CN1900304A - 代谢控制的工程化 - Google Patents
代谢控制的工程化 Download PDFInfo
- Publication number
- CN1900304A CN1900304A CNA2006101007709A CN200610100770A CN1900304A CN 1900304 A CN1900304 A CN 1900304A CN A2006101007709 A CNA2006101007709 A CN A2006101007709A CN 200610100770 A CN200610100770 A CN 200610100770A CN 1900304 A CN1900304 A CN 1900304A
- Authority
- CN
- China
- Prior art keywords
- promotor
- host cell
- metabolite
- allos
- glnap2
- 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.)
- Pending
Links
- 230000002503 metabolic effect Effects 0.000 title abstract description 4
- 238000000034 method Methods 0.000 claims abstract description 20
- 230000014509 gene expression Effects 0.000 claims description 36
- 102000004190 Enzymes Human genes 0.000 claims description 22
- 108090000790 Enzymes Proteins 0.000 claims description 22
- 239000002773 nucleotide Substances 0.000 claims description 22
- 125000003729 nucleotide group Chemical group 0.000 claims description 22
- -1 bisphosphate geranyl geraniol ester Chemical class 0.000 claims description 14
- 150000003505 terpenes Chemical class 0.000 claims description 14
- 230000001851 biosynthetic effect Effects 0.000 claims description 9
- XWRJRXQNOHXIOX-UHFFFAOYSA-N geranylgeraniol Natural products CC(C)=CCCC(C)=CCOCC=C(C)CCC=C(C)C XWRJRXQNOHXIOX-UHFFFAOYSA-N 0.000 claims description 8
- OJISWRZIEWCUBN-UHFFFAOYSA-N geranylnerol Natural products CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCO OJISWRZIEWCUBN-UHFFFAOYSA-N 0.000 claims description 8
- JNRZNAGCSGWZMY-UHFFFAOYSA-N C(C(=O)C)(=O)OP(=O)=O Chemical compound C(C(=O)C)(=O)OP(=O)=O JNRZNAGCSGWZMY-UHFFFAOYSA-N 0.000 claims description 6
- 108090000364 Ligases Proteins 0.000 claims description 6
- 102000003960 Ligases Human genes 0.000 claims description 6
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 claims description 6
- JEVVKJMRZMXFBT-XWDZUXABSA-N Lycophyll Natural products OC/C(=C/CC/C(=C\C=C\C(=C/C=C/C(=C\C=C\C=C(/C=C/C=C(\C=C\C=C(/CC/C=C(/CO)\C)\C)/C)\C)/C)\C)/C)/C JEVVKJMRZMXFBT-XWDZUXABSA-N 0.000 claims description 5
- 235000012661 lycopene Nutrition 0.000 claims description 5
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 claims description 5
- 229960004999 lycopene Drugs 0.000 claims description 5
- 239000001751 lycopene Substances 0.000 claims description 5
- ZCIHMQAPACOQHT-ZGMPDRQDSA-N trans-isorenieratene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/c1c(C)ccc(C)c1C)C=CC=C(/C)C=Cc2c(C)ccc(C)c2C ZCIHMQAPACOQHT-ZGMPDRQDSA-N 0.000 claims description 5
- 101710173432 Phytoene synthase Proteins 0.000 claims description 4
- 108010060155 deoxyxylulose-5-phosphate synthase Proteins 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 108010001545 phytoene dehydrogenase Proteins 0.000 claims description 3
- 239000002207 metabolite Substances 0.000 abstract description 45
- 229920001184 polypeptide Polymers 0.000 abstract description 24
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 24
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 24
- 230000001105 regulatory effect Effects 0.000 abstract description 17
- 230000004907 flux Effects 0.000 abstract description 8
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 50
- 108090000623 proteins and genes Proteins 0.000 description 35
- 241000894006 Bacteria Species 0.000 description 25
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 22
- 230000001580 bacterial effect Effects 0.000 description 22
- 230000000968 intestinal effect Effects 0.000 description 17
- 239000013612 plasmid Substances 0.000 description 15
- 238000012360 testing method Methods 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 238000013459 approach Methods 0.000 description 14
- 101150051351 glnL gene Proteins 0.000 description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 13
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 12
- 239000002243 precursor Substances 0.000 description 12
- 230000012010 growth Effects 0.000 description 11
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 10
- 230000003570 biosynthesizing effect Effects 0.000 description 10
- 230000004060 metabolic process Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 101150065098 glnG gene Proteins 0.000 description 8
- 101150012415 ntrC gene Proteins 0.000 description 8
- 238000010561 standard procedure Methods 0.000 description 8
- 230000033228 biological regulation Effects 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 108010072039 Histidine kinase Proteins 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 238000012239 gene modification Methods 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 229940107700 pyruvic acid Drugs 0.000 description 6
- 108090000769 Isomerases Proteins 0.000 description 5
- 102000004195 Isomerases Human genes 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 230000008034 disappearance Effects 0.000 description 5
- 230000005017 genetic modification Effects 0.000 description 5
- 235000013617 genetically modified food Nutrition 0.000 description 5
- 235000003642 hunger Nutrition 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 101150015622 pyk gene Proteins 0.000 description 5
- YVLPJIGOMTXXLP-UHFFFAOYSA-N 15-cis-phytoene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C YVLPJIGOMTXXLP-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- NUHSROFQTUXZQQ-UHFFFAOYSA-N Isopentenyl diphosphate Natural products CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- 101100002724 Thermus thermophilus aroH gene Proteins 0.000 description 4
- 101150076125 aroG gene Proteins 0.000 description 4
- 102000005936 beta-Galactosidase Human genes 0.000 description 4
- 108010005774 beta-Galactosidase Proteins 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 101150075592 idi gene Proteins 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 229930000044 secondary metabolite Natural products 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- MUCRYNWJQNHDJH-OADIDDRXSA-N Ursonic acid Chemical compound C1CC(=O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C MUCRYNWJQNHDJH-OADIDDRXSA-N 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 150000001491 aromatic compounds Chemical class 0.000 description 3
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 3
- 239000005516 coenzyme A Substances 0.000 description 3
- 229940093530 coenzyme a Drugs 0.000 description 3
- 230000003111 delayed effect Effects 0.000 description 3
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 3
- 101150056470 dxs gene Proteins 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 101150066555 lacZ gene Proteins 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 229930001119 polyketide Natural products 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 239000002132 β-lactam antibiotic Substances 0.000 description 3
- 229940124586 β-lactam antibiotics Drugs 0.000 description 3
- ATCICVFRSJQYDV-UHFFFAOYSA-N (6E,8E,10E,12E,14E,16E,18E,20E,22E,26E)-2,6,10,14,19,23,27,31-octamethyldotriaconta-2,6,8,10,12,14,16,18,20,22,26,30-dodecaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC=C(C)CCC=C(C)C ATCICVFRSJQYDV-UHFFFAOYSA-N 0.000 description 2
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- IGUZJYCAXLYZEE-RFZPGFLSSA-N 1-deoxy-D-xylulose Chemical compound CC(=O)[C@@H](O)[C@H](O)CO IGUZJYCAXLYZEE-RFZPGFLSSA-N 0.000 description 2
- YVLPJIGOMTXXLP-UUKUAVTLSA-N 15,15'-cis-Phytoene Natural products C(=C\C=C/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C YVLPJIGOMTXXLP-UUKUAVTLSA-N 0.000 description 2
- YVLPJIGOMTXXLP-BAHRDPFUSA-N 15Z-phytoene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)CCC=C(/C)CCC=C(C)C)C)C)C)C YVLPJIGOMTXXLP-BAHRDPFUSA-N 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- 241000205042 Archaeoglobus fulgidus Species 0.000 description 2
- 102000005636 Cyclic AMP Response Element-Binding Protein Human genes 0.000 description 2
- 108010045171 Cyclic AMP Response Element-Binding Protein Proteins 0.000 description 2
- GSXOAOHZAIYLCY-UHFFFAOYSA-N D-F6P Natural products OCC(=O)C(O)C(O)C(O)COP(O)(O)=O GSXOAOHZAIYLCY-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 101100310802 Dictyostelium discoideum splA gene Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 101000919269 Homo sapiens cAMP-responsive element modulator Proteins 0.000 description 2
- 241000588696 Pantoea ananatis Species 0.000 description 2
- 108091027981 Response element Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- BGWGXPAPYGQALX-ARQDHWQXSA-N beta-D-fructofuranose 6-phosphate Chemical compound OC[C@@]1(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BGWGXPAPYGQALX-ARQDHWQXSA-N 0.000 description 2
- 238000005842 biochemical reaction Methods 0.000 description 2
- 125000005340 bisphosphate group Chemical group 0.000 description 2
- 102100029387 cAMP-responsive element modulator Human genes 0.000 description 2
- 235000021466 carotenoid Nutrition 0.000 description 2
- 150000001747 carotenoids Chemical class 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- ZKLLSNQJRLJIGT-UYFOZJQFSA-N keto-D-fructose 1-phosphate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)COP(O)(O)=O ZKLLSNQJRLJIGT-UYFOZJQFSA-N 0.000 description 2
- 101150060972 lacX gene Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 2
- DTBNBXWJWCWCIK-UHFFFAOYSA-K phosphonatoenolpyruvate Chemical compound [O-]C(=O)C(=C)OP([O-])([O-])=O DTBNBXWJWCWCIK-UHFFFAOYSA-K 0.000 description 2
- 235000011765 phytoene Nutrition 0.000 description 2
- 239000003375 plant hormone Substances 0.000 description 2
- 150000003881 polyketide derivatives Chemical class 0.000 description 2
- 101150043515 pps gene Proteins 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 101150100525 pykA gene Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000008521 reorganization Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003384 small molecules Chemical group 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- JKQXZKUSFCKOGQ-JLGXGRJMSA-N (3R,3'R)-beta,beta-carotene-3,3'-diol Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C[C@@H](O)CC1(C)C JKQXZKUSFCKOGQ-JLGXGRJMSA-N 0.000 description 1
- 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 1
- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 description 1
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 241000589158 Agrobacterium Species 0.000 description 1
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 description 1
- 108700020747 Bacteria FruR Proteins 0.000 description 1
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 239000005792 Geraniol Substances 0.000 description 1
- GLZPCOQZEFWAFX-YFHOEESVSA-N Geraniol Natural products CC(C)=CCC\C(C)=C/CO GLZPCOQZEFWAFX-YFHOEESVSA-N 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- GLZPCOQZEFWAFX-UHFFFAOYSA-N KU0063794 Natural products CC(C)=CCCC(C)=CCO GLZPCOQZEFWAFX-UHFFFAOYSA-N 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- ATCICVFRSJQYDV-DDRHJXQASA-N Neurosporene Natural products C(=C\C=C\C(=C/C=C/C=C(\C=C\C=C(/CC/C=C(\CC/C=C(\C)/C)/C)\C)/C)\C)(\C=C\C=C(/CC/C=C(\C)/C)\C)/C ATCICVFRSJQYDV-DDRHJXQASA-N 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 102000013009 Pyruvate Kinase Human genes 0.000 description 1
- 108020005115 Pyruvate Kinase Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 241000589500 Thermus aquaticus Species 0.000 description 1
- JKQXZKUSFCKOGQ-LQFQNGICSA-N Z-zeaxanthin Natural products C([C@H](O)CC=1C)C(C)(C)C=1C=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC1=C(C)C[C@@H](O)CC1(C)C JKQXZKUSFCKOGQ-LQFQNGICSA-N 0.000 description 1
- QOPRSMDTRDMBNK-RNUUUQFGSA-N Zeaxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCC(O)C1(C)C)C=CC=C(/C)C=CC2=C(C)CC(O)CC2(C)C QOPRSMDTRDMBNK-RNUUUQFGSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 238000003811 acetone extraction Methods 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- JKQXZKUSFCKOGQ-LOFNIBRQSA-N all-trans-Zeaxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2=C(C)CC(O)CC2(C)C JKQXZKUSFCKOGQ-LOFNIBRQSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 235000013793 astaxanthin Nutrition 0.000 description 1
- 239000001168 astaxanthin Substances 0.000 description 1
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 description 1
- 229940022405 astaxanthin Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008238 biochemical pathway Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001746 carotenes Chemical class 0.000 description 1
- 235000005473 carotenes Nutrition 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 101150090177 cheY gene Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- OVSVTCFNLSGAMM-KGBODLQUSA-N cis-phytofluene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/C=C/C=C(C)/CCC=C(/C)CCC=C(C)C)C)C)C)C OVSVTCFNLSGAMM-KGBODLQUSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 101150056237 creB gene Proteins 0.000 description 1
- 101150112756 creC gene Proteins 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 150000004141 diterpene derivatives Chemical class 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 101150092055 envZ gene Proteins 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000008713 feedback mechanism Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- HRQKOYFGHJYEFS-RZWPOVEWSA-N gamma-carotene Natural products C(=C\C=C\C(=C/C=C/C=C(\C=C\C=C(/C=C/C=1C(C)(C)CCCC=1C)\C)/C)\C)(\C=C\C=C(/CC/C=C(\C)/C)\C)/C HRQKOYFGHJYEFS-RZWPOVEWSA-N 0.000 description 1
- 230000009395 genetic defect Effects 0.000 description 1
- 229940113087 geraniol Drugs 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 101150081163 glnA gene Proteins 0.000 description 1
- 108010064177 glutamine synthetase I Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- CATSNJVOTSVZJV-UHFFFAOYSA-N heptan-2-one Chemical compound CCCCCC(C)=O CATSNJVOTSVZJV-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 150000001261 hydroxy acids Chemical class 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-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 1
- 108060004506 lycopene beta-cyclase Proteins 0.000 description 1
- 108060004507 lycopene cyclase Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229930003658 monoterpene Natural products 0.000 description 1
- 150000002773 monoterpene derivatives Chemical class 0.000 description 1
- 235000002577 monoterpenes Nutrition 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 235000008665 neurosporene Nutrition 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 101150091444 ompR gene Proteins 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 101150068006 phoB gene Proteins 0.000 description 1
- 101150028857 phoP gene Proteins 0.000 description 1
- 101150086617 phoQ gene Proteins 0.000 description 1
- 101150022503 phoR gene Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 235000002677 phytofluene Nutrition 0.000 description 1
- OVSVTCFNLSGAMM-UZFNGAIXSA-N phytofluene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=C\C=C(/C)\C=C\C=C(C)CCC=C(C)CCC=C(C)C OVSVTCFNLSGAMM-UZFNGAIXSA-N 0.000 description 1
- ZYSFBWMZMDHGOJ-SGKBLAECSA-N phytofluene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)C=CC=C(/C)CCC=C(C)C)C)C)C)C ZYSFBWMZMDHGOJ-SGKBLAECSA-N 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 125000000830 polyketide group Chemical group 0.000 description 1
- 101150053304 pykF gene Proteins 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000001054 red pigment Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- AIBOHNYYKWYQMM-MXBSLTGDSA-N torulene Chemical compound CC(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C AIBOHNYYKWYQMM-MXBSLTGDSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- ZIUDAKDLOLDEGU-UHFFFAOYSA-N trans-Phytofluen Natural products CC(C)=CCCC(C)CCCC(C)CC=CC(C)=CC=CC=C(C)C=CCC(C)CCCC(C)CCC=C(C)C ZIUDAKDLOLDEGU-UHFFFAOYSA-N 0.000 description 1
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- NCYCYZXNIZJOKI-UHFFFAOYSA-N vitamin A aldehyde Natural products O=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-UHFFFAOYSA-N 0.000 description 1
- 101150075472 ycf27 gene Proteins 0.000 description 1
- 235000010930 zeaxanthin Nutrition 0.000 description 1
- 239000001775 zeaxanthin Substances 0.000 description 1
- 229940043269 zeaxanthin Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Control Of Positive-Displacement Air Blowers (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明特征在于在代谢物和代谢通量的调控下在细胞中生产异源分子的方法,所述方法可增加异源多肽和代谢物的合成。
Description
本申请是申请日为2000年7月27日,申请号为200510065954.1,发明名称为“代谢控制的工程化”的中国专利申请的分案申请。
技术领域
本发明涉及代谢控制的工程化,更具体地涉及工程化的宿主细胞,其可以增加异源多肽和代谢物的产量,本发明还涉及在宿主细胞中生产异源多肽和代谢物的方法。
背景技术
重组DNA技术的应用使得可工程化宿主细胞,以产生所需的化合物,如多肽和次生代谢物。在工程细胞中大规模生产多肽使得可生产具有药学用途的蛋白质和具有工业用途的酶。次生代谢物是来自自然界的产物,长久以来已知其生物学和医学重要性。由于这种分子固有的结构复杂性,传统的化学合成通常需要大量人力并使用昂贵的前体和辅因子以制备该化合物。近年来,在细胞中表达异源蛋白已经促进在生物中异源生物合成途径的工程化,以从非昂贵的起始材料生产代谢物。以此方式,已产生了许多化合物,包括聚酮化合物,β-内酰胺抗生素,单萜,类固醇,和芳香化合物。
发明内容
本发明部分基于如下发现,即可通过使用受次生代谢物如乙酰磷酸浓度调节的启动子调节多肽(如生物合成酶)的表达,而增加异源多肽和代谢物的产生。术语“异源”是指多肽或代谢物是通过人为导入的。异源多肽或代谢物可与天然存在的内源性物质相同。术语“代谢物”指是一或多个生物化学反应的产物的有机化合物。代谢物其自身可以是其它反应的前体。次生代谢物是产生自另一代谢物的代谢物。
因此本发明一方面涉及一种含有一核酸序列的细菌宿主细胞,该核酸序列包含一个启动子和一个编码异源多肽的核酸序列。细菌宿主细胞例如包括大肠杆菌,枯草芽孢杆菌,鼠伤寒沙门氏菌,根瘤土壤杆菌,嗜热水生菌,和豌豆根瘤菌细胞。此核酸序列可操纵地与由应答调节蛋白控制的启动子连接。换而言之,核酸序列与启动子的连接方式是使核苷酸序列在体外或体内表达。“启动子”是指导遗传物质转录的任何DNA片段。启动子是由应答调节蛋白控制的,例如大肠杆菌的ntrC,phoB,phoP,ompR,cheY,creB,或torB,或其它细菌菌种的同源物。另外,应答调节蛋白可以是簇合直向同源组(cluster orthologous group,COG)COG0745的另一成员,其由http://www.ncbi.nlm.nih.gov/COG/所定义(Tatusov等,核酸研究(2000);28:33-36)。在一实施方案中,启动子是与大肠杆菌ntrC结合的。术语“ntrC”是指大肠杆菌ntrC蛋白(SWISSPROT:P06713,http://www.expasy.ch/)及在其它适当细菌中的同源物。本文所用“结合”是指平衡结合常数(KD)小于100nM,优选小于1nM的物理缔合作用。一适当的启动子例如是大肠杆菌glnAp2启动子,如以GenBank登记号No.M10421公布的DNA序列的大约93-323位之间的区域(Reitzer & Magasanik(1985)美国科学院院报82:1979-1983)。此区域包括glnA基因的未翻译序列。另外,可在glnA编码序列和异源多肽的编码序列之间构建一翻译融合体。
宿主细胞被遗传修饰从而启动子由乙酰磷酸在无氮饥饿的情况下调节。例如,宿主细胞可通过缺失或突变编码组氨酸蛋白激酶的基因而遗传修饰,例如,该组氨酸蛋白激酶是COG0642的成员(如由http://www.ncbi.nlm.nih.gov/COG/所述;Tatusov等,如前),例如glnL,phoR,phoQ,creC,或envZ。在另一实施例中,组氨酸蛋白激酶对控制启动子的应答调节蛋白有特异性。组氨酸蛋白激酶可由glnL,如大肠杆菌glnL(SWISSPROT P06712,http://www.expasy.ch/)编码。
尽管宿主细胞被遗传修饰从而启动子由乙酰磷酸在无氮饥饿的情况下调节,以表达异源多肽或代谢物,但宿主细胞可在任何所需条件下繁殖,例如在氮饥饿条件,氮缺乏条件,或氮富足条件下繁殖。
由此核酸序列编码的异源多肽可以是产生代谢物所需的生物合成酶,其可以是哺乳动物蛋白,例如分泌的生长因子,单克隆抗体,或胞外基质组分。在另一实施例中,异源多肽可以是用于疫苗中的所需抗原,例如来自病毒,细菌,真菌或原生动物病原体的表面蛋白。
本发明的另一方面涉及一种试剂盒,其含有一种核酸序列,该核酸序列包括由应答调节蛋白控制的启动子。此试剂盒还任选的含有已经遗传修饰的细菌宿主细胞,从而启动子在不存在氮饥饿情况下由乙酰磷酸调节。此试剂盒还可提供使用说明。所述核酸序列可含有位于启动子3’端的限制酶多接头,这样***多接头的序列与由应答调节蛋白控制的启动子可操纵地连接。在该试剂盒的一实施方案中,启动子是大肠杆菌glnAp2启动子,细菌宿主细胞是含有glnL基因缺失或突变的大肠杆菌细胞。
本发明另一方面涉及含有第一个表达盒的宿主细胞。第一个表达盒包括启动子,如上述那些启动子,以及编码异源代谢物生物合成所需的酶的核酸序列。本文所用术语“酶”是指具有催化一个化学反应或多个反应的能力的多肽。所述核酸序列可操纵地与启动子连接,该启动子在无氮饥饿情况下由乙酰磷酸调节。宿主细胞还含有表达代谢物生物合成所需的其它酶的另外的核酸序列。这种另外的核酸序列可以是表达内源酶的内源序列,或表达异源酶的导入的序列。
在一实施例中,异源代谢物是类异戊二烯,多羟基链烷酸酯,聚酮化合物,β-内酰胺抗生素,芳香化合物,或其前体,例如上游代谢物,或其衍生物,例如下游代谢物。例如,类异戊二烯可以是类胡萝卜素,固醇,紫杉醇,二萜,赤霉素,和醌。类异戊二烯的特别例子包括二磷酸异戊酯,二磷酸二甲基烯丙酯,二磷酸香叶酯,二磷酸法呢酯,二磷酸香叶基香叶酯,和八氢番茄红素。类胡萝卜素特别例子包括β-胡萝卜素,ζ-胡萝卜素,虾青素,玉米黄质,玉米黄质-β-葡糖苷,六氢番茄红素,链孢红素,叶黄素和torulene。当所需的异源代谢物是类异戊二烯时,异源酶可以是异戊烯二磷酸异构酶,二磷酸香叶基香叶酯合酶,或1-脱氧木酮糖-5-磷酸合酶。当所需的异源代谢物是多羟基链烷酸酯时,异源酶可以是3-酮酯酰还原酶,或聚-3-羟基链烷酸酯聚合酶。
宿主细胞可以是细菌细胞,例如大肠杆菌细胞。此宿主细胞任选地通过缺失或突变一个基因而遗传修饰,该基因例如是上述编码组氨酸蛋白激酶的基因。在一特异的实施例中,宿主细胞还含有第二个表达盒,其含有与受乙酰磷酸浓度调节的启动子如glnAp2可操纵连接的编码烯醇丙酮酸磷酸合酶的核酸序列。
本发明另一方面涉及一种在宿主细胞中生产异源类异戊二烯的方法。该方法包括过表达烯醇丙酮酸磷酸合酶,和表达异源类异戊二烯合成所需的生物合成酶。在一实施方案中,在宿主细胞中编码丙酮酸激酶或烯醇丙酮酸磷酸羧化酶的基因经遗传缺失或弱化。在另一实施方案中,编码烯醇丙酮酸磷酸羧激酶的基因在宿主细胞中过表达。本发明的另一方面涉及一种在宿主细胞中生产番茄红素的方法,此方法包括表达以下异源酶:1-脱氧-D-木酮糖-5-磷酸合酶,二磷酸香叶基香叶酯合酶,八氢番茄红素合酶,和八氢番茄红素饱和酶。在此方法的一实施方案中,异戊烯二磷酸异构酶例如使用glnAp2启动子而过表达。
本发明的另一方面涉及一种核酸序列,其含有一个启动子和编码产生第一种代谢物所需的生物合成酶的序列。该启动子可操纵地与该序列连接,并由第二种代谢物调节,该第二种代谢物的浓度是生物合成第一种代谢物的前体的可用性的指示。在一实施例中,第二种代谢物是从生物合成第一种代谢物的前体中产生的废物。
在一实施方案中,第一种代谢物是多羟基链烷酸酯,例如多羟基丁酸酯,所述核酸序列编码生物合成酶,例如3-酮酯酰辅酶A(coA)还原酶,或聚-3-羟基辛酰辅酶A聚合酶。在另一种情况中,第一种代谢物是聚酮化合物,β-内酰胺抗生素,或芳香化合物。在另一种情况中,第一种代谢物是类异戊二烯,例如本文提及的类异戊二烯。所述核酸序列可编码类异戊二烯生成所需的生物合成酶,例如异戊烯二磷酸异构酶,二磷酸香叶基香叶酯合酶,1-脱氧木酮糖-5-磷酸合酶,烯醇丙酮酸磷酸合酶,二磷酸法呢酯合酶,二磷酸香叶基香叶酯合酶,八氢番茄红素合酶,八氢番茄红素脱饱和酶,或番茄红素环化酶。类异戊二烯的一个前体可以是丙酮酸。丙酮酸浓度与乙酸和乙酰磷酸浓度相关。因此,在这种情况下,第二种代谢物是乙酰磷酸。对乙酰磷酸的应答的启动子可通过应答调节蛋白控制,如以上提及的应答调节蛋白。这种启动子在特异的宿主细胞中可只应答乙酰磷酸。在一特定的实施例中,应答乙酰磷酸浓度的启动子与大肠杆菌ntrC结合,例如大肠杆菌glnAp2启动子。
启动子可以由cAMP调节。启动子可以是结合CAP(分解代谢物激活蛋白)的细菌启动子。在哺乳动物中,启动子可以是含有cAMP应答元件(CRE)的启动子,其结合CREB,CREM,或ATF-1蛋白。在酵母细胞中,启动子可以是由cAMP调节的启动子,或结合Gis1,Msn2或Msn4蛋白的启动子。启动子的其它可能的调节信号可以是果糖-1-磷酸,或果糖-6-磷酸。大肠杆菌FruR蛋白调节这种启动子。
所述核酸序列可包含在质粒中,其还可含有一个细菌复制源点,和一个选择标记。此序列还可包含酵母或其它真核生物复制源点,和适当的选择标记,并可整合入基因组中。
在宿主细胞中对生物合成异源化合物的优化,依赖于细胞生理学感应参数,并依赖于利用这些参数调节生物合成。本领域的一种标准方法是培养细胞,使用者外源性加入试剂如诱导物,以激活所需产物的生物合成所需的基因。已广泛观测到高水平诱导重组蛋白或途径导致生长延迟及降低代谢活性。(Kurland和Dong(1996)分子微生物学21:1-4)。外源性应用诱导物根据经验进行,并不监测细胞中生物合成资源的可用性。相反,天然途径依赖于反馈机制控制这种过程。在本发明中一些启动子与特异的遗传限定的宿主细胞和异源多肽的组合,令人预料到地产生高度精确的通用控制线路,其应答细胞代谢状态而调节异源多肽或代谢物合成的通量。实际上,动态控制的重组途径使产量增加,生长延迟最小化,并降低副产物形成所产生的毒性。对生理状态应答而调节基因表达也有益于其它应用,如基因治疗。
本发明的一或多种实施方案详见下述。通过以下阐述和权利要求可清楚本发明的其它特征,目的和优点。
具体实施方式
本发明提供了代谢控制工程方法,例如利用特异宿主细胞中的启动子优化蛋白质表达以生产蛋白质或合成代谢物的方法。
本发明的中心成分是一表达盒,其包含一个启动子和编码希望表达的异源多肽的核酸序列。此表达盒用本领域的标准方法构建,从而编码核酸序列可操纵地与启动子相连,例如由启动子调节。选择受细胞生理学或细胞代谢状态参数调节的启动子。有许多启动子可以使用。在一些应用中,所述表达盒包含于质粒中,如具有细菌复制源点和选择标记的细菌质粒。表达盒可用本领域的标准方法整合入细胞的基因组中。
如果表达盒用于工程异源多肽在晚期对数生长期或稳定期的调控产生,则可由此选择启动子。例如,可选择应答其水平在对数生长期或稳定期积累的小分子信号如第二信使的启动子。第二信使可以是作为生物化学途径的前体,中间体,或废物而积累的分子。在细菌中,小分子信号可以是糖酵解的中间体,如果糖-1-磷酸或果糖-6-磷酸,或糖酵解的废物,如乙酸或乙酰磷酸。在真核细胞中,cAMP浓度是熟知的营养状态信号。
表达盒中的启动子可基于大规模表达分析试验的结果选择,例如基因芯片试验。由乙酰磷酸诱导的基因可通过与从在乙酸中生长的细胞中制备的微阵列标记cDNA杂交,并将信号与参照信号对比而鉴别,参照信号例如是用从早期对数生长的细胞中制备的cDNA所得的信号。此试验可在原核或真核细胞,例如细菌,酵母,植物和哺乳动物细胞中进行。在原核生物中进行的这种试验例如见Talaat等,(2000)自然生物技术18:679-82和Oh&Liao(2000)生物技术进展16:278-86所述。一旦鉴别出在所需条件下表达的基因,其启动子可用在表达盒中。或者,可通过外源性加入所需分子(如代谢途径中的前体),或通过人工操纵试验条件(如生长至晚期对数期,或在生物合成酶过量产生同时仍生长),进行此试验。可基于诱导的基因鉴别启动子。
在一种情况下,使用表达盒在细菌细胞中工程代谢物的调控产生。可选择由第二种代谢物调节的启动子,该第二种代谢物的浓度是生物合成第一种代谢物的前体的可用性的指标。例如,如果第一种代谢物是合成自前体丙酮酸和甘油醛-3-磷酸的类异戊二烯,则第二种代谢物可以是乙酰磷酸。在富集环境中,细胞产生过量的乙酰辅酶A,这是丙酮酸的一种产物。过量的乙酰辅酶A用于产生ATP和乙酸,乙酸作为废物分泌。乙酸浓度随细胞密度提高。乙酸,乙酰辅酶A和乙酰磷酸浓度通过以下生物化学反应而相关:
(1)乙酰辅酶A+Pi乙酰磷酸+辅酶A
(2)乙酰磷酸+ADP乙酸+ATP
因此,高乙酰磷酸浓度表明过量的乙酰辅酶A和过量的丙酮酸。通过缺失或突变例如glnL而遗传修饰的宿主细胞,使ntrC功能成为乙酰磷酸依赖性的(Feng等,(1992)细菌学杂志174:6061-6070)。在此方式中,由ntrC调节的启动子,例如glnAp2启动子,可用于控制对乙酰磷酸应答的基因表达。glnAp2启动子可使用本领域标准方法获得。例如,可设计并合成与glnAp2启动子退火的引物。可使用聚合酶链反应(PCR)扩增含有glnAp2启动子的核酸片段,此片段可用于进一步构建过程。类似地,可容易地制备含有组氨酸蛋白激酶基因例如glnL缺失的大肠杆菌菌株。见Link等,(1997)细菌学杂志179:6228-6237详细阐述了一种可能的方法。编码所需异源多肽的序列可克隆入glnAp2启动子的下游,以便其可操纵地与启动子相连。然后可将具有失活的glnL基因的宿主细胞用此序列转化。培养转化的菌株,并在生长期间监测多肽的产生。在高细胞密度可观测到强的蛋白质表达,如Farmer和Liao(2000)自然生物技术18:533-537所述,其内容在此并入参考。
哺乳动物细胞可用作生产多肽或代谢物的宿主细胞。可选择例如对cAMP应答的启动子。这种启动子可含有cAMP应答元件(CRE),其结合CREB,CREM或ATF-1蛋白。使用本领域标准方法,可将所需编码序列置于启动子的控制下,并转化入哺乳动物细胞中。在一些情况中,可将此构建体***病毒中,如失活的病毒。这种应用使得在基因治疗方案中可实现由异源生物合成酶产生的蛋白质或代谢物的调控产生。植物细胞也可用作宿主细胞。同样,可选择适当的启动子,例如应答植物激素,代谢物,或产生所需代谢物的前体的启动子。启动子可通过微阵列试验鉴别。在将所需启动子与所需编码序列在适当载体中融合后,可将此构建体电穿孔入根瘤农杆菌中,然后使用本领域标准方法用于转化植物细胞。在另一实施例中,可操作酵母细胞使之在代谢控制下表达异源多肽或代谢物。例如,酿酒酵母启动子可以是由cAMP调节的启动子,例如与Gis1,Msn2,或Msn4蛋白结合的启动子。应答各种代谢条件的所有酵母基因的调控正在日益增加地充分研究。例如,DeRisi等,(1997)科学278:680-686阐述了在各种代谢条件下,近于全部的根瘤农杆菌基因的转录模式的试验。可基于这些数据选择由所需代谢物调节的启动子。本领域熟知产生酵母质粒和转化酵母的方法。
可使用上述表达方法重建各种代谢途径。例如,通过构建以下基因的表达载体,可将产生番茄红素的途径导入大肠杆菌中,所述基因为:来自大肠杆菌的dxs(编码1-脱氧-D-木酮糖-5-磷酸合酶),来自Archaeoglobus fulgidus的gps(编码二磷酸香叶基香叶酯(GGPP)合酶),和来自Erwinia uredovora的crtBI(分别编码番茄红素合酶和脱饱和酶)。这些基因可位于单个或多个质粒上,或可整合入大肠杆菌染色体中。另外,可使用任何方法过表达烯醇丙酮酸磷酸合酶,例如通过与glnAp2启动子融合而进行。异戊基二磷酸异构酶可使用任何方法过表达,例如通过与glnAp2启动子融合而进行。
在另一实施例中,可将产生多羟基链烷酸酯(PHA),例如多羟基丁酸酯的途径导入大肠杆菌中。PHA是一类具有各种热塑性和商业用途的羟酸线性聚酯。编码3-酮酯酰辅酶A还原酶和聚-3-羟基链烷酸酯聚合酶的铜绿假单胞菌基因可置于所需启动子例如glnAp2的调节下,因为乙酰辅酶A水平可表明合成PHA的前体的可用性。
不用进一步详细阐述,确信以上阐述已充分说明了本发明。以下实施例只是举例说明本发明,而无任何限制之意。文中所引用的文献均以全文并入参考。
方法
生长条件:将所有大肠杆菌全部生长于含有指定的培养基的摇瓶中,此摇瓶置于37℃水浴摇床(Model G76;New Brunswick Scientific,Edison,NJ)中。将培养物生长于基本培养基中,该培养基由含有0.5%(wt/vol)葡萄糖的M9限定盐34,或含有1.5%(wt/vol)葡萄糖的YE限定盐组成。YE限定盐(每升)由14g K2HPO4,16gKH2PO4,5g(NH4)2SO4,1g MgSO4,和1mg硫胺素组成。在550nm经分光光度法监测细胞浊度。
代谢物测定:用HPLC(Constametric 3500溶剂输送***和分光监测仪3100可变波长检测仪;LDC Analytical,Riviera Beach,FL)经保持在65℃的有机酸层析柱(Aminex HPX-87H,Bio-Rad实验室,Hercules,CA),测定乙酸,丙酮酸和其它有机酸。流动相由0.01NH2SO4组成,流速保持在0.6ml/分钟。在210nm检测层析柱峰。用Sigma试剂盒No.315-100测定葡萄糖。为定量番茄红素,将1ml细菌培养物用丙酮提取,离心,并在474nm测定上清吸光度。通过与标准曲线吸光度对比计算番茄红素浓度。
SDS-PAGE和酶分析:SDS-PAGE方法见于Laemmli(1970)自然227:680-685所述。测定β-半乳糖苷酶活性基本如Miller(1992)细菌遗传学简要课程,冷泉港实验室出版社,冷泉港NY所述。
结果
在大肠杆菌中使用glnAp2启动子与lacZ的异源融合体
乙酰磷酸水平提高可表明过量葡萄糖通量。本发明特征是宿主细胞,核酸序列,和利用乙酰磷酸作为信号调节所需代谢途径中控速酶的表达,以充分利用过量碳通量并使通量不产生毒性产物,乙酸。
为测试glnAp2作为产物表达的动态控制物的潜力,构建含有可操纵地与glnAp2启动子连接的异源lacZ基因的核酸序列。含有启动子和两个ntrC结合位点的glnAp2启动子区域,通过本领域的标准方法可易于获得。此glnAp2启动子经PCR从大肠杆菌基因组DNA中扩增出,使用正向引物5’-CAGCTGCAAAGGTCATTGCACCAAC(含有工程PvuII位点),和反向引物5’-GGTACCAGTACGT-GTTCAGCGGACATAC(含有工程KpnI位点)。这两个引物扩增公布的DNA序列16(GenBank登记号No.M10421)的93-343位之间的区域。
将glnAp2 PCR片段也克隆入pRS551的EcoRI位点,因此产生p2GFPuv,其含有在无启动子的lacZ基因之前的glnAp2。通过同源重组(Simons等,(1987)基因53:85-96),将glnAp2-lacZ区域转移至λRS45中,产生噬菌体λp2GFPuv。通过用λp2GFPuv噬菌体感染(Silhavy等,(1984),基因融合试验,冷泉港实验室出版社,冷泉港NY),将glnAp2-lacZ融合体分别整合入BW13711(lacX74)和BW18302(lacX glnL2001;Feng等,如前)的染色体中而构建JCL1595和JCL1596。此菌株含有glnL2001等位基因,其由glnL编码序列的23和182密码子之间的内部缺失组成,并预计产生无效突变(Feng等,如前)。
β-半乳糖苷酶活性的时程是在野生型和glnL突变体中测定的。glnAp2-β-半乳糖苷酶活性以时间依赖型方式增加,这类似于从glnL宿主(JCL1596)中分泌的乙酸浓度,而在等基因的野生型对照(JCL1595)中未发现启动子活性被诱导。
表1:glnAp2-lacZ的β-半乳糖苷酶活性
β-半乳糖苷酶活性(nmol/分钟/mg蛋白) | ||
6小时 | 11小时 | |
WT(JCL1595)中glnAp2-lacZ | <100 | ~100 |
glnL(JCL1596)中glnAp2-lacZ | ~700 | ~1500 |
VJS632中Plac-lacZ | ~500 | ~550 |
因此,在没有glnL的情况下,glnAp2能应答由乙酸分泌代表的过量碳通量。随着细胞接近晚指数期,生物合成需求降低,细胞开始呈现过量碳通量,这由乙酸的产生增加而表明。在这点上,在大约6小时,令人意外的glnAp2-β-半乳糖苷酶活性开始提高(~700nmol/分钟/mg蛋白,见表1),而野生型菌株(JCL1595)中glnAp2-β-半乳糖苷酶活性相对较低,并在整个期间保持恒定(~100nmol/分钟/mg蛋白,见表1)。在10小时以后,在无glnL情况下,glnAp2-β-半乳糖苷酶活性达到~1500nmol/分钟/mg蛋白,见表1。glnASp2的诱导分布图也完全与lac启动子(Plac)的分布图形成鲜明对照。菌株VJS632(lac+)中染色体Plac活性在用IPTG(异丙基硫代-β-D-半乳糖苷)诱导后迅速提高,并在细胞中达到恒定水平表达(~550nmol/分钟/mg蛋白,见表1),其不依赖于生长时期。
在大肠杆菌中使用glnAp2启动子与pps和aroG的异源融合体
将两个不同的代谢酶,烯醇丙酮酸磷酸合酶(pps)和3-脱氧-D-***庚酮糖酸(arabinoheptulosonate)-7-磷酸(DAHP)合酶(aroG)的表达置于glnAp2启动子的控制下。作为对照,将同样的这两个蛋白也用呈静置对照的tac启动子(Ptac),在相同遗传背景和环境条件下过表达。表达pps的标准方法导致生长延迟(Patnaik等,细菌学杂志174:7527-7532)。
通过将含有aroG pRW5tkt的PCR片段克隆入pJF118EH的EcoRI-BamHI位点中,构建质粒pAROG。质粒pPS706已由Patnaik等(如前)阐述。这两个质粒表达在Ptac启动子下的相应基因。将含有glnAp2启动子的PCR片段分别克隆入质粒pAROG和pPS706的EcoRV-EcoRI位点中,产生含有在glnAp2启动子控制下的相应基因的质粒p2AROG3和pPSG706。
用所有4个质粒转化宿主菌株BW18302(lacX glnL2001)。将具有相应质粒的菌株培养于M9盐-葡萄糖培养基中。在5小时后对比生长情况。
表2:过表达菌株的生长
在生长5小时后的OD550 | |
无质粒 | ~0.5 |
Ptac-aroG | ~0.5 |
glnAp2-aroG | ~0.5 |
Ptac-pps | ~0.12 |
glnAp2-pps | ~0.4 |
如前所述,用Ptac-pps过表达pps导致生长明显延迟。然而,使用glnAp2意外地产生接近正常的生长(见表2)。在15小时后,从每个菌株中分离蛋白质,并在10%SDS-PAGE凝胶上进行分析。当与Ptac启动子控制的pps基因相对比时,由glnAp2启动子控制的pps基因有至少多500%的pps蛋白质表达。另一令人惊奇的发现是,利用glnAp2启动子进行表达与利用Ptac启动子进行表达相比,在细胞的提取物中AroG蛋白也至少多300%,aroG蛋白的过表达没有公开的害处。
通过idi过表达在大肠杆菌中生产番茄红素
通过表达来自大肠杆菌的dxs基因(编码1-脱氧-D-木酮糖5-磷酸合酶),来自Archaeoglobus fulgidus的gps基因(编码二磷酸香叶基香叶酯(GGPP)合酶),和来自Erwinia uredovora的crtBI基因(分别编码八氢番茄红素合酶和脱饱和酶),在大肠杆菌中重建重组番茄红素途径。将这些基因***低拷贝数质粒pCL1920中形成pCW9,并同时过表达。
使用glnAp2启动子控制idi(异戊烯二磷酸异构酶)的表达。含有idi基因的构建体衍生自无启动子载体pJF118。***glnAp2启动子形成p2IDI。作为对照,***Ptac启动子形成pTacIDI。将这些质粒分别导入含有pCW9的glnL菌株(BW18302)中。含有p2IDI的菌株(glnAp2-idi)在含有葡萄糖的规定培养基中培养26小时后,产生100mg/L的番茄红素。与此相反,含有Ptac-idi的菌株在相同条件下,只产生少量的番茄红素(<5mg/L)。另外,p2IDI菌株比pTacIDI产生几乎少3倍的乙酸,这表明为流向乙酸的碳通量已更改为流向番茄红素。
表3 在大肠杆菌分批培养物中番茄红素形成的碳收率
在葡萄糖上番茄红素碳收率(mol C/mol C) | |
宿主(BW18302) | 0.0000 |
+pTacIDI(Ptac-idi) | 0.0003 |
+pTacIDI(Ptac-idi)/pPS184(Ptac-pps) | 0.0012 |
+p2IDI(glnAp2-idi) | 0.014 |
+p2IDI(glnAp2-idi)/pPSG184(glnAp2-pps) | 0.022 |
使用pps增加番茄红素产量
从另一相容质粒pPSG18中经glnAp2过表达pps,同时用pCL1920表达番茄红素途径其余基因(dxs,gps,crtBI)。pps和idi与番茄红素途径共表达使番茄红素的终滴度提高50%,并使产量提高3倍,从0.05mg/ml/小时提高至0.16mg/ml/小时(表3)。这与含有pTacIDI和pPS184的对比菌株(Ptac-idi+Ptac-pps)相反,它们的产量没有明显改进,并发生实际生长抑制。
生产番茄红素的其它宿主细胞
将pykF∷cat和pykA∷kan等位基因导入野生型菌株中,以产生两个单突变体菌株(JCL1610(pykF)和JCL1612(pykA)),和一个双突变体菌株(JCL1613(pykF pykA))(Ponce等,(1995)细菌杂志177:5719-5722)。双突变体菌株能达到终番茄红素滴度为大约14mg番茄红素/g干细胞,而每个单突变体菌株所得番茄红素滴度为大约2.5mg番茄红素/g干细胞。单pyk突变体产生的番茄红素水平与野生型菌株相似,大约为4mg番茄红素/g干细胞。另外,过表达Pck(烯醇丙酮酸磷酸羧激酶)使番茄红素的终滴度提高大约3倍。过表达Ppc(烯醇丙酮酸磷酸羧化酶)使番茄红素的产量降低大约30%。
其它实施方案
本发明的许多实施方案已经阐述。另外应知在不违背本发明精神和范围之下,可对本发明加以各种修改。因此,其它实施方案在所附权利要求的范围内。例如,提及的多肽和基因的所有同源物均在本发明范围内。
Claims (2)
1.一种在宿主细胞中生产异源类异戊二烯的方法,包括培养包含编码异源烯醇丙酮酸磷酸合酶的核酸序列的宿主细胞使所述的酶过表达;并在该宿主细胞中表达异源类异戊二烯合成所需的生物合成酶;以及从该宿主细胞中收获所述异源类异戊二烯。
2.一种在宿主细胞中产生番茄红素的方法,包括培养包含编码异源1-脱氧-D-木酮糖5-磷酸合酶、异源二磷酸香叶基香叶酯合酶、异源八氢番茄红素合酶、异源八氢番茄红素脱氢酶的核酸序列的宿主细胞,以及从该宿主细胞中收获番茄红素。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14580199P | 1999-07-27 | 1999-07-27 | |
US60/145,801 | 1999-07-27 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB2005100659541A Division CN100432216C (zh) | 1999-07-27 | 2000-07-27 | 代谢控制的工程化 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1900304A true CN1900304A (zh) | 2007-01-24 |
Family
ID=22514617
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNA2006101007709A Pending CN1900304A (zh) | 1999-07-27 | 2000-07-27 | 代谢控制的工程化 |
CN2006101007696A Expired - Lifetime CN1896246B (zh) | 1999-07-27 | 2000-07-27 | 代谢控制的工程化 |
CNB2005100659541A Expired - Lifetime CN100432216C (zh) | 1999-07-27 | 2000-07-27 | 代谢控制的工程化 |
CNB008108781A Expired - Lifetime CN1224698C (zh) | 1999-07-27 | 2000-07-27 | 代谢控制的工程化 |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2006101007696A Expired - Lifetime CN1896246B (zh) | 1999-07-27 | 2000-07-27 | 代谢控制的工程化 |
CNB2005100659541A Expired - Lifetime CN100432216C (zh) | 1999-07-27 | 2000-07-27 | 代谢控制的工程化 |
CNB008108781A Expired - Lifetime CN1224698C (zh) | 1999-07-27 | 2000-07-27 | 代谢控制的工程化 |
Country Status (9)
Country | Link |
---|---|
US (1) | US7122341B1 (zh) |
EP (4) | EP1947172B1 (zh) |
JP (2) | JP4685308B2 (zh) |
CN (4) | CN1900304A (zh) |
AT (1) | ATE386103T1 (zh) |
DE (1) | DE60038032T2 (zh) |
DK (4) | DK2182072T3 (zh) |
HK (3) | HK1119742A1 (zh) |
WO (1) | WO2001007567A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103232986A (zh) * | 2013-05-27 | 2013-08-07 | 青岛蔚蓝生物集团有限公司 | 一种异戊二烯生产方法 |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TW200934872A (en) * | 2008-02-05 | 2009-08-16 | Echem Hightech Co Ltd | A strain of genetically reengineered escherichia coli for biosynthesis of high yield carotenoids after mutation screening |
DK2446025T3 (en) | 2009-06-22 | 2018-10-22 | Codexis Inc | : KETOREDUCTASE-MEDIATED STEREOSELECTIVE ROAD TO ALFA CHLORAL ALCOHOLS |
WO2011100265A2 (en) | 2010-02-10 | 2011-08-18 | Codexis, Inc. | Processes using amino acid dehydrogenases and ketoreductase-based cofactor regenerating system |
CN101979587B (zh) * | 2010-10-14 | 2013-05-01 | 浙江大学 | 少动鞘氨醇单胞菌的八氢番茄红素脱氢酶基因及其应用 |
PL2721153T3 (pl) | 2011-06-16 | 2020-03-31 | The Regents Of The University Of California | Syntetyczne klastry genów |
WO2014071182A1 (en) | 2012-11-01 | 2014-05-08 | Massachusetts Institute Of Technology | Directed evolution of synthetic gene cluster |
CN106574289A (zh) * | 2014-06-13 | 2017-04-19 | 德诺芙公司 | 生产萜烯或类萜烯的方法 |
RU2769873C2 (ru) | 2015-07-13 | 2022-04-07 | Пивот Байо, Инк. | Способы и композиции для улучшения признаков растений |
EP3359664A4 (en) | 2015-10-05 | 2019-03-20 | Massachusetts Institute Of Technology | NITROGEN FIXATION WITH REINFORCED NIF CLUSTERS |
CN110799474B (zh) | 2017-01-12 | 2022-07-26 | 皮沃特生物公司 | 用于改良植物性状的方法及组合物 |
CN111587287A (zh) | 2017-10-25 | 2020-08-25 | 皮沃特生物股份有限公司 | 用于改良固氮的工程微生物的方法和组合物 |
JP2021505154A (ja) | 2017-12-07 | 2021-02-18 | ザイマージェン インコーポレイテッド | 発酵によって(6e)−8−ヒドロキシゲラニオールを生産するための設計された生合成経路 |
KR20200111172A (ko) | 2017-12-21 | 2020-09-28 | 지머젠 인코포레이티드 | 네페탈락톨 산화 환원 효소, 네페탈락톨 합성 효소, 및 네페탈락톤을 생산할 수 있는 미생물 |
CN108441510B (zh) * | 2018-03-26 | 2020-12-22 | 武汉天问生物科技有限公司 | 转基因水稻grh和黑金米的培育及grh目的基因的检测方法 |
CN108624600B (zh) * | 2018-05-22 | 2021-06-18 | 昆明理工大学 | 锌指转录因子基因RkMsn4的用途 |
WO2020006064A2 (en) | 2018-06-27 | 2020-01-02 | Pivot Bio, Inc. | Agricultural compositions comprising remodeled nitrogen fixing microbes |
KR102229379B1 (ko) * | 2019-06-11 | 2021-03-19 | 주식회사 제노포커스 | 파이토플루엔 고생산성 재조합 미생물 및 이를 이용한 파이토플루엔의 제조방법 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5429939A (en) | 1989-04-21 | 1995-07-04 | Kirin Beer Kabushiki Kaisha | DNA sequences useful for the synthesis of carotenoids |
JP2950888B2 (ja) * | 1989-04-21 | 1999-09-20 | 麒麟麦酒株式会社 | カロチノイドの合成に有用なdna鎖 |
US5530189A (en) | 1990-03-02 | 1996-06-25 | Amoco Corporation | Lycopene biosynthesis in genetically engineered hosts |
KR0178871B1 (ko) * | 1994-08-23 | 1999-04-01 | 게이사쿠 마나베 | 케토기 도입 효소, 그를 암호화하는 dna 및 케토카로테노이드를제조하는방법 |
CA2199853C (en) | 1994-09-16 | 2009-03-24 | James C. Liao | Microorganisms and methods for overproduction of dahp by cloned pps gene |
US5830692A (en) * | 1995-03-24 | 1998-11-03 | Consortium Fur Elektrochemische Industrie Gmbh | Expression system which can be regulated |
US5744341A (en) | 1996-03-29 | 1998-04-28 | University Of Maryland College Park | Genes of carotenoid biosynthesis and metabolism and a system for screening for such genes |
ES2200110T3 (es) * | 1996-12-02 | 2004-03-01 | F. Hoffmann-La Roche Ag | Produccion fermentativa de caroteniodes mejorada. |
JPH10327865A (ja) * | 1997-05-29 | 1998-12-15 | Kirin Brewery Co Ltd | カロテノイド配糖体およびその製造法 |
US20020045220A1 (en) * | 1999-10-13 | 2002-04-18 | Chaitan Khosla | Biosynthesis of polyketide synthase substrates |
-
2000
- 2000-07-27 EP EP08000125A patent/EP1947172B1/en not_active Expired - Lifetime
- 2000-07-27 CN CNA2006101007709A patent/CN1900304A/zh active Pending
- 2000-07-27 EP EP10152563.2A patent/EP2184366B1/en not_active Expired - Lifetime
- 2000-07-27 DK DK10152561.6T patent/DK2182072T3/da active
- 2000-07-27 EP EP10152561A patent/EP2182072B1/en not_active Expired - Lifetime
- 2000-07-27 AT AT00950804T patent/ATE386103T1/de not_active IP Right Cessation
- 2000-07-27 DK DK08000125.8T patent/DK1947172T3/da active
- 2000-07-27 JP JP2001521952A patent/JP4685308B2/ja not_active Expired - Lifetime
- 2000-07-27 US US10/048,186 patent/US7122341B1/en not_active Expired - Lifetime
- 2000-07-27 CN CN2006101007696A patent/CN1896246B/zh not_active Expired - Lifetime
- 2000-07-27 DK DK00950804T patent/DK1220892T3/da active
- 2000-07-27 DE DE60038032T patent/DE60038032T2/de not_active Expired - Lifetime
- 2000-07-27 EP EP00950804A patent/EP1220892B1/en not_active Expired - Lifetime
- 2000-07-27 CN CNB2005100659541A patent/CN100432216C/zh not_active Expired - Lifetime
- 2000-07-27 DK DK10152563.2T patent/DK2184366T3/da active
- 2000-07-27 CN CNB008108781A patent/CN1224698C/zh not_active Expired - Lifetime
- 2000-07-27 WO PCT/US2000/020519 patent/WO2001007567A1/en active IP Right Grant
-
2008
- 2008-12-08 HK HK08113304.6A patent/HK1119742A1/xx not_active IP Right Cessation
-
2010
- 2010-08-25 JP JP2010188609A patent/JP5419829B2/ja not_active Expired - Lifetime
- 2010-09-15 HK HK10108751.0A patent/HK1142368A1/zh not_active IP Right Cessation
- 2010-09-15 HK HK10108750.1A patent/HK1142367A1/xx not_active IP Right Cessation
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103232986A (zh) * | 2013-05-27 | 2013-08-07 | 青岛蔚蓝生物集团有限公司 | 一种异戊二烯生产方法 |
CN103232986B (zh) * | 2013-05-27 | 2014-06-18 | 青岛蔚蓝生物集团有限公司 | 一种异戊二烯生产方法 |
Also Published As
Publication number | Publication date |
---|---|
EP1947172A3 (en) | 2008-10-15 |
DK2182072T3 (da) | 2013-04-02 |
DK2184366T3 (da) | 2014-06-02 |
US7122341B1 (en) | 2006-10-17 |
CN1365386A (zh) | 2002-08-21 |
JP2010284173A (ja) | 2010-12-24 |
DK1947172T3 (da) | 2013-03-25 |
ATE386103T1 (de) | 2008-03-15 |
EP1220892A1 (en) | 2002-07-10 |
CN1224698C (zh) | 2005-10-26 |
HK1142367A1 (en) | 2010-12-03 |
EP2182072B1 (en) | 2012-12-26 |
DE60038032T2 (de) | 2009-02-05 |
JP4685308B2 (ja) | 2011-05-18 |
CN1670189A (zh) | 2005-09-21 |
EP1220892A4 (en) | 2004-04-14 |
EP2184366A2 (en) | 2010-05-12 |
CN100432216C (zh) | 2008-11-12 |
CN1896246A (zh) | 2007-01-17 |
DK1220892T3 (da) | 2008-06-09 |
EP1947172B1 (en) | 2012-12-19 |
EP2184366A3 (en) | 2010-06-02 |
EP2182072A3 (en) | 2010-06-09 |
HK1119742A1 (en) | 2009-03-13 |
JP2003508085A (ja) | 2003-03-04 |
EP2184366B1 (en) | 2014-03-12 |
EP1947172A2 (en) | 2008-07-23 |
WO2001007567A1 (en) | 2001-02-01 |
EP2182072A2 (en) | 2010-05-05 |
EP1220892B1 (en) | 2008-02-13 |
HK1142368A1 (zh) | 2010-12-03 |
CN1896246B (zh) | 2013-01-16 |
JP5419829B2 (ja) | 2014-02-19 |
DE60038032D1 (de) | 2008-03-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1224698C (zh) | 代谢控制的工程化 | |
CN108474009A (zh) | 麦芽糖依赖性降解决定子、麦芽糖响应型启动子、稳定化构建体及其在生成非分解代谢化合物中的用途 | |
CN107787361A (zh) | 生成l‑天冬氨酸衍生物的突变微生物及使用该突变微生物生成l‑天冬氨酸衍生物的方法 | |
US10648005B2 (en) | Transformed synechococcus elongatus strains having improved productivity of farnesene and use thereof | |
Wu et al. | Application of dynamic regulation to increase L-phenylalanine production in Escherichia coli | |
Wu et al. | Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803 | |
Li et al. | Heterologous production of α-Carotene in Corynebacterium glutamicum using a multi-copy chromosomal integration method | |
Shen et al. | Engineering of Escherichia coli for lycopene production through promoter engineering | |
CN109689857A (zh) | 重组细胞、以及异戊二烯或萜的生产方法 | |
KR101965330B1 (ko) | 라이코펜을 생산하는 재조합 미생물 및 이를 이용한 라이코펜의 생산방법 | |
He et al. | Two dominant selectable markers for genetic manipulation in Neurospora crassa | |
JP4528297B2 (ja) | コエンザイムq−10の向上した産生 | |
KR20200045439A (ko) | 재조합 슈도모나스 푸티다 균주 및 이를 이용한 메발론산 생산 방법 | |
US6706516B1 (en) | Engineering of metabolic control | |
US11203760B2 (en) | Gene therapy DNA vector GDTT1.8NAS12 and the method for obtaining thereof | |
TWI276684B (en) | Engineering of metabolic control | |
KR20210047992A (ko) | 알파-휴물렌 생산용 형질전환 메탄자화균 및 이의 용도 | |
KR20200023450A (ko) | 기능적 dna 서열의 안정화된 카피 수를 갖는 미생물 및 관련 방법 | |
US20240093253A1 (en) | Sustainable production of cannabinoids from simple precursor feedstocks using saccharomyces cerevisiae | |
Liao | Engineering of metabolic control | |
CN101487007B (zh) | 成团泛菌β-胡萝卜素合成操纵子及其表达载体与应用 | |
KR101123070B1 (ko) | 세포내 에너지 농도가 높은 세포 및 그의 용도 | |
KR20210068923A (ko) | 신규 이소부탄올 내성 유전자 및 그의 용도 | |
KR20190022592A (ko) | 당근 Hsp17.7 유전자를 포함하는 에스케리키아 속 미생물을 이용하여 산물을 생산하는 방법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20070124 |