CN101768617A - New technology for full-cell biosynthesis of deoxynucleoside triphosphate - Google Patents
New technology for full-cell biosynthesis of deoxynucleoside triphosphate Download PDFInfo
- Publication number
- CN101768617A CN101768617A CN201019026027A CN201019026027A CN101768617A CN 101768617 A CN101768617 A CN 101768617A CN 201019026027 A CN201019026027 A CN 201019026027A CN 201019026027 A CN201019026027 A CN 201019026027A CN 101768617 A CN101768617 A CN 101768617A
- Authority
- CN
- China
- Prior art keywords
- triphosphate
- deoxynucleoside
- whole
- synthetic
- new technology
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000001226 triphosphate Substances 0.000 title claims abstract description 34
- 235000011178 triphosphate Nutrition 0.000 title claims abstract description 34
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 238000005516 engineering process Methods 0.000 title claims abstract description 21
- 230000015572 biosynthetic process Effects 0.000 title abstract description 5
- 238000000034 method Methods 0.000 claims abstract description 38
- 210000005253 yeast cell Anatomy 0.000 claims abstract description 16
- 230000008929 regeneration Effects 0.000 claims abstract description 15
- 238000011069 regeneration method Methods 0.000 claims abstract description 15
- 230000008569 process Effects 0.000 claims abstract description 12
- 239000000758 substrate Substances 0.000 claims abstract description 9
- 230000002925 chemical effect Effects 0.000 claims abstract description 8
- 238000010168 coupling process Methods 0.000 claims abstract description 7
- 238000005859 coupling reaction Methods 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 28
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 25
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 25
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 claims description 22
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 18
- 108090000790 Enzymes Proteins 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 230000000977 initiatory effect Effects 0.000 claims description 15
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 14
- RGWHQCVHVJXOKC-SHYZEUOFSA-N dCTP Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO[P@](O)(=O)O[P@](O)(=O)OP(O)(O)=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-N 0.000 claims description 13
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 claims description 13
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 12
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims description 12
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 11
- 239000003960 organic solvent Substances 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- LTFMZDNNPPEQNG-KVQBGUIXSA-N 2'-deoxyguanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1C[C@H](O)[C@@H](COP(O)(O)=O)O1 LTFMZDNNPPEQNG-KVQBGUIXSA-N 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- NCMVOABPESMRCP-SHYZEUOFSA-N 2'-deoxycytosine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 NCMVOABPESMRCP-SHYZEUOFSA-N 0.000 claims description 8
- 229910019142 PO4 Inorganic materials 0.000 claims description 8
- 241000235648 Pichia Species 0.000 claims description 8
- GYOZYWVXFNDGLU-XLPZGREQSA-N dTMP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)C1 GYOZYWVXFNDGLU-XLPZGREQSA-N 0.000 claims description 8
- 239000010452 phosphate Substances 0.000 claims description 8
- 241000235017 Zygosaccharomyces Species 0.000 claims description 7
- KHWCHTKSEGGWEX-UHFFFAOYSA-N deoxyadenylic acid Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(O)=O)O1 KHWCHTKSEGGWEX-UHFFFAOYSA-N 0.000 claims description 7
- -1 phosphate anion Chemical class 0.000 claims description 7
- 239000004094 surface-active agent Substances 0.000 claims description 7
- 241000722885 Brettanomyces Species 0.000 claims description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 6
- LTFMZDNNPPEQNG-UHFFFAOYSA-N deoxyguanylic acid Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1CC(O)C(COP(O)(O)=O)O1 LTFMZDNNPPEQNG-UHFFFAOYSA-N 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 229910021645 metal ion Inorganic materials 0.000 claims description 6
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 5
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 4
- 238000010170 biological method Methods 0.000 claims description 4
- 150000002894 organic compounds Chemical class 0.000 claims description 4
- 241000235035 Debaryomyces Species 0.000 claims description 3
- 241000235649 Kluyveromyces Species 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 230000035699 permeability Effects 0.000 claims description 3
- 238000002525 ultrasonication Methods 0.000 claims description 3
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 claims description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 2
- KHWCHTKSEGGWEX-RRKCRQDMSA-N 2'-deoxyadenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(O)=O)O1 KHWCHTKSEGGWEX-RRKCRQDMSA-N 0.000 claims description 2
- 241000222173 Candida parapsilosis Species 0.000 claims description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 2
- 241000235036 Debaryomyces hansenii Species 0.000 claims description 2
- 229930091371 Fructose Natural products 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 claims description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 2
- 229930195725 Mannitol Natural products 0.000 claims description 2
- 244000253911 Saccharomyces fragilis Species 0.000 claims description 2
- 235000018368 Saccharomyces fragilis Nutrition 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 239000003945 anionic surfactant Substances 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 229940055022 candida parapsilosis Drugs 0.000 claims description 2
- 239000003093 cationic surfactant Substances 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 229940031154 kluyveromyces marxianus Drugs 0.000 claims description 2
- 239000000594 mannitol Substances 0.000 claims description 2
- 235000010355 mannitol Nutrition 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 239000004367 Lipase Substances 0.000 claims 1
- 102000004882 Lipase Human genes 0.000 claims 1
- 108090001060 Lipase Proteins 0.000 claims 1
- 229960000074 biopharmaceutical Drugs 0.000 claims 1
- 235000019421 lipase Nutrition 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 52
- 238000006555 catalytic reaction Methods 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 7
- 230000002503 metabolic effect Effects 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 5
- 239000005549 deoxyribonucleoside Substances 0.000 abstract description 4
- 150000004712 monophosphates Chemical class 0.000 abstract description 4
- 238000012269 metabolic engineering Methods 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 24
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 16
- 239000000243 solution Substances 0.000 description 15
- 235000011007 phosphoric acid Nutrition 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 9
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 9
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 9
- 238000003916 acid precipitation Methods 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 235000011187 glycerol Nutrition 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 238000004445 quantitative analysis Methods 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 230000004907 flux Effects 0.000 description 7
- 230000037361 pathway Effects 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 108091000080 Phosphotransferase Proteins 0.000 description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 6
- 235000021317 phosphate Nutrition 0.000 description 6
- 102000020233 phosphotransferase Human genes 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 241001192924 Parna Species 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 102000013009 Pyruvate Kinase Human genes 0.000 description 4
- 108020005115 Pyruvate Kinase Proteins 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000001186 cumulative effect Effects 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000001311 chemical methods and process Methods 0.000 description 3
- 239000005515 coenzyme Substances 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 3
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 2
- 108010041388 Ribonucleotide Reductases Proteins 0.000 description 2
- 102000000505 Ribonucleotide Reductases Human genes 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- WCVHUIPWSPEOIG-UHFFFAOYSA-N n,n-dimethylheptadecan-1-amine Chemical compound CCCCCCCCCCCCCCCCCN(C)C WCVHUIPWSPEOIG-UHFFFAOYSA-N 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical group OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 2
- 229910001414 potassium ion Inorganic materials 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 108010012839 (deoxy)nucleoside-phosphate kinase Proteins 0.000 description 1
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 description 1
- 101100113633 Arabidopsis thaliana CKL9 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 208000006558 Dental Calculus Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000005731 Glucose-6-phosphate isomerase Human genes 0.000 description 1
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 1
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 description 1
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 description 1
- 102100040468 Guanylate kinase Human genes 0.000 description 1
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- 101000614191 Homo sapiens Guanylate kinase Proteins 0.000 description 1
- XQQSWXUDAPLMKD-UHFFFAOYSA-N N,N-dimethylheptadecan-1-amine hydrobromide Chemical compound Br.CCCCCCCCCCCCCCCCCN(C)C XQQSWXUDAPLMKD-UHFFFAOYSA-N 0.000 description 1
- BACYUWVYYTXETD-UHFFFAOYSA-N N-Lauroylsarcosine Chemical class CCCCCCCCCCCC(=O)N(C)CC(O)=O BACYUWVYYTXETD-UHFFFAOYSA-N 0.000 description 1
- ALKWJXWZUTYERW-UHFFFAOYSA-L O.O.O.O.[Mn](=O)(Cl)Cl Chemical class O.O.O.O.[Mn](=O)(Cl)Cl ALKWJXWZUTYERW-UHFFFAOYSA-L 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- DSQNMNVLWGRUSL-UHFFFAOYSA-N P(=O)(=O)C(C(C)=O)(O)O Chemical compound P(=O)(=O)C(C(C)=O)(O)O DSQNMNVLWGRUSL-UHFFFAOYSA-N 0.000 description 1
- 102000001105 Phosphofructokinases Human genes 0.000 description 1
- 108010069341 Phosphofructokinases Proteins 0.000 description 1
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 1
- 108700023219 Phosphoglycerate kinases Proteins 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108010053763 Pyruvate Carboxylase Proteins 0.000 description 1
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 description 1
- 101100020443 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) CDC8 gene Proteins 0.000 description 1
- 101000666920 Streptomyces hygroscopicus subsp. limoneus Validoxylamine A 7'-phosphate phosphatase Proteins 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- 101150118153 URA6 gene Proteins 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl 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-DQQFMEOOSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 101150065716 adk1 gene Proteins 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 229940094991 herring sperm dna Drugs 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229940085991 phosphate ion Drugs 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000019832 sodium triphosphate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical class CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a new technology for synthesizing deoxynucleoside triphosphate by whole-cell biosynthesis, which takes deoxynucleoside monophosphate (dNMP) and phosphate ions as substrates, sugar as an energy donor and permeable yeast cells to react in the presence of chemical effect substances to prepare the deoxynucleoside triphosphate. The invention utilizes the principles of whole-cell catalysis and metabolic engineering, adopts chemical effect substances to regulate and control metabolic flow, establishes an efficient energy self-coupling and regeneration system, and finally realizes the efficient preparation of the deoxyribonucleoside triphosphate. The invention overcomes the defects of the traditional production process of the deoxyribonucleoside triphosphate, and has the characteristics of simple process, high conversion rate, low cost, small pollution and the like.
Description
Technical field
The present invention relates to a kind of method by the synthetic deoxynucleoside triphosphate dNTP of deoxynucleoside one phosphoric acid dNMP.
Background technology
Deoxynucleoside triphosphate is artificial-synthetic DNA's an indispensable precursor raw material, and artificial-synthetic DNA's fragment is widely used in aspects such as genetically engineered, molecular biology, life science, genomic medicine.Deoxy-ribonucleoside triphosphate is the substrate of the short reaction of various archaeal dna polymerases, is that dna sequence analysis, genetic analysis, rite-directed mutagenesis, PCR (Polymerase ChainReaction), RT-PCR, reverse transcription and dna marker react indispensable lower molecular weight biological organic molecule.And being modern molecular biology, biological chemistry and modern biomedical, dna sequence analysis, rite-directed mutagenesis, PCR and DNA chip technology study indispensable technology.Especially round pcr has characteristics such as be widely used simple to operate, and it obtains application more and more widely in fields such as gene molecule clone, protein engineering, biological medicine research and development, heredity and communicable disease diagnosis, legal medical expert's evaluation, parent-offspring's calibratings.
Along with DNA synthetic PCR in biotechnology research and the industrialization uses, the demand of dNTP is stably increasing.Have only enterprise of several family can produce deoxy-ribonucleoside triphosphate at present in the world, the method that is adopted mainly is to utilize the enzymatic or the chemical phosphorylation of thymus nucleic acid hydrolysate, the acquisition of end product requires polystep reaction, complex production process, and product yield is low.
Commercially mainly produce dNTP by chemical process, reaction is to be substrate with corresponding tributyl ammonium salt of dNMP and ortho-phosphoric acid, is catalyzer with dicyclohexylcarbodiimide (DCC), carries out in pyridine or dimethylformamide organic solvents such as (DMF).The productive rate that chemical method production obtains different dNTP is 40～80%.Yet, each dNTP component in the purification reaction mixture, its process is very complicated and cost is very big, and it need separate inactive dNMP, dNDP, DCC and ortho-phosphoric acid and by product such as dezyribonucleoside four phosphoric acid or five phosphoric acid etc.In addition, because the principle of off gas treatment is very strict, pyridine or DMF solvent must be reduced, and separate, and reclaim then.Therefore, chemical method has very big environmental pollution, and very low by its yield behind reaction and the purifying, and cost is also higher relatively.
Consider that from economy and environmental factors occurred the synthetic deoxynucleoside triphosphate of enzyme process in recent years in the world, this method has more advantage than chemical process.The reaction of two enzyme phosphorylations of synthetic needs of dNTP generates dNDP and second step generation dNTP from dNMP.Ladner and Whitesides are by the synthetic dNTP[Ladner W of isolating deoxynucleoside one phosphoric acid (dNMP) mixture from herring sperm dna, Whitesides G.Enzymatic synthesis of deoxyATP using DNAas starting material[J] .J Org Chem, 1985,50:1076-1079].In recent years, Jie Bao, people such as the Dewey D.Y.Ryu four kinds of kinase whose genes of '-deoxynucleoside monophosphate of successfully will encoding are separated from the genome of Saccharomyces cerevisiae ATCC2610 bacterial strain, be respectively the ADK1 of coding deoxydenylate kinase (AK), the GUK1 of coding deoxyguanylate kinase (GK), the URA6 of coding Deoxyribose cytidine acid kinase (CK), and the CDC8 gene of coding deoxythymidine acid kinase (TK).They arrive these gene clones in E.coli BL21 (DE3) bacterial strain, and make AK, GK, CK, TK overexpression.The conversion of deoxynucleotide kinase realization from the '-deoxynucleoside monophosphate to the deoxynucleoside diphosphate with purifying.Second step added conversion [the Bao J of pyruvate kinase (PK) realization from dNDP to dNTP that extracts from tame rabbit muscle, Ryu DDY.Total Biosynthesis of Deoxynucleoside Triphosphates UsingDeoxynucleoside Monophosphate Kinases for PCR Application[J] .Biotechnol Bioeng.2007,98 (1): 1-11].However, the productive rate of dNTP is still lower, have only about 25% [Bao J, Bruque GA, RyuDDY.Biosynthesis of deoxynucleoside triphosphates, dCTP and dTTP:Reaction mechanismand kinetics[J] .Enzyme Microb Technol.2005,36:350-356; Bao J, Ryu DDY.Biosynthesisreaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP[J] .Biotechnol Bioeng.2005,89:485-491].In addition, the man research institution of existing in the world number is is researching and developing ribonucleotide reductase, and the shortcomings such as complicated condition and enzyme stability difference but these ribonucleotide reductases that are in development mostly respond are not suitable for industrialization, are of limited application.
At present both at home and abroad the dNTP yield general not high, cost is high and a major reason of complex production process is that energy regeneration and coupling efficiency are low.Need to consume lot of energy (ATP) in the building-up process of dNTP, therefore needing two enzyme systems is the regeneration system of ATP and the synthetase series of dNTP.The regeneration system of ATP is substrate with sugar, and (EMP) realizes by glycolytic pathway, and this approach is one of most economical approach of energy regeneration, and donor ATP in the dNTP building-up process as phosphodonor and energy and exist.Therefore dNTP synthetic key just is how to set up an energy regeneration efficiently and from coupling system.Adopt yeast whole-cell catalytic technology, can overcome that substrate utilization efficient is low in the building-up process, efficiency of pcr product is low and problem such as production cost height.Simultaneously, compare with enzyme process, because use is full cell, the stability of enzyme is better, and the adaptability of organic solvent-resistant is stronger, the in-situ regeneration of easier realization energy and coenzyme.In the prior art, sugar is very low by the efficient that EMP Embden Meyerbof Parnas pathway generates ATP, can only keep the general life metabolism of yeast, strengthen the flux of EMP Embden Meyerbof Parnas pathway, overexpression substrate phosphorylation level, have only method to realize, adopt the latter more convenient, be easy to realize by genetic engineering technique or employing chemical effect substance change metabolism stream.Under the adjusting of chemical effect material, can make the metabolic flux of EMP Embden Meyerbof Parnas pathway take place obviously to change, ATP regenerated speed also is greatly improved, and when the speed of its speed and dNTP synthetic system is complementary, promptly realizes efficiently synthesizing of dNTP.
Summary of the invention
Technical problem to be solved by this invention provides the preparation method of a kind of simply and efficiently deoxynucleoside triphosphate dNTP, to overcome shortcomings such as dNTP traditional preparation process complexity, the high yield of cost is low, pollution is big.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological, with deoxynucleoside one phosphoric acid and phosphate anion is substrate, with sugar is energy donor, the yeast cell of having property of utilization is set up efficiently energy self coupling connection and regeneration system rapidly and is prepared deoxynucleoside triphosphate in the presence of the chemical effect material.
Wherein, described deoxynucleoside triphosphate dNTP is meant any one among deoxyadenosine triphosphate dATP, deoxyguanosine triphosphate dGTP, deoxycytidine triphosphate dCTP and the deoxythymidine triphosphate dTTP, and its structural formula is as follows:
Wherein, described deoxynucleoside one phosphoric acid dNMP is meant any one among deoxyadenosine monophosphate dAMP, deoxyguanosine monophosphate dGMP, deoxycytidine monophosphate dCMP and the deoxythymidine monophosphate dTMP, and its structural formula is as follows:
Wherein, the initial action concentration of described deoxynucleoside one phosphoric acid is 1～100mM; Described phosphate anion initial action concentration is 0.01～1.0M; The initial action concentration of sugar is 0.1～1.0M; Described phosphate ion can be enumerated Tripyrophosphoric acid such as ortho-phosphoric acid, tetra-sodium, tripolyphosphate, potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, inorganic phosphates such as Sodium phosphate dibasic; Described sugar is glucose, fructose, sucrose or maltose.
Wherein, described chemical effect material is meant the combination of metal ion and organic compound; Described metal ion is Mg
2+, K
+And Na
+In any one or a few; Described organic compound is the combination of any one or two kinds in acetaldehyde and the polyvalent alcohol; Mg
2+Can be inorganic magnesium salts such as sal epsom, magnesium nitrate, magnesium chloride, initial action concentration be 1～100mM; K
+Can be inorganic potassium salts such as potassium primary phosphate, dipotassium hydrogen phosphate, vitriolate of tartar, saltpetre, Repone K, initial action concentration be 1～100mM; Acetaldehyde initial action concentration is 10～80mL/L, and polyvalent alcohol can be ethylene glycol, glycerol, sorbyl alcohol or N.F,USP MANNITOL, preferred glycerol, and polyvalent alcohol initial action concentration is 5～100mL/L.
Wherein, described yeast cell is any one during yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to, preferred example can be lifted the microorganism yeast saccharomyces cerevisiae that belongs to yeast belong, bread yeast etc.; The microorganism Candida parapsilosis that belongs to mycocandida; The Ao Molieshi pichia spp that belongs to Pichia; The microorganism white torulopsis that belongs to torulopsis; The spherical Dbaly yeast of class that belongs to Debaryomyces; Belong to the Lu Shi zygosaccharomyces that zygosaccharomyces belongs to; The kluyveromyces marxianus that belongs to genus kluyveromyces; The outstanding fourth debaryomyces hansenii that belongs to Hansenula; Belong to the different Brettanomyces of Brettanomyces genus etc.
The usage quantity of yeast cell is for to press wet thallus 100～800g/L, and preferred 200～600g/L promptly is the reaction solution of 1L for cumulative volume, needs to add 100～800g wet thallus, preferably adds 200～600g wet thallus.
Zymic utilizes form to be the dry thing of yeast cell, the centrifugal cell that obtains of culture of isolated, immobilized cell, the lyophilized products of cell, commercially available yeast powder, air-dry yeast or waste yeast mud by fermentation.
Wherein, the yeast cell of having property is meant the yeast cell that the permeability changes of the cytolemma of handling by chemistry, physics or biological method is crossed, and described chemistry, physics or biological method comprise surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze enzyme process.
The tensio-active agent that uses in the surfactant method is nonionic surface active agent polyethylene oxide amines or triton x-100, cationic surfactant hexadecyl trimethylamine bromide, or anion surfactant Sarkosyl L salt, the tensio-active agent usage quantity is 0.1～50g/L, and preferred 1～20g/L is promptly during surfactant method process for producing bacterial strain, tensio-active agent is directly added reaction solution, for cumulative volume is the reaction solution of 1L, adds 0.1～50g, preferably adds 1～20g.
The organic solvent that uses in the organic solvent method is dimethylbenzene, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ethyl acetate, organic solvent concentration is 0.1～50mL/L, preferably with 1～20mL/L, when being organic solvent method process for producing bacterial strain, organic solvent is directly added reaction solution, for cumulative volume is the reaction solution of 1L, adds 0.1～50mL, preferably adds 1～20mL.
Other handles the method for cell permeability, as freeze-thaw method, ultrasonication method, aeration drying etc., after employing is handled strain cell earlier, the bacterial strain of handling well is added the mode of reaction solution again.
Being reflected in the aqueous solution of above-mentioned preparation deoxynucleoside triphosphate carried out, and reacts 2～24 hours under 5.0～9.0,20～50 ℃ of conditions of pH, preferably reacts 2～24 hours under 7.0～8.0,30 ℃ of conditions of pH.
Beneficial effect: the invention has the advantages that:
1, the present invention utilizes yeast cell to carry out catalyzed reaction, and it is abundant that yeast cell contains the enzyme class, has the potentiality of the multiple biochemical reaction of catalysis.Special yeast cell has coenzyme, and (ATP) refresh function can play a significant role aspect catalytic oxidation-reduction reaction, the phosphoric acid shift reaction for NADH, NADPH.Utilize the enzyme system of yeast cell carry out enzymic catalytic reaction produce desired product in extensive range, have good market outlook.
2, the present invention is based upon on the basis of whole-cell catalytic, its characteristics be to have overcome the additive method substrate conversion efficiency not high, be difficult to realize defectives such as energy and regenerating coenzyme.Compare with enzyme process, because cell has and keeps the complete multienzyme system of its vital movement, various enzymes are keeping residing state of original life cell and specific position again, reaction energy needed and cofactors do not need extraneous the supply, directly produce by cell, therefore can finish the multistep enzymic catalytic reaction quickly and effectively, have transformation efficiency height, cost low, and pollute little advantage.Compare with existing chemical process simultaneously, the present invention is a kind of bioconversion method that can obviously reduce environmental pollution.
3, the present invention has set up energy self coupling connection and regeneration system rapidly efficiently, it is glycolysis-(EMP) path enzyme system (hexokinase that reaction has utilized the intravital enzyme of microorganism, glucose phosphate isomerase, phosphofructokinase, zymohexase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydro-genase, phosphoglyceric kinase, phosphoglycerate phosphomutase, enolase, pyruvate kinase, pyruvic carboxylase, ethanol dehydrogenase) and dNTP synthetase series (nucleoside monophosphate kinase, nucleoside diphosphokinase) carries out catalyzed reaction, it is synthetic that the ATP that EMP Embden Meyerbof Parnas pathway produces can apply to dNTP, adding by chemical substance in addition, can accelerate the regeneration rate of ATP, form efficiently energy self coupling connection and regeneration system rapidly, make the dNTP excess accumulation.
4, the present invention utilizes the metabolic engineering principle, adds magnesium ion, potassium ion, and the composition of acetaldehyde and polyvalent alcohol, mainly plays following several respects effect:
1) regulate metabolic flux, make intrasystem pathways metabolism flow take place obviously to change, the metabolic flux that flows to glycerine significantly reduces.Metabolic flux is after regulating, and the approach of EMP make that EMP master's approach is strengthened, thereby improved energy utilization ratio, and the accumulation of dNTP is just being needed ATP energize and phosphate radical by strongly inhibited, dNTP and then be able to a large amount of accumulation.
2) accelerate NADH regeneration, keep NADH/NAD in the born of the same parents
+Ratio, the redox equilibrium of recovery cell.And the NADH oxidative pathway is turned to the ethanol fermentation approach, thereby strengthen the flux of glycolytic pathway, promote the conversion of dAMP to dATP.
3) add metal ions such as magnesium ion, potassium ion, make the cumulative speed of FDP obviously accelerate, stimulate the pyruvate kinase activity; Be because the decomposition of phosphoenolpyruvic acid on the other hand, make the regeneration of the NAD that former cause glycerolphos phate dehydrogenase catalysis phosphodihydroxyacetone causes, still take on by ethanol dehydrogenase.And acetaldehyde and polyvalent alcohol can guarantee and accelerate NADH regeneration effectively, and it is alive and stable to keep enzyme.Thus, metal ion and organism can promote mutually that both act synergistically efficiently in generation, make intrasystem pathways metabolism flow take place obviously to change, and the metabolic flux that flows to glycerine significantly reduces, and realization dNTP's is efficient synthetic.
4) enzyme of assurance yeast enzyme system is lived and is stable, helps improving the combined coefficient of dNTP.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
Yeast culture base (g/L): glucose 40, urea 2.0, potassium primary phosphate 1.5, bitter salt 0.5, Zinc vitriol 4.0 * 10
-3, ferrous sulfate 3.0 * 10
-3, four hydration Manganous chloride tetrahydrates 0.3 * 10
-3, Calcium Chloride Powder Anhydrous 1.0 * 10
-3, vitamin H 0.05 * 10
-3Yeast saccharomyces cerevisiae inoculum size 10% was cultivated centrifugal 4000rpm, 20 minutes 24 hours in 30 ℃ of following 120rpm shaking tables.Get yeast slurry ,-7 ℃ of preservations are standby.
The yeast that following examples are used all is to cultivate by above-mentioned training method.
Embodiment 2: utilize dAMP to prepare dATP.
Capacity be in the beaker of 500mL modulation by dAMP 1.49mmol, glucose 0.072mol, bread yeast mud 100g, sal epsom 14.96mmol, SODIUM PHOSPHATE, MONOBASIC 0.036mol, glycerol 3mL, hexadecyl trimethylamine. the reaction solution 300mL that brometo de amonio 0.3g and water are formed, transfer pH to 7.0 with sodium hydroxide, the stirring at low speed reaction is 6 hours under 30 ℃ of conditions, after reaction finishes, use the perchloric acid precipitation, with HPLC dATP is carried out quantitative analysis, contain dATP 2.59g/L in the conversion fluid, the yield of dATP reaches 93.6% (mol meter).
Embodiment 3: utilize dAMP to prepare dATP.
Capacity be in the beaker of 500mL modulation by dAMP 1.66mmol, glucose 0.08mol, the bread yeast mud 110g after air-dry, Repone K 8.05mmol, SODIUM PHOSPHATE, MONOBASIC 0.036mol, the reaction solution 300mL that acetaldehyde 3mL and water are formed transfers pH to 7.0 with sodium hydroxide, the stirring at low speed reaction is 6 hours under 30 ℃ of conditions, reaction is used the perchloric acid precipitation after finishing, and with HPLC dATP is carried out quantitative analysis, contain dATP 2.85g/L in the conversion fluid, the yield of dATP reaches 92.4% (mol meter).
Embodiment 4: utilize dCMP to prepare dCTP.
Capacity be in the beaker of 500mL modulation by dCMP 1.50mmol, glucose 0.068mol, yeast saccharomyces cerevisiae 90g, air-dry processing, magnesium chloride 6.89mmol, Repone K 5.37mmol, SODIUM PHOSPHATE, MONOBASIC 0.03mol, acetaldehyde 3mL, glycerol 3mL, the reaction solution 300mL that triton x-100 6g and water are formed transfers pH to 6.5 with sodium hydroxide, the stirring at low speed reaction is 18 hours under 37 ℃ of conditions, reaction is used the perchloric acid precipitation after finishing, and with HPLC dCTP is carried out quantitative analysis, contain dCTP 0.71g/L in the conversion fluid, the yield of dCTP reaches 25.6% (mol meter).
Embodiment 5: utilize dCMP to prepare dCTP.
Capacity be in the beaker of 500ml modulation by dCMP 1.61mmol, glucose 0.076mol, Lu Shi zygosaccharomyces 100g, multigelation 3 times, Repone K 8.05mmol, SODIUM PHOSPHATE, MONOBASIC 0.03mol, the reaction solution 300mL that glycerol 3mL and water are formed, transfer pH to 6.5 with sodium hydroxide, the stirring at low speed reaction is 18 hours under 37 ℃ of conditions, after reaction finishes, use the perchloric acid precipitation, with HPLC dCTP is carried out quantitative analysis, contain dCTP 0.80g/L in the conversion fluid, the yield of dCTP reaches 26.9% (mol meter).
Embodiment 6: utilize dGMP to prepare dGTP.
Capacity be in the beaker of 500ml modulation by dGMP 1.30mmol, glucose 0.065mol, Lu Shi zygosaccharomyces 90g, sal epsom 16.62mmol, SODIUM PHOSPHATE, MONOBASIC 0.03mol, acetaldehyde 3mL, hexadecyl trimethylamine. the reaction solution 300mL that brometo de amonio 0.3g and water are formed, transfer pH to 6.5 with sodium hydroxide, the stirring at low speed reaction is 12 hours under 37 ℃ of conditions, after reaction finishes, use the perchloric acid precipitation, with HPLC dGTP is carried out quantitative analysis, contain dGTP 0.99g/L in the conversion fluid, the yield of dGTP reaches 45.0% (mol meter).
Embodiment 7: utilize dGMP to prepare dGTP.
Capacity be in the beaker of 500ml modulation by dGMP 1.44mmol, glucose 0.068mol, yeast saccharomyces cerevisiae 90g, air-dry processing, sal epsom 14.96mmol, SODIUM PHOSPHATE, MONOBASIC 0.03mol, ethylene glycol 3mL, the reaction solution 300mL that triton x-100 6mL and water are formed transfers pH to 6.5 with sodium hydroxide, the stirring at low speed reaction is 12 hours under 37 ℃ of conditions, reaction is used the perchloric acid precipitation after finishing, and with HPLC dGTP is carried out quantitative analysis, contain dGTP1.13g/L in the conversion fluid, the yield of dGTP reaches 46.4% (mol meter).
Embodiment 8: utilize dTMP to prepare dTTP.
Capacity be in the beaker of 500ml modulation by dTMP 1.50mmol, glucose 0.09mol, different Brettanomyces 150g, sal epsom 14.96mmol, SODIUM PHOSPHATE, MONOBASIC 0.036mol, acetaldehyde 3mL, the reaction solution 300mL that toluene 3mL and water are formed, transfer pH to 8.0 with sodium hydroxide, the stirring at low speed reaction is 10 hours under 30 ℃ of conditions, after reaction finishes, use the perchloric acid precipitation, with HPLC dTTP is carried out quantitative analysis, contain dTTP 0.84g/L in the conversion fluid, the yield of dTTP reaches 34.8% (mol meter).
Embodiment 9: utilize dTMP to prepare dTTP.
Capacity be in the beaker of 500ml modulation by dTMP 1.64mmol, glucose 0.10mol, yeast saccharomyces cerevisiae 100g, air-dry processing, magnesium chloride 6.89mmol, Repone K 8.05mmol, SODIUM PHOSPHATE, MONOBASIC 0.036mol, acetaldehyde 3mL, glycerol 3mL, the reaction solution 300mL that acetone 6mL and water are formed transfers pH to 7.0 with sodium hydroxide, the stirring at low speed reaction is 10 hours under 30 ℃ of conditions, reaction is used the perchloric acid precipitation after finishing, and with HPLC dTTP is carried out quantitative analysis, contain dTTP 0.95g/L in the conversion fluid, the yield of dTTP reaches 36.0% (mol meter).
Claims (12)
1. the new technology of the synthetic deoxynucleoside triphosphate of a whole-cell biological, it is characterized in that with deoxynucleoside one phosphoric acid and phosphate anion be substrate, with sugar is energy donor, the yeast cell of having property of utilization is set up efficiently energy self coupling connection and regeneration system rapidly and is prepared deoxynucleoside triphosphate in the presence of the chemical effect material.
2. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1, it is characterized in that described deoxynucleoside triphosphate is meant any one among deoxyadenosine triphosphate dATP, deoxyguanosine triphosphate dGTP, deoxycytidine triphosphate dCTP and the deoxythymidine triphosphate dTTP, its structural formula is as follows:
3. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1, it is characterized in that described deoxynucleoside one phosphoric acid is meant any one among deoxyadenosine monophosphate dAMP, deoxyguanosine monophosphate dGMP, deoxycytidine monophosphate dCMP and the deoxythymidine monophosphate dTMP, its structural formula is as follows:
4. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1, the initial action concentration that it is characterized in that described deoxynucleoside one phosphoric acid is 1～100mM; Described phosphate anion initial action concentration is 0.01～1.0M; The initial action concentration of sugar is 0.1～1.0M; Described sugar is glucose, fructose, sucrose or maltose.
5. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1 is characterized in that described chemical effect material is meant the combination of metal ion and organic compound; Described metal ion is Mg
2+And K
+In the combination of any one or two kinds; Described organic compound is the combination of any one or two kinds in acetaldehyde and the polyvalent alcohol; Mg
2+Initial action concentration is 1～100mM, K
+Initial action concentration is 1～100mM, Na
+Initial action concentration is 1～100mM, and acetaldehyde initial action concentration is 10～80mL/L, and polyvalent alcohol initial action concentration is 5～100mL/L.
6. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 5 is characterized in that described polyvalent alcohol is ethylene glycol, glycerol, sorbyl alcohol or N.F,USP MANNITOL.
7. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1, it is characterized in that described yeast cell is any one during yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to, the zymic usage quantity is by wet thallus 100～800g/L.
8. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 7 is characterized in that described yeast cell is yeast saccharomyces cerevisiae, Candida parapsilosis, bread yeast, Ao Molieshi pichia spp, white torulopsis, the spherical Dbaly yeast of class, Lu Shi zygosaccharomyces, kluyveromyces marxianus, outstanding fourth debaryomyces hansenii or different Brettanomyces.
9. according to the new technology of the synthetic deoxynucleoside triphosphate of claim 1,7 or 8 described whole-cell biologicals, the yeast cell that it is characterized in that described having property is meant the yeast cell that the permeability changes of the cytolemma of handling by chemistry, physics or biological method is crossed, and described chemistry, physics or biological method comprise surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze enzyme process.
10. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 9, it is characterized in that the tensio-active agent that uses in the described surfactant method is nonionic surface active agent, cationic surfactant or anion surfactant, working concentration is 0.1～50g/L.
11. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 9, it is characterized in that the organic solvent that uses in the described organic solvent method is dimethylbenzene, toluene, lipase, acetone or ethyl acetate, working concentration is 0.1～50ml/L.
12. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1 is characterized in that being reflected in the aqueous solution of described preparation deoxynucleoside triphosphate carry out, and reacts 2～24 hours under 5.0～9.0,20～50 ℃ of conditions of pH.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010190260277A CN101768617B (en) | 2010-02-04 | 2010-02-04 | Method for synthesizing deoxynucleoside triphosphate through whole-cell biosynthesis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010190260277A CN101768617B (en) | 2010-02-04 | 2010-02-04 | Method for synthesizing deoxynucleoside triphosphate through whole-cell biosynthesis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101768617A true CN101768617A (en) | 2010-07-07 |
| CN101768617B CN101768617B (en) | 2012-06-27 |
Family
ID=42501717
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010190260277A Expired - Fee Related CN101768617B (en) | 2010-02-04 | 2010-02-04 | Method for synthesizing deoxynucleoside triphosphate through whole-cell biosynthesis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101768617B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168123A (en) * | 2011-01-10 | 2011-08-31 | 吕朝阳 | Novel method for preparing deoxyribonucleoside triphosphate (dNMP) |
| CN102199644A (en) * | 2011-04-15 | 2011-09-28 | 江苏省中国科学院植物研究所 | Genetic engineering preparation method of cytidine triphosphate |
| CN102199643A (en) * | 2011-03-04 | 2011-09-28 | 苏州天马医药集团天吉生物制药有限公司 | Preparation method of citicoline |
| CN111349672A (en) * | 2020-04-13 | 2020-06-30 | 南京曼蕊生物科技有限公司 | Biosynthesis process for preparing deoxyribonucleoside triphosphate |
| CN112143766A (en) * | 2020-09-24 | 2020-12-29 | 天津全和诚科技有限责任公司 | Biosynthesis method for efficiently preparing nucleoside triphosphate |
| CN114940985A (en) * | 2022-04-20 | 2022-08-26 | 苏州酶泰生物科技有限公司 | Protein with activity of deoxyadenosine diphosphate kinase and acetate kinase and application thereof |
-
2010
- 2010-02-04 CN CN2010190260277A patent/CN101768617B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168123A (en) * | 2011-01-10 | 2011-08-31 | 吕朝阳 | Novel method for preparing deoxyribonucleoside triphosphate (dNMP) |
| CN102199643A (en) * | 2011-03-04 | 2011-09-28 | 苏州天马医药集团天吉生物制药有限公司 | Preparation method of citicoline |
| CN102199644A (en) * | 2011-04-15 | 2011-09-28 | 江苏省中国科学院植物研究所 | Genetic engineering preparation method of cytidine triphosphate |
| CN102199644B (en) * | 2011-04-15 | 2014-12-17 | 江苏省中国科学院植物研究所 | Genetic engineering preparation method of cytidine triphosphate |
| CN111349672A (en) * | 2020-04-13 | 2020-06-30 | 南京曼蕊生物科技有限公司 | Biosynthesis process for preparing deoxyribonucleoside triphosphate |
| CN112143766A (en) * | 2020-09-24 | 2020-12-29 | 天津全和诚科技有限责任公司 | Biosynthesis method for efficiently preparing nucleoside triphosphate |
| CN114940985A (en) * | 2022-04-20 | 2022-08-26 | 苏州酶泰生物科技有限公司 | Protein with activity of deoxyadenosine diphosphate kinase and acetate kinase and application thereof |
| CN114940985B (en) * | 2022-04-20 | 2024-03-19 | 苏州酶泰生物科技有限公司 | Protein with deoxyadenosine diphosphate kinase and acetate kinase activities and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101768617B (en) | 2012-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101768617B (en) | Method for synthesizing deoxynucleoside triphosphate through whole-cell biosynthesis | |
| CN101230373B (en) | Preparation method of S-adenosylmethionine | |
| US9416385B2 (en) | Method for microbial production of cyclic adenosine 3′, 5′-monophosphate | |
| CN102220400B (en) | Method for synthesis of glutathione in vitro | |
| CN1896264A (en) | Preparation method of nucleoside triphosphate | |
| CN104109701A (en) | Adenosine triphosphate preparation method | |
| WO2023273959A1 (en) | Adenosine-involved fully enzymatic synthesis method for nmn | |
| CN102605027B (en) | Enzymatic preparation method of oxidized coenzyme II | |
| Tiwari et al. | Novel cold temperature active β-glucosidase from Pseudomonas lutea BG8 suitable for simultaneous saccharification and fermentation | |
| WO2023273960A1 (en) | Method for semisynthesis of nmn involving adenosine | |
| Valino et al. | New biocatalysts for one pot multistep enzymatic synthesis of pyrimidine nucleoside diphosphates from readily available reagents | |
| CN101538598B (en) | Preparation method of citicoline | |
| EP0385486B1 (en) | Process for producing fructose-1,6-diphosphate | |
| CN101724670B (en) | Method for co-production of chiral hydroxy ester from uridine phosphinylidyne compounds | |
| CN101792786A (en) | Method for synthesizing cytidine phosphoryl compound by directional catalysis | |
| CN115433750A (en) | Preparation method of nicotinamide mononucleotide | |
| CN110904063A (en) | Fermentation process of nucleoside phosphorylase and application method thereof | |
| CN113717886B (en) | Bacillus coagulans and method for producing 2' -deoxyadenosine by catalysis thereof | |
| CN104830930B (en) | A kind of production method of 2 '-deoxyguanosine of nucleoside medicine intermediate | |
| CN101775415B (en) | Method for synthesizing phosphorylcholine by whole-cell biocatalysis | |
| CN101230372B (en) | Method for synthesizing uridine diphosphate-N-acetylglucosamine through whole-cell biocatalysis | |
| WO2014146242A1 (en) | Enzymatic preparation method for oxidized coenzyme ii | |
| CN103540537A (en) | Preparation method of uridine triphosphate | |
| CN102732488A (en) | Construction method and application of fusion of NADH oxidase gene and dehydrogenase genes | |
| CN102465162A (en) | ATP (adenosine triphosphate) regeneration system and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120627 Termination date: 20130204 |