pathways.
- the pathways disclosed herein may additionally comprise one or more polynucleotides coding for enzymes in any known pathway that catalyze a conversion of dihydroxyacetone-phosphate to pyruvate, PEP to oxaloacetate, or pyruvate to malate.
- the disclosure contemplates the modification ⁇ e.g., engineering) of one or more of the enzymes provided herein.
- modification may be performed to redesign the substrate specificity of the enzyme and/or to modify ⁇ e.g., reduce) its activity against others substrates in order to increase its selectivity for a given substrate.
- one or more enzymes as provided herein may be engineered to alter ⁇ e.g., enhance including, for example, increase its catalytic activity or its substrate specificity) one or more of its properties.
- sequence alignment and comparative modeling of proteins may be used to alter one or more of the enzymes disclosed herein.
- Homology modeling or comparative modeling refers to building an atomic-resolution model of the desired protein from its primary amino acid sequence and an experimental three-dimensional structure of a similar protein. This model may allow for the enzyme substrate binding site to be defined, and the identification of specific amino acid positions that may be replaced to other natural amino acid in order to redesign its substrate specificity.
- variants or modified sequences having substantial identity or homology with the polynucleotides encoding enzymes as disclosed herein may be utilized in the practice of the disclosure. Such sequences can be referred to as variants or modified sequences. That is, a polynucleotide sequence may be modified yet still retain the ability to encode a polypeptide exhibiting the desired activity. Such variants or modified sequences are thus equivalents in the sense that they retain their intended function. Generally, the variant or modified sequence may comprise at least about 40%-60%, preferably about 60%-80%, more preferably about 80%-90%, and even more preferably about 90%-95% sequence identity with the native sequence.
- a microorganism may be modified to express including, for example, overexpress, one or more enzymes as provided herein.
- the microorganism may be modified by genetic engineering techniques (i.e., recombinant technology), classical microbiological techniques, or a combination of such techniques and can also include naturally occurring genetic variants to produce a genetically modified microorganism. Some of such techniques are generally disclosed, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press.
- a genetically modified microorganism may include a microorganism in which a polynucleotide has been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect of expression (e.g., over-expression) of one or more enzymes as provided herein within the microorganism.
- Genetic modifications which result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene.
- Addition of cloned genes to increase gene expression can include maintaining the cloned gene(s) on replicating plasmids or integrating the cloned gene(s) into the genome of the production organism. Furthermore, increasing the expression of desired cloned genes can include operatively linking the cloned gene(s) to native or heterologous transcriptional control elements.
- the expression of one or more of the enzymes provided herein are under the control of a regulatory sequence that controls directly or indirectly the expression of the enzyme in a time-dependent fashion during a fermentation reaction.
- a microorganism is transformed or transfected with a genetic vehicle such as, an expression vector comprising an exogenous polynucleotide sequence coding for the enzymes provided herein.
- Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may typically, but not always, comprise a replication system (i.e. vector) recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and may preferably, but not necessarily, also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment.
- a replication system i.e. vector
- Expression systems may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome -binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, mRNA stabilizing sequences, nucleotide sequences homologous to host chromosomal DNA, and/or a multiple cloning site.
- ARS autonomously replicating sequence
- Signal peptides may also be included where appropriate, preferably from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.
- the vectors can be constructed using standard methods (see, e.g., Sambrook et ah, Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989; and Ausubel, et ah, Current Protocols in Molecular Biology, Greene Publishing, Co. N.Y, 1995).
- polynucleotides of the present disclosure including polynucleotides coding for one or more of the enzymes disclosed herein is typically carried out in recombinant vectors.
- Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes, episomal vectors and gene expression vectors, which can all be employed.
- a vector of use according to the disclosure may be selected to accommodate a protein coding sequence of a desired size.
- a suitable host cell is transformed with the vector after in vitro cloning manipulations.
- Host cells may be prokaryotic, such as any of a number of bacterial strains, or may be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells including, for example, rodent, simian or human cells.
- Each vector contains various functional components, which generally include a cloning site, an origin of replication and at least one selectable marker gene. If given vector is an expression vector, it additionally possesses one or more of the following: enhancer element, promoter, transcription termination and signal sequences, each positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a polypeptide repertoire member according to the disclosure.
- Vectors may contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells.
- the sequence may be one that enables the vector to replicate independently of the host chromosomal DNA and may include origins of replication or autonomously replicating sequences.
- origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
- the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria
- the 2 micron plasmid origin is suitable for yeast
- various viral origins ⁇ e.g. SV 40, adenovirus
- the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as COS cells.
- a cloning or expression vector may contain a selection gene also referred to as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium.
- Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate, hygromycin, thiostrepton, apramycin or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.
- the replication of vectors may be performed in E. coli ⁇ e.g., strain TBI or TGI, DH5a, ⁇ , JM110).
- An E. coli-selectable marker for example, the ⁇ -lactamase gene that confers resistance to the antibiotic ampicillin, may be of use.
- These selectable markers can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19, or pUC119.
- Expression vectors may contain a promoter that is recognized by the host organism.
- the promoter may be operably linked to a coding sequence of interest. Such a promoter may be inducible or constitutive.
- Polynucleotides are operably linked when the polynucleotides are in a relationship permitting them to function in their intended manner.
- Promoters suitable for use with prokaryotic hosts may include, for example, the a-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, the erythromycin promoter, apramycin promoter, hygromycin promoter, methylenomycin promoter and hybrid promoters such as the tac promoter. Moreover, host constitutive or inducible promoters may be used. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno sequence operably linked to the coding sequence.
- Viral promoters obtained from the genomes of viruses include promoters from polyoma virus, fowlpox virus, adenovirus ⁇ e.g., Adenovirus 2 or 5), herpes simplex virus (thymidine kinase promoter), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus ⁇ e.g., MoMLV, or RSV LTR), Hepatitis-B virus, Myeloproliferative sarcoma virus promoter (MPSV), VISNA, and Simian Virus 40 (SV40).
- Heterologous mammalian promoters include, e.g., the actin promoter, immunoglobulin promoter, heat-shock protein promoters.
- the early and late promoters of the SV40 virus are conveniently obtained as a restriction fragment that also contains the SV40 viral origin of replication (see, e.g., Fiers et al, Nature, 273: 113 (1978); Mulligan and Berg, Science, 209: 1422-1427 (1980); and Pavlakis et al, Proc. Natl. Acad. Sci. USA, 78:7398-7402 (1981)).
- the immediate early promoter of the human cytomegalovirus (CMV) is conveniently obtained as a Hind III E restriction fragment (see, e.g., Greenaway et al, Gene, 18:355-360 (1982)).
- a broad host range promoter such as the SV40 early promoter or the Rous sarcoma virus LTR, is suitable for use in the present expression vectors.
- a strong promoter may be employed to provide for high level transcription and expression of the desired product.
- the eukaryotic promoters that have been identified as strong promoters for high-level expression are the SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus long terminal repeat, and human cytomegalovirus immediate early promoter (CMV or CMV IE).
- the promoter is a SV40 or a CMV early promoter.
- the promoters employed may be constitutive or regulatable, e.g., inducible.
- exemplary inducible promoters include jun, fos and metallothionein and heat shock promoters.
- One or both promoters of the transcription units can be an inducible promoter.
- the GFP is expressed from a constitutive promoter while an inducible promoter drives transcription of the gene coding for one or more enzymes as disclosed herein and/or the amplifiable selectable marker.
- the transcriptional regulatory region in higher eukaryotes may comprise an enhancer sequence.
- enhancer sequences from mammalian genes are known e.g., from globin, elastase, albumin, a-fetoprotein and insulin genes.
- a suitable enhancer is an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the enhancer of the cytomegalovirus immediate early promoter (Boshart et al.
- the enhancer sequences may be introduced into the vector at a position 5' or 3' to the gene of interest, but is preferably located at a site 5' to the promoter.
- Yeast and mammalian expression vectors may contain prokaryotic sequences that facilitate the propagation of the vector in bacteria. Therefore, the vector may have other components such as an origin of replication ⁇ e.g., a nucleic acid sequence that enables the vector to replicate in one or more selected host cells), antibiotic resistance genes for selection in bacteria, and/or an amber stop codon which can permit translation to read through the codon. Additional eukaryotic selectable gene(s) may be incorporated.
- the origin of replication is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known, e.g., the ColEl origin of replication in bacteria.
- SV40 SV40
- polyoma adenovirus
- VSV or BPV adenovirus origin
- SV40 origin may typically be used only because it contains the early promoter
- the cloning site may be a multiple cloning site, e.g., containing multiple restriction sites.
- the plasmids may be propagated in bacterial host cells to prepare DNA stocks for subcloning steps or for introduction into eukaryotic host cells.
- Transfection of eukaryotic host cells can be any performed by any method well known in the art. Transfection methods include lipofection, electroporation, calcium phosphate co-precipitation, rubidium chloride or polycation mediated transfection, protoplast fusion and microinjection.
- the transfection is a stable transfection.
- the transfection method that provides optimal transfection frequency and expression of the construct in the particular host cell line and type, is favored. Suitable methods can be determined by routine procedures.
- the constructs are integrated so as to be stably maintained within the host chromosome.
- Vectors may be introduced to selected host cells by any of a number of suitable methods known to those skilled in the art.
- vector constructs may be introduced to appropriate cells by any of a number of transformation methods for plasmid vectors.
- standard calcium-chloride-mediated bacterial transformation is still commonly used to introduce naked DNA to bacteria (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), but electroporation and conjugation may also be used (see, e.g., Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).
- yeast or other fungal cells For the introduction of vector constructs to yeast or other fungal cells, chemical transformation methods may be used (e.g., Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Transformed cells may be isolated on selective media appropriate to the selectable marker used. Alternatively, or in addition, plates or filters lifted from plates may be scanned for GFP fluorescence to identify transformed clones.
- Plasmid vectors may be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation (see, e.g., Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.).
- Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture.
- LipofectAMINETM Life Technologies
- LipoTaxiTM LipoTaxiTM
- the host cell may be capable of expressing the construct encoding the desired protein, processing the protein and transporting a secreted protein to the cell surface for secretion. Processing includes co- and post-translational modification such as leader peptide cleavage, GPI attachment, glycosylation, ubiquitination, and disulfide bond formation.
- Immortalized host cell cultures amenable to transfection and in vitro cell culture and of the kind typically employed in genetic engineering are preferred. Examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 derivatives adapted for growth in suspension culture, Graham et al., J.
- monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals N.Y. Acad. Sci., 383:44-68 (1982)); PEER human acute lymphoblastic cell line (Ravid et al. Int.
- MRC 5 cells MRC 5 cells; FS4 cells; human hepatoma line (Hep G2), human HT1080 cells, KB cells, JW-2 cells, Detroit 6 cells, NIH-3T3 cells, hybridoma and myeloma cells.
- Embryonic cells used for generating transgenic animals are also suitable ⁇ e.g., zygotes and embryonic stem cells).
- Suitable host cells for cloning or expressing polynucleotides ⁇ e.g., DNA) in vectors may include, for example, prokaryote, yeast, or higher eukaryote cells.
- Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B.
- E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli XI 776 (ATCC 31,537), E. coli JM110 (ATCC 47,013) and E. coli W3110 (ATCC 27,325) are suitable.
- eukaryotic microbes such as filamentous fungi or yeast may be suitable cloning or expression hosts for vectors comprising polynucleotides coding for one or more enzymes.
- Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms.
- a number of other genera, species, and strains are commonly available and useful herein, such as
- K. lactis K. fragilis
- K. bulgaricus ATCC 16,045)
- K. wickeramii ATCC 24,178
- K. waltii ATCC
- Pichia pastors EP 183,070
- Candida Trichoderma reesia
- Neurospora crassa Neurospora crassa
- Schwanniomyces such as Schwanniomyces occidentalis
- filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
- suitable host cells for expression may be derived from multicellular organisms.
- invertebrate cells include plant and insect cells.
- Numerous baculo viral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silk moth) have been identified.
- a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells.
- Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, tobacco, lemna, and other plant cells can also be utilized as host cells.
- Examples of useful mammalian host cells are Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells/- DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al, J. Gen Virol. 36: 59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, (Biol. Reprod.
- monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
- Host cells are transformed or transfected with the above-described expression or cloning vectors for production of one or more enzymes as disclosed herein or with polynucleotides coding for one or more enzymes as disclosed herein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
- Host cells containing desired nucleic acid sequences coding for the disclosed enzymes may be cultured in a variety of media.
- Commercially available media such as Ham's
- DMEM Modified Eagle's Medium
- 5,122,469; WO90103430; WO 87/00195; or U.S. Patent Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides
- growth factors such as insulin, transferrin, or epidermal growth factor
- salts such as sodium chloride, calcium, magnesium, and phosphate
- buffers such as HEPES
- the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- Such polynucleotides may be modified ⁇ e.g., genetically engineered) to modulate ⁇ e.g.
- the polynucleotides may be modified to change the substrate specificity of the encoded enzyme ⁇ e.g., a polynucleotide that codes for an enzyme with specificity for a substrate may be modified such that the enzyme has specificity for an alternative substrate).
- Preferred microorganisms may comprise polynucleotides coding for one or more of the enzymes as set forth in any one of Tables 1 - 12 and Figure 1 - 12. [0002] Enzymes, and polynucleotides encoding same, for catalyzing the conversions in
- Tables 1-12 and Figures 1-12 are categorized in Table 13-25, respectively, by Enzyme
- Table 17 Exemplary genes coding for ⁇ enzymes in Table 5 and Figure 5.
- Table 20 Exemplary g enes coding for enzyn les in Table 8 and Figure 8.
- Table 22 Exemplary genes coding for e nzymes in Table 10 and Figure 10.
- Table 23 Exemplary £ >enes coding for enzymes in Table 1 1 and Figure 1 1.
- Table 24 Exemplary g enes coding for enzymes in Table 12 and Figure 12.
- Butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3- propanediol may be produced by contacting any of the disclosed genetically modified microorganisms with a fermentable carbon source.
- Such methods may preferably comprise contacting a fermentable carbon source with a microorganism comprising one or more polynucleotides coding for enzymes in a pathway that catalyze the conversion of the fermentable carbon source into any of the intermediates provided in either of Tables 1-12 or Figures 1-12 and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion one or more of the intermediates provided in Figures 1-12 (Tables 1-12) to butadiene and one or more of 1,3-butanediol, 1,4- butanediol, and/or 1,3-propanediol in a fermentation media; and expressing the one or more polynucleotides coding for the enzymes in the pathway that catalyzes the conversion of the fermentable carbon source into one or more of the intermediates provided in Figures 1-12 (Tables 1-12) and one or more polynucleotides coding for enzymes in a pathway that catalyze
- oxidation-reduction (redox) reactions For example, during fermentation, glucose is oxidized in a series of enzymatic reactions into smaller molecules with the concomitant release of energy. The electrons released are transferred from one reaction to another through universal electron carriers, such Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NAD(P)), which act as cofactors for oxidoreductase enzymes.
- NAD Nicotinamide Adenine Dinucleotide
- NAD(P) Nicotinamide Adenine Dinucleotide Phosphate
- glucose is oxidized by enzymes using the oxidized form of the cofactors (NAD(P)+ and/or NAD+) as cofactor thus generating reducing equivalents in the form of the reduced cofactor (NAD(P)H and NADH).
- NAD(P)+ and/or NAD+ the cofactors
- NAD(P)H and NADH the reduced cofactor
- redox-balanced metabolism is required, i.e., the cofactors must be regenerated by the reduction of microbial cell metabolic compounds.
- Microorganism-catalyzed fermentation for the production of natural products is a widely known application of biocatalysis.
- Industrial microorganisms can affect multistep conversions of renewable feedstocks to high value chemical products in a single reactor.
- Products of microorganism-catalyzed fermentation processes range from chemicals such as ethanol, lactic acid, amino acids and vitamins, to high value small molecule pharmaceuticals, protein pharmaceuticals, and industrial enzymes.
- the biocatalysts are whole-cell microorganisms, including microorganisms that have been genetically modified to express heterologous genes.
- Some key parameters for efficient microorganism-catalyzed fermentation processes include the ability to grow microorganisms to a greater cell density, increased yield of desired products, increased amount of volumetric productivity, removal of unwanted co- metabolites, improved utilization of inexpensive carbon and nitrogen sources, adaptation to varying fermenter conditions, increased production of a primary metabolite, increased production of a secondary metabolite, increased tolerance to acidic conditions, increased tolerance to basic conditions, increased tolerance to organic solvents, increased tolerance to high salt conditions and increased tolerance to high or low temperatures. Inefficiencies in any of these parameters can result in high manufacturing costs, inability to capture or maintain market share, and/or failure to bring fermented end-products to market.
- compositions of the present disclosure can be adapted to conventional fermentation bioreactors (e.g., batch, fed-batch, cell recycle, and continuous fermentation).
- a microorganism e.g., a genetically modified microorganism as provided herein is cultivated in liquid fermentation media (i.e., a submerged culture) which leads to excretion of the fermented product(s) into the fermentation media.
- the fermented end product(s) can be isolated from the fermentation media using any suitable method known in the art.
- formation of the fermented product occurs during an initial, fast growth period of the microorganism. In one embodiment, formation of the fermented product occurs during a second period in which the culture is maintained in a slow-growing or quiescent state. In one embodiment, formation of the fermented product occurs during more than one growth period of the microorganism. In such embodiments, the amount of fermented product formed per unit of time is generally a function of the metabolic activity of the microorganism, the physiological culture conditions (e.g., pH, temperature, medium composition), and the amount of microorganisms present in the fermentation process.
- the physiological culture conditions e.g., pH, temperature, medium composition
- the fermentation product is recovered from the periplasm or culture medium as a secreted metabolite.
- the fermentation product is extracted from the microorganism, for example when the microorganism lacks a secretory signal corresponding to the fermentation product.
- the microorganisms are ruptured and the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions may then be separated if necessary.
- the fermentation product of interest may then be purified from the remaining supernatant solution or suspension by, for example, distillation, fractionation, chromatography, precipitation, filtration, and the like.
- the microorganism cells (or portions thereof) may be used as biocatalysts or for other functions in a subsequent process without substantial purification.
- Butadiene is gaseous at room temperature or in fermentative conditions (20-45°C), and their production from a fermentation process results in a gas that could accumulate in the headspace of a fermentation tank, and be siphoned and concentrated.
- Butadiene may be purified from fermentation of gases, including gaseous alcohol, C0 2 and other compound by solvent extraction, cryogenic processes, distillation, fractionation, chromatography, precipitation, filtration, and the like.
- butadiene produced via any of the processes or methods disclosed herein may be converted to polybutadiene.
- butadiene produced via methods disclosed herein may be polymerized with other olefins to form copolymers such as acrylonitrile-butadiene-styrene (ABS), acrylonitrile-butadiene (ABR), or styrene-butadiene (SBR) copolymers, BR butyl rubber (RB), poly butadiene rubber (PBR), nitrile rubber and polychloroprene (Neoprene),.
- ABS acrylonitrile-butadiene-styrene
- ABR acrylonitrile-butadiene
- SBR styrene-butadiene copolymers
- RB BR butyl rubber
- PBR poly butadiene rubber
- nitrile rubber and polychloroprene Neoprene
- 1 ,3-butanediol; 1 ,4-butanediol; and/or 1 ,3-propanediol may be used in the manufacture of chemicals/compounds or may be used as intermediates in the production of other industrially useful chemicals/compounds.
- 1 ,3-butanediol and 1 ,4-butanediol may be dehydrated to form butadiene.
- 1 ,4-butanediol may be used in the production of tetrahydrofuran (e.g. , an intermediate of spandex and other performance polymer production) and polybutylene terephthalate (PBT) resins for engineering plastics.
- 1 ,4-butanediol may also be used in the manufacture of gamma- butyrolactone, polyurethane elastomers, and biodegradable polymers such as polybutylene succinate.
- 1 ,3-propanediol may be used as a building block in the production of polymers such as polytrimethylene terephthalate and can be formulated into a variety of industrial products including composites, adhesives, laminates, coatings, moldings, aliphatic polyesters, copolyesters. 1 ,3-propanediol may also be used as a solvent, an antifreeze, or in wood paint. Without further description, it is believed that one of ordinary skill in the art may, using the preceding description and the following illustrative examples, make and utilize the agents of the present disclosure and practice the claimed methods. The following working examples are provided to facilitate the practice of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.
- Example 1 Modification of microorganism for production of butadiene and one or more of
- a microorganism such as a bacterium is genetically modified to produce butadiene and one or more of 1 ,3-butanediol; 1 ,4-butanediol; and/or 1 ,3-propanediol from a fermentable carbon source including, for example, glucose.
- a microorganism may be genetically engineered by any methods known in the art to comprise: i.) one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of the fermentable carbon source to crotonyl-CoA, acryloyl- CoA and acetyl-CoA, 3-hydroxypropionyl-CoA and acetyl-CoA, and/or formyl-CoA and acetoacetyl-CoA intermediates and one or more polynucleotides coding for enzymes in a pathway that catalyze a conversion of crotonyl-CoA, acryloyl-CoA and acetyl-CoA, 3- hydroxypropionyl-CoA and acetyl-CoA, and/or formyl-CoA and acetoacetyl-CoA intermediates to butadiene; and ii) one or more polynucleotides coding for enzymes
- a microorganism that lacks one or more enzymes for the conversion of a fermentable carbon source to butadiene and 1 ,3-butanediol; 1 ,4-butanediol; and/or 1 ,3-propanedioladienol
- enzymes e.g., one or more functional enzymes that are catalytically active
- a microorganism that lacks one or more enzymes (e.g., one or more functional enzymes that are catalytically active) for the conversion of a fermentable carbon source to butadiene and 1 ,3-butanediol; 1 ,4-butanediol; and/or 1 ,3-propanediol.
- Example 2 Fermentation of glucose by genetically modified microorganism to produce butadiene and one or more of 1,3-butanediol; 1,4-butanediol; and/or 1,3- propanediol
- a genetically modified microorganism, as produced in Example 1 above, may be used to ferment a carbon source producing butadiene and succinate, adipate, and/or butanol.
- a previously-sterilized culture medium comprising a fermentable carbon source (e.g., 9 g/L glucose, 1 g/L KH2P04, 2 g/L (NH4)2HP04, 5 mg/L FeS04'7H20, 10 mg/L MgS04 » 7H20, 2.5 mg/L MnS04 » H20, 10 mg/L CaC12 » 6H20, 10 mg/L CoC12 » 6H20, and 10 g/L yeast extract) is charged in a bioreactor.
- a fermentable carbon source e.g., 9 g/L glucose, 1 g/L KH2P04, 2 g/L (NH4)2HP04, 5 mg/L FeS04'7H20, 10 mg/L MgS04 » 7H20, 2.5 mg/L MnS04 » H20, 10 mg/L CaC12 » 6H20, 10 mg/L CoC12 » 6H20, and 10 g/L yeast extract
- anaerobic conditions are maintained by, for example, sparging nitrogen through the culture medium.
- a suitable temperature for fermentation e.g., about 30 °C
- a near physiological pH e.g., about 6.5
- the bioreactor is agitated at, for example, about 50 rpm. Fermentation is allowed to run to completion.
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
La présente invention concerne généralement des micro-organismes (par exemple micro-organismes n'étant pas d'origine naturelle) qui comprennent un ou plusieurs polynucléotides codant pour des enzymes dans une voie qui catalyse une conversion d'une source de carbone (par exemple, une source de carbone fermentable) en butadiène et un ou plusieurs parmi le 1,3-butanediol, le 1,4-butanediol, et/ou le 1,3-propanediol et l'utilisation de tels micro-organismes pour la production de butadiène et d'un ou plusieurs parmi le 1,3-butanediol, le 1,4-butanediol, et/ou le 1,3-propanediol.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261716034P | 2012-10-19 | 2012-10-19 | |
US61/716,034 | 2012-10-19 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2014063156A2 true WO2014063156A2 (fr) | 2014-04-24 |
WO2014063156A3 WO2014063156A3 (fr) | 2014-06-26 |
Family
ID=50488906
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2013/065957 WO2014063156A2 (fr) | 2012-10-19 | 2013-10-21 | Micro-organismes modifiés et leurs procédés d'utilisation pour produire du butadiène et un ou plusieurs parmi le 1,3-butanediol, le 1,4-butanediol et/ou le 1,3-propanediol |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2014063156A2 (fr) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017168161A1 (fr) * | 2016-03-30 | 2017-10-05 | Zuvasyntha Limited | Enzyme modifiée |
US10487342B2 (en) | 2014-07-11 | 2019-11-26 | Genomatica, Inc. | Microorganisms and methods for the production of butadiene using acetyl-CoA |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100196977A1 (en) * | 2008-12-30 | 2010-08-05 | Chotani Gopal K | Methods of producing isoprene and a co-product |
US20110300597A1 (en) * | 2010-05-05 | 2011-12-08 | Burk Mark J | Microorganisms and methods for the biosynthesis of butadiene |
US20130189753A1 (en) * | 2011-06-17 | 2013-07-25 | Invista North America S.A R.L. | Methods for biosynthesizing 1,3 butadiene |
-
2013
- 2013-10-21 WO PCT/US2013/065957 patent/WO2014063156A2/fr active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100196977A1 (en) * | 2008-12-30 | 2010-08-05 | Chotani Gopal K | Methods of producing isoprene and a co-product |
US20110300597A1 (en) * | 2010-05-05 | 2011-12-08 | Burk Mark J | Microorganisms and methods for the biosynthesis of butadiene |
US20130189753A1 (en) * | 2011-06-17 | 2013-07-25 | Invista North America S.A R.L. | Methods for biosynthesizing 1,3 butadiene |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10487342B2 (en) | 2014-07-11 | 2019-11-26 | Genomatica, Inc. | Microorganisms and methods for the production of butadiene using acetyl-CoA |
US11371063B2 (en) | 2014-07-11 | 2022-06-28 | Genomatica, Inc. | Microorganisms and methods for the production of butadiene using acetyl-coA |
WO2017168161A1 (fr) * | 2016-03-30 | 2017-10-05 | Zuvasyntha Limited | Enzyme modifiée |
Also Published As
Publication number | Publication date |
---|---|
WO2014063156A3 (fr) | 2014-06-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10273505B2 (en) | Modified microorganisms and methods of making butadiene using same | |
WO2013192183A1 (fr) | Microorganismes modifiés et procédés de coproduction de butadiène avec du 1-propanol et/ou du 1,2-propanediol | |
US20140134687A1 (en) | Modified microorganisms and methods of using same for producing butadiene and succinate | |
US20150064760A1 (en) | Modified microorganism and methods of using same for producing butadiene and 1-propanol and/or 1,2-propanediol | |
US10774317B2 (en) | Engineered enzyme having acetoacetyl-CoA hydrolase activity, microorganisms comprising same, and methods of using same | |
EP3149187B1 (fr) | Micro-organismes modifiés comprenant un système optimisé pour l'utilisation d'oligosaccharides et procédés pour les utiliser | |
EP2673355A2 (fr) | Cellules et procédés de production d'acide isobutyrique | |
US20150211024A1 (en) | Methods for production of a terpene and a co-product | |
WO2014063156A2 (fr) | Micro-organismes modifiés et leurs procédés d'utilisation pour produire du butadiène et un ou plusieurs parmi le 1,3-butanediol, le 1,4-butanediol et/ou le 1,3-propanediol | |
WO2015002977A1 (fr) | Microorganismes modifiés et procédés d'utilisation de ces derniers pour la coproduction anaérobie d'isoprène et d'acide acétique | |
WO2014099927A1 (fr) | Micro-organismes modifiés et procédés d'utilisation de ceux-ci pour produire de l'isoprène, du 2-méthyl-1-butanol, du 2-méthyl-1,3-butanediol, et/ou du 2-méthyl-but-2-én-1-ol | |
US20150240264A1 (en) | Methods for co-production of a terpene, succinate and hydrogen |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13847581 Country of ref document: EP Kind code of ref document: A2 |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 13847581 Country of ref document: EP Kind code of ref document: A2 |