MXPA02001712A - Process for the biological production of 1,3-propanediol with high titer - Google Patents
Process for the biological production of 1,3-propanediol with high titerInfo
- Publication number
- MXPA02001712A MXPA02001712A MXPA/A/2002/001712A MXPA02001712A MXPA02001712A MX PA02001712 A MXPA02001712 A MX PA02001712A MX PA02001712 A MXPA02001712 A MX PA02001712A MX PA02001712 A MXPA02001712 A MX PA02001712A
- Authority
- MX
- Mexico
- Prior art keywords
- propanediol
- glycerol
- activity
- gene
- polypeptide
- Prior art date
Links
- YPFDHNVEDLHUCE-UHFFFAOYSA-N 1,3-Propanediol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 title claims abstract description 364
- 229920000166 polytrimethylene carbonate Polymers 0.000 title claims abstract description 182
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims abstract description 67
- 229940035437 1,3-propanediol Drugs 0.000 claims abstract description 181
- 241000588724 Escherichia coli Species 0.000 claims abstract description 124
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 73
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 70
- 244000005700 microbiome Species 0.000 claims abstract description 62
- 238000006243 chemical reaction Methods 0.000 claims abstract description 60
- 230000024881 catalytic activity Effects 0.000 claims abstract description 47
- AKXKFZDCRYJKTF-UHFFFAOYSA-N 3-hydroxypropanal Chemical compound OCCC=O AKXKFZDCRYJKTF-UHFFFAOYSA-N 0.000 claims abstract description 41
- 230000003197 catalytic Effects 0.000 claims abstract description 40
- 101700047726 ABGX Proteins 0.000 claims abstract description 21
- 101710008404 GAPDH Proteins 0.000 claims abstract description 21
- 101700057718 dpaB Proteins 0.000 claims abstract description 21
- 101700077654 yorZ Proteins 0.000 claims abstract description 17
- 101700085544 dpaA Proteins 0.000 claims abstract description 16
- 108060003436 dhaR Proteins 0.000 claims abstract description 14
- 101700034989 dhaT Proteins 0.000 claims abstract description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 388
- 230000000694 effects Effects 0.000 claims description 138
- 229920001184 polypeptide Polymers 0.000 claims description 83
- 239000000758 substrate Substances 0.000 claims description 59
- 150000001413 amino acids Chemical class 0.000 claims description 49
- 102000004316 Oxidoreductases Human genes 0.000 claims description 47
- 108090000854 Oxidoreductases Proteins 0.000 claims description 47
- 150000007523 nucleic acids Chemical class 0.000 claims description 44
- 230000035772 mutation Effects 0.000 claims description 33
- 108020004707 nucleic acids Proteins 0.000 claims description 28
- XKKIRYQCIQNQAC-UHFFFAOYSA-N C(CC[OH+][O-])O Chemical compound C(CC[OH+][O-])O XKKIRYQCIQNQAC-UHFFFAOYSA-N 0.000 claims description 26
- 101700033219 GPD1 Proteins 0.000 claims description 23
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 claims description 23
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 claims description 23
- 108091022084 Glycerol dehydrogenases Proteins 0.000 claims description 22
- AWUCVROLDVIAJX-UHFFFAOYSA-N Glycerol 3-phosphate Chemical compound OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 claims description 21
- 241000588748 Klebsiella Species 0.000 claims description 21
- 108010025885 glycerol dehydratase Proteins 0.000 claims description 20
- 101700059607 DAR1 Proteins 0.000 claims description 18
- 229940120503 Dihydroxyacetone Drugs 0.000 claims description 17
- RXKJFZQQPQGTFL-UHFFFAOYSA-N Dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 claims description 17
- 108091000071 Glycerol Kinase Proteins 0.000 claims description 16
- 102000002387 Glycerol kinases Human genes 0.000 claims description 16
- 230000000295 complement Effects 0.000 claims description 15
- 102000005924 Triose-phosphate isomerases Human genes 0.000 claims description 12
- 108020003073 Triose-phosphate isomerases Proteins 0.000 claims description 12
- 108010065027 Propanediol Dehydratase Proteins 0.000 claims description 11
- 241000193403 Clostridium Species 0.000 claims description 10
- 241000235070 Saccharomyces Species 0.000 claims description 10
- 150000004676 glycans Polymers 0.000 claims description 10
- 150000002772 monosaccharides Chemical class 0.000 claims description 10
- 229920001542 oligosaccharide Polymers 0.000 claims description 10
- 150000002482 oligosaccharides Polymers 0.000 claims description 10
- 229920001282 polysaccharide Polymers 0.000 claims description 10
- 239000005017 polysaccharide Substances 0.000 claims description 10
- 150000004804 polysaccharides Polymers 0.000 claims description 10
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 9
- 101700013235 GPD2 Proteins 0.000 claims description 9
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 9
- 241000235648 Pichia Species 0.000 claims description 8
- 238000009396 hybridization Methods 0.000 claims description 7
- 241000607142 Salmonella Species 0.000 claims description 6
- 206010039447 Salmonellosis Diseases 0.000 claims description 6
- 241000588722 Escherichia Species 0.000 claims description 5
- 241000187747 Streptomyces Species 0.000 claims description 5
- 101710025070 gapdh-2 Proteins 0.000 claims description 5
- 230000001105 regulatory Effects 0.000 claims description 5
- 241000228212 Aspergillus Species 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 101710044675 DHAPRD Proteins 0.000 claims description 4
- 241000235035 Debaryomyces Species 0.000 claims description 4
- 102100006961 GPD2 Human genes 0.000 claims description 4
- 101700001004 GPP1 Proteins 0.000 claims description 4
- 102000004195 Isomerases Human genes 0.000 claims description 4
- 108090000769 Isomerases Proteins 0.000 claims description 4
- 241000235395 Mucor Species 0.000 claims description 4
- 241000589516 Pseudomonas Species 0.000 claims description 4
- 241000235346 Schizosaccharomyces Species 0.000 claims description 4
- 241000588914 Enterobacter Species 0.000 claims description 3
- 241000186660 Lactobacillus Species 0.000 claims description 3
- 229940039696 Lactobacillus Drugs 0.000 claims description 3
- 241000235017 Zygosaccharomyces Species 0.000 claims description 3
- 101710025069 yplQ Proteins 0.000 claims description 3
- 101710010461 Gapdh1 Proteins 0.000 claims description 2
- 101700004979 IRX10 Proteins 0.000 claims description 2
- 101710010416 IRX10L Proteins 0.000 claims description 2
- 101710010904 PCBD2 Proteins 0.000 claims description 2
- 101700037537 glpD Proteins 0.000 claims description 2
- 101700057982 glpD1 Proteins 0.000 claims description 2
- 101700083505 glpO Proteins 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 108091006028 chimera Proteins 0.000 claims 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims 2
- 102100016606 GPD1 Human genes 0.000 claims 1
- 101700000203 GPP2 Proteins 0.000 claims 1
- 241000589350 Methylobacter Species 0.000 claims 1
- 230000004043 responsiveness Effects 0.000 claims 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 69
- 239000008103 glucose Substances 0.000 abstract description 68
- 101710025050 MK0970 Proteins 0.000 abstract description 16
- 241000588747 Klebsiella pneumoniae Species 0.000 abstract description 14
- 229940045505 Klebsiella pneumoniae Drugs 0.000 abstract description 14
- 230000002068 genetic Effects 0.000 abstract description 9
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 130
- 229960005150 glycerol Drugs 0.000 description 122
- 235000011187 glycerol Nutrition 0.000 description 122
- 210000004027 cells Anatomy 0.000 description 86
- 101710013423 dhaAF Proteins 0.000 description 77
- 238000000855 fermentation Methods 0.000 description 75
- 230000004151 fermentation Effects 0.000 description 74
- 229960001031 Glucose Drugs 0.000 description 68
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-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 description 67
- 235000001727 glucose Nutrition 0.000 description 66
- BAWFJGJZGIEFAR-NNYOXOHSSA-N Nicotinamide adenine dinucleotide 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 55
- 239000002609 media Substances 0.000 description 49
- 102000004190 Enzymes Human genes 0.000 description 47
- 108090000790 Enzymes Proteins 0.000 description 47
- 239000007858 starting material Substances 0.000 description 45
- 230000014509 gene expression Effects 0.000 description 42
- 239000000047 product Substances 0.000 description 39
- 229940052665 NADH Drugs 0.000 description 35
- GNGACRATGGDKBX-UHFFFAOYSA-N Dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 32
- 235000018102 proteins Nutrition 0.000 description 31
- 102000004169 proteins and genes Human genes 0.000 description 31
- 108090000623 proteins and genes Proteins 0.000 description 31
- 239000000499 gel Substances 0.000 description 27
- 229920001850 Nucleic acid sequence Polymers 0.000 description 26
- 235000001014 amino acid Nutrition 0.000 description 26
- 239000002773 nucleotide Substances 0.000 description 22
- 125000003729 nucleotide group Chemical group 0.000 description 22
- 230000001419 dependent Effects 0.000 description 20
- 238000004166 bioassay Methods 0.000 description 19
- 101700066745 gldA Proteins 0.000 description 19
- 230000012010 growth Effects 0.000 description 19
- 229950006238 nadide Drugs 0.000 description 19
- 230000001131 transforming Effects 0.000 description 19
- 101700013246 GK Proteins 0.000 description 18
- 101710042253 GK2 Proteins 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 238000010276 construction Methods 0.000 description 16
- 101710004291 egsA Proteins 0.000 description 16
- 230000015572 biosynthetic process Effects 0.000 description 15
- 239000000284 extract Substances 0.000 description 15
- 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 14
- 229960005091 Chloramphenicol Drugs 0.000 description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 14
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 14
- 101700003317 TPI1 Proteins 0.000 description 14
- 238000010367 cloning Methods 0.000 description 14
- 101700029304 dhaB Proteins 0.000 description 14
- -1 glycerol Chemical class 0.000 description 14
- 101700079498 tapA Proteins 0.000 description 14
- 101710031202 tpiA1 Proteins 0.000 description 14
- 101710031201 tpiA2 Proteins 0.000 description 14
- 101700056631 yqhD Proteins 0.000 description 14
- 108020004705 Codon Proteins 0.000 description 13
- 108010021809 EC 1.1.1.1 Proteins 0.000 description 13
- 102000007698 EC 1.1.1.1 Human genes 0.000 description 13
- 230000004075 alteration Effects 0.000 description 13
- 238000005755 formation reaction Methods 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- 239000006142 Luria-Bertani Agar Substances 0.000 description 12
- 238000007792 addition Methods 0.000 description 12
- 238000003379 elimination reaction Methods 0.000 description 12
- 101700043757 eryH Proteins 0.000 description 12
- 101700005315 lerI Proteins 0.000 description 12
- SBUJHOSQTJFQJX-NOAMYHISSA-N Kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 11
- 229940088594 Vitamin Drugs 0.000 description 11
- 229960000318 kanamycin Drugs 0.000 description 11
- 235000013343 vitamin Nutrition 0.000 description 11
- 239000011782 vitamin Substances 0.000 description 11
- 150000003722 vitamin derivatives Chemical class 0.000 description 11
- 229930003231 vitamins Natural products 0.000 description 11
- 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 10
- 241000894006 Bacteria Species 0.000 description 10
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L MgCl2 Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 229910001868 water Inorganic materials 0.000 description 10
- 229930003270 Vitamin B Natural products 0.000 description 9
- 229960000723 ampicillin Drugs 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 230000001580 bacterial Effects 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 238000006467 substitution reaction Methods 0.000 description 9
- 235000019156 vitamin B Nutrition 0.000 description 9
- 239000011720 vitamin B Substances 0.000 description 9
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 8
- 229960003669 Carbenicillin Drugs 0.000 description 8
- FPPNZSSZRUTDAP-UWFZAAFLSA-N Carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 8
- 108010011958 EC 1.1.1.202 Proteins 0.000 description 8
- BJHIKXHVCXFQLS-UYFOZJQFSA-N Fructose Natural products OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 8
- 229930003779 Vitamin B12 Natural products 0.000 description 8
- 235000010633 broth Nutrition 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 8
- 125000003346 cobalamin group Chemical group 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 235000019163 vitamin B12 Nutrition 0.000 description 8
- 239000011715 vitamin B12 Substances 0.000 description 8
- 230000002759 chromosomal Effects 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 239000010452 phosphate Substances 0.000 description 7
- 235000000346 sugar Nutrition 0.000 description 7
- 101700019681 yghD Proteins 0.000 description 7
- 229940041514 Candida albicans extract Drugs 0.000 description 6
- 239000006137 Luria-Bertani broth Substances 0.000 description 6
- XJLXINKUBYWONI-NNYOXOHSSA-N Nicotinamide adenine dinucleotide phosphate 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 6
- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 description 6
- 229940081969 Saccharomyces cerevisiae Drugs 0.000 description 6
- 239000003054 catalyst Substances 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 230000002255 enzymatic Effects 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000000543 intermediate Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000000051 modifying Effects 0.000 description 6
- 230000036961 partial Effects 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 5
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N Ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 230000001413 cellular Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 230000000875 corresponding Effects 0.000 description 5
- 230000001808 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 230000002503 metabolic Effects 0.000 description 5
- 235000021317 phosphate Nutrition 0.000 description 5
- 238000005215 recombination Methods 0.000 description 5
- 230000003362 replicative Effects 0.000 description 5
- 230000000754 repressing Effects 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 150000008163 sugars Chemical class 0.000 description 5
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (-)-propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 description 4
- LXJXRIRHZLFYRP-VKHMYHEASA-L (R)-2-Hydroxy-3-(phosphonooxy)-propanal Natural products O=C[C@H](O)COP([O-])([O-])=O LXJXRIRHZLFYRP-VKHMYHEASA-L 0.000 description 4
- UNFWWIHTNXNPBV-WXKVUWSESA-N Actinospectacin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 4
- 229920001405 Coding region Polymers 0.000 description 4
- 239000005715 Fructose Substances 0.000 description 4
- MNQZXJOMYWMBOU-UHFFFAOYSA-N Glyceraldehyde Chemical compound OCC(O)C=O MNQZXJOMYWMBOU-UHFFFAOYSA-N 0.000 description 4
- 102000030951 Phosphotransferases Human genes 0.000 description 4
- 108091000081 Phosphotransferases Proteins 0.000 description 4
- 229960004063 Propylene glycol Drugs 0.000 description 4
- IKHGUXGNUITLKF-UHFFFAOYSA-N acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 239000005515 coenzyme Substances 0.000 description 4
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 4
- 230000009089 cytolysis Effects 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 125000005639 glycero group Chemical group 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 125000001165 hydrophobic group Chemical group 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 230000002934 lysing Effects 0.000 description 4
- 230000001404 mediated Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000006011 modification reaction Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- KWYUFKZDYYNOTN-UHFFFAOYSA-M potassium hydroxide Inorganic materials [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 4
- 235000013772 propylene glycol Nutrition 0.000 description 4
- 108091007521 restriction endonucleases Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 229960000268 spectinomycin Drugs 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241001515965 unidentified phage Species 0.000 description 4
- 230000035899 viability Effects 0.000 description 4
- 210000000349 Chromosomes Anatomy 0.000 description 3
- 241000588923 Citrobacter Species 0.000 description 3
- 241000195493 Cryptophyta Species 0.000 description 3
- SBJKKFFYIZUCET-JLAZNSOCSA-N Dehydro-L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-JLAZNSOCSA-N 0.000 description 3
- 229940023064 Escherichia coli Drugs 0.000 description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K Iron(III) chloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N L-serine Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L MANGANESE CHLORIDE Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 3
- 230000035633 Metabolized Effects 0.000 description 3
- 229910021380 MnCl2 Inorganic materials 0.000 description 3
- 229960000344 Thiamine hydrochloride Drugs 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000009632 agar plate Methods 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 230000000692 anti-sense Effects 0.000 description 3
- 230000031018 biological processes and functions Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 235000011148 calcium chloride Nutrition 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 230000000977 initiatory Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl β-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 239000011565 manganese chloride Substances 0.000 description 3
- 235000002867 manganese chloride Nutrition 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- BDAGIHXWWSANSR-UHFFFAOYSA-M methanoate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000008057 potassium phosphate buffer Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000002829 reduced Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 3
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 3
- 239000011747 thiamine hydrochloride Substances 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000002103 transcriptional Effects 0.000 description 3
- 238000001419 two-dimensional polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Natural products NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 229960000643 Adenine Drugs 0.000 description 2
- OIRDTQYFTABQOQ-SXVXDFOESA-N Adenosine Natural products Nc1ncnc2c1ncn2[C@@H]3O[C@@H](CO)[C@H](O)[C@@H]3O OIRDTQYFTABQOQ-SXVXDFOESA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 229960005261 Aspartic Acid Drugs 0.000 description 2
- OWMVSZAMULFTJU-UHFFFAOYSA-N Bis-tris methane Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- 229940077731 Carbohydrate nutrients Drugs 0.000 description 2
- 241000588919 Citrobacter freundii Species 0.000 description 2
- 241000193469 Clostridium pasteurianum Species 0.000 description 2
- 229920002676 Complementary DNA Polymers 0.000 description 2
- 229940104302 Cytosine Drugs 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N Cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N DEOXYTHYMIDINE 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
- 238000007399 DNA isolation Methods 0.000 description 2
- UDPGUMQDCGORJQ-UHFFFAOYSA-N Ethephon Chemical compound OP(O)(=O)CCCl UDPGUMQDCGORJQ-UHFFFAOYSA-N 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 229960002989 Glutamic Acid Drugs 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N HEPES Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 229960000310 ISOLEUCINE Drugs 0.000 description 2
- 241000588915 Klebsiella aerogenes Species 0.000 description 2
- 241000588749 Klebsiella oxytoca Species 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 240000001929 Lactobacillus brevis Species 0.000 description 2
- 235000013957 Lactobacillus brevis Nutrition 0.000 description 2
- 239000007993 MOPS buffer Substances 0.000 description 2
- 230000036740 Metabolism Effects 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N N'-amino-N-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N Oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
- IUGYQRQAERSCNH-UHFFFAOYSA-N Pivalic acid Chemical compound CC(C)(C)C(O)=O IUGYQRQAERSCNH-UHFFFAOYSA-N 0.000 description 2
- ZCCUUQDIBDJBTK-UHFFFAOYSA-N Psoralen Chemical compound C1=C2OC(=O)C=CC2=CC2=C1OC=C2 ZCCUUQDIBDJBTK-UHFFFAOYSA-N 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 229920000978 Start codon Polymers 0.000 description 2
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-L Sulphite Chemical class [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N Thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- SRBFZHDQGSBBOR-SQOUGZDYSA-N Xylose Natural products O[C@@H]1CO[C@@H](O)[C@@H](O)[C@@H]1O SRBFZHDQGSBBOR-SQOUGZDYSA-N 0.000 description 2
- VBJHPXDIVMXHJU-UHFFFAOYSA-N Zeocin Chemical compound N=1C(C=2SC=C(N=2)C(=O)NCCCCN=C(N)N)CSC=1CCNC(=O)C(C(O)C)NC(=O)C(C)C(O)C(C)NC(=O)C(C(OC1C(C(O)C(O)C(CO)O1)OC1C(C(OC(N)=O)C(O)C(CO)O1)O)C=1[N]C=NC=1)NC(=O)C1=NC(C(CC(N)=O)NCC(N)C(N)=O)=NC(N)=C1C VBJHPXDIVMXHJU-UHFFFAOYSA-N 0.000 description 2
- 108010084455 Zeocin Proteins 0.000 description 2
- GYQWVHROHDZXRS-UHFFFAOYSA-N [1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]azanium;carbonate Chemical compound OC(O)=O.OCC(N)(CO)CO.OCC(N)(CO)CO GYQWVHROHDZXRS-UHFFFAOYSA-N 0.000 description 2
- ZKJOXOJMGXFSPF-QYZPTAICSA-N [[(2R,3R,4R,5R)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate;hydrate Chemical compound O.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 ZKJOXOJMGXFSPF-QYZPTAICSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- 101710025952 adhT Proteins 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- UGFAIRIUMAVXCW-UHFFFAOYSA-N carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 2
- 229910002091 carbon monoxide Inorganic materials 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000004690 coupled electron pair approximation Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 101700002483 ddrA Proteins 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 150000002009 diols Chemical class 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 238000007429 general method Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 230000002414 glycolytic Effects 0.000 description 2
- 239000001963 growth media Substances 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 150000007857 hydrazones Chemical class 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000002209 hydrophobic Effects 0.000 description 2
- XLSMFKSTNGKWQX-UHFFFAOYSA-N hydroxyacetone Chemical compound CC(=O)CO XLSMFKSTNGKWQX-UHFFFAOYSA-N 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000035786 metabolism Effects 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000003287 optical Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000003204 osmotic Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000023 polynucleotide Polymers 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 239000001184 potassium carbonate Substances 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 101700051431 pqn-8 Proteins 0.000 description 2
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000002194 synthesizing Effects 0.000 description 2
- YXFVVABEGXRONW-UHFFFAOYSA-N toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000000844 transformation Methods 0.000 description 2
- 238000005429 turbidity Methods 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- VEMLQICWTSVKQH-BTVCFUMJSA-N (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal;propane-1,2,3-triol Chemical compound OCC(O)CO.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O VEMLQICWTSVKQH-BTVCFUMJSA-N 0.000 description 1
- OVRAJPVCMXSZJC-RYRWEOBNSA-N (2S,3R,4S,5S,6R)-4-amino-2-[(1S,2S,3R,4S,6R)-4,6-diamino-3-[(2R,4R,5S,6R)-4,5-dihydroxy-6-methyloxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-6-(hydroxymethyl)oxane-3,5-diol Chemical compound C1[C@@H](O)[C@H](O)[C@@H](C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OVRAJPVCMXSZJC-RYRWEOBNSA-N 0.000 description 1
- PXOPDYTVWWQZEK-DPQCFBEESA-N (5Z,9Z,14Z)-2,3,7,8,12,13,17,18,19,22-decahydro-1H-corrin Chemical compound N1=C(\C=C\2N=C(\C=C\3NC(/CC/3)=C\3)CC/2)CCC1C1CCC/3=N1 PXOPDYTVWWQZEK-DPQCFBEESA-N 0.000 description 1
- FHPQRKDCCAFWMX-UHFFFAOYSA-N 1-hydroxypropan-2-one;propane-1,2,3-triol Chemical compound CC(=O)CO.OCC(O)CO FHPQRKDCCAFWMX-UHFFFAOYSA-N 0.000 description 1
- RZYHXKLKJRGJGP-UHFFFAOYSA-N 2,2,2-trifluoro-N,N-bis(trimethylsilyl)acetamide Chemical compound C[Si](C)(C)N([Si](C)(C)C)C(=O)C(F)(F)F RZYHXKLKJRGJGP-UHFFFAOYSA-N 0.000 description 1
- RVCKCEDKBVEEHL-UHFFFAOYSA-N 2,3,4,5,6-Pentachlorobenzyl alcohol Chemical compound OCC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl RVCKCEDKBVEEHL-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- PJWIPEXIFFQAQZ-PUFIMZNGSA-N 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@H](O)CC(=O)C(O)=O PJWIPEXIFFQAQZ-PUFIMZNGSA-N 0.000 description 1
- HBYDCZLFVCBQJW-UHFFFAOYSA-N 3-hydroxypropanal;propane-1,3-diol Chemical compound OCCCO.OCCC=O HBYDCZLFVCBQJW-UHFFFAOYSA-N 0.000 description 1
- CHDDAVCOAOFSLD-UHFFFAOYSA-N 3-phosphonopyruvic acid Chemical compound OC(=O)C(=O)CP(O)(O)=O CHDDAVCOAOFSLD-UHFFFAOYSA-N 0.000 description 1
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 1
- QYNUQALWYRSVHF-ABLWVSNPSA-N 5,10-Methylenetetrahydrofolate Chemical compound C1N2C=3C(=O)NC(N)=NC=3NCC2CN1C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 QYNUQALWYRSVHF-ABLWVSNPSA-N 0.000 description 1
- IHFQGEDFEXGPFR-UHFFFAOYSA-N 5-[4-(4-heptylbenzoyl)phenyl]pentanoic acid Chemical compound C1=CC(CCCCCCC)=CC=C1C(=O)C1=CC=C(CCCCC(O)=O)C=C1 IHFQGEDFEXGPFR-UHFFFAOYSA-N 0.000 description 1
- BLEMLGBVQSNDOD-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CN=C2[C]1C(Cl)=C(Br)C=C2 BLEMLGBVQSNDOD-AEOCFKNESA-N 0.000 description 1
- ZJTJUVIJVLLGSP-UHFFFAOYSA-N 7,8-dimethylisoalloxazine Chemical compound N1C(=O)NC(=O)C2=C1N=C1C=C(C)C(C)=CC1=N2 ZJTJUVIJVLLGSP-UHFFFAOYSA-N 0.000 description 1
- 101700007462 ADH1A Proteins 0.000 description 1
- 102100000356 ADH1A Human genes 0.000 description 1
- 101700080270 ADH2 Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- FRHBOQMZUOWXQL-UHFFFAOYSA-L Ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 description 1
- 241000272441 Asteroma Species 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 241000178343 Butea superba Species 0.000 description 1
- 101700086558 CELA1 Proteins 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate dianion Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 210000003763 Chloroplasts Anatomy 0.000 description 1
- 241000511343 Chondrostoma nasus Species 0.000 description 1
- 241001112696 Clostridia Species 0.000 description 1
- FEESAGIUMZGLMF-JFYQDRLCSA-M Cobinamide Chemical compound [Co+3].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@H](O)C)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O FEESAGIUMZGLMF-JFYQDRLCSA-M 0.000 description 1
- 102000016917 Complement C1s Human genes 0.000 description 1
- 108010028774 Complement C1s Proteins 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L Copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- FNZLKVNUWIIPSJ-UHNVWZDZSA-N D-ribulose 5-phosphate Chemical compound OCC(=O)[C@H](O)[C@H](O)COP(O)(O)=O FNZLKVNUWIIPSJ-UHNVWZDZSA-N 0.000 description 1
- 101700035028 DAR2 Proteins 0.000 description 1
- 101700058746 DDR1 Proteins 0.000 description 1
- 101700005454 DDR2 Proteins 0.000 description 1
- 230000003682 DNA packaging Effects 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- OJIYIVCMRYCWSE-UHFFFAOYSA-M Domiphen bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CCOC1=CC=CC=C1 OJIYIVCMRYCWSE-UHFFFAOYSA-M 0.000 description 1
- 241000195634 Dunaliella Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000701867 Enterobacteria phage T7 Species 0.000 description 1
- 108050001049 Extracellular protein Proteins 0.000 description 1
- 101710025698 FASLG Proteins 0.000 description 1
- 240000001441 Fragaria vesca Species 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 229960002737 Fructose Drugs 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102100012694 GAL Human genes 0.000 description 1
- 101700002119 GAL Proteins 0.000 description 1
- 101700014301 GALK1 Proteins 0.000 description 1
- 101710037135 GAPC2 Proteins 0.000 description 1
- 101710037116 GAPC3 Proteins 0.000 description 1
- 101710025049 GAPDG Proteins 0.000 description 1
- 102100006425 GAPDH Human genes 0.000 description 1
- 101710037084 GLDC Proteins 0.000 description 1
- GYHNNYVSQQEPJS-UHFFFAOYSA-N Gallium Chemical compound [Ga] GYHNNYVSQQEPJS-UHFFFAOYSA-N 0.000 description 1
- 101710013484 Gld Proteins 0.000 description 1
- 229960002442 Glucosamine Drugs 0.000 description 1
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 1
- 101710007246 H3-3A Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 101700009603 HIS3 Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 108090001042 Hydro-Lyases Proteins 0.000 description 1
- 102000004867 Hydro-Lyases Human genes 0.000 description 1
- 241000411968 Ilyobacter Species 0.000 description 1
- 241001055489 Klebsiella pneumoniae ATCC 25955 Species 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- GUBGYTABKSRVRQ-UUNJERMWSA-N Lactose Natural products O([C@@H]1[C@H](O)[C@H](O)[C@H](O)O[C@@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 GUBGYTABKSRVRQ-UUNJERMWSA-N 0.000 description 1
- 241000392000 Licina Species 0.000 description 1
- 210000004185 Liver Anatomy 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- JDSHMPZPIAZGSV-UHFFFAOYSA-N Melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 description 1
- 229920000877 Melamine resin Polymers 0.000 description 1
- 108020004999 Messenger RNA Proteins 0.000 description 1
- 230000036091 Metabolic activity Effects 0.000 description 1
- 210000004080 Milk Anatomy 0.000 description 1
- 102000002247 NADPH Dehydrogenase Human genes 0.000 description 1
- 108010014870 NADPH Dehydrogenase Proteins 0.000 description 1
- 229910017912 NH2OH Inorganic materials 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 101710017470 NT5C3B Proteins 0.000 description 1
- SNICXCGAKADSCV-JTQLQIEISA-N Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- 229960002715 Nicotine Drugs 0.000 description 1
- 210000004940 Nucleus Anatomy 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- 229920000272 Oligonucleotide Polymers 0.000 description 1
- 101700060831 PGP Proteins 0.000 description 1
- 102100001725 PGP Human genes 0.000 description 1
- 101700002320 PIR Proteins 0.000 description 1
- 101710042137 PRSS1 Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 241001192924 Parna Species 0.000 description 1
- 241000863392 Pelobacter Species 0.000 description 1
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Chemical compound C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 1
- DTBNBXWJWCWCIK-UHFFFAOYSA-N Phosphoenolpyruvic acid Natural products OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 241000920340 Pion Species 0.000 description 1
- 229950008679 Protamine sulfate Drugs 0.000 description 1
- 102000014961 Protein Precursors Human genes 0.000 description 1
- 108010078762 Protein Precursors Proteins 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 210000003705 Ribosomes Anatomy 0.000 description 1
- 101710044706 SFA1 Proteins 0.000 description 1
- 101700027726 SPE1 Proteins 0.000 description 1
- 240000004552 Saccharomyces cerevisiae var. diastaticus Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 241000588717 Shimwellia blattae Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 210000003491 Skin Anatomy 0.000 description 1
- 206010041235 Snoring Diseases 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 102100006017 TAC3 Human genes 0.000 description 1
- 101700070528 TAC3 Proteins 0.000 description 1
- 102100009594 TPI1 Human genes 0.000 description 1
- 101700000513 TYRP1 Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 229940104230 Thymidine Drugs 0.000 description 1
- 229940113082 Thymine Drugs 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N Trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 101710042194 Trpgamma Proteins 0.000 description 1
- 240000004922 Vigna radiata Species 0.000 description 1
- 235000010721 Vigna radiata var radiata Nutrition 0.000 description 1
- 235000011469 Vigna radiata var sublobata Nutrition 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 229960003487 Xylose Drugs 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- WCDYMMVGBZNUGB-ORPFKJIMSA-N [(2R,3R,4S,5R,6R)-6-[[(1R,3R,4R,5R,6R)-4,5-dihydroxy-2,7-dioxabicyclo[4.2.0]octan-3-yl]oxy]-3,4,5-trihydroxyoxan-2-yl]methyl 3-hydroxy-2-tetradecyloctadecanoate Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](COC(=O)C(CCCCCCCCCCCCCC)C(O)CCCCCCCCCCCCCCC)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H]2OC[C@H]2O1 WCDYMMVGBZNUGB-ORPFKJIMSA-N 0.000 description 1
- JDZUTRNFTHMPMQ-VOPDUKPCSA-N [(2R,3S,4R,5R)-3-acetyloxy-5-[(2S,3S,4R,5R)-3-acetyloxy-4,5-dihydroxyoxan-2-yl]oxy-2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienoxy]oxan-4-yl] acetate Chemical compound CC(=O)O[C@H]1[C@H](OC(C)=O)[C@H](OC/C=C(C)/CC/C=C(C)/CCC=C(C)C)OC[C@H]1O[C@H]1[C@@H](OC(C)=O)[C@H](O)[C@H](O)CO1 JDZUTRNFTHMPMQ-VOPDUKPCSA-N 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002378 acidificating Effects 0.000 description 1
- 239000000999 acridine dye Substances 0.000 description 1
- HGINCPLSRVDWNT-UHFFFAOYSA-N acrylaldehyde Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 230000001476 alcoholic Effects 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 125000004429 atoms Chemical group 0.000 description 1
- 230000037358 bacterial metabolism Effects 0.000 description 1
- 244000052616 bacterial pathogens Species 0.000 description 1
- 238000002869 basic local alignment search tool Methods 0.000 description 1
- 238000010256 biochemical assay Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000037348 biosynthesis Effects 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 150000003842 bromide salts Chemical class 0.000 description 1
- KAKZBPTYRLMSJV-UHFFFAOYSA-N butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- CREMABGTGYGIQB-UHFFFAOYSA-N carbon carbon Chemical compound C.C CREMABGTGYGIQB-UHFFFAOYSA-N 0.000 description 1
- 150000001722 carbon compounds Chemical class 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000001925 catabolic Effects 0.000 description 1
- 101710025091 cbbGC Proteins 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000005465 channeling Effects 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- ZIHHMGTYZOSFRC-UWWAPWIJSA-M cobamamide Chemical compound C1(/[C@](C)(CCC(=O)NC[C@H](C)OP(O)(=O)OC2[C@H]([C@H](O[C@@H]2CO)N2C3=CC(C)=C(C)C=C3N=C2)O)[C@@H](CC(N)=O)[C@]2(N1[Co+]C[C@@H]1[C@H]([C@@H](O)[C@@H](O1)N1C3=NC=NC(N)=C3N=C1)O)[H])=C(C)\C([C@H](C/1(C)C)CCC(N)=O)=N\C\1=C/C([C@H]([C@@]\1(CC(N)=O)C)CCC(N)=O)=N/C/1=C(C)\C1=N[C@]2(C)[C@@](C)(CC(N)=O)[C@@H]1CCC(N)=O ZIHHMGTYZOSFRC-UWWAPWIJSA-M 0.000 description 1
- 235000006279 cobamamide Nutrition 0.000 description 1
- 239000011789 cobamamide Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000003636 conditioned culture media Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000005824 corn Nutrition 0.000 description 1
- WUPRCGRRQUZFAB-DEGKJRJSSA-N corrin Chemical group N1C2CC\C1=C\C(CC/1)=N\C\1=C/C(CC\1)=N/C/1=C\C1=NC2CC1 WUPRCGRRQUZFAB-DEGKJRJSSA-N 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000001082 cryoprotectant Effects 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 150000001923 cyclic compounds Chemical class 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 230000001809 detectable Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 101710014338 eglS Proteins 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- ANUSOIHIIPAHJV-UHFFFAOYSA-N fenticlor Chemical compound OC1=CC=C(Cl)C=C1SC1=CC(Cl)=CC=C1O ANUSOIHIIPAHJV-UHFFFAOYSA-N 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000007863 gel particle Substances 0.000 description 1
- 101700038358 glpF Proteins 0.000 description 1
- 101700079581 glpX Proteins 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- LXJXRIRHZLFYRP-UHFFFAOYSA-N glyceraldehyde 3-phosphate Chemical compound O=CC(O)COP(O)(O)=O LXJXRIRHZLFYRP-UHFFFAOYSA-N 0.000 description 1
- 108010000361 glycerol 2-dehydrogenase (NADP+) Proteins 0.000 description 1
- 125000000267 glycino group Chemical group [H]N([*])C([H])([H])C(=O)O[H] 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 101700042169 gspH Proteins 0.000 description 1
- 101700004055 gspI Proteins 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- KJUGUADJHNHALS-UHFFFAOYSA-O hydron;2H-tetrazole Chemical compound C1=NN=[NH+]N1 KJUGUADJHNHALS-UHFFFAOYSA-O 0.000 description 1
- YUDNQQJOVFPCTF-UHFFFAOYSA-L hydroxyacetone phosphate(2-) Chemical compound CC(=O)COP([O-])([O-])=O YUDNQQJOVFPCTF-UHFFFAOYSA-L 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000004313 iron ammonium citrate Substances 0.000 description 1
- 235000000011 iron ammonium citrate Nutrition 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229920002106 messenger RNA Polymers 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- BAVYZALUXZFZLV-UHFFFAOYSA-N methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000006151 minimal media Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- MEFBJEMVZONFCJ-UHFFFAOYSA-N molybdate Chemical compound [O-][Mo]([O-])(=O)=O MEFBJEMVZONFCJ-UHFFFAOYSA-N 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000012666 negative regulation of transcription by glucose Effects 0.000 description 1
- 229930015196 nicotine Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001894 non-coding RNA Polymers 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N oxane Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N p-acetaminophenol Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 101700078455 pduC Proteins 0.000 description 1
- 101700065331 pduD Proteins 0.000 description 1
- 101700032916 pduE Proteins 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000000737 periodic Effects 0.000 description 1
- 150000003003 phosphines Chemical class 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000865 phosphorylative Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 108091008117 polyclonal antibodies Proteins 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 229940002612 prodrugs Drugs 0.000 description 1
- 230000002035 prolonged Effects 0.000 description 1
- ULWHHBHJGPPBCO-UHFFFAOYSA-N propane-1,1-diol Chemical class CCC(O)O ULWHHBHJGPPBCO-UHFFFAOYSA-N 0.000 description 1
- NBBJYMSMWIIQGU-UHFFFAOYSA-N propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 108010012557 prothrombin complex concentrates Proteins 0.000 description 1
- 239000011802 pulverized particle Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000036159 relative stability Effects 0.000 description 1
- 230000000284 resting Effects 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 230000000717 retained Effects 0.000 description 1
- 230000002441 reversible Effects 0.000 description 1
- 235000019231 riboflavin-5'-phosphate Nutrition 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- NESXXUAWVHXRGX-UHFFFAOYSA-M sodium;1-carboxyethenyl hydrogen phosphate Chemical compound [Na+].OP(O)(=O)OC(=C)C([O-])=O NESXXUAWVHXRGX-UHFFFAOYSA-M 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000003068 static Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 230000002588 toxic Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 101710035147 ura3 Proteins 0.000 description 1
- 230000003612 virological Effects 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002912 waste gas Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 101710013713 xcpT Proteins 0.000 description 1
- 101700044806 yiiQ Proteins 0.000 description 1
- 101700000117 yiiR Proteins 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
Abstract
The present invention provides an improved method for the biological production of 1, 3-propanediol from a fermentable carbon source in a single microorganism. In one aspect of the present invention, an improved process for the conversion of glucose to 1,3-propanediol is achieved by the use of an E. coli transformed with the Klebsiella pneumoniae dha regulon genes dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ, all these genes arranged in the same genetic organization as found in wild type Klebsiella pneumoniae. In another aspect of the present invention, an improved process for the production of 1,3-propanediol from glucose using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, and a dehydratase reactivation factor compared to an identical process using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, a dehydratase reactivation factor and a 1,3-propanediol oxidoreductase (dhaT). The dramatically improved process relies on the presence in E. Coli of a gene encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol.
Description
PROCESS FOR L? BIOLOGICAL PRODUCTION OF 1,3-PROPANODIOL FIELD OF THE INVENTION This invention comprises a process for the bioconversion of a fermentable coal source to 1,3-propanediol by a simple microorganism. BACKGROUND OF THE INVENTION 1,3-propanediol is a monomer which has a potential utility in the production of polyester fibers and in the manufacture of polyurethanes and cyclic compounds.
Various chemical routes for 1,3-propanediol are known, for example, ethylene oxide can be converted to 1,3-propanedicol on a catalyst, in the presence of phosphines, water, carbon monoxide, hydrogen and an acid, the catalytic hydration in solution phase of acrolein followed by reduction, or from compounds such as glycerol, which react in the presence of carbon monoxide and hydrogen on catalysts that has atoms of the group
VIII of the periodic table Although it is possible to generate 1, 3-propanediol by these methods, they are expensive and generate waste streams that contain environmental contaminants. It is known more than a century ago that 1,3-propanediol can be produced from fermentation
Rβ: 135575
of glycerol. Bacterial strains capable of producing 1,3-propanediol have been found, for example, in the groups Ci trobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, La ctobacillus, and Pelobacter. In the case studied, the glycerol is converted to 1,3-propanediol, in a 2-step reaction sequence catalyzed by enzymes. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) and water, equation 1. In the second stage, 3-HPA is reduced to 1,3-propanediol by an oxidized reductase linked to NAD + equation 2. 1,3-propanediol is not further metabolized and as a result, glycerol? 3-HPA + H20 (equation 1)
3-HPA + NADH + H +? 1, 3-pr? Panodiol + NAD + (equation 2) accumulates in the media, The overall reaction consumes a reducing equivalent in the form of a cofactor, reduced ß-nicotinamide adenine dinucleotide (NADH), which is oxidized to the nicotine dinucleotide amide adenine (NAD +) In the Klebsiella pneu onia, Ci trobacter freundii, and Clos tridi um pasteurianum, the genes encoding the three structural units of glycerol dehydratase [dhaBl -3 or dhaB, C and E) are located adjacent to a gene which encodes 1,3-propanediol oxide reductase specific. { dha T) (see figure 1). Although the genetic organization
differs in some way between these microorganisms, these genes are grouped in-. an group that also comprises orfX and orfZ (genes that encode a reactivation factor of dehydratase for glycerol dehydratase) as well as orfY or orfW (genes of unknown function). The specific 1, 3-propanediol oxidereductase are known. { dha T) of these microorganisms, for belonging to the family of alcohol dehydrogenases type III; each shows a conserved portion of the linkage to the iron and has a preference for the interconversion of 1,3-propanediol linked to NAD + / NADH and 3-HPA. However, the interconversion of 1,3-propanediol linked to NAD + / NADH and 3-HPA is also catalyzed by alcohol dehydrogenase which does not bind specifically to dehydratase enzymes (eg, baker's yeast alcohol dehydrogenase and of the horse's liver (EC1.1.1.1)), although with less efficient kinetic parameters. Glicei ol dehydratase (E.C.4.2.1.30) and diol (1,2-propanediol) dehydratase (E.C..2.1.28) are related, but different 3 enzymes are encoded by different genes. The genes of the dihydratase dehydratase of Klebsiella oxytoca and Salmon ella typhimuri? M are similar to the genes of glycerol dehydratase, and bind in a group comprising the genes analogous to orf and orfZ
propanodiol oxide reductase. { dha T), glycerol dehydrogenase. { dhaD), and.- ihydroxyacetone kinase. { dhaK) are encompassed by the regution of dha. The dha reguión, in Klebsiella pneumoniae and Ci trobacter freundii, also encompass a gene coding for a transcriptional activating protein (d aR). The regulons of dha of Ci trobacter and Klebsiella s have been expressed in Escherichia coli and have been shown to convert glycerol to 1,3-propanediol. No chemical or biological method described above for the production of 1,3-propanediol is well suited for production on an industrial scale, since the chemical processes are energy intensive and the biological processes are limited to the relatively low concentration of the mat. costly starting waste, glycerol. These drawbacks can be overcome with a method that requires a <; Low energy level and a non-expensive starting material such as carbohydrates or sugars, or by increasing the metabolic efficiency of a glycerol process. The development of any method will require the ability to manipulate the genetic machinery responsible for the conversion of sugars to glycerol and glycerol to 1,3-propanediol. The biological processes for the preparation of glycerol are known. The abundance of producers
of glycerol are yeasts, but some bacteria different from fungi and. algae are also known. Both bacteria and yeast produce glycerol by converting glucose or other carbohydrates through the path of fruiting-1, 6-bisphosphate in glycolysis, or in the trajectories of Embden Meyerhof Parnas, while closing algae convert dissolved carbon dioxide or carbonate into the chloroplasts within the 3-carbon intermediates of the Calvin cycle. In a series of steps, the 3-carbon intermediate, phosofoglyceric acid, is converted to the 3-phosphate glyceraldehyde which can be easily interconverted to its isomer c-sto, dihydroxyacetone phosphate and finally to glycerol. Specifically, the bacteria Bacill us licheniformis and Lactobaci. lus lycopersi ca, synthesize glycerol, and the production of glycerol is found in the halotolerant algae of the species Dunaliella and Asteromas gracilis for protection against high concentrations of external salt. Similarly, various osmotolerant yeasts synthesize glycerol as a protective measure. Most strains of Saccharomyces produce some glycerol 1 during alcoholic fermentation, and this can be increased physiologically by the application of osmotic ion. At the beginning of
This century, commercial production of glycerol was achieved by the use of Saccharomyces cultures to which "management reagents" were added such as sulfites or alkalis. Through the formation of an inactive complex, the management agents block or inhibit the conversion of acetaldehyde to ethanol, thus, the equivalents that reduce the excess (NADH) are available or are "managed" towards the DHAP for the reduction to produce glycerol. This method is limited by partial inhibition of yeast growth, which is due to sulfites. This limitation can be partially overcome} by the use of alkalis that create equivalents in excess of (NADH) with a different mechanism. In this practice, the alkalis initiate a disproportion of Cannizarrc to produce ethanol and acetic acid from two equivalents of acetaldehyde. The gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and formed in sequence from d B S. Diasta ticus (Wang et al, J.Bact 176, 7091-7095 (1994) .The DAR1 gene was cloned into a transfer vector and used to transform E. coli where the expression produces an active enzyme. Wang et al. (Supra) recognize that DAR1 is regulated by the cellular osmotic medium but does not suggest the way in which the gene can
to be used to enrich the production of 1,3-propanediol in a recombinant microorganism. Other enzymes of glycerol 3-phosphate dehydrogenase have been isolated, for example, sn-glycerol-3-phosphate dehydrogenase has been cloned and sequenced from Saccharomyces cerevisiae (Larason et al., Mol.Microbiol.10, 1101 (1993)) and Albertyn et al., (Mol.Cel.] Biol. 14, 4135 (1994)), teach the cloning of GPDI encoding a glycerol-3-phosphate dsehydrogenase from Saccharomyces cerevisiae.
As Wang et al. (Supra), both Albertyn et al. And Larason et al. Recognize the osmo-sensitivity of the regulation of this gene, but do not suggest how the gene can be used in the production of 1, 3- propanediol in a recombinant microorganism. As with G3PDH, sle has isolated glycerol 3-phosphate from Saccharomyces cerevisiae and the protein identified as encoded by the GPP1 and GPP2 genes (Norbeck et al., J. Biol. Chem. 271,
13875 (1996)). Like the genes that encode the G3PDH gene, it seems that GPP2 is osmosensitive. Although it is desirable to convert a simple microorganism from a source of fermentable carbon different from glycerol or dihydroxyacetone to the 1,3-
glycerol dehydratase, does not produce 1, 3-propanediol glucose or xylose in the absence of exogenous glycerol Efforts to improve the yield of 1,3-propanediol from glycerol, have been reported where co-substrates capable of supplying equivalents of reduction, typically fermentable sugars, are included in the process. Improvements have been claimed in yields for the resting cells of Ci trobacter freundii and Klebsiella pneumoniae DSM 4270 which co-ferment glycerol and glucose (Gottschalk et al., Supra).; and Tran-Dinh et al., DE 3734 764); but not for growing cells of
Klebsiella pneumoniae ATCC 25955 which co-ferments glycerol and glucose, which do not produce 1,3-propanediol (I-T. Tong, Ph.D. Thesis, University of Wisconsin-Madison (1992)). Increasing yields have been reported by the co-fermentation of glycerol and glucose or fructose by an Escheri chia coli recombmanjte; Nevertheless; 1,3-propanediol is not produced in the absence of glycerol (Tong et al., supra.). In these systems, the simple microorganisms use the carbohydrate as a source of NADH that dries, while energy and carbon are supplied for the maintenance or growth of the cells. These descriptions suggest that the
sugars do not enter the carbon stream that produces 1,3-propanediol. Recently however, the conversion of carbon substrates other than glycerol or dihydroxyacetone to 1,3-propanediol by a simple microorganism expressing a dehydratase enzyme have been described (US Patents 5,686,276, WO 9821339, WO 9928480; 98211341 (US 6013494)). A specific deficiency in the biological processes that lead to the production of 1,3-propanediol from glycerol or glucose, has been the low concentration of the product that is reached through fermentation, thus, it requires an intensive separation process and energy to obtain the 1,3-propanediol from an aqueous fermentation broth. The fermentations in otes or in batches fed glycerol for 1,3-propanediol, have led to final concentrations of 65 g / 1 by Clostridium.
Butyri cum (Saint-Amans et al., Biotechnology Letters 16, 831 (1994)), 71 g / 1 of Clostridium um butyricum mutants (Abad-Andaloussi et al., Appl. Environ Microbiol. 6144 13 (1995)), 61 g / 1 of Klebsiella pneu omae (Oman et al., Appl Microbiol. Biotechnol 33, 121 (19 90)), and 35 g / 1 of Citrobacter freundi i (Oman et al., supra). The
fermentations of the 1,3-propanediol gucose, which exceed the concentration obtained from fermentations with glycerol, have not been described. The problem that remains to be solved is the way to produce 1, 3-propanediol biologically, with high concentration and a simple microorganism, from a low-cost carbon substrate such as glucose and other sugars. 1,3-propanediol requires glycerol as a substrate for a sequential reaction in do: stages, in which a dehydratase enzyme (typically a dehydratase-dependent coenzyme Bi2) converts to glycerol in an intermediate 3-hydroxypri-propadehyde, which is then reduced to 1,3-propanedic 1 by an NADH- (or NADPH) -dependent oxide reductase. The complexity of the requirement of the cofact.or, requires the use of a whole cell catalyst for an industrial process that uses this reaction sequence for the production of 1,3-propanediol. BRIEF DESCRIPTION OF THE INVENTION Applicants have solved the established problem, and the present invention provides the bioconversion of a fermentable charcoal body, directly to 1,3-prop.-indole at a concentration significantly higher than that previously obtained and
with the use of a simple microorganism. Glucose is used as a model substrate and E. coli is used as a model host. In one aspect of this invention, recombinant E. coli expresses a group of genes (comprising genes encoding the activity of dehydratase, a factor of reactivation of dehydratase, a 1,3-propanediol oxide reductase. ), a glycerol 1,3 phosphate eshidrogenasa, and a glycerol
3-phosphatase) which converts glucose to 1,3-propanediol in concentrations approaching those of glycerol for fermentations of 1,3-propanediol. In another aspect of this invention, the elimination of the functional gene dha T in this E. Recombinant coli results in a significantly higher concentration of 1,3-propanediol from glucose. This unexpected increase in the concentration, results in an improved economy and thus, an improved process for the production of 1,3-propanediol from glucose. In addition, the present invention can generally be applied to include any carbon substrate that is readily converted to 1) glycerol, 2) dihydroxyacetone, 3) compjests C3 in the oxidation state of glycerol (eg, glycerol 3-phosphate), or 4) C3 compounds in the oxidation state of dihydroxyacetone (for example, hydroxyacetone phosphate)
Aerobacter, Lactobacillus Aspergillus, Saccharomyces, Schizosa ccharomyces, Zygosaccharomyces, Pichia,
Kl uveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methy wolf cte r, Salmonella, Bacillus, Aerobacter, Streptomyces, Escherichia, and Pseudomonas. E. coli is the preferred host. Accordingly, the present invention provides a recombinant microorganism, useful for the production of 1,3-propanediol comprising: (a) at least one gene encoding a polypeptide having a glycerol 3-phosphate dehydrogenase activity; (b) at least one gene encoding a polypeptide having glycerol 3-phosphatase activity; (c) at least one gene encoding a polypeptide having dehydratase activity; (d) at least one gene encoding a dehydratase reactivation factor; (e) at least one endogenous gene encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol, wherein no dha T functional gene coding for 1,3-propanediol oxide reductase is present, Preferred modality is in recombinant microorganism
(preferably E. coli) where no dha T gene is present. Optionally, the recombinant microorganism may comprise mutations (e.g., deletion mutations or dot mutations) in endogenous genes.
selected from the group consisting of: (a) a gene encoding a polypeptide having glycerol kinase activity; (b) a gene encoding a polypeptide has glycerol dehydrogenase activity; (c) a gene encoding a polypeptide having triosephosphate isomerase activity. In another embodiment, the invention includes a process for the production of 1,3-propanediol which comprises: (a) appropriate reactions, a recombinant E. coli comprising a dah regimen and lacking a functional dha T gene which encodes a 1,3-propanediol oxide reductase activity, with at least one carbon source, wherein the carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and simple carbon substrates; (b) optionally recovering the 1,3-propanediol produced in (a). The invention also provides a process for the production of 1,3-propanocyan comprising: (a) contacting the recombinant organism of the present invention, with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and simple carbon substrates whereby 1,3-propanediol is produced; and (b)
optionally recovering 1, 3-propanediol produced in (a). Similarly, the invention aims to provide a process for the production of 1,3-propanediol from a recombinant microorganism comprising: (a) contacting a recombinant microorganism with at least one carbon source, the recombinant microorganism < 2 comprises: (i) at least one gene encoding a polypeptide having a dehydratase activity. (ii) at least one geh encoding a reactivation factor for Ja dehydratase, (iii) at least one endogenous gene encoding a specific catalytic activity sufficient to convert the 3-nidroxypropionaldehyde to 1,3-propanediol; wherein no functional dha T gene encoding: a to 1,3-propanediol oxide reductase is present; the carbon source selected from the group consisting of glycerol and dihydroxyacetone, wherein 1,3-propanediol is produced and (b) optionally recovering the 1,3-ptopanediol produced in (a) Still another aspect of the invention is provided for co-feeding the carbon substrate. In this production mode of 1,3-
propane diol the steps so: n: (a) contact of a recombinant E. coli with a first carbon source and with a second carbon source, the recombinant E. coli comprises (i) at least one exogenous gene encoding a polypeptide which has dehydratase activity; (ii) at least one exogenous gene encoding a dehydratase reactivation factor; (iii) at least one exogenous gene encoding a non-specific catalytic activity sufficient to convert the three hydroxypropionaldehyde to 1,3-propanediol wherein no dha T functional gene encoding the activity of 1,3-propanediol oxide reductase is present in E. coli, wherein the first carbon source is selected from the group consisting of glycerol and dihydroxyacetone, and the second carbon source is selected from the group consisting of: monosaccharides, oligosaccharides, polysaccharides and simple carbon substrates and ( b) the 1,3-propanediol produced in a) is optionally recovered. The co-feeding can be sequential or simultaneous. Recombinant E. coli used in a co-feeding modality may also comprise
(a) a set of exogenous genes consisting of (i) at least one gene encoding a polypeptide having glycerol 3 dehydrogenase activity; (ii) at least one gene encoding a polypeptide having activity of
glycerol 3-phosphatase; (iii) at least a subset of genes encoding the genes of dhaR genes, orfY, orfX, orfW, dhaBl, dhaB2, dhaB3 and orfZ, and (b) a set of endogenous genes, each gene having a mutation that inactivates to the gene, the set consists of: (i) a gene encoding a polypeptide having glycerol kinase activity; (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity; and (iii) a gene encoding a polypeptide having triosephosphate isomerase activity. Useful E. coli recombinant strains include the recombinant strain of E co. KLP23 comprising: (a) a set of two endogenous genes each gene has a mutation that inactivates the gene, the set consisting of: (i) a gene encoding a polypeptide having a glycerol kinase activity; and (ii) a gene encoding a polypeptide having a glycerol dehydrogenase activity; (b) at least one exogenous gene encoding a polypeptide having glycerol 3-phosphate acid activity; (c) at least one exogenous gene encoding a polypeptide having glycerol dephosphatase activity; (d) a plasmid pKP32 and a recombinant strain of E. coli RJ8 comprising (a) a set of three endogenous genes, each gene having a mutation that inactivates the gene, the conjugate consists of (i) a gene that
encodes a polypeptide having a glycerol kinase activity; (ii) a gene encoding a polypeptide having glycerol dehydrogenase activity, and (iii) a gene encoding a polypeptide having a triosephosphate isomerase activity Other useful embodiments include a recombinant E. coli comprising: (a) a set of exogenous genes consisting of d: (i) at least one gene encoding a polypeptide having dehydratase activity; (ii) at least one gene encoding a polypeptide having glycerol 3-phosphate dehydrogenase activity; ) at least one gene encoding a polypeptide having glycerol 3-phosphatase activity;
(iv) at least one gene encoding a dehydrotatase reactivation factor; and (b) at least one endogenous gene coding for non-specific catalytic activity to convert 3-hydroxypropionaldehyde to
1,3-propanediol, wherein no functional dha T gene encoding the activity of 1,3-propanediol oxide reductase is present in recombinant E. coli. Another embodiment is a recombinant E. coli comprising: (a) a set of exogenous genes consisting of (i) at least one gene encoding a polypeptide having glycerol 3-phosphate activity
dehydrogenase; (ii) at least one gene encoding a polypeptide having glycerol 3-phosphatase activity; and (iii) at least one subset of genes encoding the gene products of dhaR, orfY, orfX, orfW, dhaBl, dhaB2, dhaB3 and orfZ, and (b) at least one endogenous gene encoding a non-specific catalytic activity to convert 3-hydroxypropionaldehyde to 1,3-propanediol, wherein no functional dhaT gene encoding the activity of 1,3-propynediol oxide reductase is present in recombinant coli. This embodiment also includes a process using a recombinant coli which also comprises a set of endogenous genes, each gene has a mutation that inactivates the gene, the set consists of: (a) a gene encoding a polypeptide having glycerol activity kinase;
(b) a gene encoding a polypeptide, having glycerol dehydrogenase activity, and (c) a gene that modifies a polypeptide having triosephosphate isomerase activity. This embodiment still further includes a process for the 1,3-propanediol bioproduction comprising: (a) contacting, under appropriate conditions, the immediately described recombinant E. coli, with at least one carbon source selected from the group consisting of of monosaccharides, oligosaccharides, polysaccharides and
carbon-carbon substrates, whereby 1,3-propanediol is produced and; (b) r < optionally take up the 1,3-propanediol produced in e. (to) . And it also includes an additional process for the bioproduction of 1,3-pr Dipanediol which comprises: (a) contacting the recombinant coli of the immediately described modalities further comprising: (i) at least one exogenous gene encoding a polypeptide which has a dehydratase effect; (ii) at least one exogenous gene encoding a dehydcatase reactivation factor; (iii) at least one endogenous gene encoding non-specific catalytic activity to convert 3-hydroxypropionaldehyde to 1,3-propanediol with a carbon source selected from the group consisting of glycerol and hydroxyacetone at the MS, and (b) optionally recovering to 1,3-propanediol produced in (a) Brief description of the drawings, sequence descriptions and biological deposits The invention can be understood more fully from the following detailed description, figures, the accompanying sequence descriptions, and the biological deposits that are part of this application. Figure 1 presents] The organization of genes within the dha regution sequence of sub clone pHK28-26.
showing the endogenous activity of the E. coli oxide reductase (non-specific catalytic activity) on a native gel. The 68 sequence descriptions and the attached sequence listing will comply with the rules governing nucleotide and / or amino acid sequence descriptions in patent applications, as set forth in 37 C.F.R. § 1,821-1,825 ("Requirements for Patent
Applications Containing Nu leotide Sequences and / or Amino Acid Sequence Disclosures-the Sequence rules ") and will be consistent with the World Intellectual Property Organization (WIPO) standard, ST2.5 (1998) and the requirements of the sequence listing of the EPO and PCT (rules 5.2 and 49.5 (a-bis), and section 208 and annex C of the Administration Instructions.) The sequence descriptions contain the one-letter code for the nucleotide sequence characters, and the coding codes. 3 letters for amino acids as defined in accordance with the IUPAC-IYUB standards described in Nucleic Acids Res. 13,302 1-3030 (1985) and in Biochemical Journal 219,345-373 (1984) which are incorporated herein by reference. ID N0: 1 contains the nucleotide sequence determined from a 12.1 kb EcoRI-SalI fragment of pKPl (a DNA containing cosmids from the
Reference to the Designation Date of the deposit identification of the International Depositary Depositary DH50C of B. coli ATCC 69789 April 18, 1995 transfo mada containing a portion of the Klebsiella genome encoding the glycerol dehydratase enzyme DH5a of E. coli ATCC 69790 April 18, 1995 transformed containing the cosmid pKP4 containing a portion of the Klebsiella genome encoding a diol dehydratase E enzyme. col ± MSP33.6 ATCC 98598 November 25, 1997
RJF lOm de E. col ± ATCC 98597 November 25, 1997 mutant glpK
The deposits will remain in the indicated international depository for at least 30 years, and will be made available to the public when a patent is granted describing them. The availability of a deposit does not constitute a license to practice
invention object in derogation of patent rights granted by government action. As used herein, "ATCC" refers to the international depository of the American Type Culture Collection located at 10810 University BIvd .., Manassas, VA 20110-2209 USA. "ATCC Number" is the access number for crops in deposit with the ATCC Detailed description of the invention The present invention provides an improved process for the bioconversion of a fermentable carbon source, directly 1, 3-propanediol, using a simple microorganism. The method is characterized by a concentration, improved cell viability and performance, as well as a decrease in cell lysis during fermentation. The present invention1 is based in part on the observation that the fermentation processes of 1,3-propanediol comprising 1,3-propanediol oxide reductase. { dha T) are characterized by high levels of 3HPA and other aldehydes and ketones in the medium, which correlates with a lack of cellular viability. The present invention is also based, in part, on the unexpected finding that model hosts, E. coli, is capable of converting 3-HPA to 1,3-propanediol, by an endogenous catalytic activity not
specific, capable of converting 3-hydroxypropionaldehyde to 1,3-propanediol. The present invention is also based in part on the unexpected finding that an E. coli fermentation process comprising this non-specific catalytic activity and lacking a functional dha T results in increased cell viability during fermentation and it provides higher concentrations and / or yields of 1,3-propanediol, than a fermentation process comprising a dha T functions]. In one aspect, the gliferol is a model substrate, the microorganism has a mutation in the wild type dha T such that there is no 1,3-propanediol oxide reductase activity, which comprises a catalytic activity not specific enough to convert the 3-hydroxypropionaldehyde to 1,3-propanediol. In another aspect, glucose is a model substrate and recombinant E. coli is a model host. In this regard, E. coli comprises a non-specific endogenous catalytic activity sufficient to convert 3-hydroxypropionaldehyde 1,3-propanediol. In one embodiment, the non-specific catalytic activity is an alcohol dehydrogenase. In one aspect, the present invention provides a recombinant E. coli that expresses a group of genes that
comprise (a) at least 1 a gene encoding a polypeptide having glycerol phosphate dehydrogenase activity; (b) at least one gene encoding a polypeptide having glycerol 3-phosphatase activity; (c) at least one gene encoding a polypeptide having a dehydratase activity; (d) at least one gene encoding a reactivation factor in the dehydratase; (e) at least one endogenous gene encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol; the use of this microorganism converts glucose to 1,3-propanediol at a high concentration. In another aspect of this invention, the elimination of the dha T functional gene in this recombinant E. coli provides an unexpected higher concentration of 1,3-propanediol from glucose than previously appreciated. The present invention provides an improved method for the biological production of 1,3-propanediol, from a fermentable carbon source in a simple microorganism. In one aspect of the present invention, an improved process for the conversion of glucose to 1,3-propanediol is achieved by the use of a recombinant microorganism comprising a host E. coli transformed with the Klebsiella dha regimen.
pneumoniae genes from dhaR, orfY, dha T, orfX, orfW, dhaBl, dhaB2, dhaB3, and orfZ, all these genes placed in the same genetic organization as found in the wild type Klebsiella pneumoniae. The concentration obtained for the fermentation process is significantly higher than any previously reported concentration for a similar fermentation. This improvement is based on the us30 of plasmid pDT29 as described in example 6 and example 7. In another aspect of the present invention, an additional improved process for the production of 1,3-propanediol from glucose is achieved, using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, and a reactivation factor of dehydratase compared to a process using a recombinant E. coli containing genes encoding G3PDH, a G3P phosphatase , a dehydratase, a reactivation factor of the dehydratase and also a functional Dha T. The dramatically improved process is based on an endogenous gene that encodes a non-specific catalytic activity, expected to be an alcohol dehydarogenase that is present in E. coli. The dramatic improvement in the process is evident as an increase in the 1,3-propanediol concentration,
as illustrated in Examples 7 and 9. The improvement in the process is also evident as a decrease in cell lysis as determined by the extra cellular concentration of soluble proteins in the fermentation broth. This aspect of the invention is illustrated in Figure 2. Additionally, the improvement in the process is evident as the prolonged cell viability during the course of the fermentation. This aspect of the invention is illustrated in Figure 3. In addition, improvement in the process is also evident as an increase in performance. In E. coli expressing a 1,3-propanediol oxide reductas a. { dha T) (for example, E. coli KLP23 transformed with plasmid pDT29), glycerol can be metabolized to a product different from 3-HPA. In direct contrast, in E coli that does not express a 1,3-propanediol oxide reductase. { dhaT) (e.g., E. coli KLP23 transformed with plasmid pKP32), glycerol is not metabolized to a product other than 3-HPA. That this crypt: .ca trajectory is attributed to the presence or absence of a functional dhaT, is demonstrated by the lower glycerol yield from glucose as illustrated in Figure 4. As used herein, the following terms can be used for the interpretation of the claims and the description.
The terms "glyceryl-3-phosphate dehydrogenase" and
"G3PDH", refer to a polypeptide responsible for an activity of enzymes that catalyze the conversion of dihydroxyacetone phosphate. { DHAP) to glycerol-3-phosphate (G3P). The G3PDH in VÍVD can be NADH; NADPH; or dependent on FAD. When specifically referring to a glycerol-3-phosphafo dehydrogenase specific co-factor, the terms, "NADH-dependent glycerol-3-phosphate dehydrogenase", "NADPH-dependent glycerol-3-phosphate dehydrogenase" and "glycerol-3" -FDA-dependent phosphate dehydrogenase "will be used. As is generally the case with NADPH dependent and NADH-dependent glycerol-3-phosphate dehydrogenase, NADH and NADPH can be used interchangeably
(for example by the ger.coded by gpsA), the terms NADPH-dependent glycerol-3-phosphate dehydrogenase and NADH dependent will be used interchangeably. The enzyme dependent on NADH (EC
1. 1.1.8) is encoded for example by various genes including GPDI (GenBank Z74071x2), or GPD2 (GenBank Z35169xl), or GPD3 (Genbank G984182), or DAR1 (Genbank Z74071x2). The NADPH-dependent enzyme (EC 1.1.1.94!) Is encoded by gpsA (Genbank U321643, (cds 197911-196892) G466746 and L45246) The FAD-dependent enzyme (EC 1.1.99.5) is encoded pbr GUT2 (GenBank Z47047x23), or
glpD (GenBank G147838), or glpABC (GenBank M20938) see WO 9928480 and references therein, which are incorporated herein by reference) The terms "glycero 1-3-phosphatase", "sn-glycerol-3-phosphatase", or " d, 1-glycerc 1 phosphatase ", and" G3P phofatase "refer to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol-3-phosphatase and water to glycerol1 and inorganic phosphate. G3P phosphatase is encoded by example, by GPP1 (GenBank Z47047x25), or GPP2 (GenBaiik U18813xll) (see WO 9928480 and references therein, which are incorporated herein by reference). The term "glycero kinase" refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol and ATP to glycerol-3-phosphate and ADP. The high-energy phosphate ATP donor can be replaced by physiological substitutes (eg, phosphono pyruvate). Glycerol kinase is encoded, for example, by G1JT1 (GenBank U11583xl9) and glpK (GenBank L19201) (see WO 9928480 and references therein, which are incorporated herein by reference). The term "glycerol dehydrogenase" refers to a polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol to dihydroxyacetone (E.C.1.1.1.6) or glycerol to glyceraldehyde (E.C.1.1.1.72)
A polypeptide responsible for an enzyme activity that catalyzes the conversion of glycerol hydroxyacetone is also referred to as "dihydroxyacetone reductase". Glycerol dehydrogenase may depend on NADH (E.C.1.1.1.6), NADH (E.C.1.1.1.72), or other cofactors (eg, E.C.1.1.99.2). NADH-dependent glycerol dehydrogenase is encoded, for example, by gldA (GenBank U00006) (see WO 9928480 and references therein, which are incorporated herein by reference.) The term "dehydratase enzyme" or "dehydratase" will refer to any activity of enzymes, which catalyzes the conversion of a glycerol molecule to a 3-hydroxypropionaldehyde product For the purposes of the present invention, dehydratase enzymes include a glycerol dehydratase (EC4.2.1.30) and a diol dehydratase (EC4.2. 1.28) having preferred substrates of glycerol and 1,2-propanediol respectively The genes for the dehydratase enzymes have been identified in Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium, and Klebsiella oxytoca In each case, the dehydratase is It consists of 3 sub units: the major sub unit or "a", the medium subunit or "ß", and the small sub unit or "?". Due to a wide variation in a none nclature of genes used in the
literature, a comparative table is provided in table 1 to facilitate identification. Genes are also described by way of example, Daniel et al, (FEMS Microbiol, Rev. 22, 553 (1999)) and Toraya and Mori (J. Biol. Chem. 274, 3372 (19S 9)). With reference to the table
1, genes encoding the major subunit or "a" of glycerol dehydratase include the genes dhaBl, gldA, and dhaB; genes encoding the sub-mediated unit or "ß" include dhaB2, gldB, and dhaC; the genes that code the small sub unit or "?" which include dhaB3, gldC, and dhaE.Also as a reference in table 1, the genes encoding the large subunit or "a" of the diol dehydratase including pduC and pddA, the genes of the sub-media or "ß" "include pduD and pddB; genes encoding the small subunit or"? "include pduE and pddC.
Table 1: Comparative table of gene names and GenBank references for dehydratase and functions linked to dehydratase GEN FUNCTION:
FUNCTION OF THE GEN:
20
Glycerol and diol dehydratases are subject to inactivation by mechanism-based inactivation by glycerol and some other substrates (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999)). The term "dehydratase reactivation factor" refers to those proteins responsible for the reactivation of dehydratase activity. The terms "rehydration activity of dehydratase", "reactivation of dehydratase activity" or "regeneration of dehydratase activity", refers to the conversion phenomenon of a dehydratase, which is not capable of catalyzing a substrate to one capable of catalyzing a substrate to the phenomenon of inhibiting the inactivation of a dehydratase, or to the phenomenon of inhibiting the inactivation of a dehydratase, or to the phenomenon of extending the average useful life of the dehydratase enzyme in vivo. Two proteins have been identified as being involved in the dehydratase reactivation factor (see WO 982134KUS 6013494) and references therein, which are hereby incorporated by reference; Daniel and collaborators, supra; Toraya and Mori, J. Biol,
Chem. 274, 3372 (1999); and Tobimatsu et al., J. Bacteriol. 181, 4110 (1999. With reference to table 1, the genes that encode one of the proteins that include
• > rfZ, dhaB4, gdrA, pduG and ddrA. Also with reference to Table 1, the genes encoding the second of the two proteins include orfX, orf2b, gdrB, pduH and ddrB. The terms "1,3-propanediol oxide reductase '" 1,3-propanediol dehydrog = nasa "or" Dha T "refer to the polypeptide responsible for an enzyme activity that is capable of catalyzing the interconversion of 3-HPA and 1 , 3-propanediol, with the proviso that the genes encoding such activity are found to be physically or transcriptionally linked to a dehydratase enzyme in its natural environment (this is wild type), for example, the gene is within of a dha regution as is the case of dha T of Klebsiella pneumoniae With reference to table 1, the genes encoding 1,3-propanediol oxide reductase include the dha T of Klebsiella pneumoniae, Ci trobacter freundii, and Clostridium Each of these genes encodes a polypeptide that belongs to the family of type III alcohol dehydrogenases that show a conserved portion of the iron snoring and have preference for the interconnection linked with NAD + / NADH of 3-HP A and 1,3-propanediol (Johnson and Lin, J. Bacteriol. 169, 2050 (1987); Daniel et al., J. Bacteriol. 177, 2151 (1995); and Leurs and collaborators,
FEMS Microbiol. Lett. 154,337 (1997). Enzymes with similar physical properties have been isolated from
Lactobacillus brevis and La ctobacillus bucheri (Veiga from Dunha and Foster, Appl. Environ.Microbiol. 58, 2005 (1992)). The term "regution dha" refers to a set of associated genes or open reading structures that encode various biological activities, including but not limited to a dehydratase activity, a reactivation activity, and a 1,3-propanediol oxide. reductase. Typically a regution of dha, comprises the open reading structures dhaR, orfY, dha T, orfX, orfW, dhaBl, dhaB2, dhaB3, and orfZ, as described herein. The term "non-specific catalytic activity" refers to the polypeptides responsible for an enzyme activity, which is sufficient to catalyze the interconnection of 3-HPA and 1,3-propanediol and exclusively excludes 1,3-propanediol oxide reductase. Typically, these enzymes are alcohol dehydrogenases.
Such enzymes can utilize different NADVNADH cofactors, including but not limited to flavins such as FAD or FMN. A gene for a non-specific alcohol dehydrogenase is found, for example, to be encoded
endogenously and functionally expressed within the microorganism E. coli KL, P23. The terms "function" or "enzyme function" refer to the datealitic activity of an enzyme in the alteration of the energy required to carry out a specific chemical reaction. It is understood that such an activity can be applied to a reaction in equilibrium, where the production of the product or substrate can be achieved under appropriate conditions. { The terms "substra or carbon" and "carbon source" refer to a carbon source capable of metabolizing by host microorgaisms in the present invention, and particulate selected carbon sources: "and" proteins "are used interchangeably. of the group consisting of monosaccharides, oligosaccharides, polysaccharides, and substrates of a carbon or mixtures thereof The terms "host cell" or "host microorganisms" refer to a microorganism capable of receiving external or heterologous genes and of expressing those genes for produce a gene product, the terms "gene e > "Erno" and "external DNA", "heterologous gene" and "heterologous DNA" refer to a natural genetic material for an organism, which has been placed
within a host microorganism by various means. The gene of interest may be a gene that occurs naturally, a mutated gene or a synthetic gene. The terms "transformation" and "transfection"; they refer to the acquisition of new genes in a cell after the incorporation of the nucleic acid. Acquired genes can be integrated into chromosomal DNA, or introduced as extrachromosomal replicating frequencies. The term "transformant" refers to the product of a transformation. The term "genetically altered" refers to the process of changing hereditary material by transformation or mutation. The terms "recombinant microorganism" and "transformed host" refer to any microorganism that has been transformed with external or heterologous genes c additional copies of homologous genes The recombinant microorganisms of the present invention express genes encoding glycerol-3-phosphate dehydrogenase (GPDI), glycerol-3-phosphatase (GPP2), glycerc 1 dehydratase (dhaBl, d aB2 and dnaB3), reactivation factor dehydratase (.orfZ and orfX), and optionally 1,3-propanediol oxide reductase { dha T) for the production of 1,3-propanediol from unsuitable carbon substrates.
Preferred embodiment is an E. coli transformed with these genes but lacking a functional Dha T. A host microorganism other than E. coli can also be transformed to contain the genes described and the gene for non-specific catalytic activity for the interconversion of 3-HPA and 1,3-propanediol, specifically excluding 1,3-propanediol oxide reductase. { dha T) Gene "refers to a nucleic acid fragment that expresses a specific protein including the regulatory sequences that precede (non-coding 5 ') and that follow (non-coding 3') the coding region. "and" wild type "refers to a gene as it is found in nature with its own regulatory sequences. The terms "coding" and "coding" refer to the process by which a gene, through the mechanisms of transcription and translation, produce an amino acid sequence. Is it understood that the process of coding a specific amino acid sequence includes DNA sequences that may involve base changes, which it does not provoke? a change in the encoded amino acid, or that involve base changes that can alter one or more amino acids, but do not affect the functional properties of the protein encoded by the
DNA sequence. It will be understood therefore that the invention encompasses more than the specific exemplary sequences. The term "isolated" refers to a protein or DNA sequence that is separated from at least one component by which they naturally associate. An "isolated nucleic acid molecule" is an RNA or DNA polymer that has a double or single strand , which optionally contains altered or non-natural, synthetic nucleotide bases. An isolated nucleic acid molecule in the form of a DNA polymer can comprise one or more segments of cDNA, genomic DNA or synthetic DNA. "Substantially similar" refers to nucleic acid molecules wherein changes in one or more nucleotide bases result in the substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. "substantially similar" also refers to nucleic acid molecules, wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid molecule to mediate the alteration of gene expression by anti-sense or anti-sense technology. co-suppression. "Substantially similar" also refers to modifications of acid molecules
s. . . , "" Isfe
nucleic acid of the current invention (such as deletion or insertion of one or more nucleotide nases) that do not substantially affect the functional properties of the resulting transcript, against the ability to mediate the alteration of gene expression by antisense or co-antisense technology. deletion, or alteration of the functional properties of the resulting protein molecule. The invention encompasses more than the specific exemplary sequences. For example, it is well known in the art that alterations in a gene that result in the production of a chemically equivalent amino acid at a given site, but that do not affect the functional properties of the encoded protein, are common. For the purposes of the present invention, substitutions are defined as exchanges within? Of the following five groups 1. Lightly polar, non-polar, small aliphatic residues: Ala, Ser, Thr (Pro, Gly) Polarly-charged residues, polar and its amides: Asp, Asn, Glu, Gln; 3. Positively charged, polar residues: His, Arg, Lys; Large non-polar aliphatic wastes: Met, Leu, Lie, Val (Cys); Y
Large aromatic residues: Phe, Try, Trp. Thus, a codon for the amino acid of alanine, a hydrophobic amino acid, can be replaced by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes that result in the substitution of a negatively charged residue for another (such as aspartic acid for glutamic acid) or a positively charged residue for another (such as licina for arginine) can also be expected to produce a functionally equivalent product. In many cases, changes in nucleotides resulting in the alteration of the N-terminal and C-terminal portions of the protein molecule would not be expected to alter the activity of the protein. Each of the proposed modifications is within the routine nature of the art, as is the determination of the retention of the biological activity of the coded products. In addition, the skilled technician recognizes that the substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under severe conditions (O.lx SSC, 0.1% SDS, 65 ° C and washed with 2x SSC, 0.1% SDS followed by O.lx SSC, 0.1% SDS), with the
sequences exemplified here. Preferred substantially similar nucleic acid fragments of the present invention are those fragments of nucleic acid whose DNA sequences are at least 80% identical to the DNA sequence of the nucleic acid fragments reported herein. The most preferred nucleic acid fragments, are at least 90% identical to the DNA sequence of the nucleic acid fragments reported here. More preferred are nucleic acid fragments that are at least 95% identical to the DNA sequence of the nucleic acid fragments reported herein. A nucleic acid fragment is "hybridized" to another nucleic acid fragment, such as a cDNA, DNA genomic, or RNA, when a single strand form of the nucleic acid fragment can form base pairs complementary to the ot: or nucleic acid fragment under the appropriate conditions of temperature and ionic strength of the solution. Hybridization conditions of the wash are well known and exemplified in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989) particularly chapter 11 and table 11.1 from there (incorporated here completely as
reference). The ionic temperature resistance conditions determine the "hybridization severity." For the preliminary excision of the homologous nucleic acids, the low stringency hybridization conditions correspond to a Tm of 55 °, for example SSC can be used. 5x, C 1% SDS, 0.25% milk, and no formamide, or 30% formamide, 5x SSC, 0.5% SDS.The improved conditions of severity correspond to a Tm of formamide, with 5x or with 6x SSC. Hybridization requires that the two nucleic acids contain complementary sequences, although it depends on the severity of the hybridization, the non-equivalencies between the bases are possible.The appropriate severity to hybridize nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art The greater the degree of similarity or homology between two nucleotide sequences, the higher the value of Tm for the nucleic acid hybrids that has that sequence. The relative stability (corresponding to a higher Tm) of the nucleic acid hybridization decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids of more than 100 nucleotides in length, equations have been derived to calculate Tn (SE Sambrook et al., Supra, 9.50-9.51). For hybridization with acids
shorter nuclei for example oligonucleotides, the position of the correspondences becomes more important and the length of the ol: .gonucleotide determines its specificity (see Sambroo > k et al, supra, 11.7-11.8). In one embodiment, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably, a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably about 20 nucleotides and more preferably the length is 30 mer nucleotides, In addition, the skilled technician will recognize that the temperature and salt concentration in the wash solution can be adjusted as necessary in accordance with factors such as the length of a probe. A "substantial portion" refers to an amino acid or nucleotide sequence that sufficiently comprises an amino acid sequence of a polypeptide or the nucleotide sequence of a gene, to result in the putative identification of that polypeptide or gene, either by evaluation manual of the sequence by someone with skill in the art, or by a comparison of computer-automated sequences and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol,
here they are reported, can now use a substantial or total portion of the sequences described for purposes known to those skilled in the art. In this way, the current invention comprises the complete sequences as reported in the sequence listing and annex, as well as a portion of those sequences as defined above. The term "complementary" describes the relationship between the bases of nucleotides that they can hybridize one to the other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. In this manner, the current invention also includes isolated nucleic acid molecules that are complementary to the complete sequences as reported in the attached sequence listing, as well as those substantially similar nucleic acid sequences. The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequence. In art, identity "also means the degree of sequence relationship between the sequence of polypeptides or polynucleotides, as the case may be, as determined by the coupling between strings between such sequences.
it can easily calculate "identity" and "similarity" by known methods, including but not being requested those described in: Computational Molecular Biology; Les, A.M., Ed; Oxford University Press: New York, 1988; Biocomputing: Informatics and Genome Projects; Smith, C .W., Ed .; Academic Press: New York, 1993; Computer Anal; rsis of Sequence Data, Part 1; Griffin, A.M. and Griffin, H.G., Eds .; Humana Press: New Jersey, 1994; Sequence Ana lysis in molecular Biology; von - Heinje, G., ed .; Academic Press: New York.1987; and Sequence
Analysis Primer; Gribskov, M. and Devereux, J., Eds .; Stockton Press: New York, 1991. The preferred methods for determining the identity are designed to give the greatest coupling between the 1 s sequences tested. The methods for determining identity and similarity are codified in publicly available computer programs. The methods of the preferred computation programs for determining the identity and similarity between two sequences, include but are not limited to, the Pileup GCG program found in the GCG program package, using the e.lgorithm of Needlman and Wunsch with their values by standard elimination of the penalty of creation of space = 12 and the penalty for extension of space = 4 (Devereux et al., Nucleic acids Res. 12: 387--395 (1984)), BLASTP, BLASTIN, Y
at least 95% identical to the 3rd nucleotide reference sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or a nucleotide number of up to 5% of the total nucleotides in the Reference nucleotide sequence can be inserted within the reference sequence. These mutations of the reference sequence can occur at the 5 'and 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions disseminated either individually between the nucleotides in the reference sequence, or in one or more contiguous groups center of the reference sequence. Analogously, for a polypeptide having an amino acid sequence having at least, for example, 95% identity with the amino acid reference sequence, it is preferred that the amino acid sequence of the polypeptide be identical to the reference sequence, except that the polypeptide sequence may include up to five amino acid alterations per 100 amino acids of the reference amino acid. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to an amino acid reference sequence, up to 5% of
the amino acid residues in the reference sequence with another amino acid, or an amino acid number of up to 5% of the total amino acid residues in the reference sequence can be inserted within the reference sequence. These alterations of the reference sequence may occur at the positions of the amino or carboxy terminal :. of the amino acid reference sequence or anywhere in those terminal positions, either individually scattered between the residues in the reference sequence or in one or more contiguous groups within the reference sequence. The term "homologous" refers to a natural protein or polypeptide that naturally occurs in a given host cell. The invention includes microorganisms that produce homologous proteins by means of recombinant DNA technology. The term "homology, in percent" refers to the degree of identity of the amino acid sequence between polypeptides. When a first amino acid sequence is identical to a second amino acid sequence, then the first and second amino acid sequence show 100% homology. The homology between one of the two polypeptides is a direct function of the total number of coupling amino acids at a given position in any sequence, for example,
if half of the total number of amino acids in either of the two sequences are the same, then the two sequences are said to show 50% homology, "codon degeneracy" refers to the divergence in the genetic code that allows the variation of the nucleotide sequence without affecting the amino acid sequence of the encoded polypeptide. Thus, the present invention refers to any nucleic acid molecule that encodes all or a substantial portion of the amino acid sequence as set forth in SEQ ID NO: 57. The skilled technician is well informed of the codon deviation "shown by a specific host cell in the use of the nucleotide codon, to specify a given amino acid." Therefore, when synthesizing a gene for enhanced expression in a host cell, it is desirable to design the gene in such a way that its codon usage frequency approximates the frequency of the host cell's preferred codon usage: modifications to the sequence, such as deletions, insertions, or substitutions in the sequence that produce silent changes, which do not substantially affect the functional properties of the resulting protein molecule are also contemplated, for example, alteration in the sequences is contemplated.
of genes, which reflect the degeneration of the genetic code, or that result in the production of a chemically and uivalent amino acid at a given site. Thus, a codon for the amino acid of alanine, a hydrophobic amino acid, can be replaced by a codon encoding other less hydrophobic residues such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes that result in the substitution of a negatively charged residue for another, such as aspartic acid for glutamic acid or a positively charged residue for another, such as lysine for arginine, can also be expected produce a biologically equivalent product. The nucleotide changes that result in the alteration of the C-terminal and N-terminal portions of the protein molecule would not be expected to alter the activity of the protein either. In some cases, it may be desirable to actually make mutants of the sequence, in order to study the effect of the alteration on the biological activity of the protein. Each of the proposed modifications are well within the routine skill in the art, as is the determination of the retention of biological activity in the encoded products. In addition, the authorized technician recognizes that the sequences covered by this
invention, are also defined by their ability to hybridize under severe conditions (O.lx SSC, 0.1% SDS, 65 ° C), with the sequences exemplified here, The term "expressionf refers to the translation and transcription of the gene product that encodes the sequence of the gene product The terms "plasmid", "vector" and "cassette" refer to a chromosomal element x, which often carries genes that are not part of the cell's central metabolism, and usually in the form of circular double-stranded DNA molecules, such elements can be autonomously replicating sequences, integrating sequences of genomes, nucleotide or phage sequences, linear or circular, of single-stranded DNA or RNA, derived from any source, in which has bound or recombined a number of nucleotide sequences or within a single construct, which is capable of introducing a promoter fragment and a DNA sequence for a selected gene product do, along with the appropriate 3 'untranslated sequence within a cell. The "transformation cassette" refers to a specific vector that contains an external gene, and that has elements besides the external gene that facilitate the transformation of a particular host cell. The "expression cassette" refers to a specific vector
that contains an external gene and that has elements besides the external gene, that allow the enriched expression of that gene in an external host. Construction of Recombinant Organisms Recombinant organisms containing the necessary genes that will encode the enzymatic path for the conversion of a substrate or carbon to 1,3-propanediol can be constructed using techniques well known in the art. The genes that encode glycerol-3-phosphate dehydrogenase (GPD1), glycerol-3-phosphatase (GPP2), glycerol dehydratase (dhaBl, dhaB2, and dnaB2), factor of reactivation of dehydratase. { orfZ and orfX) and 1,3-propanediol oxide reductase. { dha T) were isolated from a natural host such as Klebsiella or Saccharomyces and used to transform host strains such as E. coli DH5a, ECL707, AA200, or KLP23. Isolation of genes Methods for obtaining the desired genes from a bacterial genome are common and well known in the art in molecular biology. For example, if the sequence of the gene is known, appropriate genomic collections 3 can be created by digestion of restriction endonuclases, and can be separated by exclusion with probes complementary to the desired sequence of genes. Once the sequence is isolated,
appropriate restriction adjacent to the cos region of the cosmid vector. The vectors are cosmids that contain the linearized external DNA, then react with a DNA packaging vehicle such as a bacteriophage. During the packing process, the cos sites unfold and the external DNA is packed into the head portion of the bacterial viral particle. These particles are then used to transfect appropriate host cells such as E. coli. Once injected into the cell, the external DNA circulates under the influence of the sticky cos terminals. In this manner, large segments of external DNA can be introduced and expressed in recombinant host cells. Isolation and cloning of genes that encode glycerol dehydratase. { dha l, dhaB2 and d ab3), reactivation factors of the dehyd genaase. { orfZ and orfX), and 1,3-propanediol dehydrogenase (dha T) The cosmid vectors cosmid transformation methods were used within the context of the present invention to clone large segments of genomic DNA from bacterial genera known to possess genes capable of processing glycerol to 1,3-propanediol. Specifically, the genomic DNA of K. pneumoniae was isolated by methods well known in the art.
and it was digested with the restriction enzyme Sau3A for its insertion into the cosmid neighbor supéreos 1 and was packed using the Gigapackll packaging extracts.
After the construction of the E. coli XLl-blue vector
MR, cells were transformed with the cosmid DNA. The transformants were removed by exclusion for their ability to convert gl.Lcerol to 1,3-propanediol, by growing the cells in the presence of glycerol and analyzing the media for the formation of 1,3-propanediol. Two of the positive transformants of 1,3-propanediol were analyzed and the cosmids pKP1 and pKP2 were named. The formation of DNA sequence revealed a broad homology to the glycerol dehydratase gene of C. freundii, demonstrating that these transformants contained the DNA that encoded the glycerol dehydratase gene
Other positive transformants of 1,3-propanediol were analyzed and the cosmids were designated pKP4 and pKP5. The formation of DNA sequences revealed that these cosmids carried DNA encoding a diol dehydratase gene,
Although the current invention uses the isolated genes within a Klebsiella cosmid, alternate sources of dehydratase genes and desiratase reactivation factor genes include but are not limited to Ci trobacter, Clostridia and Salmonella (see table).
1)
Genes encoding G3PDH and G3P phosphatase The present invention provides genes suitable for the expression of G3PDH and G3P phosphatase activity in a host cell. The genes encoding G3PDH are known. For example, GPD1 has been isolated from Saccharomyces and has the base sequence given by SEQ ID NO: 53, which encodes the amino acid sequence given in SEQ ID NO: 54 (Wang et al., Supra). Similarly, the G3PDH activity of Saccharomyces encoded by GPD2 has also been isolated (Eriksson et al., Mol,
Microbiol. 17.95 (1995)). For the purposes of the present invention, it is contemplated that any gene encodes a polypeptide responsible for NADH-dependent G3PDH activity, whether appropriate where the activity is capable of catalyzing the conversion of dihydroxy acetone phosphate (DHAP) to glycerol-3-phosphate or (G3P). Furthermore, it is contemplated that any gene encoding the amino acid sequence of the NADH-dependent G3PDH corresponding to the DAR1, GPD1, GPD2, GPD3 and gpsA genes will be functional in the present invention wherein said amino acid sequence can comprise substitutions, eliminations or additions of amino acids that do not alter the function of the enzyme. The authorized person will appreciate
that the genes encoding G3PDH isolated from other sources will also be appropriate for use in the present invention. The genes of G3P phosphatase are known. For example, GPP2 has been isolated from Saccharomyces cerevisiae and has the base sequence given by SEQ ID NO: 55, which encodes the amino acid sequence given in SEQ ID NO: 56 (Norbeck et al., J. Biol. Chem. 271 , 13875 (1996)). For the purposes of the present invention, any gene encoding an activity of the G3P phosphatase is appropriate for use in the method wherein that activity is capable of catalyzing the conversion of glycerol-3-phosphate plus water to glycerol plus inorganic phosphate. in addition, any gene encoding the amino acid sequence of G3p phosphatase corresponding to the GPP2 and GPP1 genes will be functional in the present invention including any amino acid sequence encompassing its substitutions, deletions or additions of amino acids that do not alter the function of the enzyme. G3P phosphatase. The skilled person will appreciate that the genes encoding G3P phosphatase isolated from other sources will be appropriate for use in the present invention.
Host cells The host cells suitable for the recombinant production of 1,3-propanediol can be prokaryotic and eukaryotic, and are limited only by the ability of the host cell to express the active enzymes for the path of 1,3-propanediol. The appropriate host cells will be bacteria such as; Ci trobacter, Clostridiun :, Klebsiella, Aerobacter,
Lactobaccillus, Aspergillus, Saccharomyces, Schizosa ccharomyces, Zygosaccharomyces, Pichia,
Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor,
Torulopsis, Methylobacte Escherichia, Salmonella,
Bacillus, Streptomyces and Pseudomonas. E. coli, E. blattae, Klebsiella, Ci trobacter and Aerobacter are preferred in the present invention. The microorganisms can be converted to a 1, 3-propanediol producer of high concentration by using the following general protocol: 1. Determine the presence in a potential host organism of an endogenous activity similar to dha T, allowing permanent concentration of a toxic or inhibitory level of 3-HPA in the presence of 1, 3-propanediol 1-2M. If such activity exists in the potential host organism, carry out a mutagenesis
appropriate to eliminate or inactivate this activity. The confirmation of a non-functional or eliminated activity similar to dha T can be detected by the lack of accumulation of 3-HPA in the presence of 1,3-propanediol 1-2M. Express the appropriate genes for (a) glycerol production, if glycerol is not the carbon source, (b) glycerol dehydratase and the associated maintenance system and (c) yqhD. The considerations that would need to be taken into account with respect to certain microorganisms refer to the expression or repression of endogenous enzymes similar to dha T under the conditions for the production of 1,3-propanediol. This may also include the presence of glycerol glucose or anaerobism, expression cassettes and vectors. The present invention provides a variety of expression and transformation vectors and cassettes, suitable for cloning, transformation and expression of G3PDH, G3P phosphates, dehydratase and the reactivation factor of dehydratase within an appropriate host cell. The appropriate vectors will be those that are compatible with the microorganism used. The appropriate vectors can be derived, for example, from
from a bacterium a virus (such as bacteriophage T7 or phage derived M-13), a cosmid, a yeast or a plant. The protocols for obtaining and using such vectors are known to those in the art
(Sambrook et al., Molecular Cloning: A
Laboratory Manual-Volumene IS 1,2,3 (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989)). Typically, the vector or cassette contains sequences that direct the transcription and translation of the appropriate gene, a selection marker, and sequences that allow for autonomous replication or chromosomal integration. Appropriate vectors comprise a region of the gene harboring the transcriptional initiation controls, and a 3 'region of the DNA fragment that controls the end: transcriptional ion. It is most preferred when both control regions are derived from genes homologous to the cell The transformed host. Such control regions need not be derived from the native genes to the specific species chosen as the production host. The initiation control regions or promoters, which are useful for driving the expression of the G3PDH and G3P phosphatase genes (DAR1 and DAR2 respectively), in the desired host cell are numerous and familiar to those skilled in the art.
Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GALIO, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO and TPI (Useful for expression in Saccharomyces) AOXl (useful for expression in Pichia) and lac, trp,? PL,? PR, T7 (Useful for expression in E. coli) Regions of control of the termination can also be derived from multiple genes natural to the preferred hosts, Optionally, a termination site may be unnecessary, however it is more preferred if it is included, For the effective expression of the current enzymes, the DNA encoding the enzymes is operatively linked through codons of initiation to the selected expression control regions so that the expression results in the formation of appropriate messenger RNA. Particularly useful in the present invention are the vectors pDT29 and pKP32 which are designed for be in conjunction with pAH48. The essential elements of pDT29 and pKP32 are derived from the regulon dha T isolated from Klebsiella pneumoniae. The pDT29 contains the open reading frames dhaR, orfY, dha T, orfX, orfW, dhaBl, dhaB2 and dhaB3, nucleotide of the sequences of which are contained within SEQ ID NO: l. The pKP32 contains the
same set of open reading structures as found in pDT29, from the same source, with the difference that pKP32 lacks dha T. pAH48 is the vehicle used for the intrusion of DAR1 and GPP2 genes into the host cell and more specifically comprises the DAR1 and GPP2 genes isolated from Saccharomyces cerevisiae. Transformation of appropriate hosts and gene expression for the production of 1,3-propanediol
Once the appropriate cassettes are constructed, they are used to transform the appropriate host cells. The introduction of the cassette containing the genes encoding G3PDH, G3P phosphatase, dehydratase, and the reactivation factor of dehydratase within the host cell, can be achieved by known methods such as for example transformation (eg, using cells permeabilized with calcium, electroporation), or by transfection using a recombinant phage virus (Samtrook et al., supra). In the current invention cassettes were used to transform E. coli as fully described in the general methods and examples, Mutants In addition to the exemplified cells, it is contemplated that the current method is capable of making use of cells
that have single or multiple mutations, specifically designed to enrich the production of 1,3-propanediol. Cells that normally divert a carbon supply inventory into nonproductive trajectories, or that they show an important repression of catabolites, can be mutated to avoid these phenotypic deficiencies. For example, many wild-type cells are subjected to the repression of catabolics from glucose and by-products in the media, and mutant strains of these wild-type organisms, capable of the production of 1,3-propanediol, are contemplated. are resistant to glucose repression would be particularly useful in the present invention. Methods for creation of mutants are common and well known in the art. For example, wild-type cells can be exposed to a variety of agents such as radiation or chemical methods and then separated by exclusion for the desired phenotype. When mutations are created through either ultraviolet (UV) or ionizing radiation, they can be used.
The shortwave UV wavelengths suitable for genetic mutations will fall within the 200 nm and 300 nm range where 254 nm is preferred. UV radiation at this wavelength, causes changes mainly
within the nucleic acid sequence from guanidine and cytosine for adenine and thymidine. Since all cells have DNA repair mechanisms that would repair most of the UV-induced mutations, agents such as caffeine and other inhibitors can be added to interrupt a repair process and maximize the number of effective mutations. Long-wave UV mutations that use light in the range of 300nm to 400nm are also possible but are generally not as effective as short-wave UV light, unless used in conjunction with various activators such as psoralen dyes that interact with DNA, mutagenesis with chemical agents is also effective for the generation of mutants, and commonly the substances used inject chemicals that affect non-replicating DNA such as HN02 and NH2OH, as well as agents that affect replicating DNA as acridine dyes, nctables for causing mutations in the structure. The specific methods for the creation of mutants using chemical radiation agents is well documented in the art. See for example, Thomas D. Brook i | n biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. , or Deshpande, Mukun V.,
Appl. Biochem. Biotechnpl. 36, 227 (1992), which is incorporated herein by reference. After mutagenesis has occurred, mutants having the desired phenotype can be selected by a variety of methods. Separation by random exclusion is more common where mutagenized cells are selected for their ability to produce the desired intermediate product,
Alternatively, selective isolation of mutants can be carried out by growing a mutagenized population on selective media sn where only resistant colonies can develop. Methods for the selection of mutants are highly developed and well known in the art of industrial biology. See for example Brock, supra; DeMancilha et al., Food Chem. 14,313 (1984). The removal of the undesirable enzyme activity can also be carried out by disruption of the gene encoding the enzyme. Such methods are known to those skilled in the art and are exemplified in example 4 and example 8. Alterations in the production trajectory of the
1, 3-propanediol Trajectories representative of enzymes. The production of 1,3-propane thiol from glucose is
It can be carried out by the following series of stages. This series is representative of a variety of trajectories known to those skilled in the art and illustrated in Figure 5. Glucose is converted into a series of enzymatic steps of the glycolytic path for dihydroxyacetone phosphate. { DHAP) and 3-phosphate glyceraldehyde (3-PG) is then formed glycerol by hydrolysis of DHAP to dihydroxyacetone. { DHA) followed by reduction, or reduction of DHAP to glycerol-3-phosphate
(G3P89 followed by hydrolysis) The hydrolysis step can be catalyzed by any number of cellular phosphatases, which are known to be non-specific with respect to their substrates, or the activity can be introduced into the recombinant host. It can be catalyzed by an NAD + (or NADP +) bound host enzyme or the activity can be introduced into the host by recombination.It is notable that the dha regution contains a glycero] dehydrogenase (EC1.1.1.6) that catalyzes the reversible reaction of equation 3. Glycerol? 3-HPA + H20 (equation 1)
3-HPA + NADH + H + - 1,3-propanediol + NAD + (equation 2) Glycerol + NAD +? DHA + NADH + H + (equation 3)
The glycerol is converted to 1,3-propanediol by means of the intermediate 3-hydroxypropionaldehyde (3-HPA) as described in detail above. The intermediary
3-HPA is produced from the glyce-rol, equation 1, by a dehydratase enzyme that can be encoded by the host or can be introduced into the host by recombination. This dehydratase can be glycerol dehydratase (E.C.4.2.1.30), shidratase diol (E.C.4.2.1.28) or any other enzyme capable of catalyzing this transformation. The glycero dehydratase, but not the diol dehydratase, is encoded, by the regution dha. 1,3-propanediol is produced from 3-HPA, equation 2, by an NAD + _ (or NADP +) bound host enzyme or the activity can be introduced into the host by recombination. This final reaction in the production of 1,3-propanediol can be catalyzed by 1,3-propanediol dehydrogenase (E.C.1.1.1.202) or other: hydrogenated alcohol. Mutations and trans iormations that affect car t canalization. A variety of mutant microorganisms, comprising variations in the path of the prodrug of 1,3-propanediol will be useful in the present invention. For example, the introduction of a triosephosphate isomerase (tpi-) mutation within the organism of the present invention is an example of the use of a mutation to improve performance by channeling carbon. Triosephosphate isomerase is the enzyme responsible for the conversion of DAHP to 3-phosphoglyceraldehyde and as such
it allows the deviation of the carbon flux of the main path form from glucose to glycerol and 1,3-propanediol (figure 5). A? Ií, mutation by elimination
(tpi-) enriches the overall metabolic efficiency of the desired trajectory over that described in the art,
Similarly, mutations that block the alternating trajectories for the intermediates of the 1,3-propanediol production path would be useful for the present invention. For example, the elimination of the glycero] kinase prevents glycerol, formed from G3P by the action of G3P phosphatase, from being converted back to G3P at the expense of ATP (FIG. 5). Also, the removal of glycerol dehydrogenase (e.g., gldA) prevents the glycerol, formed from DHAP by the glycerol 3-phosphate dehydrogenase dependent on NADH, from being converted to dihydroxyacetone (Figure 5). The mutations can be directed towards a structural gene in order to prevent or improve the activity of an enzymatic activity, or it can be directed to a regulatory gene, including promoter regions and ribosome binding sites, in order to modulate the expression level of an enzymatic activity. It is thus contemplated that transformations and mutations can be combined in order to control the particular activities of the enzyme for the
enrichment of 1,3-propanediol production. Thus, it is within the scope of the present invention, the anticipation of modifying pions of a whole cell catalyst leading to an increasing production of
1,3-propanediol. The present invention utilizes a preferred path for 1,3-prop > anodiol from a sugar substrate, where the carbon flux moves from glucose to DHAP, G3P, glycerol, 3-HPA and finally to 1.3 propanediol. Current production strains have been engineered to maximize the metabolic efficiency of the trajectory by incorporating various elimination mutations that prevent the diversion of carbon to nonproductive compounds. Glycerol can deviate from the 2 HPA conversion by transformation to DHA or G3P by means of the glycerol. 1 dehydrogenase or the glycerol kinase as described above (figure 5). In this way, the current production strains contain deletion mutations in the gldA and glpK genes. Similarly DHAP can be diverted to 3-PG by the triosephosphate isomerase, and thus the current production of microorganism also contains an elimination mutation in this gene. The current method additionally incorporates a dehydratase enzyme for the conversion of glycerol to BHPA, which functions in set
with the reactivation factor encoded by orfX and orfZ of the dha regution (figure 59). Although conversion of 3HPA to 1,3-propanediol is typically carried out by 1,3-propanediol oxide reductase, the current method uses a non-specific catalytic activity that produces higher concentrations and yields of the final product, 1,3-propanediol (f: .gura 5). In such a process, 1,3-propanediol concentrations of less than 10g / l are reached, where that expects concentrations of 200 g / 1, Alternatively, an improved process for 1,3-propanediol can use glycerol or dihydroxyacetone as substrate, wherein the pathway comprises only the three substrates, glycerol - »3HPA - 1,3-propanediol. In such a process, the oxide reductase is again removed in favor of the non-specific catalytic activity (expected to be a dehydrogenase alcohol), however, the need for deletion mutations is nullified by the energy considerations of adding glycerol to the culture. In such a process, 1, 3-propanediol concentrators of at least 71 g / 1 are reached where concentrations of 200 g / l are expected. Similarly, it is within the scope of the invention to provide mutants in microorganisms of
wild type, which have been modified by the elimination or mutation of dha T activity to create improved producers of 1,3-propanediol. For example, microorganisms that naturally contain all the elements of the dha regu- lation can be manipulated to activate the dha T gene that encodes the 1,3-propanediol oxide redu :: rate activity. These microorganisms will be expected to produce higher yields and concentrations of 1,3-propanocyl, mediated by the presence of an endogenous catalytic activity that is expected to be an alcohol dehydrogenase. Examples of such microorganisms include but are not limited to the species Klebsiella, Ci trobacter, and Clostridium. Carbon substrates and media. The fermentation media in the present invention should contain appropriate carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as a, lactose or sucrose, polysaccharides such as Imidon or cellulose or mixtures thereof and unpurified mixtures of renewable raw materials such as, for example, pee of cheese whey, direct corn liquor, sugar beet molasses and barley malt. Additionally, the carbon substrate can also be substrates of a carbon such as dioxide
carbon or methanol for which metabolic conversion has been demonstrated within key biochemical intermediaries. The production of glycerol from simple carbon sources (eg methanol, formaldehyde or formate) has been reported in methylotrophic yeasts (K. Yamada et al., Agrie. Biol. Chem. 53 (2), 541-543 ( 1989)) and in bacteria (Hunter et al., Biochemistry 24, 4148-4155 (1985)). These microorganisms can assimilate simple carbon compounds, in the range of a state of oxidation of methane to formate and produce glycerol. The trajectory of carbon assimilation can be through ribulose monophosphate, through serine or xylulose monophosphate (Gottschalk, Bacterial Metabolism, Second Edition, Springer-Verlag New York (1986)). The path of ribulose monophosphate involves the condensation of formiate with ribulose-5-phosphate to form a 6-carbon sugar that becomes fructose and eventually the 3-carbon product glyceraldehyde-3-phosphate. Similarly, the trajectory of serine assimilates the compound of a carbon within the glycolytic path by means of methylene tetrahydrofolate. In addition to the one and two carbon substrates, methyl nitrogenic microorganisms are also known
use a number of compounds that contain other carbons such as melamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic washes are known for using the carbon of methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth CL Compd., [Int.Sy p.], 7th (1993), 415-32. es) Murrell, J. Collin; Kelyl Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine < Oleic acid (Sulter et al., Arch. Micr. biol. 153 (5), 485-489 (1990)) Thus, it is contemplated that the carbon source used in the present invention can encompass a wide variety of substrates containing carbon, and will be limited only by the choice of microorganism or process, Although it is contemplated that all substrates and mixtures (co-feed) of the aforementioned coals are appropriate in the present invention, the preferred carbon substrates are glucose, fructose, sucrose, or methanol where the process is intended to introduce an endogenous glycerol, and glycerol or dihydroxyacetone, where the process anticipates a glycerol or hydroxyacetone feed. In addition to an appropriate carbon source, the fermentation media must contain minerals, you go out,
cofactors, buffer solutions, and other suitable components, known to those skilled in the art, suitable for the growth of crops and promotion of the enzymatic path necessary for the production of 1,3-propanediol. Particular attention is given to the salts of Co (II) and / or vitamin B? 2 or precursors thereof. Adenosyl-cobalamin (coenzyme Bi2) is an essential cofactor for the activity of dehydratase. The synthesis of coenzyme Bi2 is found in prokaryotes, some of which are able to synthesize the de novo compound, for example, the species Escherichia blata, Klebsiella, Citrobacter, and Clostridium, while others may lead to out partial reactions. E. coli for example can not manufacture the corrin ring structure, but can catalyze the conversion of cobinamide to corrinoid and can introduce the 5'-deoxyadenosyl group. Thus, it is known in the art that a precursor of co-enzyme B? 2, such as vitamin Bi2 needs to be supplied in fermentations of E. coli. Additions of vitanin B? 2 to fermentations of E. coli can be add continuously, at a constant speed or be staged to coincide with the generation of the cell mass, or they can be added in
single or multiple bolus additions. Preferred vitamin BÜ2 (mg) ratios fed to the cell mass (OD550) are from 0.06 to 0.60. The most preferred ratios of vitamin B 2 (mg) fed to the cell mass (OD 550) are from 0.12 to 0.48. Although vitamin B12 is added to the transformed E. coli of the present invention, it is contemplated that other microorganisms capable of de novo biosynthesis of Bi2, are also suitable production cells and the addition of B? 2 to these microorganisms will be unnecessary . Culture conditions Typically the cells grow at 35 ° C in appropriate media. Preferred growth media in the present invention are commercially prepared common media such as Luria Bertani broth (LB), Sabouraud dextrose broth (SD) or broth yeast medium
(YM) Other defined synthetic growth media can also be used. The appropriate medium for growth of the particular microorganism will be known to one skilled in the art of microbiology or the science of fermentation. The use of agents known to modulate the repression of catabolites directly or indirectly. , for example, adenosine 2 ': 3'-cyclic monophosphate, can also be incorporated into the
means of reaction. Sim.Llarly, the use of agents known to modulate the enzymatic activities (eg, methyl viologen) leading to the enrichment of 1,3-propanediol production, can be used in conjunction with or as an alternative to genetic manipulations, Appropriate pH ranges for fermentation are between a pH of 5.0 to a pH of 9.0, where pH 6.0 is preferred up to pH 8.0 as the initial condition. Reactions can be carried out under aerobic or anaerobic conditions where anaerobic or microaerobic conditions are preferred. Feeding batch fermentations can be executed with carbon feed for example glucose, limited or in excess. Continuous and batch fermentations: The current process uses a fermentation batch method. Classical batch fermentation is a closed system in which the composition of the media is fixed at the beginning of fermentation, and is not subject to artificial alterations during fermentation. Thus, at the beginning of the fermentation, the media are inoculated with the microorganism or desired microorganisms and the fermentation is allowed to happen without adding anything to the fermentation.
system. Typically, however, "batch" fermentation is batch with respect to the addition of carbon source and attempts are often made on control factors such as pH and oxygen concentrations. In batch systems, the biomass and metabolite compositions of the system change constantly up to the moment when the fermentation is suspended. Within batch cultures, the cells moderate through a logic phase from moderate to static until a logical phase of high growth and finally to a stationary phase where it decreases stops the rapidity of growth. If they are untreated, the cells in the stationary phase will eventually die. The cells in the logical phase are generally responsible for the bulk of the production of the final product or intermediary. A variation in the standard batch system is the fed batch system. The fermentation processes of the batch c: >N food are also suitable in the present invention, and comprise a typical intermittent system with the exception that the substrate is added in increments as the fermentation progresses. Feeding batch systems are useful when the repression of catabolites is inhibited to inhibit the metabolism of cells, and where
It is desirable to have limited amounts of substrate in the media. The measurement of the current substrate concentration in the fed batch systems is difficult and is therefore estimated on the basis of the changes in factors that are measured such as pH, dissolved oxygen and the relative pressure of the waste gases such as C02. Batch and batch fermentation with feed are common and well known in the art and examples can be found in Brock, supra. Although the present invention is carried out in batch form, it is contemplated that the method would be adaptable to continuous fermentation methods. Continuous fermentation is an open system in which continuously defined fermentation media are added to a bioreactor, and an equal amount of conditioned media for processing is simultaneously separated. Continuous fermentation generally maintains the cultures at a high constant density where the cells are mainly in the growth of the logical phase. Continuous fermentation allows the modulation of a factor or any number of factors that affect cell growth or concentration of the terminal product. For example, a method will maintain a limited nutrient such as the carbon source or the nitrogen level at a fixed rate, and allow moderate
the other parameters. In other systems, various factors that affect growth can be altered continuously while maintaining cell concentration constant, measured by the turbidity of the media. The continuous systems try to maintain conditions of growth in permanent regime, and thus the cellular loss due to the means that are removed, must be balanced against the rapid growth of the cells in the fermentation The methods of modulation of nutrients and growth factors For continuous fermentation processes, as well as techniques for maximizing the rapidity of product formation, they are well known in the art of industrial microbiology and a variety of methods are detailed by Brock supra. It is contemplated that the present invention may be practiced using continuous or batch fed, batch processes, and that any known mode of fermentation would be appropriate. Additionally, it is contemplated that the cells can be immobilized on substrates as whole cell catalysts and subjected to fermentation conditions for the production of 1,3-propanediol. Identification and purification of 1,3-propanediol Methods for the pi )ification of 1,3-propanediol from fermentation media are known in the art.
For example, propane diols can be obtained from cell media by subjecting the reaction mixture to extraction with an organic solvent, distillation and column chromatography (U.S. 5,356,812). A particularly good organic solvent for this process is cyclohexane (U.S. 5,008,473). The 1,3-propanediol can be identified directly by sending the media to a high pressure liquid chromatography (HPLC) analysis. Preferred in the present invention is a method wherein the fermentation media is analyzed on an analytical ion exchange column using a mobile phase of 0.01 N sulfuric acid in an isocratic form, EXAMPLES GENERAL METHODS The procedures for phosphorylation, bound and Transformations are well known in art. Suitable techniques for use in the following examples can be found in 3ambrook, J. et al., Molecular Cloning: A Laboratory Manual, Second Edition,
Cold Spring Harbor Labóratery Press (1989). Suitable materials and methods for the maintenance and growth of bacterial cultures are well known in the art. The techniques suitable for use in the following examples can be found in
Manual of Methods for General Bacteriology (Phillipp
Gerhardt, R. G. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs
Phillips, eds), American Society for Microbiology,
Washington, D.C. (1994 or Thomas D. Brock in
Biotechnoloqy: A Textbook of Industrial Microbioloqy,
Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. All reagents and materials used for growth and maintenance of bacterial cells were obtained from Aldrich C nemicals (Milwaukee, Wl), DIFCO Laboratories (Detroit, MI) GIBCO / BRL (Gaithersburg, MD), or Sigma Chemical Company (St. Louis , MO) unless otherwise specified, The meaning of the abbreviations is as fol:
"h" means hours, "mn" means minutes, "sec" means seconds, "d" means days, "mL" means milliliters, "L" means liters, 50 amp is 50 μg / mL ampicillin and LB-50 amp It is Luria-Bertani broth that contains 50 μg / mL of ampicillin. Within the tables the folng abbreviations are used. "With." Conversion, Sel. "is selectivity based on carbon and" nd "is not detectable The strains and vectors used and constructed in the folng examples are listed in the folng table:
PLASMID CEPA ELIMINATION ORF / GEN
KLP23 qldA glpK RJ8m gldA glpK Tpi pAH48 GPP2 DARl pDT29 dhaR orfY dha T orfX orfW dhaBl dhaB2 dhaB 3 orfZ pKP32 dhaR orfY orfX orfW dhaBl dhaB2 dhaB3 orfZ
ENZYME TESTS Tests for dehydratase enzymes: The activity of dehydratase in the cell-free extracts was determined using glycerol or 1,2-propanediol as a substrate. Typically, cell-free extracts were prepared by cell disruption using a French press, fold by centrifugation of cell debris. The assay, based on the reaction of aldehydes with methylbenzo-2-thiazolon hydrazone, has been described by Forage and Foster (Biochim, Biophys, Acta 569, 249 (1979)). Honda et al. (J. Bacteriol., 143, 1458 (1980)) describe an assay that measures the reactivation of dehydratases. The dehydratase activity was determined in whole cells treated with toluene, with or without ATP, using glycerol or 1,2-propanediol as a substrate. The reactivation was determined by the ratio of product formation against the addition of ATP. Product formation (3-HPA or propionaldehyde when using glycerol or 1,2-pro-anodiol as the substrate respectively) was measured directly using HPLC, or indirectly using the methylbenzo-2-thiazolon hydrazone reagent. Alternatively, product formation was determined by coupling the conversion of the aldehyde with its respective alcohol using an alcohol
dehydrogenase linked to NADH and observing the appearance of NADH. Assays for 1, 3-pi: opanediol oxide reductase: The activity of L, 3-propanediol oxidoreductase, sometimes referred to as 1,3-propanediol dehydrogenase, was determined for free cell extracts in solution or in gels in bars using 1,3-propanediol and NAD + as substrates has been described
(Johnson and Lin, J. Báraiol., 169, 2050 (1987)). Alternatively, the conversion of 3-HPA and
NADH at 1, 3-propanediol and NAD +, due to the disappearance of
NADH The gel-bar assay has the potential advantage of separating the activity of 1,3-propanediol oxide reductase. { dha T) of that of nonspecific alcohol dehydrogenases by virtue of size separation. The native molecular weights of 1,3-propanediol oxide reductases. { dha T) of the Citrobacter frendii, Klebsiella? neumoniae, and Clostridium pasteurianum, are unusually large, in the order of 330,000 to 440,000 Daltons. Lactobacillus brevis and Lactobacilus buchneri containing 1,3-propanediol oxide reductases associated with dehydratase, with properties similar to those of 1, i3-propanediol oxide reductases known. { dha T).
Assays for glycerol 3-phosphate dehydrogenase activity: A procedure as modified bewas used from a method published by Bell et al.
(J. Biol. Chem. 250, 7153 (1975)). This method involved the incubation of a sample of cell-free extract in a cell containing 0.2 mM NADH, dihydroxyacetone phosphate (DHAP), 2.0 mM and enzymes in Tris / HCl, 0.1 M, buffer solution cor. pH 7.5 with 5 mM DTT, in a total volume of 1 mL at 3Q ° C. Rapidly supporting the reaction of the enzyme and NADH was first determined at 340 nm for at least 3 minutes. The second substrate, DHAP, was subsequently added and the change in absorbance over time was observed for at least 3 minutes. The activity of G3PDH was defined by subtracting the speed of backing from the gross speed. Assay for the activity of glycerol 3-phosphatase: The assay for enzyme activity was carried out by incubating the extract with an organic phosphate substrate in a magnesium buffer solution and bis-Tris or MES, pH 6.5. The substrate that was used was 1-a-glycerol phosphate or d 1-a-glycerol phosphate. The final concentrations of the reagents in the assays are: buffer solution (20 mM, bis-Tris or 50 mM
MONTH); MgCl2 (10 mM); and substrate (20 mM). If the total protein in the sample was lower and there was no visible precipitation with an acid shutdown, the sample was conveniently tested in the cell. This method involved the incubation of a sample of enzymes in a cell containing a substrate 20 mM (50 μL, 200 mM), 50 mM MES, 10 mM MgCl2, pH 6.5 buffer solution. The final volume of the phosphatase assay was 0.5 mL. The sample containing the enzyme was added to the reaction sample; the contents of the tank were mixed and then the tank was placed in a circulating water bath at a T of 37 ° C from 5 to 120 min, the length of time depending on whether the activity of the phosphatase sn the enzyme sample It was from 2 to 0.02 U / mL. The enzymatic reaction was quenched by the addition of an acidic molybdate reagent (0.4 mL). After the Fiske reagent was added
Subbarow (0.1 mL) and distilled water (1.5 mL), the solution was mixed and allowed to develop. After 10 minutes, to allow full color development, the absorbances of the samples were read at 660 nm using a Cary 219 UV / vis spectrophotometer. The amount of inorganic phosphate released was compared to a standard curve that was prepared by using an inorganic phosphate inventory solution (0.65 mM) and preparing 6
standards with final inorganic phosphate concentrations in the range of 0.026 to 0.130 μmol / mL, Assay for glycerol kinase activeness: An appropriate amount of enzymes, typically a cell-free crude extract, was added to a reaction mixture that contained 40 mM ATP, 20 mM MgSO4,
21 mM, glycerol uniformly labeled with 13C (99%,
Cambridge Isotope Laboratories), and 0.1 M Tris-HCl, pH 9 for 75 min at 25 ° C, The conversion of glycerol to glycerol 3-phosphate was determined by 113 JC-NMR (125 MHz): glycerol (63.11 ppm, d, J = 41 Hz and 72.66 ppm, t, J = 41
Hz); glycerol 3-phosphate (2.93 ppm, d, J = 41 Hz, 65.31 ppm, br d, J = 43 Hz, and 72.66 ppm, dt, J = 6.41 Hz). Assay of the glycerol of? Shidrogenase linked to NADH: The activity of glycerol dehydrogenase linked to
NADH (gldA) in 1-ibral extracts of cells from E. coli strains was determined after separation with proteins by a non-denaturing polyacrylamide gel electrophoresis. The conversion of glycerol plus NAD + to dihydroxyacetone plus NADH was coupled with the conversion of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) in a deep-colored formazan, using as mediator phenazine methosulfate (PMS) (Tang et al., J. Bacteriol., 140, 182 (1997)).
The electrophoresis was carried out in duplicate by normal procedures using natural gels: 8-16% TG, 1.5 mm, 15-track gels from Novex, San
Diego, CA). The residual glycerol was separated from the gels by washing 3 times with 50 mM Tris or potassium carbonate buffer solution, pH for 10 min. Duplicate gels with or without glycerol (approximately a final concentration of 0.16 M) were developed in 15 mL of assay solution containing 50 mM Tris or potassium carbonate, pH S, ammonium sulfate 60 mg, NAD + 75 mg, MTT 1.5 mg and PMS 0.5 mg. The presence or absence of glycerol dehydrogenase activity bound to NADH in strains of E. coli (gldA) was also determined, following electrophoresis with polyacrylamide gel, by reaction with polyclonal antibodies raised for the purified glycerol dehydrogenase of K. pneumoniae (dhaD). Isolation and identification of 1,3-propanediol The conversion of glycerol to 1,3-propanediol was observed by HPLC. Analysis will be carried out using standard techniques and materials available to someone with skill in the art of chromatography. An appropriate method uses a Waters Maximum 820 HPLC system using UV detection (2 LO nm) and Rl. The samples were injected onto a Shodex SH-1011 column (8 mm x 300 mm,
acquired from Waters, Milfprd, MA) equipped with a Shodex SH-1011P pre-column (6 mm x 50 mm), with the temperature controlled at 50 ° C, using H2S04 0.01 N as a mobile pass at a flow of 0 05 mL / min .. When the quantitative analysis is desired, the samples were prepared with a known amount of trimethylacetic acid as an external standard. Typically, the glucose retention times (Rl detection) glycerol, 1,3-propanediol (Rl detection) and trimethylacetic acid (Rl and UV detection) were 15.27 min, 20.67 min, 26.08 min, and 35.03 min, respectively. The production of 1,3-propanediol was confirmed by GC / MS. The analyzes were carried out using standard techniques and materials available to someone with skill in the art of GC / MS. An appropriate method uses a Hewlett Packard 5890 series II gas chromatograph, coupled with a Hewlett Packard 5971 (El) mass selective detector and an HP-INNOWax column (30 m long, 0.25 mm internal diameter, 0.25 thick). of micron film). The retention time and the mass spectrum of the 1,3-propanediol generated was compared to that of the 1,3-propanediol authentic (m / e: 57,58). An alternative method for GC / MS involves the derivation of the sample. 1 μl of sample (for example culture supernatant) was added to 30 μL of acid
concentrated perchloric (70% v / v). After mixing, the sample was frozen and lyophilized. A 1: 1 mixture of bis (trimethylsilyl) trifluoroacetamide: pyridine (300 μL) was added to the lyophilized material, mixed vigorously and placed at 65 ° C for 1 hour. The sample was clarified of insoluble material by centrifugation. The resulting liquid was divided into two phases, the upper one of which was used for < 1 analysis The sample was chromatographed on a DB-5 column (48 m, 0.25 mm I.D., 0.25 μm, film thickness, from J &W Scientific) and the retention time and mass spectrum of the derivative of
1,3-propanediol obtained from the culture supernatants was compared to that obtained from the authentic standards. The mass spectra of 1,3-propanediol derived from TMS, contain the characteristic ions of
205, 177, 130 and 115 AMU. Cell Lysis: Cell lysis was estimated by measuring the concentration of soluble extracellular protein in the fermentation broth. Fermenter samples were centrifuged in a desktop centrifuge (typically, 3-5 min a
12,000 rpm in an Eppendorf micro-centrifuge model 5415C) in order to separate the cells. The resulting supernatant was analyzed for protein concentration or the Bradford method using a reagent
commercially available (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA). Feasibility: Cell viability! cells obtained from the fermentor on non-selective LB agar plates were determined by plate formation, at appropriate dilutions. The cell viability in the fermenter experiments is compared to 1 using the ratio of viable cells per mL of fermentor broth divided by OD550 (AU). EXAMPLE 1 CLONING AND TRANSFORMATION OF GUEST CELLS FROM E. COLI WITH DNA COMFORT FOR THE EXPRESSION OF 1, 3-PROPANODIOL
Means: Synthetic medium S12 was used in the separation by exclusion of bacterial transformants, for the ability to make 1,3-propanediol. The S12 medium contains: 10 mM ammonium sulfate, 50 mM potassium phosphate buffer, pH 7.0, 2 mM MgCl2, 0.7 mM CaCl2, 50 μM MnCl2, 1 μM FeCl3, 1 μM ZnCl, 1.7 μM CuS04, 2.5 μM CoCl2. , Na2Mo04 2.4 μM, and 2 μM, thiamine hydrochloride. The medium A used for the growth of the fermentation consisted of: 10 mM ammonium sulfate, 50 mM MOPS / KOH buffer, pH 7.5, solution
mM potassium phosphate buffer, pH 7.5; 2 mM MgCl2, 7.7 M CaCl20, 50 μM MnCl2, 1 μM FeCl3, 1 μM ZnCl, CuS04
1. 72 μM, CoCl2 2.53 μM, Na2'Mo02 2.42 μM, 2 μM thiamine hydrochloride; Yeast extract 0.01%, casamino acids 0.01%; vitamin B12 0.8 μg / mL and 50 μg / mL amp. The middle
A was supplemented with 0.2% glycerol or 0.2% glycerol plus 0.2% D-glucose as required, Cells: La Klebsiella pn umoniae ECL2106 (Ruch et al., J. Bacterial, 124, 348 (1975)), also known in the literature as K. Aerogenes or Aerobacter aerogenes, was obtained from ECC Lin (Harvard Medical School, Cambridge, MA) and remained as a laboratory culture. Klebsiella pneumomae ATCC 25955 was purchased from American Type Culture Colleption (Manassas, VA). E. coli DH5a was purchased from Gibco / BRL and transformed with cosmid DNA isolated from Klebsiella pneumomae ATCC 25955 containing a gene encoding glycerol or the enzyme diol dehydratase. The cosmids containing the glycerol dehydratase were identified as pKP1 and pKP2 and the cosmid containing the diol dehydratase enzyme was identified as pKP4. The cells
transformed DH5a were identified as DH5a-pKP1, DH5a-pKP2 and DH5a-pKP4 ECLI707 was obtained from E. coli (Sprenger et al., J. Gen. Microbiol., 135, 1255 (1989)) from E.C.C. Lin (Harvard Medical School, Cambridge, MA) and similarly transformed with the cosmid DNA of Klebsiella pneumoniae. These transformants were identified as ECL707-pKP1 and ECL 707-pKP2, which contained the glycerol dehydratase gene and ECL707-pKP4 which contained the diol dehydratase gene. The AA200 from E. coli containing a mutation in the tpi gene (Anderson et al, J. Gen. Microbiol.
329 (1970)) was purchased from the genetic inventory center of E. coli, Yale University (New Haven, CT) and transformed with the cosmid DNA of Klebsiella to give the recombinant microorganisms AA200-pKP1 and AA200-pKP2, which contained glycerol dehydratase gene, and the
AA200-pKP4, which contained the diol dehydratase gene,
DH5a: Six transformation plates containing approximately 1000 coloriies of XLl-blue MR from E. coli, transfected with DNA from K. pneumoniae, were washed with 5 ml LB medium and centrifuged. The bacteria were pelleted and resuspended in LB + glycerol 5 mL medium. An aliquot (50 μL) was inoculated into
a 15 mL tube that cushions synthetic medium S12 with 0.2% glycerol + 400 ng per mL of vitamin B? 2 + yeast extract 0.001% plus 50 amp. The tube was filled with the medium to the top and wrapped with film and incubated at 30 ° C. A slight turbidity was observed after 48 hours. The aliquots, analyzed for distribution of products as described above to
78 h and 132 h, they were posaltivas for 1, 3-propanediol, the last points of time contained increasing amounts of 1, 3-propanediol The bacteria, which tested positive for the production of 1, 3-propanociol, were serially diluted and they were placed in plates on LB50 amp plates, in order to isolate the simple colonies. 48 single colonies were isolated and checked for the production of 1,3-propanediol. The cosmid DNA was isolated from 6 independent clones and transformed into strain DH5a of
E. coli. Transformants were checked again for the production of 1,3-propanediol. Two transformants were further characterized and designated as DH5a-pKP1 and DH5a-pKP2. A fragment of EcqRI-Sali from 12.1 kb of pKPl, subcloned into pIB 31 (IBI Biosystem, New Heven, CT), was formed into sequences and was named pHK28-26 (SEQ ID NO: 1). The sequence formation revealed the places
in bases 8724-9308; the open reading frame dhaB3 encoding the gamma subunit of glycerol dehydratase is found in bases 9311-9736; the open reading frame dhaBX, which encodes a dehydratase reactivation protein, is found in bases 9749-11572; and a fragment of the open reading frame for lpF encoding a glycoprotein-restoring facilitator protein is found in bases 11626-12145. Single colonies of XLl-blue MR of E. coli transfected with the packed cosmid DNA of K. pneumoniae were inoculated into microtiter wells containing 200 μL of S15 medium
(10 mM ammonium sulfate, potassium phosphate buffer, pH 7.C, 1 mM, MOPS / KOH buffer, pH 7.0, 50 mM, MgCl2, 2 mM, CaCl2, 0.7 mM;
MnCl2, 50 μM; FeCl3, 1 μM L- ZnCl, 1 μM; CuSO4, 1.72 μM;
CoCl2, 2.53 μM; Na2Mo04.2.42 μM; and 2 μM thiamine hydrochloride) + 0.2% glycerol + 400 ng / mL vitamin B12 + 0.001% yeast extract + 50 μg / mL ampicillin. In addition to the microtiter wells, a master plate containing LB-50 amp was also inoculated. After 96 hours, 100 μL was withdrawn and centrifuged in a Rainin microfuge tube containing a membrane filter.
0.2 micron nylon. The bacteria were retained and the filtrate was processed for HPLC analysis. Positive clones demonstrating 1,3-propanediol production were identified after exclusion by exclusion of approximately 240 colonies. They were identified
3 positive clones, two of which had grown in
LB-50 amp and one of the puales had not done it. A simple colony, isolated from one of the two positive clones that grew in LB-50 amp and verified for the production of 1,3-propanediol was designated pKP4. He
Cosmid DNA was isolated from E. coli strains containing pKP4 and the E. coli strain DH5a was transformed. An independent transformant, designated as DHa-pKP4, was verified for the production of 1,3-propanediol, ECL707. The ECL707 strain of E. coli was transformed with the cosmid DNA of K. pneumoniae, which corresponded to one of pKPl, kPK2, pKP4, or to the vector Supéreos only and named
ECL707-pKPl, ECL707-pKP2, ECL707-pKP4, and ECL707-SC, respectively. ECL707 is defective in glpK, gld, and ptsD that encodes glycerol kinase dependent on ATP, glycerol dehydrogenase linked to NAD +, and enzyme II for dihydroxyacetone of the phosphoenolpyruvate-dependent phosphoten- traase system respectively,
* (Number of postitive isolates / number of isolates tested.) EXAMPLE 2 ENGINEERING OF GLYCEROL KINASE MUTANTS OF FM5
OF E. COLI FOR THE PRODUCTION OF GLYCEROL FROM GLUCOSE. Construction of the plasmid | Integration step for the substitution of the gjlicerol kinase gene in FM5 of E. coli: Genomic DNA of FM5 E. coli (ATCC 53911) was prepared using the Puregene DNA Isolation Kit (Gentra)
Systems, Minneapolis, MN). A DNA fragment of one kb, containing glpF parcia L and glycerol kinase genes
(glpK) was amplified by PCR (Millus and Faloona, Methods
Enzymol. 155, 335 (1997)) from the FM5 genomic DNA using the primers SEQ ID NO: 2 and SEQ ID NO: 3. A 1. kb DNA fragment containing the partial genes glpK and glpX, was amplified by PCR to from the FM5 genomic DNA using the primers SEQ ID NO: 4 and SEQ
ID NO: 5. A Muñí site was incorporated into the primer SEQ ID NO: 4. The 5 'termination of the primer SEQ ID NO: 4 was the reverse complement of the primer SEQ ID NO: 3 to allow the subsequent extension of the overlap. of PCR. The splicing of genes by the extension technique of overlap
(Horton et al., BioTechniques 8, 528 (1990))
used to generate a 2.1 kb fragment by PCR using the two above PC fragments as templates and primers SEQ ID NO: 2 and SEQ ID NO: 5. This fragment represents a 0.8 kb deletion of the central region of the glpK gene of 1.5 kb. In total, this fragment had flank regions of 1. 0 kb and 1.1 kb on each side of the Muñí cloning site within the partial glpK) to allow replacement of the chromosomal gene by homologous recombination. The above 2.1 kb PCR fragment is completed in its base pairs (using mung bean huclease) and cloned into the complete PCR vector in its base pairs using the Zero base pair completion PCR cloning kit (Invitroge n, San Diego, CA) to produce the 5.6 kb pRNIOO plasmid containing the Zoecind and kanamycin resistance genes. The 1.2 kb HincII fragment from pLoxCatl (unpublished results), which contains a chloramphenicol resistance gene flanked by the sites of the bacteriophage Pl loxP
Snaith et al., Gene 166, 173 (1995)), is used to interrupt the glpK fragment in plasmid pRNlOO by ligating it to plasmid pRNlOO digested with Muñí (and ending in blunt tips) to produce plasmid pRN101-l of 6.9 kb. A 376 bp fragment containing the R6K origin was amplified by PCR from the vector
pGP704 (Miller and Mekalanos, J. Bacteriol., 170, 2575-2583 (1988) using the primers of SEQ ID NO: 6 and SEQ ID NO: 7, completed in their base pairs and ligated to the Asp718-AatII fragment of: 5.3 kb (which is completed in its base pairs) from pRN101-l to produce plasmid pRN102-l of 5 7 kb, which contains chloramphenicol and kanamycin resistance genes Substitution of the region of the ColEl origin in pRN101 -l with the origin R6K to generate pRN102-l, also involves the elimination of most of the zeocin resistance gene.The host for replication pRN102-l was E. coli SY327 (Miller and Mekalanos, J. Bacteriol., 170, 2575-2583 (1988)) which contains the pir gene necessary for the function of origin R6K Engineering of the mutant R'.liFlOm of glycerol kinase with an interruption in the chloramphenicol resistance gene: The FM5 of E coli was electrotransformed with the non-replicating integration plasmid pRN102-l and the transformants that were resistant to chloramphenicol
(12.5 μg / mL) and kanamycin sensitive (30 μg / mL) were further separated by exclusion for a non-use of glycerol in a minimal M9 medium containing 1 mM glycerol. A digest of the EcoRI of the genomic DNA of one of the mutant talis, RJFlOm, when
probing with the intact glpK gene by means of Southern analysis (Southern, J. Mol. Biol. 98, 503-517 (1975)) indicated that it was a double cross member (replacement of the glpK gene), since the two expected bands of 7.9 kb and 2.0 kb were observed, due to the presence of an additional EcoRI site within the chloramphenicol resistance gene. The wild-type control produced the expected simple band of 9.4 kb. A 13 C NMR analysis of the RJFlOm mutant confirmed that it was unable to convert glycerol labeled with 13C and ATP to glycerol-3-phosphate. The glpK mutant was further analyzed by genomic PCR using the combinations of the primers of SEQ ID NO 8 and SEQ ID NO: 9, SEQ ID NO:
and SEQ ID NO: 11, and SEQ ID NO: 8, and SEQ ID NO: 11 that resulted in the fragment DS expected by PCR of 2.3 kb, 2.4 kb and 4.0 kb respectively. Wild type control produced the expected band of 3.5 kb with the primers of SEQ ID NO: 8 and SEQ ID NO: 11. The RJFlOm mutant of glpK was electrotransformed with plasmid pAH48 to allow the production of glycerol from glucose. The mutant of E. coli RJFlOm of glpK has been deposited with the ATCC under the terms of the Budapest treaty on November 24, 1997.
Engineering of the glycerol kinase mutant RJF10 with a separate interruption skin chloramphenicol resistance gene: After overnight growth on a YENB medium (0.75% in yeast extract, 0.8% nutrient broths) at 37 ° C, if RJFlOm of E. coli in a suspension of water, was electrotransformed with the plasmid pJW168 (unpublished results) containing the bacteriophage Cre Pl recombinase gene under the control of the IPTG-inducible lacUV5 promoter, a pSClOl replicon sensitive to temperature and a gene for resistance to ampicillin. After growth in SOC medium at 30 ° C, transformants were selected at 30 ° C (temperature authorized for replication of pJW168) on an LB agar medium supplemented with carbenicillin (50 μg / mL) and IPTG
(1 mM). Two serial transfers were carried out overnight at colonies accumulated at 30 ° C, on fresh medium of LB agar supplemented with carbenicillin and
IPTG, in order to allow the extirpation of the chromosomal chloramphenicol resistance gene by means of recombination in loxP sites mediated by the Cre recombinase (Hoess and Abremski, J. Mol. Biol. 181, 351-362 (1985)). The resulting colonies were replicated on the LB agar medium supplemented with carbenicillin and IPTG and LB agar supplemented with chloramphenicol (12.5
μg / mL) to identify colonies that were resistant to carbenicillin and to the separation of the indicator marker gene, sensitive to chloramphenicol. A culture at 30 ° C was used overnight in one of these colonies to inoculate 10 mL of LB medium. With the growth
° C to OD (600 nm) of 0.6 AU, the culture was incubated at 37 ° C overnight. Various dilutions were plated on a pre-heated LB agar medium, and plates were incubated overnight at 42 ° C (the temperature not authorized for replication of pJW168). The resulting colonies were replicated on the LB agar medium and the LB agar medium supplemented with carbenicillin (75 μg / mL) to identify colonies that were sensitive to carbenicillin, indicating the loss of the plasmid pJW168. One such glpK mutant, RJF10, was further analyzed by genomic PCR using primers of SEQ ID NO: 8 and SEQ ID NO: 11 and yielded the expected band of 3.0 kb confirming the ablation of the marker gene. The non-use of glycerol by the RJF10 mutant was confirmed by the lack of growth in a minimal M9 medium containing 1 mM glycerol. The RJF10 mutant of glpK was electrotransformed with plasmid pAH48 to allow glycerol production of glucose.
with 204 bps in the 3 'direction of the gldA translational start codon and terminates at 178 bps from the 5' address of the gldA translational stop codon, and contains the kan insert, was isolated from pKPl3 by PCR using the primers SEQ ID NO: 16 and SEQ ID NO: 17, which incorporate the terminal sites Sphl and Xbal respectively, subcloned between the Sphl and Xbal sites in pMAK705 (Genencor International, Palo Alto, CA), to generate pMP33. The FM5 of E. coli was transformed with pMP33 and selected on 20 μg / mL of kan at 30 ° C, which is the allowable temperature for the replication of pMAK705. A colony was expanded overnight at 30 ° C in liquid media supplemented with 20 μg / mL of kan. Approximately, 32,000 cells were plated on 20 μg / mL of kan and ss incubated for 16 hours at 44 ° C, which is the restriction temperature for the replication of pMAK705. Transformants growing at 44 ° C have a plasmid integrated within the chromosome that occurs at a frequency of about 0.0001. Analyzes of POR and Southern blotting (E.M. Southern, J. Mol. Biol. 98, 503-517 (1975)) were used to determine the nature of the chromosomal integration events in the transformants. Western blot analysis (Towbin et al., Proc. Nati Acad. Sci. 76.4350 (1979)) was used to determine whether the
Glycerol dehydrogenase protein, the product of gldA, is produced in the transformants. An activity assay was used to determine if the activity of glycerol dehydrogenase remained! in the transformants. The activity in the glycerol dehydrogenase bands on the natural gels was determined by coupling the conversion of glycerol plus NAD + to dihydroxyacetone plus NADH to the conversion of a tetrazolium dye, MTT
[3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] to a deep colored formazan, with methosulfatc of phenazine as mediator. Glycerol dehydrogenase also requires the presence of 30 mM ammonium sulfate and 100 mM Tris, pH 9 (Tang et al, J. Bacter ol. 140, 182 (1997)). From 8 transformants analyzed 6 it was determined that they were agonists to the gldA. The MSP33.6 of E. coli has been deposited with the ATCC under the terms of the Budapest Treaty on
November 24, 1997 EXAMPLE 4 CONSTRUCTION OF A STRAWBERRY OF E. COL! WITH AGENIC GENES
ß And gldA. A DNA fragment qe 1.6 kb containing the gene of gldA and including 228 bps of the 5 'address of the translational start codon and 220 bps of the 3' DNA address of the cc of translational stop,
was isolated from E. coli by PCR using the primers SEQ ID NO: 18 and SEQ ID NO: 19, which incorporate the terminal sites Sphl and Xbal respectively and were cloned between the sphl and Xbal sites of pUC18, to generate pQN2. The pQN2 was cut at the unique Salí and Ncol sites with the gldA gene, the terminals were rinsed with Klenow and re-ligated resulting in a 109 bps elimination in the middle of the gldA and the regeneration of a single site Salí, for ger: erar pQN4. A 1.2 kb DNA fragment containing the jen that conferred resistance to kanamycin (kan) and flanked by the loxP sites, was isolated from pLoxKan2 (Gen ncor International, Palo Alto,
CA) as a Stul / Xhol fragment, the ends were rinsed with Klenow and subcloned into pQN4 at the SalI site after rinsing with Klenow to generate pQN8. A 0.4 kb DNA fragment containing the R6K origin of replication was isolated from pGP704 (Miller and Mekalatos, J. Bacteriol, 370, 2575-2583 (1988)) by PCR using the primers SEQ ID NO: 20 and SEQ ID NO: 21, which incorporate the Sphl / Xbal terminal sites respectively, and were ligated to the 2.8 kb DNA fragment of Sphl / Xbal containing the gldA :: kan cassette from pQN8, to generate pKP22. A DNA fragment of a kb containing the gene conferring resistance to chloramphenicol (cam), and flanked by the loxP sites
isolated from pLoxCat2 (Genencor International, Palo Alto, CA) as in the Xbal fragment and subcloned into pKP22 at the Xbal site to generate pKP23. The RJF10 strain of E. coli (see example 2), which is glpK-, was transformed with pKP23 and the transformants with the kanRcamS phenotype were isolated, indicating a double cross integration that is confirmed by the Southern spotting analysis. The gel activity assays of glycerol dehydrogenase
(as described in Example 3) demonstrate that active glycerol dehydrogenase was not present in these transformants. The kan marker was separated from the chromosome using the Cre producer plasmid pJW168, as described in example 2, to produce the strain
KLP23. Several isolates with the kanS phenotype demonstrate that there is no glycerol dehydrogenase activity, and the southern stain analysis confirmed the loss of the kan marker. EXAMPLE 5 CONSTRUCTION OF PLASMIDE AND CONSTRUCTION OF CEPA FOR THE EXPRESSION OF GLYCEROL 3-PHOSPHATE DEHYDROGENASE (PARÍ AND / OR GLYCEROL 3-PHOSPHATASE (GPP2; Construction of expression cassettes for glycerol
3-phosphatase (gpp2): The chromosome V lambda clone 6592 of Saccharomyces cerevisiae (GenBank, accession number # U18813xll) is
obtained from ATCC. The glycerol 3-phosphate phosphatase gene (GPP2) was cloned by cloning from the lambda clone as the target DNA using synthetic primers (SEQ ID NO: 22 with SEQ ID NO: 23) incorporating a BamHI-RBS-Xbal site in the 5 'termination and a Smal site at the 3' termination. The product was subcloned into pCR-Script (Stratagene, Madison, Wl) and the Srfl site to generate plasmid pAH15 containing GPP2. Plasmid pAH15 contains the GPP2 gene in the inactive orientation for the expression of the lac promoter in pCR-Script SK +. The BamHI-Smal fragment from a pAH15 containing the GPP2 gene was inserted into the pBl LjueScriptII SK + to generate plasmid pAH19. PAH19 contains the GPP2 gene in the correct orientation for the expression of the lac promoter. The Xbal-Pstl fragment of pAH19 contains the GPP2 gene, is inserted into pPHOX2 p) to create the plasmid pAH21. PAH21 / DH5a is the expression plasmid Construction of expression cassettes for glycerol
3-phosphate dehydrogenase (IpARl): DAR1 was isolated by PCR cloning from S genomic DNA. cerevisiae using synthetic primers (SEQ ID NO: 24 with SEQ ID NO: 25). Successful cloning by PCR places a Nco I site at the 5 'end of DAR1 where the ATG within Ncol is the methionine initiator DAR1. At the 3 'termination of DAR1, a site
BamHI is introduced after the terminator of the translation. PCR fragments are digested with Ncol +
BamHI, and cloned into the same sites within the pTtc | 99A expression plasmid (Pharmacia, Piscataway, NJ) to give pDARlA. In order to create a better ribosome binding site at the 5 'end of DAR1, a Spel-RBS-NcoI linker obtained by combining the complementary base pairs of the synthetic primers (SEQ ID NO: 26 with SEQ ID NO: 27 ) is inserted into the Ncol site of the pDARlA to create pAH40. Plasmid pAH40 contains the new RBS and DAR1 gene in the correct orientation for the expression of the trc promoter of pTrc99A (Pharmacia, Piscataway, NJ). The NcoI-BamHI fragment of pDARlA and a second set of the Spel-RBS-NcoI ligator obtained by the combination of the complementary base parss of the synthetic primers (SEQ ED NO: 28 with SEQ ID NO: 29) are inserted into the s itio Spel-BamHI from pBC-SK + (Stratagene, Madison, Wl to create plasmid pAH42, Plasmid pAH42 contains a chloramphenicol resistant gene, Construction of expression cassettes for darl and
9PP2: The expression cassette for DAR1 and GPP2 was assembled from the individual subclones DAR1 and GPP2 above
described, using standard methods of molecular biology.
The BamHI-PstI fragment of pAH19 containing the ribosomal binding site (RBS) and the GPP2 gene were inserted into pAH40 to create pAH43. The BamHI-PstI fragment of pAH19 containing the RBS and GPP2 gene was inserted into pAH42 to create pAH45. The ribosome binding site at the 5 'end of GPP2 is modified as follows. A BamHI-RBS-Spel linker obtained by the synthetic complement primers in the base pairs GATCCAGGAAACAGA (SEQ ID NO: 30) with
CTAGTCTGTTTCCTG (SEQ ID: 31 '), for the Xbal-PstI fragment of pAH19 containing the GPP2 gene, is inserted into the BamHI-PstI site of pAH40 to create pAH48. Plasmid pAH48 contains the DAR1 gene, the modified RBS and the GPP2 gene in the correct orientation for the expression of the trc promoter of pTrc99A (Pharmacia, Piscataway, NJ).
Transformation of E. coli: The plasmids described herein were transformed into DH5a from E. coli, FM5 and KLP23 using standard molecular biology techniques. The transformants were verified by their DNA RFLP pattern. EXAMPLE 6 CONSTRUCTION OF EXPRESSION PLASMIDS FOR USE IN THE
TRANSFORMATION OF ESCHERICHIA COLI WITH GENES OF EL
KLEPS REGULATION [EELLA PNEUMONIAE dha
Construction of the expression vector pTacIQ: The expression vector of E. coli pTaqlQ, was prepared by inserting the laclq gene (Farabaugh, Nature 274 (5673)
765-769 (1978)) and the tac promoter (Amann et al., Gene 25, 167-178 (1983)) within the EcoRI restriction endonuclease site of pBR322 (Sutcliffe, Cold Spring Harb, Symp. Quant. Biol. 43, 77-90 (1979).) A multiple cloning site and a terminator sequence (SEQ ID NO: 32 replaces the pBR322 sequence from EcoRI to Sphl.) Subcloning of glycerol dehydratase genes
(dhaBl, 2.3, X): The open reading structure for the dhaB3 gene was amplified from pHK28-26 by PCR using primers (SEQ ID NO: 33 and SEQ ID NO: 34) that incorporate an EcoRI site in the 5 'ending and an Xbal site at the 3' ending. The product was subplotted into pLitmus29 (New England Biplab, Inc., Beverly, MA) to generate plasmid pDfÍAB3 containing dhaB3. The region that contains the complete coding region for dhaBl, dhaB2, dhaB3 and dhaBX of the dhaB operon of pHK28-26 was cloned into pBluescriptIIKS + (Stratagene, La Jolla, CA) using the restriction enzymes Kpnl and EcoRI to create the pM7 plasmid
The dhaBX gene was separated by digesting plasmid pM7 with Apal and Xbal, purifying the 5.9 kb fragment and ligating it with a 325-bp Apal-Xbal fragment of plasmid pDHAB3 to create pMll containing dhaBl, dhaB2 and dhaB3. The open reading structure for the dhaBl gene was amplified from pHK28-26 by PCR using the primers (SEQ ID NO: 35 and SEQ ID NO: 36) that incorporate a HindIII site and a consensus ribosome binding site. at the 5 'end and an Xbal site at the 3' end. The product was subcloned into pLitmus28 (New England Biolab, Inc., Bpverly, MA) to generate plasmid pDTl containing dhaBl, a Notl-Xba1 fragment! of the pMll containing part of the dhaBl gene, the dhβ2 gene and the dhaB3 gene, was inserted into pDTl to create the dhaB expression plasmid pDT2. The HindIII-Xbal fragment containing the dhaB genes (1,2,3) of pDT2 was inserted into pTacIQ to create pDT3. Subcloning of the 1,3-propanediol dehydrogenase gene
. { dha T): The Kpnl-Sac I fragment of pHK28-26 containing the 1,3-propanediol dehydrogenase gene. { dha T) was subcloned into pBluescriptII KS + creating the pAHl plasmid. The dha T gene was amplified by PCR, from pAHl as DNA
of template and synthetic primers (SEQ ID NO: 37 with
SEQ ID NO: 38) that incorporate an Xbal site at the 5 'end and a BamHI site at the 3' end. The product was subcloned into pCR-Script (Stratagene) at the Srfl site to generate the pAH4 and pAH5 plasmids containing dha T. The pAH4 plasmid containing the dha T gene in the correct orientation for the expression of the lac pCR-Script promoter and pAH5, contains the dha T gene in the opposite orientation. The Xbal-BamHI fragment of pAH4 containing the dha T gene was inserted into pTacIQ to generate the pAH8 plasmid. The HindII-BamHI fragment from pAH8 containing the RBS and the dha T gene was inserted into pBluescriptIIKS + to create pAHll, Construction of an expression cassette for dha T and dhaB (1,2,3): A cassette 1 was assembled from expression for dhaB (1,2,3) from the individual subclones dhaB (1,2,3) and dha T previously described using standard methods of molecular biology. A Spel-Sacl fragment containing the dhaB genes (1,2,3) of pDT3 was inserted into pAHll at the Spel-Sacl sites to create pAH24. A Sall-Xbal linker
(SEQ ID NO: 39 and SEQ ID NO>: 40) was inserted into pAH5 which was digested with the Sall-Xbal restriction enzymes to create pDT16. The linker destroys the Xbal site. The 1 kb Sall-Mlul fragment from pDT16 was inserted after
inside pAH24 replacing the existing Sall-Mlul fragment to create pDT18. PDT21 was constructed by inserting the Sall-Notl fragment from pD '18 and the Notl-Xbal fragment from pM7 into pCL1920 (SEQ ID NO: 41). The sequence of the glucose promoter isomerase from Streptomyces (SEQ ID NO:
42) was cloned by PCR and inserted into the sites
EcoRI-HinDIII from pLitmu $ 28 to construct pDT5 pCL1925 was constructed by inserting the EcoRI-PvuII fragment from pDT5 into the EcoRI-PvuI site of pCL1920. PDT24 was constructed by cloning the HinDIII-MluII fragment from pDT21 and the Mluíl-Xbál fragment from pDT21 within the HinDIII-Xbal sites of pCLL925. Construction of an expression cassette for dha T and dhaB. { 1, 2, 3, X): pDT21 was constructed by inserting the Sall-Notl fragment of pDT18 and the Notl-Xbal fragment of? M7 into pCL1920 (SEQ ID NO: 41). The sequence of the glucose promoter isomerase of S treptomyces (SEQ ID NO: 42) was cloned by PCR and inserted into the EcoRI-HinDIII sites of pLitmus28 to construct pDT5. PCL1925 was constructed by inserting the EcoRI-PvuII fragment of pDT5 into the EcoRI-PvuI site of pCL1920. PDT24 was constructed by cloning the HinDIII-MluII fragment from pDT21 and the Mlul-Xbal fragment from pDT21 within the HinDIII-Xbal sites of pCL 925.
Construction of an expression cassette for dhaR, orfY, dha T, orfX, orfW and dhaB (l, 2,3, X) pDT29 was constructed by inserting the Sacl-EcoRI fragment of pHK28-26 into the SacI-EcoRI sites of PCL1925. Construction of an expression cassette for dhaR, orfY, orfX, orfW, and dhaB (1,2,3, X) A derivative of plasmid pDT29 was constructed in the first 5 tones and the last 5 codons (plus the stop codon) of the dha T gene, it was ttooddooss eexxcceeppttuuaannddoo llccjs They were eliminated by a technique known as PCR-mediated overlap extension. by pDT29 as a template, 2 primary PCR products were generated using the following primers: SEQ ID NO: 43 = 5'GAC GCA AC GTA TTC CGT CGC3 '; SEQ ID NO: 44 = 5'ATG AGC T T CGT ATG TTC CGC CAG GCA TTC
TGA GTG TTA ACG3 '; SEQ ID NO: 45 = 5 'GCC TGG CGG AAC ATA CGA TAG CTC ATA ATA TAC3'; SEQ ID NO: 46 = 5'CGG GGC GCt GGG CCA GTA CTG3 'SEQ ID NO: 45 was paired with SEQ ID NO: 46, to generate a product of 931 base pairs and encompass nucleic acid including dhaBl 5 '(for a single Scal site), all the orfY, and the first five codons
of dha T. SEQ ID NO: 43 was paired with SEQ ID NO: 44 to generate a product 1348 base pairs that included the nucleic acid that includes the last five codons (plus the stop codon) of dha T, all the orfX, all the orfW, and the dhaR 5 '(to a single SapI site). The 15 bases at the 5 'end of SEQ ID NO: 44, constitute one end which is the inverse complement of a 15 base portion of SEQ ID NO: 45. Similarly, the 11 bases at the 5' end of the SEQ ID NO: 45 constitute one end which is the inverse complement of an 11 base portion of SEQ ID NO: 44. Thus, the two primary PCR products were joined after complement 1 < s base pairs (by means of an overlap of the 2 'end base pairs) and spread by PCR to generate a tercei nucleic acid product of 2253 base pairs. This third product by PCR was digested with SapI and Scal and ligated into pDT29 which was also digested with SapI and Scal, to generate the plasmid pKP32 which is identical to pDT2, except for the large elimination within the structure within the dha T EJ SMPLO 7 CONVERSION OF GLUCOSE TO 1 [3-propanediol USING A CEPA
OF E. COLI KLP23 / pAH48 / pDT 29 AND THE IMPROVED PROCESS USING KLP23 / pAH48 / pKP32
Pre-culture: KLP23 / pAH48 / pDT29 and KLP23 / pAH48 / pKP32 are pre-cultured to seed a fermentor in 2YT medium (10 g / L yeast extract, 16 g / L tryptone, and 10 g / L NaCl) containing 200 mg / L of carbenicillin
(or ampicillin) and 50 μg / L of spectinomycin. KLP23 / pAH48 / pKP32 is identical to KLP23 / pAH48 / pDT29 except that it is removed if dha T. Cultures were started from frozen batches (10% DMSO as cryoprotectant) in 500 mL of a medium in an Erlenmeyer flask of 2 liters, which grow at 35 ° C on a shaker at 250 rpm until an OD550 of approximately 1 L or AU is reached and used to seed the fermenter. Fermentor medium The following components were sterilized together in the fermenting vessel: 45 g of KH2P0, 12 g of citric acid, 12 g of MgSO4 * 7H20, 30 g of yeast extract, 2.0 g of ammonium ferric citrate, 5 mL of Mazu DF204 as antifoam, 1.2 g of CaCl2 »2H20, and 7.3 mL of sulfuric acid. The pH was raised to 6.8 with 20-28% NH4OH and 1 SD following compounds were added: 1.2 g of carbenicillin or ampicil ina, 0.30 g of spectinomycin,
60 mL of a solution of trace elements and glucose (from a feed of 60-67% by weight).
After the inoculation the volume was 6.0 L and the glucose concentration f | ue of lOg / L. The solution of the elements in traces contía: citric acid. H20 (4.0),
MnS04 »H20 (3.0), NaCl (l.O) FeS0» 7H20 (0.10), CoCl2 «6H20
(0.10), ZnS04 «7H20 (0.10), CuS04» 5H20 (0.010), H3B03
(0.0010), and Na2Mo04 »2H20 (0 010) Fermentapion growth: A fermentor was prepared in a stirred tank of 15
L with the medium described above. The temperature was controlled at 35 ° C and aqueous ammonia (20-28% by weight) was used to control p-1 to 6.8. The initial values for the air flow rate (set as minimum values of between 6 and 12 standard liters per minute) and the speed of the agitator (set at minimum values between 350 and 690 rpm) were set so that the control dissolved oxygen (DO) was initiated when the OUR values reached approximately 140 mmol / L / h. The back pressure was controlled at 0.5 bar. The DO control was set at 10%. Except for minor courses, the glucose was maintained between 0 g / L and LO g / L with a diet of
60% or 67% by weight. Vitamin B12 or coenzyme was added
Bi2 as seen below, Fermentation with KLP23 / pAH48 / pDT29: A summary of the representative fermentation of the conversion of glucose, 3-propanediol (1,3-PD) using
the strain of E. coli KLP23 / pAH48 / pDT29 is given in table 4. Vitamin Bi2 (0.075 g / L, 500 mL) was fed, starting 3 hours after inoculation at a rate of 16 mL / h. The yield of 1,3-propanediol was 24% by weight (g 1,3-propanediol / g of glucose consumed) and a concentration of 68 g / L of 1,3-propanediol was obtained. TABLE 4 Summary of the representative fermentation, of the conversion of glucose to 1 L 3-propanediol (1, 3, -PD) using the E. coli strain KLP23 / pAH48 / pDT29 Time OD550 OD (%) Glucose Glice ol 1, 3-PD
() (AU) (g / L) (g / L) (g / L) 0 0 150 12.9 0.0 0 6 17 80 8.3 3.1 1 12 42 53 2.8 12.5 9 18 98 9 5.7 12.6 32
24 136 11 32.8 12.0 51
148 10 12.3 13.3 62
32 152 11 12.5 14.3 65
38 159 11 1.5 17.2 68
Similar results are obtained with an identical feed of vitamin Bi2 at two times the concentration or additions of vitamin Bl2 through
of the fermentation time course. The highest concentration obtained was 77 g / L Improved fermentation with LP23 / pAH48 / pKP32 A summary of the fermentation representative of the conversion of glucose to 1,3-propanediol (1,3-PD) using the E. coli strain KLP23 / p &H48 / pKP32 is given in Table 5. Vitamin Bi2 (0.150 g / L, 500 mL) was fed, starting 3 hours after inoculation at a rate of 16 mL / h. After 36 hours, approximately 2 L of the fermentation broth was purged in order to allow the continuous addition of the glucose feed. The yield of 1,3-propanediol was 26% by weight (g 1,3-propanediol / g of glucose consumed) and a concentration of 112 g / L of 1,3-propanediol was obtained. TABLE 5 Summary of representative fermentation of improved conversion of glu cose to 1,3-propanediol (1,3-PD) using a strain of coli KLP23 / pAH48 / oKP32 Time OD550 OD (%) Glucose Glice ol 1,3 -P.S
(h) (AU) (g / L) (g / L) (g / L) 0 0 148 12.8 0.0 0 6 22 84 6.9 3.3 0
12 34 90 9.7 10.4 7
18 66 43 9.3 5.9 24
24 161 9 0.2 2.5 46
200 10 0. 2 6. 0 67 36 212 10 1. 2 9. 7 88 42 202 2 0. 1 15. 5 98 48 197 12 1. 2 23. 8 112
Similar results are obtained with an identical feed of vitamin Bi2 at half the concentration or with bolus additions of vitamin Bi2 through the time course of the fermentation. The highest obtsnide concentration was 114 g / L. EXAMPLE 8 PREPARATION BY ENGINEERING THE TRIOSEFOSPHATE MUTANT
ISOMERASA OF E. COLI KLP23 FOR PERFORMANCE
ENRICHED 1, 3-PROPANODIOL FROM GLUCOSE The construction of the plasmid for the replacement of the triosephosphate isomerase gene in E. coli KLP23 The E. col and KLP23 of genomic DNA was prepared using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). A DNA fragment of a kb containing cdh and the 3 'end of the triosephosphate isomerase genes [tpiA) was amplified by PCR (Mullis and Faloona, Methods Enzymol., 155, 335-350 (1987)) from a KLP23 genomic DNA using the primers SEQ ID NO: 47 and SEQ ID NO: 48. A 1.0 kb DNA fragment containing the 5 'terminus of tpiA,
yiiQ, and the 5 'termination of yiiR genes, was amplified by PCR from the KLP23 genomic DNA using the primers SEQ ID NO: 49 and SEQ ID NO: 50. A Scal site was incorporated into the primer SEQ ID NO: 49 The 5 'terminus of the primer SEQ ID NO: 49 was the reverse complement of the primer SEQ ID NO: 48 to allow the extension of subsequent overlap by PCR. The splicing of genes by the overlap extension technique (Horton et al., E ioTechniques 8, 528-535 (1990)) was used to generate a 2 kb fragment by PCR using the two previous PCR fragments as templates and primers SEQ ID NO: 47 and SEQ ID NO: 50. This fragment represented a 73% deletion of the tpiA structural gene of 768 base pairs. In total, this fragment had flanking regions of 1.0 kb on each side of the Scal cloning site (within the partial tpiA) to allow replacement of the chromosomal gene by homologous recombination.
The previous 2.0 kb PCR fragment, complete in its base pairs, was cloned into a complete PCR-vector in its base pairs using the Zero base pair completion PCR cloning kit (Invitrogen, San Diego, CA) to yield the 5.5 kb pRN106-2 plasmid containing kanamycin and zeocin resistance genes. The 1.2 kb HincII fragment from pLoxCatl (unpublished results), which contained a gene from
Resistance to chloramphenilcol flanked by the bacteriophage Pl loxP sites (Snaith et al., Gene 166, 173-174 (1995)), was used to interrupt the tpiA fragment in plasmid pRN106-2 by ligating it with the plasmid digested by Scal pRN106- 2 to yield a 6.8 kb pRN107-l plasmid. Engineering of the triosephosphate isomerase RJ8m mutant by linear transformation eg DNA Using pRN107-l as a template and the primers SEQ ID NO: 47 and SEQ ID NO: 50, the 3.2 kb fragment containing the tpiA flanking regions was amplified by PCR. and the cassette loxP-CmR! loxP and extracted with gel. He
KLP23 from E. coli was electrotransformed with up to 1 μg of this linear DNA fragment of 3.2 kb and transformants that were resistant to chloramphenicol (12.5 μg / mL) and sensitive to kanamycin (30 μg / mL) were separated by further exclusion on M9 minimal media for poor utilization of glucose on 1 mM glucose, for normal use of gluhonate on 1 mM gluconate, and to ensure the phenotype of non-use of glycerol from guest KLP23 on 1 mM glycerol. A digest of
EcoRI of genomic DNA from one such mutant,
RJ8m, when placed in probes with the intact tpiA gene by means of Southern analysis (Southern, J. Mol. Biol. 98, 503-517 (1975)) indicates that it was a member
double cross (I replace tpiA genes) since the two expected bands of i. 6 kb and 3.0 kb were observed, due to the presence of an additional EcoRI site within the chloramphenicol resistance gene. As expected, the KLP23 host and wild-type FM5 controls produced simple bands of 8.9 kb and 9.4 kb respectively. This tpiA mutant was further analyzed by genomic PCR using the primers SEQ ID NO: 51 and SEQ ID NO: 52, which resulted in the expected PCR fragment of 4.6 kb whereas for the same primer pair, the KLP23 host and the strains Wild-type FM5 both yielded the expected PCR fragment of 3.9 kb. When the cell-free extracts of the tpiA mutant RJ8m and the host KLP23 were tested for tpiA activity using glyceraldehyde 3-phosphate: or substrate, no activity was observed with the RJ8m. The RJ8m mutant of tpiA was electrotransformed with pJlasmid pAH48 to allow the production of glycerol from glucose and also with both plasmids pAH48 and pDT29 or pKP32 to result in the production of 1,3-propanediol from glucose. The chloramphenicol resistance marker was removed from RJ8m to give RJ8
the strain of E. coli RJ8 / pAH48 / pDT29 is given in Table 6. Vitamin B: 2 was provided as bolus additions of 2, 16 and 16 mg at 2, 8, and 26 h, respectively. The yield of 1,3-propanediol was 35% by weight (g of 1,3-propanediol / g of glucose consumed) and a concentration of 50.1 g / L of 1,3-propanediol were obtained. TABLE 6 Summary of the representative fermentation of the conversion of glucose to 1,3-propanediol (1,3-PD) using E. coli strain RJ8 / pAH48 / pDT29 Time OD550 OD (% Glucose Glice or 1.3- P.S.
(h) (AU) (g / L) (g / L) (g / L) 0 0 140 10.6 0.1 0.0 6 5 107 11.1 0.5 0.4
16 90 8.5 1.7 1.3
14 25 86 1.8 2.4 5.9
19 38 53 3.5 5.9 15.4
53 38 0.1 9.2 26.7
31 54 10 4.5 7.4 39.0
37 37 23 17.2 6.0 45.0
43 21 13 9. 9 7.7 50.1
Improved fermentation with RJ8 / pAH48 / pKP32 A summary of the representative fermentation of 1; conversion of glucose to, 3-propanediol (1,3-PD) using the E. coli strain RJ8 / pAH4 8 / pKP32 is given in table 7. Vitamin Bi2 was supplied as bolus additions of 48 and 16 mg to approximately 26 and 44 hours respectively. The yield of 1,3-propanediol was
36% by weight (g of 1,3-propanediol / g of glucose consumed) and a concentration of 129 g / L of 1,3-propanediol was obtained TABLE 7 Summary of the representative fermentation of the improved glucose conversion to 1,3-propanediol (1,3-PD) using the E. coli strain RJ8 / pAH48 / pKP32
Time OD550 OD (%) Glucose Glycerc > 1 1,3-PD
(h) (AU) (g / L) (g / L) (g / L) 0 0 150 12.6 0.1 0 6 12 113 6.0 2.6 0 12 24 99 0.0 10.6 0 18 51 76 2.4 28.9 0 24 78 82 2.4 44.2 5 30 114 70 3.8 26.9 33 36 111 72 0.0 20.0 57 42 139 65 0.1 21.9 69 48 157 36 0.1 22.4 79
55 158 25 0.2 21.4 94 64 169 14 0.1 15.8 113
72 169 12 0.1 13.4 119
74 162 14 0.1 14.8 129
EXI IMPLEMENT 10 IDENTIFICATION OF THE NON-SPECIFIC ACTIvITIES OF E. COLI
(yghD) IN THE PROCESS B IEJORADO DE 1, 3-PROPANODIOL
Demonstration of non-specific catalytic activity in fermentations that produce 1, 3-propanediol with the improved catalyst: An essay of sperm cells for the activity of the
1, 3-propanediol dehydrogenase was used to demonstrate that the catalytic activity is not specific in E. coli.
Time dependent 3-HPA was observed in the flasks containing the cells recovered from the fermenter either pre or post-addition of vitamin B12. In direct contrast, significant levels of 1,3-prop-diol were observed only in those flasks containing the cells recovered from the addition to the posterior fermentor of vitamin B12. Detection of non-specific catalytic activity in cell-free extracts An activity staining assay with a native gel was used to demonstrate 'non-specific catalytic activity in cell-free extracts. Cells were recovered, pre and post-addition with vitamin B? 2, representative fermentation of 10-L using recombinant strains of E. coli which contained glycerol production plasmids and production of 1,3-propanediol, pAH48 and p P32, respectively; and the cell-free extracts were prepared by disrupting cells using a French press. Cell-free extracts, a preparation of 1,3-propanediol dehydrogenase from Klebs iella pneumoniae. { dha T), and molecular weight standards were applied to and run on native gradient polyacrylamide gels,
The gels were then exposed to the substrates of 1,3-propanediol and NAD + or ethanol and NAD +. As expected in
the gels where 1, 3-propanediol was the substrate, a dha T activity staining was observed which migrated on the native gel at approximately 340 Kdla. This activity was observed only in the lanes where 1, 3-propanediol dehydrogenase of pure Klebsiella pneumoniae was applied. In contrast, where 1,3-propanediol was the substrate and cell-free extracts subsequent to vitamin B 2 were applied, a non-specific catalytic activity was observed at approximately 90 Kdal. When using ethanol as a substrate, neither the dha T band nor the non-specific catalytic activity band were visible, but a separate band was found before and after the addition of vitamin B 2 to approximately 120 Kdal. This new band most likely represents an alcohol dehydrogenase with specificity towards ethanol as a substrate as is typically found in all organisms. This native gel assay, in which the proteins are separated by molecular weight before the enzymatic assay stage, offers greater sensitivity and accuracy in measuring the reduction of 1,3-propanediol in those constructs with low activity and where the activity is likely to be different from alcohol dehydrogenases with a specificity towards ethane as a substrate, which have been characterized for E. coli and
they are found in all organisms. The dehydrogenase assay works on the principle that dehydrogenase catalyses the transfer of electrons from 1,3-propanediol (or other alcohols) to NAD +. PMS (phenazine methosulfate) is then coupled to the transfer of electrons between NADH and the dye tretazolium bromide (MTT, 3-4,5-dimethylthiasol-2-yl] -2,5 diphenyltetrasolium bromide) which forms a precipitate in the gel. After a few hours, to rinse the substrates overnight, the gels are washed to separate the reagents and the soluble dye. An insoluble blue dye is formed in the bands where the gel where there is an active dehydrogensa. Various aspects of the assay have been described by Johnson and Lin (J. Bacteriol., 169: 2050 (1987)). Purification and identification of the non-specific catalytic activity in E. co j A large-scale partial purification of the non-specific catalytic activity was carried out in cells harvested from the excrement of a typical production run of 1,3-propanediol, as is described in the improved process using KLP23 / pAH48 / pKP32 of Example 7. The cell pellet was washed (16 g) and resuspended three times in 20 ml of a 50 Hepes buffer solution, Mm ph 7.5. The
cells in the suspension by sonication. The cell-free extract was obtained by centrifugation (15 min, 20,000 x g, 10 ° C) and the supernatant was further clarified by adding 250 mg of protamine sulfate with stirring on ice. The supernatant obtained by centrifugation (20 min, 20,000 x g, 10 ° C) was fractionated by passing through a Superdex® 200 preparative grade column (6 x 60 cm) equilibrated with Hepes buffer solution. The fractions of 10 ml each were collected, and an aliquot of each was concentrated 25 times using Centricon® membranes of PM 10,000 cut before assay by gel activity staining. The non-specific catalytic activity was identified in fractions 107-11, and the peak activity in fractions 108-109. A larger aliquot (7 ml each) of fractions 108 and 109 be concentrated 50 times and loaded onto all tracks of a native 12-track gel. The gel was cut in halves and one half was stained for dehydrogenase activity where a dark blue band appeared that represented by non-specific catalytic activity. The unstained gel was aligned from top to bottom with the dyed gel and a band was cut over the undyed cfel which corresponds to the band of non-specific catalytic activity. The gel cassette was pulverized and the soluble protein was extracted
immerse the pulverized particles in 2D loading buffer in 0.5 ml, heating at 95 ° C for 5 minutes, and centrifugation to separate the gel particles. The supernatant was loaded onto an isoelectric focus cassette (IEF) for a two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using the conditions described for 2D-PAGE of the E. coli extracts from the Swiss database 2D ( http: // www. expasy. ch / ch2d l_; Tonella et al., Electrophoresis 19: 1960-1971 (1998)). The gel was transferred to an electro stained PVDF membrane. The membrane was stained for protein 3 using colloidal blue gel staining. The stained dyeing used to obtain the identity of the non-specific catalytic activity is shown in Figure 6. The spots are identified using standard techniques for the formation of amino-termination peptide sequences. Only one single spot (spot A) encoded the activity of the oxide reductase. 19 cycles of spot A (FIG. 6) produced a 100% identity correspondence by the FAATA search tool with the amino terminus of yqhD, an open reading structure of E. coli with a putative activity of oxide reductase. The complete amino acid sequence for the protein encoded by yqhD is given in SÚQ ID NO: 57; sequence
The corresponding DNA is given in SEQ ID NO: 58. The yqhD gene has a 40% identity with the dhaB gene in Clostridium, a butadiene-dependent transferase of the NADH 2. Disruption of the yqhD gene in E. coli KLP23: Biochemical assays and formation of amino acid sequences at the amino terminus suggest that non-specific catalytic activity can be encoded by the E. coli and qhD gene. This gene of unknown function encodes a hypothetical reduct asa oxide and contains two alcohol dehydrogenase signatures also found in the 1,3-propanediol of shidrogensa of Klebsiella pneumoniae and Ci trobacter freundii encoded by the gene of dha T. To create disruption? in this gene, the yqhD and a 5 'flanking DNA sequence of 830 base pairs and a 3' flanking DNA sequence of 906 base pairs of E. coli KLP23 (example 4) were amplified in genomic DNA in a PCR using polymerase Táq and the following primers: (SEQ ID NO: 59) 5'-GCGGTACCGTTGCTCGACGCTCAGGTTTTCGG -3 '(SEQ ID NO: 60) 5' GCGAGCTCGACGCTTGCCCTGATCGAGTTTTGC-3 'The reaction was carried out at 94 ° C for 1 minute, 50 ° C for one minute and 72 ° C for 3 minutes for 35 cycles followed by an inal extension at 72 ° C for 5 minutes. The resulting DNA fragment 3.7 Kb was purified,
digested with Sacl and Kp > nl and ligated to similarly digest pBluescriptl KS (+) (Stratagene) by 16 H at 16 ° C. The ligated DNA was used to transform E. coli
DH5a (Gibco / BRL) and the expected plasmid pJSP29, was isolated from a transformant demonstrating a white colony color on Lb agar (Difco) containing X-gal (40 μg / mL) and ampicillin (100 μg / mL). Plasmid pJSP29 was digested with AflII and Ndel to release a DNA fragment of 409 base pairs qu <; it comprises 363 base pairs of the yqhD gene and 46 base pairs of the 3 'flanking DNA sequence. The 5.350 base pair recombinant DNA fragment was purified and ligated to the 1,374 base pair DNA AflII / Ndlel DNA fragment containing the kanamycin resistance gene of pLoxKan2 (Genecor International, Palo Alto, CA) by 16 to 16 ° C. The ligated DNA was used to transform E. coli DH5a and plasmid blue expected pJSP32, was isolated from a transformant selected on LB agar medium containing kanamycin (50 μq / ml9. Plasmid pJSP32 was digested with KpnI blue and S acl and the disruption cassette yqhD of 3,865 base pairs, was purified and ligated to a similarly digested pGP704 (Miller and Mekalanos, J. Bacteriol,
170: 2575-2583 (1988) for 16 h at 16 ° C. The ligated DNA was used to transform E. coli SY327 (Miller and Mekalanos, J. Bacterial 170: 2575-2583 1988)) and the expected plasmid
pJSP32 was isolated from a selected transformant on an LB agar medium containing kanamycin (50μg / mL). Plasmid pJSP32 was transformed into E. coli KLP23 and transformants were selected on LB agar containing kanamycin (50 μg / mL). Of the resistant transformants of kanamycin 200, separated by exclusion, two showed that the ampicillin-sensitive phenotype expected for a double trans-double recombination event results in the replacement of the yqhD gene with the cassette of disruption: n and qhD. Disruption of the yqhD gene was confirmed by PCR using these two transformants isolated as the template and the following sets of primer pairs genomic DNA: Set # 1 (SEQ ID NO: 61) 5 '-GCGAGCTC (ACGCTTGCCCTGATCGAGTTTTGC-3'
(SEQ ID NO: 62) 5 '-CAGCTGGCAATTCCGGTTCG-3' Set # 2: (SEQ ID NO: 63) 5 '-CCCAGCTGGCAATTCCGGTTCGCTTGCTGT-3' (SEQ ID NO: 64) 5 '-GGCGACCCGACGCTCCAGACGGAAGCTGGT-3' Set # 3 ( SEQ ID NO: 65) 5 '-CCGCAAGATTCACGGATGCATCGTGAAGGG-3' (SEQ ID NO: 66) 5 '-CGCCTTCTT | GACGAGTTCTGAGCGGGA-3' daub Conj # 4 (SEQ ID NO: 67) 5 '-GGAATTCATGAACAACTTTAATCTGCACAC-3'
(SEQ ID NO: 68) 5 '-GTTTGAGG: GTAAAAAGCTTAGCGGGCGGC-3' The reactions were done using the Expand High Fidelity Polymerase (Boehrenger Manheim) or Platinum PCR Supermix containing Taq polymerase (Gibco / BRL) at 94 ° C Polymerase for minute, 50 ° C for one min, and 72 ° C for 2 min for 35 cycles followed by a final extension at 72 ° C for 5 min. The resulting PCR products were analyzed by gel electrophoresis in 0.1% agarose (W / V). The results summarized in Table 8 confirm the disruption of the yghD gene transformants, TABLE '8 Expected size set (bp) size primers Disruption wild type observed yghD yghD (bp) 1,200 without product ~ 1,200 1,266 without product ~ 1,266 2,594 without product - 2,594 without product 1,189 -900 Disruption with yghD eliminates the 3 'termination of the yqhD, including 46 base pairs of the 3' flanking DNA sequence. The deletion separates 363 base pairs from the coding sequence yqhD 3 'corresponding to 121 amino acids. A stop codon is present in the 3 'direction of 15 base pairs of the
yqhD coding sequence remaining in the cassette of resistance to kanamicin pAH48 and pKP32 plasmids were co-transformed into E. coli KLP23 (yghD) and transformants containing plasmids were selected on LB agar containing ampicillin (lOOμg / mL) and spectinomycin (50μg / mL). A representative transformant was tested for its ability to convert glucose to 1,3-propanediol in 10 L fermentations in the presence or absence of vitamin B1. Demonstration that yghD is required for the important production of 1, 3-propanediol from the strain of
E. coli KLP23 / pAH48 / pKP32 The fermentations for the production of 1,3-propanediol were carried out essentially as described in example 7, with the E. coli strain and KLP23 (yghD-) / ÁH48 / pKP32 with object of testing the effect of disruption yqhD on the production of 1,3-propanediol. Representative fermentation of 10L using the aginic activity of non-specific allytic activity, strain E. coli KLP23 (yghD-) / pAH48 / pKP3 2, is shown in Table 9. The organism accumulates permamently cell mass and glycerol up to adding vitamin B 2 giving the OD550 exceeds 30A (10.4 h). Vitamin B? 2 was added as a
bolus addition of 8 mg to 10.4 h and then continuously fed vitamin B12 at a rate of
1. 32 mg / h. The fourth hour that followed the addition of B 2, the glucose consumption was decreased, the oxygen utilization rate fell and there was no additional increase in the optical density. The fermentation of the glucose was stopped and the concentration of glucose in the tank accumulated. The highest concentration of 1,3-propanediol obtained was 0.41 g / L. The organism was verified by its viability to form plates in a series of dilution of the cells on agar plates containing ampicillin and spectinomycin. The plates were incubated for 24 h at
° C in the incubator. There were no viable colonies on the plate of the E. coli fermentation KLP23. { yqhD-) / pAH48 / pKP32, table 11. In contrast, the cell suspension of a control tank to which vitamin Bi2 was not added, continued to accumulate the mass of cells and glycerol until the 10 L tank was added. filled due to the complete addition of the glucose feed solution (table 10). A determination of viability of the agar plates by dilution series of the cell suspension at the end of this fermentation showed a viable cell count that was consistent with the total number of cells estimated by the optical density value (table 11)
TABLE 9 Summary of the representative fermentation of the failed conversion of gluεose to 1,3-propanediol (1,3-PD) using the E. coli strain LP23 (yghD-) / pAH48 / pKP32. Time OD550 OD glucose glycerol 1,3-PD (h) (AU) (%) (g Vi) (g / D (g / D
0 0.4 150 11 0.05 0 2.3 3.0 134 10 0.13 0
4. 3 10.8 85.0 8 1.41 0 8.3 23.1 81.8 0 10.0 0 16.3 37.2 149 13 21.4 0.41 18.3 47.6 149 18 21.6 0.39 20.3 39.6 149 24 22.3 0.42 23.8 33.6 148 25 22.0 0.41 TABLE 10 Summary of the representative fermentation of the conversion of glucose to glycerol using the E. coli strain KPL23 (yghD-) / pAH48 / pKP32. Time (h) OD550 (AU) DO (%) Glucose (g / D Glycerol (g / 1) 0 0.2 148 9.5 0.06 2.2 2.8 128 8.9 0.13 4.2 10.4 58.5 7.0 1.4 8.2 21.6 57.6 2.7 11.2 16.2 76.8 10.7 0 40.5
. 2 117 10.2 0 52.9
23. 7 154 8.5 0 63.9
36. 2 239 10.1 0.1 122 TABLE 11 Representative summary of plaque viability counts from the terminal points of glucose fermentations is the E. coli strain KLP23 (yghD -) / pAH48 / pKP23 in the absence and presence of vitamin Bi2. Vitamin B12 time (h) to '550 (AU:) viable Terminal accounts (cfu / ml no 36.2 23.9 2.1E11 yes 23.8 33.6 0 YES 23.8 41.2 0
LIST OF SEQUENCES < 110 > He. du Pont de Nemours and Company P Prroocceessoo p raarraa l laa P Prroodduucccciioonn BBiioollóóagiiccaa ddßß < 120 > 1,3-Propanediol High Concentration
< 130 > BC1020 PCT < 140 > < 141 > < 150 > 60 / 149,534 < 1S1 > 1999-08-08 < 160 > 68 < 170 > Microsoft Office 97 < 210 > 1 < 211 > 12145 < 212 > DNA < 213 > Klebsiella pneumoniae < 400 > 1 gtcgaccacc acggtggtga ctttaatgcc gctctcatgc agcagctcgg tggcggtctc 60 aaaattcagg atgtcgccgg tatagttttt gataa.tcagc aagacgcctt cgccgccgtc 120 aatttgcatc gcgcattcaa acattttgtc cggcgtcggc gaggtgaata tttcccccgg 180 acaggcgccg gagagcatgc cctggccgat atagccgcag tgcatcggtt catgtccgct 240 gccgccgccg gagagcaggg ccaccttgcc agccaccggc gcgtcggtgc gggtcacata 300 cagcgggtcc tgatgcaggg tcagctgcgg atgggcttta gccagcccct gtaattgttc 360 attcagtaca tcttcaacac ggttaatcag ctttttcatt attcagtgct ccgttggaga 420 aggtccgatg ccgcctctct gctggcggag gcggtcatcg cgtaggggta tcgtctgacg 480? tggagcgtg cctggcgata tgatgattct ggctgagcgg acgaaaaaaa gaatgccccg 540 acgatcgggt ttcattacga aacattgctt cctgattttg tttctttatg gaacgttttt 600 gctgaggata tggtgaaaat gcgagctggc gcgctttttt tcttctgcca taagcggcgg 660 tcaggatagc cggcgaagcg ggtgggaaaa aattttttgc tgattttctg ccgactgcgg 720 gagaaaaggc ggtcaaacac ggaggattgt aagggcatta tgcggcaaag gagcggatcg 780 ggatcgcaat cctgacagag actagggttt tttgttccaa tatggaacgt aaaaaattaa 840 cctgtgtttc to atcagaac aagatttttt aaaaaggcga tgttccctgc cggccctaca 900 gtgatcgcac tgctccggta cgctccgttc aggccgcgct tcactggccg gcgcggataa 960 cgccagggct catcatgtct acatgcgcac ttatttgagg gtgaaaggaa tgctaaaagt 1020 tattcaatct ccagccaaat atcttcaggg tcctgatgct gctgttctgt tcggtcaata 1080 tgccaaaaac ccggcggaga gcttcttcgt catcgctgac gatttcgtaa tgaagctggc 1140 gggagagaaa gtggtgaatg gcctgcagag ccacgatatt cgctgccatg cggaac GGTT 1200 taacggcgaa tgcagccatg cggaaatcaa ccgtctgatg gcgattttgc aaaaacaggg 1260 ctgccgcggc gtggtcggga tcggcggtgg taaaaccctc gataccgcga aggcgatcgg 1320 ttactaccag aagctgccgg tggtggtgat cccgaccatc gcctcgaccg atgcgccaac 1380 cagcgcgctg tcggtgatct acaccgaagc gggcgagttt gaagagtatc tgatctatcc 1440 gaaaaacccg gatatggtgg tgatggacac ggcgattatc gccaaagcgc cggtacgcct 1500 gctggtctcc ggcatgggcg atgcgctctc cacctggttc gaggccaaag cttgctacga 1560 tgcgcgcgcc accagcatgg ccggaggaca gtccaccgag gcggcgctga gcctcgcccg 1620 cctgtgctat gatacgctgc tggcggaggg cgaaaaggcc cgtctggcgg cgcaggccgg 1680 ggtagtgacc gaagcgctgg agcgcatcat cgaggcgaac acttacctca gcggcattgg 1740 ctttgaaagc agtggcctgg ccgctgccca tgcaatccac aacggtttca ccattcttga 1800 agagtsccat cacctgtatc acggtgagaa agtggccttc ggtaccctgg cgcagctggt 1860 gctgcagaac agcccgatgg acgagattga aacggtgcag ggcttctgcc agcgcgtcgg 1920 cctgccggtg acgctcgcgc agatgggcgt caaagagggg atcgacgaga aaatcgccgc 1980 ggtggcgaaa gctacctgcg cggaagggga aaccatccat aatatgccgt ttgcggtgac 2 040 cccggagagc gtccatgccg ctatcctcac cgccgatctg ttaggccagc agtggctggc 2100 gcgttaattc gcggtggcta aaccgctggc ccaggtcagc 2160 ggtttttctt tctcccctcc ggcagtcgct gccggagggg ttctctatgg tacaacgcgg aaaaggatat gactgttcag 2220 actcaggata ccgggaaggc ggtctcttcc gtcattgccc agtcatggca ccgctgcagc 2280 aagtttatgc agcgcgaaac ctggcaaacg ccgcaccagg cccagggcct gaccttcgac 2340
tccatctgtc ggcgtaaaac cgcgctgctc accatcggcc aggcggcgct ggaagacgcc 2400 tgggagttta tggacggccg cccctgcgcg ctgtttattc ttgatgagtc cgcctgcatc 2460 ctgagccgtt gcggcgagcc gcaaaccctg gcccagctgg ctgccctggg atttcgcgac 2520 ggcagctatt? tgcggagag cattatcggc acctgcgcgc tgtcgctggc cgcgatgcag 2580 ggccagccga tcaacaccgc cggcgatcgg cattttaagc aggcgctaca gccatggagt 2640 ttttgctcga cgccggtgtt tgataaccac gggcggctgt tcggctctat ctcgctttgc 2700 tgtctggtcg agcaccagtc cagcgccgac ctctccctga cgctggccat cgcccgcgag 2760 gtgggtaact ccctgcttac cgacagcctg ctggcggaat ccaaccgtca cctcaatcag 2820 atgtacggcc tgctggagag catggacgat ggggtgatgg cgtggaacga acagggcgtg 2880 tcaatgttca ctgcagtttc ggcggcgaga ctgctgcatc ttgatgctca ggccagccag 2940 gggaaaaata tcgccgatct ggtgaccctc ccggcgctgc tgcgccgcgc catcaaacac 3000 gcccgcggcc tgaatcacgt cgaagtcacc tttgaaagtc agcatcagtt tgtcgatgcg 3060 gtgatcacct taaaaccgat tgtcgaggcg caaggcaaca gttttattct gctgctgcat 3120 agatgcggca ccggtggagc gctgatgacc agccagctcg gtaaagtcag ccacaccttt 3180 gagcag ATGT ctgccgacga tccggaaacc cgacgcctga tccactttgg ccgccaggcg 3240 gcgcgcggcg gcttcccggt gctactgtgc ggcgaagagg gggtcgggaa agagctgctg 3300 ttcacaatga agccaggcta aagcgaacgg gcgggcggcc cctacatctc cgtcaactgc 3360 cagctatatg ccgacagcgt gctgggccag gactttatgg gcagcgcccc taccgacgat 3420 gaaaatggtc gcctgagccg ccttgagctg gccaacggcg gcaccctgtt tctggaaaag 3480 atcgagtatc tggcgccgga gctgcagtcg gctctgctgc aggtgattaa gcagggcgtg 3540 ctcacccgcc tcgacgcccg gcgcctgatc ccggtggatg tgaaggtgat tgccaccacc 3600 accgtcgatc tggccaatct ggtggaacag aaccgcttta gccgccagct gtactatgcg 3660 ctgcactcct ttgagatcgt catcccgccg ctgcgcgccc gacgcaacag tattccgtcg 3720 ctggtgcata accggttgaa gagcctggag aagcgtttct cttcgcgact gaaagtggac 3780 gatgacgcgc tggcacagct ggtggcctac tcgtggccgg ggaatgattt tgagctcaac 3840 agcgtcattg agaatatcgc catcagcagc gacaacggcc acattcgcct gagtaatctg 3900 tcttttccga ccggaatatc gcggccgggc ggggatagcg cgtcatcgct gctgccggcc 3960 agcctgactt ttagcgccat cgaaaaggaa gctattattc acgccgcccg ggtgaccagc 4020 gggcgggtgc to ggagatgtc gcagctgctc aatatcggcc gcaccaccct gtggcgcaaa 4080 atgaagcagt acgatattga cgccagccag ttcaagcgca agcatcaggc ctagtctctt 4140 cgattcgcgc catggagaac agggcatccg acaggcgatt gctgtagcgt ttgagcgcgt 4200 cgcscagcgg atgcgcgcgg tccatggccg tcagcaggcg ttcgagccga cgggactggg 4260 tgcgcgccac gtgcagctgg gcagag? CGA gattcctccc cgggatcacg aactgtttta 4320 ctcggccata acgggccgct ttgcggtcga taagccgctc cagggcggtg atctcctctt 4380 cgccgatcgt ctggctcagg cgggtcaggc cccgcgcatc gctggccagt tcagccccca 4440 gcacgaacag atatggtgca cgtctgctga ggctttcccg cagcccggcg tcgcgggtcg 4500 tggcgtagca gacgcccagc tgggatatca gttcatcgac ggtgccgtag gcctcgacgc 4560 gaatatggtc tttctcgatg cggctgccgc cgtacagggc ggtggtgcct ttatccccgg 4620 tgcgggtata gatacgatac attcagtttc tctcacttaa cggcaggact ttaaccagct 4680 gcccggcgct ggcgccgagc gtacgcagtt gatcgtcgct atcggtgacg tgtccggtag 4740 ccagc? GCGC gtccgccggc agctgggcat gagtgagggc tatctcgccg gacgcgctga 4800 gcccgatacc cacccgcagg ggcgagcttc tggccgccag ggcgcccagc gcagcggcgt 4860 caccgcctcc gtcatag gtt atggtctggc aggggacccc ctgctcctcc agcccccagc 4920 acagctcatt gatggcgccg gcatggtgcc cgcgcg atc gtaaaacagg cgtacgcctg 4980 gcggtgaaag cgacatgacg gtcccctcgt taacactcag aatgcctggc ggaaaatcgc 5040 ggcaatctcc tgctcgttgc ctttacgcgg gt cgagaac gcattgccgt cttttagagc 5100 catctccgcc atgtagggga agtcggcctc ttttaccccc agatcgcgca gatgctgcgg 5160 tccatcgaca aataccgata gacgcgtgat agcggcgatg gctttttccg ccgcgtcgag 5220 agtggacagt ccggtgatat tttcgcccat cagttcagcg atatcggcga atttctccgg 5280 gttggcgatc aggttgtagc gcgccacatg cggcagcagg acagcgttgg ccacgccgtg 5340 cggcatgtcg tacaggccgc ccagctggtg cgccatggcg tgcacgtagc cgaggttggc 5400 gttattgaaa gccatcccgg ccagcagaga agcataggcc atgttttccc gcgcctgcag 5460 attgctgccg agggccacgg cctggcgcag gttgcgggcg atgaggcgga tcgcctgcat 5520 ggcsgcggcg tccgtcaccg ggttagcgtc tttggagata taggcctcta cggcgtgggt 5580 cagggcatcc atcccggtcg ccgcggtcag ggcggccggt ttaccgatca tcagcagtgg 5640 atcgttgata gagaccgacg gcagtttgcg ccagctgacg atcacaaact tcactttggt 5700 ttcggtgttg gtcaggacgc ag tggcgggt gacctcgctg gcggtgccgg cggtggtatt 5760 gaccgcgacg ataggcggca gcgggttggt cagggtctcg attccggcat actggtacag 5820 atcgccctca tgggtggcgg cgatgccgat gcctttgccg caatcgtgcg ggctgccgcc 5880 gcccacggtg acgatgatgt cgcactgttc gcggcgaaac acggcgaggc cgtcgcgcac 5940 gttggtgtct ttcgggttcg gctcgacgcc gtcaaagatc gccacctcga tcccggcctc 6000 ccgcagataa tgcagggttt tgtccaccgc gccatcttta attgcccgca ggcctttgtc 6060 ggtgaccagc agggc tttt tcccccccag cagctggcag cgttcgccga ctacggaaat 6120 ggcgttgggg ccaaaaaagt taacgtttgg caccagataa tcaaacatac gatagctcat 6180
aatatacctt ctcgcttcag gttataatga ggaaaaacaa tccagggcgc actgggctaa 6240 taattgatcc tgctcgaccg taccgccgcc aacgccgacg gcgccaatta cctgctcatt 6300 aaaaataact ggcaggccgc cgccaaaaa aataattcgc tgttggttgg ttagctgcag 6360 accgtacaga gattgtcctg gctggaccgo tgacgtaatt tcatgggtac cttgcttcag 6420 gctgcaggcg ctccaggctt tattcaggg aatatcgcag ctggagacga aggcctcgtc 6480 catccgctgg ataagcagcg tgttgcctcc gcggtcaact acggaaaaca ccaccgccac 6540 gttgatctca gtggcttttt tttccaccgc cgccgccatt tgctgggcgg cggccagggt 6600 gattgtctga acttgttggc tcttgttcat cattctctcc cgcaccagga taacgctggc 6660 gcgaatagtc agtagggggc gatagtaaaa aactattacc attcggttgg cttgctttat 6720 ttttgtcagc gttattttgt cgcccgccat gatttagtca atagggttaa aatagcgtcg 6780 gaaaaacgta attaagggcg ttttttatta attgatttat atcattgcgg gcgatcacat 6840 tttttatttt tgccgccgga gtaaagtttc atagtgaaac tgtcggtaga tttcgtgtgc 6900 cgaaattaaa caaattgaaa tttatttttt tcaccactgg ctcatttaaa gttccgctat 6960 tgccggtaat ggccgggcgg caacgacgct ggcccggcgt attcgctacc gtctgcggat 7020 ttcacctt tt gagccgatga acaatgaaaa gatcaaaacg atttgcagta ctggcccagc 7080 gccccgtcaa tcaggacggg ctgattggcg agtggcctga agaggggctg atcgccatgg 7140 acagcccctt tgacccggtc tcttcagtaa aagtggacaa cggtctgatc gtcgaactgg 7200 acggcaaacg ccgggaccag tttgacatga tcgaccgatt tatcgccgat tacgcgatca 7260 acgttgagcg cacagagcag gcaatgcgcc tggaggcggt ggaaatagcc cgtatgctgg 7320 tggatattca cgtcagccgg gaggagatca ttgccatcac taccgccatc acgccggcca 7380 aagcggtcga ggtgatggcg cagatgaacg tggtggagat gatgatggcg ctgcagaaga 7440 tgcgtgcccg ccggaccccc tccaaccagt gccacgtcac caatctcaaa gataatccgg 7500 tgcagattgc cgctgacgcc gccgaggccg ggatccgcgg caggagacca cttctcagaa 7560 cggtcggtat cgcgcgctac gcgccgttta acgccctggc gctgttggtc ggttcgcagt 7620 gcggccgccc cggcgtgttg acgcagtgct cggtggaaga ggccaccgag ctggagctgg 7680 gcatgcgtgg cttaaccagc tacgccgaga cggtgtcggt ctacggcacc gaagcggtat 7740 ttaccgacgg cgacgatacg ccgtggtcaa aggcgttcct cgcctcggcc tacgcctccc 7800 aatgcgctac gcgggttgaa acctccggca ccggatccga agcgctgatg ggctattcgg 7860 agagcaagtc gat gctctac ctcgaatcgc gctgcatctt cattactaaa ggcgccgggg 7920 ttcagggact gcaaaacggc gcggtgagct gtatcggcat gaccggcgct gtgccgtcgg 7980 gcattcgggc ggtgctggcg gaaaacctga tcgcctctat gctcgacctc gaagtggcgt 8040 ccgccaacga ccagactttc tcccactcgg atattcgccg caccgcgcgc accctgatgc 8100 agatgctgcc gggcaccgac tttattttct ccggctacag cgcggtgccg aactacgaca 8160 acatgttcgc cggctcgaac ttcgatgcgg aagattttga tgattacaac atcctgcagc 8220 gtgacctgat ggttgacggc ggcctgcgtc cggtgaccga ggcggaaacc attgccattc 8280 gccagaaagc ggcgcgggcg atccaggcgg ttttccgcga gctggggctg ccgccaatcg 8340 ccgacgagga ggtggaggcc gccacctacg cgcacggcag caacgagatg ccgccgcgta 8400 acgtggtgga ggatctgagt gcggtggaag agatgatgaa gcgcaacatc accggcctcg 8460 atattgtcgg cgcgctgagc cgcagcggct ittgaggatat cgccagcaat attctcaata 8520 tgctgcgcca gcgggtcacc ggcgattacc cgcagacctc ggccattctc gatcggcagt 8580 tcgaggtggt gagtgcggtc atgactatca aacgacatca ggggccgggc accggctatc 8640 gcatctctgc cgaacgctgg gcggagatca aaaatattcc gggcgtggtt cagcccgaca 8700 aggcggta ccattgaata tt cctgtgcaac agacaaccca aattcagccc tcttttaccc 8760 tgaaaacccg cgagggcggg gtagcttctg cgatgaacg cgccgatgaa gtggtgatcg 8820 gcgtcggccc tgccttcgat aaacaccagc Btcacactct gatcgatatg ccccatggcg 8880 cgatcctcaa agagctgatt gccggggtgg aagaagaggg gcttcacgcc cgggtggtgc 8940 gcattctgcg cacgtccgac gtctccttta ggcctggga tgcggccaac ctgagcggct 9000 cggggatcgg catcggtatc cagtcgaagg ggaccacggt catccatcag cgcgatctgc 9060 tgccgctcag caacctggag ctgttctccc aggcgccgct gctgacgctg gagacctacc 9120 ggcagattgg caaaaacgct gcgcgctatg cgcgcaaaga gtcaccttcg ccggtgccgg 9180 tggtgaacga tcagatggtg cggccgaaat ttatggccaa tttcatatca agccgcgcta 9240 aagagaccaa acatgtggtg caggacgccg agcccgtcac cctgcacatc gacttagtaa 9300 gggagtgacc atgagcgaga aaaccatgcg cgtgcaggat tatccgttag ccacccgctg 9360 cccggagcat atcctgacgc ctaccggcaa accattgacc gatattaccc tcgagaaggt 9420 gctctctggc gaggtgggcc cgcaggatgt gcggatctcc cgccagaccc ttgagtacca 9480 gccgagcaga ggcgcagatt tgcagcgcca j: gcggtggcg cgcaatttcc gccgcgcggc 9540 ggagcttatc gccattcctg acga gcgcat tctggctatc tataacgcgc tgcgcccgtt 9600 ccgctcctcg caggcggagc tgctggcgat cgccgacgag ctggagcaca cctggcatgc 9660 gacagtgaat gccgcctttg tccgggagtc -gcggaagtg tatcagcagc ggcataagct 9720 gcgtaaagga agctaagcgg aggtcagcat ccgttaata gccgggattg atatcggcaa 9780 cgccaccacc gaggtggcgc tggcgtccga tacccgcag gcgagggcgt ttgttgccag 9840 cgggatcgtc gcgacgacgg gcatgaaagg acgcgggac aatatcgccg ggaccctcgc 9900 cgcgctggag caggccctgg cgaaaacacc gtggtcgatg agcgatgtct ctcgcatcta 9960 tcttaacgaa gccgcgccgg tgattggcga Gtggcgatg gagaccatca ccgagaccat 10020
tatcaccgaa tcggtcataa tcgaccatga cccgcagacg ccgggcgggg tgggcgttgg 10080 cgtggggacg actatcgccc tcgggcggct ggcgacgctg ccggcggcgc agtatgccga 10140 ggggtggatc gtactgattg acgacgccgt cgatttcctt gacgccgtgt ggtggctcaa 10200 gaccggggga tgaggcgctc teaacg ggt ggcggcgatc ctcaaaaagg acgacggcgt 10260 gctggtgaac aaccgcctgc gtaaaaccct gccggtggtg gatgaagtga cgctgctgga 10320 gcaggtcccc gagggggtaa tggcggcggt ggaagtggcc gcgccgggcc aggtggtgcg 10380 gatcctgtcg aatccctacg ggatcgccac cttcttcggg ctaagcccgg aagaga cca 10440 ggccatcgtc cccatcgccc gcgccctgat tggcaaccgt tccgcggtgg tgctcaagac 10500 cccgcagggg gatgtgcagt cgcgggtga cccggcgggc ttagcggcga aacctctaca 10560 aaagcgccgc ggagaggccg atgtcgccga gggcgcggaa gccatcatgc aggcgatgag 10620 cgcctgcgct ccggtacgcg acatccgcg? cgaaccgggc acccacgccg gcggcatgct 10680 tgagcgggtg cgcaaggtaa tggcgtccct gaccggccat gagatgagcg cgatatacat 10740 ccaggatctg ctggcggtgg atacgtttat tccgcgcaag gtgcagggcg ggatggccgg 10800 cgagtgcgcc atggagaatg ccgtcgggat ggcggcgatg gtgaaagcgg atcgtctgca 10860 aatgcaggtt atcgcccgcg aactgagcgc ccgactgcag accgaggtgg tggtgggcgg 10920 aacatggcca cgtggaggcc tcgccggggc gttaaccact cccggctgtg cggcgccgct 10980 ggcgatcctc gacctcggcg ccggctcgac ggatgcggcg at gtcaacg cggaggggca 11040 gataacggcg gtccatctcg ccggggcggg gaatatggtc ttaaaaccga agcctgttga 11100 gctgggcctc gaggatcttt cgctggcgga agcgataaaa aaatacccgc tggccaaagt 11160 ggaaagcctg ttcagtattc gtcacgagaa tggcgcggtg gagttctttc gggaagccct 11220 gtgttcgcca cagcccggcg aagtggt ta catcaaggag ggcgaactgg tgccgatcga 11280 taacgccagc ccgctggaaa AAATT TKWG cgtgcgccgg caggcgaaag agaaagtgtt 11340 tgtcaccaac tgcctgcgcg cgctgcgcca ggtctcaccc ggcggttcca ttcgcgatat 11400 cgcctttgtg gtgctggtgg gcggctcatc gctggacttt gagatcccgc agettatcae 11460 ggaagccttg tcgcactatg gcg tggtcgc cgggcagggc aatattcggg gaacagaagg 11520 gccgcgcaat gcggtcgcca ccgggctgct actggccggt caggcgaatt aaacgggcgc 11580 tcgcgccagc ctctctcttt aacgtgctat ttcaggatgc cgataatgaa ccagacttct 11640 accttaaccg ggcagtgcgt ggccgagttt cttggcaccg gattgetcat tttcttcggc 11700 gcgggctgcg tcgctgcgct gcgggtcgcc ggggccagct ttggtcagtg ggagatcagt 11760 attatctggg gccttggcgt cgccatggcc atctacctga cgg cggtgt ctccggcgcg 11820 cacctaaatc cggcggtgac cattgccctg tggctgttcg cctgttttga acgccgcaag 11880 gtgctgccgt ttattgttgc ccagacggcc ggggccttct gcgccgccgc gctggtgtat 11940 gggctctatc gccagctgtt tetegat tt gaacagagtc ageatategt gcgcggcact 12000 gccgccagtc ttaacctggc cggggtcttt tccacgtacc cgcatccaca tatcactttt 12060 atacaagcgt ttgccgtgga gaccaccatc acggcaatcc tgatggcgat gatcatggcc 12120 12145 ctgaccgacg acggcaacgg aatte
< 210 > 2 < 211 > 22 < 212 > DNA < 213 > Unknown < 220 > < 223 > Description of the Artificial sequence: starter < 220 > < 223 > starter < 400 > 2 g tttctgtg ctgcggcttt ag 22
< 210 > 3 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: starter < 220 > < 223 > starter < 400 > 3 tggtcgagga tccacttcac ttt 23
< 210 > 4 < 211 > 51 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Sequence Description Artificial Primer < 220 > < 223 > starter < 400 > 4 aaagtgaagt ggatcctcga ccaattggát ggtggcgcag tagcaaacaa t 51
< 210 > 5 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the yes primer < 220 > < 223 > starter < 400 > 5 ggatcaccgc cgcagaaact acg 23
< 210 > 6 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: starter < 220 > < 223 > starter < 400 > 6 ctgtcagccg ttaagtgttc ctgtg 25
< 210 > 7 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial sequence: starter < 220 > 223 > starter < 400 > 7 cagttcaacc tgttgatagt acg 23
< 210 > 8 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the s < < 220 > < 223 > starter < 400 > 8 atgagtcaaa catcaacctt 20
< 210 > 9 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence Primer < 220 > < 223 > starter < 400 > 9 atggagaaaa aaatcactgg 20
< 2I0 > 10 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < < 500 > 10 ttacgccccg ccctgccact 20
< 210 > 11 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 11 tcagaggatg tgcacctgca 20
< 210 > 12 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 12 cgagcatgcc gcatttggca ctactc 26
< 210 > 13 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 13 gcgtctagag taggttattc ccactcttg 29
< 210 > 14 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 14 gaagtcgacc gctgcgcctt atccgg 26
< 210 > 15 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 15 cgcgtcgacg tttacaattt caggtggc 28
< 210 > 16 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S < 220 > < 223 > starter < 400 > 16 gcagcatgct ggactggtag tag 23
< 210 > 17 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer
< 220 > < 223 > starter < 400 > 17 cagtctagag ttattggcaa acctacc 27
< 210 > 18 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 18 gatgcatgcc cagggcggag acggc 25
< 210 > 19 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: c bador < 220 > < 223 > starter < 400 > 19 c aacgattg ttctctagag aaaatgtcc 29
< 210 > 20 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 20 cacgcatgca gttcaacctg ttgatagtac 30
< 210 > 21 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 21 gcgtctagat ccttttaaat taaaaatg 28
< 210 > 22 < 211 > 51
< 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 2 3 > starter < 400 > 22 gcgcggatcc aggagtctag aattatggga ttgactacta aacctctatc t 51
< 210 > 23 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 23 gatacgcccg ggttaccatt tcaacagatcj gtcctt 36
< 210 > 24 < 211 > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S < 220 > < 223 > starter < 400 > 24 ttgataatat aaccatggct gctgctgctg atag 34
< 210 > 25 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S < 220 > < 223 > starter < 400 > 25 gtatgatatg ttatcttgga tccaataaat ctaatcttc 39
< 210 > 26 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: cel ador < 220 > < 223 > primer
< 400 > 26 catgactagt aaggaggaca attc 24
< 210 > 27 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 27 catggaattg tcctccttac tagt 24
< 210 > 28 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S primer < 220 > < 223 > starter < 400 > 28 ctagtaagga ggacaattc 19
< 210 > 29 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 29 catggaattg tcctcctta 19
< 210 > 30 < 211 > 15 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence Primer < 220 > < 2 3 > starter < 400 > 30 gatccaggaa acaga 15
< 210 > 31 < 211 > 15 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of the Artificial Sequence Primer < 220 > < 223 > starter < 400 > 31 ctagtctgtt tcctg 15
< 210 > 32 < 211 > 94 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: sequence terminator
< 220 > < 223 > Sequence finder < 400 > 32 agcttaggag tctagaatat tgagctcgaa ttcccgggca tgcggtaccg gatccagaaa 60 aaagcccgca cctgacagtg cgggcttttt tttt 94
< 210 > 33 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S primer < 220 > < 223 > starter < 400 > 33 ggaattcaga tctcagcaat gagcgagaaa accatgc 37
< 2 I 0 > 34 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S primer < 220 > < 223 > starter < 400 > 34 gctctagatt agcttccttt acgcagc 27
< 210 > 35 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S primer < 220 > < 223 > primer
< 400 > 35 ggccaagctt aaggaggtta attaaatg'aa aag 33
< 210 > 36 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence; starter < 220 > < 223 > starter < 400 > 36 gctctagatt attcaatggt gtcggg 26
< 210 > 37 < 211 > 42 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the primer Si <220 > < 223 > starter < 400 > 37 gcgccgtcta gaattatgag ctatcgtatg tttgattatc tg 42
< 210 > 38 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Se < 220 > < 223 > starter < 400 > 38 tctgacacgg gatcctcaga atgcctggcg gaaaat 36
< 210 > 39 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S < 220 > < 223 > link < 400 > 39 tegaeg aat t caggagga 18
< 210 > 40 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence
catgaaattg aacacctgag acaacttgtc acagctcaac agtcacacat agacagcctg 2580 aaacaggcga tgctgcttat cgaatcaaag ctgccgacaa cacgggagcc agtgacgcct 2640 cccgtgggga aaaaatcatg gcaattctgg1 aagaaatagc gctttcagcc ggcaaaccgg 2700 ctgaagccgg atctgcgatt ctgataacaa actagcaaca ccagaacagc ccgtttgcgg 2760 gcagcaaaac ccgtgggaat taattcccct gctcgcgcag gctgggtgcc aagctctcgg 2820 gtaacatcaa ggcccgatcc ttggagcccc tgccctcccg cacgatgatc gtgccgtgat 2880 cgaaatccag atccttgacc cgcagttgca aaccctcact gatccgcatg cccgttccat 2940 acagaagctg ggcgaacaaa cgatgctcgc cttccagaaa accgaggatg cgaaccactt 3000 catccggggt cagcaccacc ggcaagcgco gcgacggccg aggtcttccg atctcctgaa 3060 gccagggcag atccgtgcac agcaccttgc cgtagaagaa cagcaaggcc gccaatgcct 3120 gacgatgcgt ggagaccgaa accttgcgct cgttcgccag ccaggacaga aatgcctcga 3180 cttcgctgct gcccaaggtt gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa 3240 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac 3300 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca 3360 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag 3420 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg 3480 gcagtcgccc taaaacaaag ttaaacatca tgagggaagc ggtgatcgcc gaagtatcga 3540 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg 3600 cggctccgca tacatttgta gtggatggcg gcctgaagcc acacagtgat attgatttgc 3660 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt 3720 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg 3780 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag 3840 aatggc AGCG caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc 3900 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg 3960 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa 4020 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt 4080 gtacagcgca cccgcatttg gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact 4140 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt 4200 atcttggaca agaagaagat cgcttggcct cgcscgcaga tcagttggaa gaatttgtcc 4260 actacgtgaa aggcgagatc accaaggtag atgtctaaca tcggcaaata attcgttcaa 4320 gccgacgccg cttcgcggcg cggcttaact caagcgttag atgcactaag cacataattg 4380 ctcacagcca aactatcagg tcaagtctgc ttttattatt tttaagcgtg cataataagc 4440 ttgggagata cctacacaaa tatcatgaaa ggctggcttt ttcttgttat cgcaatagtt 4500 gscgaagtaa tcgcaacatc cgcattaaaa tctagcgagg gctttacta 4549
< 210 > 42 < 211 > 199 < 212 > DNA < 2 - 3 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: glucose isomerase promoter < 220 > < 223 > promoter < 400 > 42 gaattcacta gtcgatctgt gctgtttgcc acggtatgca gcaccagcgc gagattatgg 60 gctcgcacgc tcgactgtcg gacgggggca ctggaacgag aagtcaggcg agccgtcacg 120 cccttgacaa tgccacatcc tgagcaaata attcaaccac taaacaaatc aaccgcgttt 180 cccggaggta accaagctt 199
< 210 > 43 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > primer
< 400 > 43 gacgcaacag tattccgtcg c 21
< 210 > 44 < 211 > 42 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 220 > < 223 > Primer < 400 > 44 atgagctatc gtatgttccg ccaggcattc tgagtgttaa cg 42
< 210 > 45 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 220 > < 223 > Primer < 400 > 45 gcctggcgga acatacgata gctcataatá tac 33
< 210 > 46 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 220 > < 223 > Primer < 400 > 46 cggggcgctg ggccagtact g 21
< 210 > 47 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Primer < 220 > < 223 > starter < 220 > < 223 > Primer < 400 > 47 tcaaacccgg tggtttctcg cgaccggg 28
< 210 > 48 < 211 > 28
< 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 48 ctcagccgga tatcgacggc gcgctggt 28
< 210 > 49 < 211 > 60 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > starter < 400 > 49 accagcgcgc cgtcgatatc cggctgagta ctcaacacct gccagctctt tacgcaggtt 60
< 210 > 50 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S < 220 > < 223 > starter < 400 > 50 cagcatgcct gcgaaccaca ggcctatc 28
< 210 > 51 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the S < 220 > < 223 > starter < 400 > 51 atgaacaagt ggggcgtagg gttaacat 28
< 210 > 52 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: primer < 220 > < 223 > primer
< 400 > 60 gcgagctcga cgcttgccct gatcgagttt tgc 33
< 210 > 61 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence Primer < 400 > 61 gcgagctcga cgcttgccct gatcgagttt tgc 33
< 210 > 62 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence Primer < 4 00 > 62 cagctggcaa ttccggttcg 20
< 210 > 63 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 63 cccagctggc aattccggtt cgcttgctgt 30
< 210 > 64 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 64 ggcgacccga cgctccagac ggaagctggt 30
< 210 > 65 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 65 ccgcaagatt cacggatgca tcgtgaaggg 30
< 210 > 66 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Sequence Artificial Sequence < 400 > 66 cgccttcttg acgagttctg agcggga 27
< 210 > 67 < 211 > 30 < 212 > DNA < 21 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Sequence < 400 > 67 ggaattcatg aacaacttta atctgcacac 30
< 210 > 68 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Sequence < 400 > 68 gtttgaggcg taaaaagctt agcgggcggc 30
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.
Claims (3)
- CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A nucleated acid fragment encoding a nonspecific catalytic activity, for the conversion of 3-hydroxypropionaldehyde to 1-3 propanediol, and characterized because it is selected from the group consisting of (a) an isolated fragment of nucleic acid encoding all or a substantial portion of the amino acid sequence of SEQ ID NO, 57; (b) an isolated fragment of nucleic acid that is substantially similar to an isolated fragment of nucleic acid encoding all or a substantial portion of the amino acid sequence of SEQ ID NO: 57; (c) an amino acid fragment of nucleic acid encoding a polypeptide of at least 387 amino acids having at least 80% with the amino acid sequence of SEQ ID NO: 57; (d) an isolated fragment of nucleic acid that hybridizes with (a) under hybridization conditions of 0. IX SSC, 0.1% SDS, 65 ° C and washed with 2X SSC, 0.1% SDS followed by 0.1 X SSC, 0.1% SDS; Y; (e) an isolated nucleic acid fragment that is complementary to (a), (b), (c) or (d) 2. The isolated nucleic acid fragment characterized in that it is set forth in SEQ ID NO: 58. 3 A polypeptide encoded by the isolated nucleic acid fragment characterized in that it is in accordance with claim 1. The polypeptide according to claim 3, characterized in that it is set forth in SEQ ID NO: 57. 5. A chimeric gene, characterized in that it comprises an isolated fragment of nucleic acid according to claim 1, operatively linked to appropriate regulatory sequences 6. A microorganism transformed with the chimeric gene of claim 5, characterized in that the microorganism is selected from the group consisting of Ci trobacter , Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobact r, Salmonella, Bacillus, Aerobacter, Streptomyces, Escherichia, and Pseudomonas A recombinant microorganism useful for the production of 1-3 propanediol, characterized in that it comprises: (a) at least one gene encoding a polypeptide having a dehydratase activity, (b) at least one ger encoding a reactivation factor of 1 a dehydratase, and (c) at least one exogenous gene encoding a non-specific catalytic activity for converting 3-hydroxypropionaldehyde to 1,3-propanediol; wherein no dha T functional gene encoding the activity of 1,3-propanediol oxide reductase is present in the recombinant micro-aniganism and the microorganism is selected from the group consisting of Ci trobacter, Enterobacter Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, andgosaccharomyces, Pichia, Kluveromyces, Candida, Ha isenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella, Bacillus, Aerobacter, Streptomyces, Escherichia, and Pseudomonas 8. The recombinant microorganism according to claim 7, characterized in that it further comprises: 12. The recombinant microorganism according to claim 8 vi, characterized in that the recombinant microorganism converts a carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and simple carbon substrates to 1,3-p? -panediol. 13. The recombinant microorganism according to claim 7, characterized in that the microorganism recombines and converts the carbon source selected from the group consisting of glycerol and dihydroxyacetone to 1,3-propanediol, 14. The recombinant microorganism according to claim 8. u 11, characterized in that the gene encoding a polypeptide having glycerol-3-phosphate dehyd :: ogenase activity, is selected from the group consisting of GPD1, GPD2, GPD3, DAR1, gpsA, GUT2, glpD and glpABC. 15. The recombinant microorganism according to claim 8 or 11, characterized in that the gene encoding a polypeptide having glycerol-3-phosphatase activity is selected from the group consisting of GPP1 and GPP2. 16. The recombinant microorganism according to claims 7 or 11, characterized in that the gene encodes a polypeptide having an activity of dehydratase, is selected from the group consisting of a glycerol dehydratase and binds. diol dehydratase 17. The recombinant microorganism according to claims 7, 8 or 11, characterized in that the gene encoding a polypeptide having a dehydratase activity is isolated from Klebsiella, Ci trobacter or Clostridium species. 18. A recombinant E. coli, characterized in that it comprises: (a) a set of exogenous genes consisting of: (i) at least one gene encoding a polypeptide having dehydratase activity (ii) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, and (iii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity, and (iv) at least one gene encoding a factor of reactivation of dehydratase, and (b) at least one endogenous gene encoding a non-specific catalytic activity to convert 3-hydroxypropionaldehyde to 1,3- propanediol; in which no dha T functional gene encoding the activity of 1,3-propanediol oxide reductase is present in recombinant E. coli, 19. A compiling E. coli, characterized in that it comprises: (a) a set of Exogenous genes that consist of: (i) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; and (ii) at least one gene encoding a polypeptide having glycerol-3-phosphatase responsiveness; and (iii) at least a subset of genes encoding the products of dhaR, orfY, orfX, orfW, dhaBl, d aB2, dhaB3 and orfZ, and (c) at least one endogenous gene encoding a non-specific catalytic activity to convert to 3-hydroxypropionaldehyde to 1,3-propanediol, wherein no functional dhaT gene encoding the activity of 1,3-propanediol oxidereductase is present in recombinant E. coli, 20. E. recombinant coli according to claim 19, characterized in that it also comprises a set of endogenous genes, each gene has a mutation that inactivates the gene, the set consists of: (a) a gene that encodes a polypeptide that has glycerol zymose activity; (b) a gene encoding a polypeptide having glycerol activity of shidrogenase; (c) a gene encoding a polypeptide having triosephospht isomerase activity; 21. A process for the bioproduction of 1,3-propanediol, characterized in that it comprises: (a) making contact with, or appropriate conditions of, the recombinant E. coli of claims 19 or 20, with at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and simple carbon substrates whereby 1,3-propanediol is produced; and (b) optionally recovering the 1,3 propanediol produced in (a) 22. A process for the bioproduction of 1,3-propanediol, characterized in that it comprises: (a) contacting the recombinant E. coli of claim 19 or 20 or the recombinant E. coli of claims 19 or 20, < which further comprises (i) at least one exogenous gene encoding a polypeptide having a dehydratase activity, [ii) at least one sxogen gene that codes for a reactivation factor of dehhiidratase. (iv) at least one endogenous gene encoding a non-specific catalytic activity for converting 3-hydroxypropionaldehyde to 1,3-propanediol; with at least one carbon source selected from the group consisting of glycerol and dihydroxyacetone, and (b) optionally recovering the 1,3-propanediol produced in (a) 23. A process for the production of 1,3-propanediol, comprising: contacting the recombinant E. coli with a first carbon source and with a second carbon source, the recombinant E. coli comprises: (i) at least one exogenous gene encoding a polypeptide having an α! dehydratase activity (ii) at least one sxogen gene encoding a dehydratase reactivation factor. (iii) at least one endogenous gjen encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol; characterized in that no functional dha T gene encoding the activity of 1,3-propanediol oxide reducatsa, is present in recombinant E. coli and wherein the first carbon source is selected from the group consisting of glycerol and dihydroxyacetone, and the second carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and substrates simple carbon; and (b) optionally recovering 1,3-propanediol produced in (a). 24. The process according to claim 23, characterized in that the recombinant E. coli further comprises: (a) a set of exogenous genes consisting of: [i) at least one gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; and (ii) at least one gene encoding a polypeptide having glycerol-3-phosphatase activity; and (iii) at least a subset of genes encoding the gene products of dhaR, orfY, orfX, orfW, dhaBl, dhaB2, dhaB3 and orfZ, and (b) a set of endogenous genes, each gene having a mutation that inactivates to the gene, the set consists of: i) a gene encoding a polypeptide having glycerol kinase activity; (ii) a gene encoding a polypeptide having glycerol delshidrogenase activity; and (iii) a gene encoding a polypeptide having triosephosphate isomerase 25 activity. The pDT29 vector, characterized in that it comprises a set of genes dhaR, orfY, dha T, orfX, orfW, dhaBl, dhaB2, dhaB3 and orfZ as is set forth in SEQ ID N0: 1. 26. The vector pKP32, characterized in that it comprises a set of genes dhaR, orfY, orfX, orfW, dhaBl, dhaB2, dhaB3 and orfZ as set forth in SEQ ID N0: 1 27. A strain of E. coli KLP23, characterized in that it comprises : (a) a set of two endogenous genes, each gene has a mutation that inactivates the gene, the set consists of (i) a gene that encodes a polypeptide that has glycerol kinase activity; and (ii) a gene encoding Read to a polypeptide having glycerol dehydrogenase activity; b) at least one exogenous gene encodes a polypeptide having glycerol-3-phosphate dehydrogenase activity; (c) at least one exogenous gene encodes a polypeptide that has glycerol-3-phosphatase activity; Y (d) a pKP32 plasmid. 28. A reconbinant strain of E. coli RJ8, characterized in that it comprises: (a) a set of three endogenous genes, each gene has a mutation that inactivates the gene, the set consists of: (i) a gene encoding a polypeptide which has glycerol kinase activity; ii) a gene encoding a polypeptide having glycerol activity of shidrogenase; (iii) a gene encoding a polypeptide having triosephosphate isomerase activity; 29. A process for the production of 1,3-propanediol, characterized in that it comprises: (a) contacting, under appropriate conditions, a recombinant E. coli comprising a dha regution and lacking a functional dha T gene coding for the activity of 1,3-propanediol oxide reductase with at least one carbon source, wherein the carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and simple carbon substrates; and (b) optionally recovering 1,3-propanediol produced in (a).
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US60/149,534 | 1999-08-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA02001712A true MXPA02001712A (en) | 2003-11-07 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6514733B1 (en) | Process for the biological production of 1,3-propanediol with high titer | |
US6432686B1 (en) | Method for the production of 1,3-propanediol by recombinant organisms comprising genes for vitamin B12 transport | |
AU725012B2 (en) | Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism | |
AU735080B2 (en) | Method for the recombinant production of 1,3-propanediol | |
US6428767B1 (en) | Method for identifying the source of carbon in 1,3-propanediol | |
KR100550203B1 (en) | Method for The Production of Glycerol by Recombinant Organisms | |
IL129723A (en) | Method for the production of glycerol by recombinant microorganisms | |
EP1586647A1 (en) | Process for the biological production of 1,3-propanediol with high titer | |
MXPA02001712A (en) | Process for the biological production of 1,3-propanediol with high titer | |
MXPA00010723A (en) | Method for the production of 1,3-propanediol by recombinant organisms comprising genes for vitamin b12 transport | |
AU7156500A (en) | Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism | |
MXPA99004405A (en) | Method for the production of 1,3-propanediol by recombinant organisms | |
MXPA99004337A (en) | Method for the recombinant production of 1,3-propanediol |