CN103571835B - 一种生产紫杉二烯的系统和微生物及其应用 - Google Patents
一种生产紫杉二烯的系统和微生物及其应用 Download PDFInfo
- Publication number
- CN103571835B CN103571835B CN201310595448.8A CN201310595448A CN103571835B CN 103571835 B CN103571835 B CN 103571835B CN 201310595448 A CN201310595448 A CN 201310595448A CN 103571835 B CN103571835 B CN 103571835B
- Authority
- CN
- China
- Prior art keywords
- taxadiene
- plasmid
- microorganism
- replicon
- gene
- 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.)
- Active
Links
- 244000005700 microbiome Species 0.000 title claims abstract description 50
- 235000016408 Podocarpus macrophyllus Nutrition 0.000 title claims 3
- 244000162450 Taxus cuspidata Species 0.000 title claims 3
- 235000009065 Taxus cuspidata Nutrition 0.000 title claims 3
- 150000001993 dienes Chemical class 0.000 title claims 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 49
- 101150071502 ERG12 gene Proteins 0.000 claims abstract description 6
- 101150045041 ERG8 gene Proteins 0.000 claims abstract description 6
- 101150079299 MVD1 gene Proteins 0.000 claims abstract description 6
- 101150006429 atoB gene Proteins 0.000 claims abstract description 6
- 239000013612 plasmid Substances 0.000 claims description 54
- 241000894006 Bacteria Species 0.000 claims description 5
- 101150075592 idi gene Proteins 0.000 claims description 5
- 230000000968 intestinal effect Effects 0.000 claims 2
- FRJSECSOXKQMOD-HQRMLTQVSA-N Taxa-4(5),11(12)-diene Chemical compound C1C[C@]2(C)CCC=C(C)[C@H]2C[C@@H]2CCC(C)=C1C2(C)C FRJSECSOXKQMOD-HQRMLTQVSA-N 0.000 abstract description 64
- 230000037361 pathway Effects 0.000 abstract description 39
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 abstract description 35
- 229930012538 Paclitaxel Natural products 0.000 abstract description 14
- 230000015572 biosynthetic process Effects 0.000 abstract description 14
- 229960001592 paclitaxel Drugs 0.000 abstract description 14
- 238000003786 synthesis reaction Methods 0.000 abstract description 14
- 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 abstract description 14
- 230000001131 transforming effect Effects 0.000 abstract description 4
- KJTLQQUUPVSXIM-ZCFIWIBFSA-M (R)-mevalonate Chemical compound OCC[C@](O)(C)CC([O-])=O KJTLQQUUPVSXIM-ZCFIWIBFSA-M 0.000 abstract 2
- 241000672609 Escherichia coli BL21 Species 0.000 abstract 1
- 239000002773 nucleotide Substances 0.000 abstract 1
- 125000003729 nucleotide group Chemical group 0.000 abstract 1
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 description 33
- 241000588724 Escherichia coli Species 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 11
- 238000010586 diagram Methods 0.000 description 10
- 229940023064 escherichia coli Drugs 0.000 description 10
- CBIDRCWHNCKSTO-UHFFFAOYSA-N prenyl diphosphate Chemical compound CC(C)=CCO[P@](O)(=O)OP(O)(O)=O CBIDRCWHNCKSTO-UHFFFAOYSA-N 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 8
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 108010014539 taxa-4(5),11(12)-diene synthase Proteins 0.000 description 7
- 102100039291 Geranylgeranyl pyrophosphate synthase Human genes 0.000 description 6
- 108010066605 Geranylgeranyl-Diphosphate Geranylgeranyltransferase Proteins 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 5
- 241000233866 Fungi Species 0.000 description 5
- OINNEUNVOZHBOX-XBQSVVNOSA-N Geranylgeranyl diphosphate Natural products [P@](=O)(OP(=O)(O)O)(OC/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)O OINNEUNVOZHBOX-XBQSVVNOSA-N 0.000 description 5
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical compound CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 5
- 101100286286 Dictyostelium discoideum ipi gene Proteins 0.000 description 4
- 102100023072 Neurolysin, mitochondrial Human genes 0.000 description 4
- 241000015728 Taxus canadensis Species 0.000 description 4
- 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 4
- 230000003321 amplification Effects 0.000 description 4
- DIOQZVSQGTUSAI-UHFFFAOYSA-N decane Chemical compound CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 4
- 101150014423 fni gene Proteins 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 150000003505 terpenes Chemical class 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- NPNUFJAVOOONJE-ZIAGYGMSSA-N β-(E)-Caryophyllene Chemical compound C1CC(C)=CCCC(=C)[C@H]2CC(C)(C)[C@@H]21 NPNUFJAVOOONJE-ZIAGYGMSSA-N 0.000 description 4
- OINNEUNVOZHBOX-QIRCYJPOSA-K 2-trans,6-trans,10-trans-geranylgeranyl diphosphate(3-) Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\COP([O-])(=O)OP([O-])([O-])=O OINNEUNVOZHBOX-QIRCYJPOSA-K 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000187747 Streptomyces Species 0.000 description 3
- 241000202349 Taxus brevifolia Species 0.000 description 3
- OJFDKHTZOUZBOS-CITAKDKDSA-N acetoacetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 OJFDKHTZOUZBOS-CITAKDKDSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- -1 isoprenyl Chemical group 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- WFCGYZPOBJZGDF-UHFFFAOYSA-N 2-methylbuta-1,3-diene;phosphono dihydrogen phosphate Chemical compound CC(=C)C=C.OP(O)(=O)OP(O)(O)=O WFCGYZPOBJZGDF-UHFFFAOYSA-N 0.000 description 2
- MARPHFYISMSXDB-FOLKQPSDSA-N 5-[2-[3-[[(2r)-4-[[[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]oxy-2-hydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethylsulfanyl]-3-(hydroxymethyl)-5-oxopentanoic acid Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CC(CO)CC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MARPHFYISMSXDB-FOLKQPSDSA-N 0.000 description 2
- OVMSOCFBDVBLFW-VHLOTGQHSA-N 5beta,20-epoxy-1,7beta,13alpha-trihydroxy-9-oxotax-11-ene-2alpha,4alpha,10beta-triyl 4,10-diacetate 2-benzoate Chemical compound O([C@@H]1[C@@]2(C[C@H](O)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)O)C(=O)C1=CC=CC=C1 OVMSOCFBDVBLFW-VHLOTGQHSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010000775 Hydroxymethylglutaryl-CoA synthase Proteins 0.000 description 2
- 101000895629 Synechococcus sp. (strain ATCC 27264 / PCC 7002 / PR-6) Geranylgeranyl pyrophosphate synthase Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- NPNUFJAVOOONJE-UHFFFAOYSA-N beta-cariophyllene Natural products C1CC(C)=CCCC(=C)C2CC(C)(C)C21 NPNUFJAVOOONJE-UHFFFAOYSA-N 0.000 description 2
- NPNUFJAVOOONJE-UONOGXRCSA-N caryophyllene Natural products C1CC(C)=CCCC(=C)[C@@H]2CC(C)(C)[C@@H]21 NPNUFJAVOOONJE-UONOGXRCSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 108091000116 phosphomevalonate kinase Proteins 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- CABVTRNMFUVUDM-VRHQGPGLSA-N (3S)-3-hydroxy-3-methylglutaryl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C[C@@](O)(CC(O)=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 CABVTRNMFUVUDM-VRHQGPGLSA-N 0.000 description 1
- OKZYCXHTTZZYSK-ZCFIWIBFSA-N (R)-5-phosphomevalonic acid Chemical compound OC(=O)C[C@@](O)(C)CCOP(O)(O)=O OKZYCXHTTZZYSK-ZCFIWIBFSA-N 0.000 description 1
- 244000101408 Abies amabilis Species 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 101100178203 Arabidopsis thaliana HMGB3 gene Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150091750 HMG1 gene Proteins 0.000 description 1
- 102000055207 HMGB1 Human genes 0.000 description 1
- 108700010013 HMGB1 Proteins 0.000 description 1
- 101150021904 HMGB1 gene Proteins 0.000 description 1
- 102000004286 Hydroxymethylglutaryl CoA Reductases Human genes 0.000 description 1
- 108090000895 Hydroxymethylglutaryl CoA Reductases Proteins 0.000 description 1
- 102000002284 Hydroxymethylglutaryl-CoA Synthase Human genes 0.000 description 1
- 102100028889 Hydroxymethylglutaryl-CoA synthase, mitochondrial Human genes 0.000 description 1
- 108010065958 Isopentenyl-diphosphate Delta-isomerase Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108700040132 Mevalonate kinases Proteins 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 241000606701 Rickettsia Species 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- UJARNTWLSVMUJF-HHNKTFFCSA-N Taxine II Natural products C1([C@@H](CC(=O)O[C@@H]2C([C@@H]3[C@@]([C@H]([C@H](OC(C)=O)C4=C(C)C(=O)C[C@H](C4(C)C)[C@H]3OC(C)=O)OC(C)=O)(C)CC2)=C)N(C)C)=CC=CC=C1 UJARNTWLSVMUJF-HHNKTFFCSA-N 0.000 description 1
- 241001116500 Taxus Species 0.000 description 1
- 102000005488 Thioesterase Human genes 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 229930014667 baccatin III Natural products 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 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
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 125000002350 geranyl group Chemical group [H]C([*])([H])/C([H])=C(C([H])([H])[H])/C([H])([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- OINNEUNVOZHBOX-KGODAQDXSA-N geranylgeranyl diphosphate Chemical compound CC(C)=CCC\C(C)=C/CC\C(C)=C\CC\C(C)=C\CO[P@@](O)(=O)OP(O)(O)=O OINNEUNVOZHBOX-KGODAQDXSA-N 0.000 description 1
- 125000002686 geranylgeranyl group Chemical group [H]C([*])([H])/C([H])=C(C([H])([H])[H])/C([H])([H])C([H])([H])/C([H])=C(C([H])([H])[H])/C([H])([H])C([H])([H])/C([H])=C(C([H])([H])[H])/C([H])([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000006317 isomerization reaction Methods 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
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 102000002678 mevalonate kinase Human genes 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
- 108020002982 thioesterase Proteins 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种生产紫杉二烯的系统和微生物及其应用,属于合成生物学领域。生产紫杉二烯的系统包含甲羟戊酸途径和紫杉二烯合成相关基因或其功能等同体。甲羟戊酸途径的相关基因为来源于大肠杆菌BL21的<i>atoB</i>、<i>idi</i>和来源于酿酒酵母INVSC1的<i>erg13</i>、<i>thmg1</i>、<i>erg12</i>、<i>erg8</i>、<i>mvd1</i>;紫杉二烯合成的相关基因<i>ggpps</i>和<i>ts</i>基因核苷酸序列如SEQ?ID?NO.1和2所示。生产紫杉二烯的微生物为含有生产紫杉二烯的系统的微生物,通过将该系统转化到微生物中得到生产紫杉二烯的微生物。本发明构建的微生物能稳定产生紫杉二烯的培养物,为后续进一步利用微生物产生紫杉醇奠定了基础,具有广阔的应用前景。
Description
技术领域
本发明属于合成生物学领域,涉及一种生产紫杉二烯的系统和微生物及其应用。
背景技术
1963年美国科学家首次从一种太平洋杉树皮和木材中分离到了紫杉醇的粗提物,并发现其对鼠肿瘤细胞有很高活性。后来通过Ⅱ-Ⅲ期临床研究发现,紫杉醇主要适用于卵巢癌和乳腺癌,对肺癌、大肠癌、黑色素瘤、头颈部癌、淋巴瘤、脑瘤也都有一定疗效,是现在最有潜能且商业应用最为成熟的一种抗癌药。现今获得紫杉醇的方法主要有三种:(1)最传统的从天然植物树皮中提取,(2)从植物叶片等组织中提取中间产物巴卡亭III等,随后通过化学合成生产紫杉醇,(3)利用植物组织培养后提取紫杉醇。这三种都是依赖于植物源的方式获得产物,因而价格一直很高,而且随着逐年的砍伐,可利用的红豆杉等植物已经越来越少,这很大程度的限制了利用该药物去治疗更多患者。
自从1993年,首次报道从太平洋红豆杉树木中分离到一株能独立生物合成产生紫杉醇的内生真菌以来,国内外就有大量的文献专注于解决紫杉醇的生物合成以及相关的紫杉烷类化合物这一问题。同时,另外一些科研工作者致力于阐明紫杉醇的合成途径,但目前仍有至少五步反应的功能基因还未克隆出来,在这些酶中,研究者一致认为催化牻牛儿基牻牛儿基焦磷酸合成紫杉二烯的紫杉二烯合酶(Taxadienesynthase,TS)是合成紫杉二烯的第一个决定性步骤。
紫杉醇属于异戊二烯类超家族化合物,至今已报道有超过30000种被鉴定为异戊二烯类。而异戊二烯类超家族化合物都来源于相同的两个中间产物:异戊烯焦磷酸(IPP,isopentenyldiphosphate)和二甲基烯丙基焦磷酸(DMAPP,dimethylallyldiphosphate)。自然界中存在两个异戊二烯合成途径来提供IPP和DMAPP这两种通用的萜类化合物前体:(1)甲羟戊酸途径(MVA途径),主要存在于高等真核生物(动物,植物线粒体,真菌和酵母等)和部分细菌中,(2)非甲羟戊酸途径(DXP或MEP途径),主要存在于植物质体、原生生物和大多数细菌中,而链霉菌、部分植物等同时含有MVA和MEP途径。近年来,利用改造微生物內源的MEP或MVA途径,或者导入改造的外源MEP或MVA途径生产萜类化合物引起了人们的广泛兴趣。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足,提供一种生产紫杉二烯的系统。
本发明的另一目的在于提供一种生产紫杉二烯的微生物。
本发明的另一目的在于提供上述生产紫杉二烯的系统或微生物的应用。
本发明的再一目的还在于提供一种提高生产紫杉二烯能力的方法。
本发明的目的通过下述技术方案实现:
一种生产紫杉二烯的系统,包含甲羟戊酸途径和紫杉二烯合成相关基因或其功能等同体(同功能基因)中的一种或多种。所述的甲羟戊酸途径的相关基因包括(1)将乙酰辅酶A缩合为乙酰乙酰辅酶A的基因atoB或其功能等同体、(2)将乙酰辅酶A和乙酰乙酰辅酶A缩合为HMG-CoA的基因erg13或其功能等同体、(3)将HMG-CoA还原为甲羟戊酸的基因thmg1或其功能等同体、(4)将甲羟戊酸磷酸化为甲羟戊酸-5-磷酸的基因erg12或其功能等同体、(5)将甲羟戊酸-5-磷酸磷酸化为甲羟戊酸-5-焦磷酸的基因erg8或其功能等同体、(6)将甲羟戊酸-5-焦磷酸脱羧生成异戊烯焦磷酸的基因mvd1或其功能等同体和(7)将异戊烯焦磷酸异构为二甲基烯丙基焦磷酸的基因idi或其功能等同体;所述的紫杉二烯合成的相关基因包括(8)将异戊二烯焦磷酸和二甲基烯丙基焦磷酸缩合为牻牛儿基牻牛儿基焦磷酸的基因牻牛儿基牻牛儿基焦磷酸合酶ggpps基因或其功能等同体和(9)将牻牛儿基牻牛儿基焦磷酸环化为紫杉二烯的紫杉二烯合酶ts基因或其功能等同体。
优选的,所述的甲羟戊酸途径相关基因或其功能共同体和紫杉二烯合成相关基因或其功能共同体被设置在同一个载体或相互不同的载体上。由此,可以进一步提高采用该系统转化微生物的效率。
所述的甲羟戊酸途径的相关基因优选为来源于大肠杆菌BL21(DE3)的atoB(CP001509.3:2216470-2217654)、idi(CP001509.3:2863926-2864474)和来源于酿酒酵母INVSC1的erg13(329138949:19060-20535)、thmg1(329138949:115734-117239)、erg12(329138949:684467-685798)、erg8(329138949:712316-713671)、mvd1(329138953:701895-703085)。
所述的紫杉二烯合成的相关基因优选为来源于Taxuscanadensis(加拿大红豆杉)经过密码子优化的牻牛儿基牻牛儿基焦磷酸合酶ggpps基因(序列如SEQIDNO.1所示)和来源于Taxusbrevifolia(短叶红豆杉)经过密码子优化的紫杉二烯合酶ts基因(序列如SEQIDNO.2所示)。
一种生产紫杉二烯的微生物为含有上述生产紫杉二烯的系统的微生物。通过将生产紫杉二烯的系统转化到微生物中得到生产紫杉二烯的微生物,通过表达该微生物的基因组中所包含的外源基因,可以使该微生物过表达甲羟戊酸途径或牻牛儿基牻牛儿基焦磷酸合酶或紫杉二烯合酶的至少一种。
优选的,所述的生产紫杉二烯的微生物为将甲羟戊酸途径相关基因或其功能共同体和紫杉二烯合成相关基因或其功能共同体中的一种或多种整合到微生物基因组中。
所述的微生物为选自真核微生物、原核微生物和病毒的至少一种。
优选的,所述的微生物为选自细菌、真菌、放线菌、螺旋体、支原体、衣原体、立克次体、病毒和酵母的至少一种。
优选的,所述的微生物为丝状真菌。
优选的,所述的微生物为酵母。
优选的,所述微生物为大肠杆菌。
优选的,所述微生物为芽孢杆菌。
优选的,所述微生物为链霉菌。
优选的,所述微生物不含內源的甲羟戊酸途径。
更优选的,所述微生物含有內源的甲羟戊酸途径。
优选的,所述的生产紫杉二烯的微生物为含有质粒pMH1、质粒pFZ81和质粒pXC02的大肠杆菌;所述的质粒pMH1启动子为lac启动子,复制子为p15A复制子,包含atoB、erg13和thmg1基因,pMH1的序列(不含骨架载体序列)如SEQIDNO.3所示;所述的质粒pFZ81启动子为lac启动子,复制子为pBBR1MCS复制子,包含erg12、erg8、mvd1和idi基因,pFZ81的序列(不含骨架载体序列)如SEQIDNO.4所示;所述的质粒pXC02启动子为T7强启动子,复制子为pSC101低拷贝复制子,包含ggpps和ts基因,pXC02的序列(不含骨架载体序列)如SEQIDNO.5所示。
更优选的,所述的生产紫杉二烯的微生物为含有质粒pMH1、质粒pFZ81和质粒pFZ131的大肠杆菌;所述的质粒pFZ131包含与pXC02相同的ggpps和ts基因,启动子为T7强启动子,只是复制子为pBBR322高拷贝复制子。
上述生产紫杉二烯的系统或微生物在生产紫杉二烯中的应用。
一种提高生产紫杉二烯能力的方法为增加上述系统中任一基因或其功能等同体的表达。
合成生物学与代谢工程等的发展使得在目标微生物中表达外源基因成为可能,通过选择合适的表达元件能很好的完成外源基因在选定微生物中的表达与功能重建。本发明以大肠杆菌这一不含甲羟戊酸途径的微生物为例,通过导入甲羟戊酸途径相关基因或功能共同体、牻牛儿基牻牛儿基焦磷酸合酶与紫杉二烯合酶成功的在大肠杆菌体内合成了紫杉二烯。这表明即使微生物不含甲羟戊酸途径亦可利用外源导入的甲羟戊酸途径来用于合成紫杉二烯等萜类化合物,这类微生物可以是大肠杆菌,芽孢杆菌等含有MEP途径的微生物。相对于不含甲羟戊酸途径的微生物,对酵母,丝状真菌和链霉菌等含有內源甲羟戊酸途径的微生物进行甲羟戊酸途径的改造则更为便捷,可通过表达部分或者全部相关基因亦或是全部外源的相关基因以达到改造甲羟戊酸途径的目的,因此本发明所建立的甲羟戊酸途径改造与对牻牛儿基牻牛儿基焦磷酸合酶及紫杉二烯合酶的改造完全可以通过选用合适的表达元件在其他微生物中进行重现。目前还无法利用常用微生物直接生产紫杉醇,而利用微生物生产紫杉二烯这一重要的紫杉醇合成中间产物能为今后微生物生产紫杉醇提供一定的指导。
本发明相对于现有技术具有如下的优点及效果:本发明利用外源的甲羟戊酸途径获得了稳定生产紫杉二烯的大肠杆菌。本发明构建的微生物能稳定产生紫杉二烯的培养物,为后续进一步利用微生物生产紫杉醇奠定了基础,具有广阔的应用前景。
附图说明
图1是甲羟戊酸途径示意图。
图2是紫杉二烯合成途径示意图。
图3是质粒pMH1示意图。
图4是质粒pFZ81示意图。
图5是质粒pFZ131示意图。
图6是质粒pXC02示意图。
图7是T2菌株摇瓶发酵产物GC-MS分析结果图。
图8是T2与T4菌株生产紫杉二烯的GC-MS分析结果比较。
图9是紫杉二烯定量标准曲线;其中,A是紫杉二烯的标准曲线,Y=-31766.7+5576.48*X,R∧2=0.9911,W:Equal;B是内标物的标准曲线,Y=-11808.6+11273.7*X,R∧2=0.9977,W:Equal。
图10是T4菌株紫杉二烯发酵结果。
具体实施方式
下面结合实施例及附图对本发明做进一步详细的描述,但不应理解为对本发明的限制。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
通过从大肠杆菌和酿酒酵母基因组DNA中扩增相应基因构建了用于大肠杆菌表达的甲羟戊酸途径和紫杉二烯合成途径,并实现了重组大肠杆菌T2和T4稳定生产紫杉二烯,相关反应途径见图1和图2。
实施例中所用到的引物如表1所示:
表1引物列表
实施例1甲羟戊酸途径质粒构建
用Qiagen公司的BloodandCellCultureDNAMiniKit纯化获得大肠杆菌BL21(DE3)基因组DNA和酿酒酵母INVSC1基因组DNA。
质粒pMH1含有甲羟戊酸途径前三个基因:来源于大肠杆菌BL21(DE3)的atoB基因(乙酰乙酰辅酶A硫酯酶),来源于酿酒酵母INVSC1的erg13(HMG-CoA合酶,3-羟甲基-戊二酸单酰辅酶A合酶)和thmg1(HMG-CoA还原酶,3-羟甲基-戊二酸单酰辅酶A还原酶,删除了HMG1的跨膜区域)。质粒pFZ81含有甲羟戊酸途径后四个基因:来源于酿酒酵母INVSC1的erg12(甲羟戊酸激酶),erg8(甲羟戊酸-5-磷酸激酶)和mvd1(甲羟戊酸-5-焦磷酸激酶),来源于大肠杆菌BL21(DE3)的idi(异戊烯焦磷酸异构酶)基因。所有基因均通过PCR扩增获得,所用引物见表1。
具体构建方法如下:
(1)质粒pMH1的构建
首先利用simplecloning方法(You,C.,etal.,2012.SimpleCloningviaDirectTransformationofPCRProductDNAMultimertoEscherichiacoliandBacillussubtilis.Appliedandenvironmentalmicrobiology.78,1593-1595.),将pBBR1MCS质粒的复制子替换为来源于pMSD15质粒的p15A复制子。以质粒pBBR1MCS为模板用引物pBBR1MCS1-p15A-f和pBBR1MCS1-p15A-r进行扩增获得pBBR1MCS片段,同时p15A复制子用引物p15A-f和p15A-r扩增(引物序列见表1)获得p15A片段,经PCR产物纯化后用Nanodrop测定DNA浓度,然后将20ngPCR扩增的p15A片段和等摩尔的pBBR1MCS片段混合,经过一轮PCR扩增,扩增条件为:98℃,2min预变性,然后30个PCR循环98℃,20s;60℃,20s;72℃,6min,最后72℃充分延伸10min。随后转化大肠杆菌XL1-blue获得质粒pBBR1MCS/p15A。
用引物pBBR1MCS1-f和pBBR1MCS1-r以pBBR1MCS/p15A为模板扩增pMH1质粒骨架,同时用AtoB-f,AtoB-r;ERG13-f,ERG13-r;tHMG1-f,tHMG1-r为引物扩增相应基因。经PCR产物纯化之后,取50ngpMH1质粒骨架和等摩尔的各基因扩增产物混合,并用去离子水调整体积到5μL,随后加入至15μL的Gibson缓冲液(Gibson,D.G.,2011.EnzymaticassemblyofoverlappingDNAfragments.MethodsEnzymol.498,349-361.)中混匀,50℃反应1h后转化大肠杆菌XL1-blue,挑取克隆,并将阳性克隆测序获得质粒pMH1,该质粒示意图如图3所示,序列(不含骨架载体序列)如SEQIDNO.3所示。该质粒控制基因表达的为中等强度的lac启动子,质粒复制子为p15A复制子。
(2)质粒pFZ81的构建
用引物pBBR1MCS2-f和pBBR1MCS2-r以pBBR1MCS-2为模板扩增pFZ81质粒骨架,同时用ERG12-f,ERG12-r;ERG8-f,ERG8-r;MVD1-f,MVD1-r;Idi-f,Idi-r为引物扩增相应基因。经PCR产物纯化之后,取50ngpFZ81质粒骨架和等摩尔的各基因扩增产物混合,并用去离子水调整体积到5μL,随后加入至15μL的Gibson缓冲液中混匀,50℃反应1h后转化大肠杆菌XL1-blue,挑取克隆,并将阳性克隆测序获得质粒pFZ81,该质粒示意图如图4所示,序列(不含骨架载体序列)如SEQIDNO.4所示。该质粒控制基因表达的为中等强度的lac启动子,质粒复制子为pBBR1MCS复制子。
实施例2紫杉二烯产生代谢途径质粒构建
将来源于Taxuscanadensis经过密码子优化的牻牛儿基牻牛儿基焦磷酸合酶ggpps基因(序列如SEQIDNO.1所示)和来源于Taxusbrevifolia经过密码子优化的紫杉二烯合酶ts基因进行基因合成(序列如SEQIDNO.2所示),随后分别用引物GS-duet-f,GS-duet-r和TS-duet-f,TS-duet-r扩增,同时载体pETduet-1用duet-TG-f,duet-TG-r进行扩增,在用Gibson方法将三个片段连接到一起,获得质粒pFZ131(图5)。质粒pFZ131的复制子经过simplecloning方法替换为pSC01复制子,具体操作如下:质粒pFZ131用引物duet-pSC101-f,duet-pSC101-r扩增,用pSC101-f,pSC101-r引物以pSC101质粒为模板扩增pSC101复制子,再将两个片段连起来获得质粒pXC02,该质粒所用引物见表1,质粒示意图如图6所示,序列(不含骨架载体序列)如SEQIDNO.5所示。
实施例3紫杉二烯生产菌株的构建
将甲羟戊酸途径的两个质粒pMH1和pFZ81同时转入大肠杆菌BL21(DE3)中获得BL21(DE3)/pMH1/pFZ81,随后将pXC02转化进入BL21(DE3)/pMH1/pFZ81中,获得紫杉二烯生产菌株T2。将pFZ131转化进入BL21(DE3)/pMH1/pFZ81中,获得紫杉二烯生产菌株T4。
实施例4T2与T4菌株摇瓶发酵生产紫杉二烯
挑取十个T2或者T4菌株的单菌落到含2%(V/V)甘油的LB培养基中(同时含有100μg/mL氨苄青霉素、50μg/mL卡那霉素和34μg/mL氯霉素),于30℃,220rpm过夜培养,随后按1%(V/V)接种量接种到新鲜的同一培养基中30℃,220rpm继续培养至OD600约为0.6~0.8时,加入终浓度为0.1mM的IPTG进行诱导表达,加入诱导剂3小时后,加入1/5培养基体积的正癸烷进行双相发酵。诱导表达后根据设定的时间点进行取样,每次取样为正癸烷相1mL,培养基5mL,正癸烷相8000g离心3min后收集上清进行GC-MS分析。
GC-MS分析条件为:
GC-MS为安捷伦7890A气相色谱配备5975MS,气相色谱柱为HP-5柱(30m*0.32mm*25μm),每次分析进样1μL,GC条件为50℃维持1分钟,10℃/分钟的速率升温到220℃,再在220℃维持10分钟。结果如图7所示,与Ajikumar等人报道GC-MS谱图对比(Ajikumar,P.K.,etal.,2010.IsoprenoidpathwayoptimizationforTaxolprecursoroverproductioninEscherichiacoli.Science.330,70-74.),表明T2和T4菌株能产生紫杉二烯。并且T4生产紫杉二烯的能力明显高于T2菌株(图8)
结论:本发明中构建的T2和T4菌株为一种可产生异戊二烯焦磷酸(IPP)和二甲烯丙基焦磷酸(DMAPP)的微生物,并能够将IPP,DMAPP转化为牻牛儿基牻牛儿基焦磷酸(geranylgeranyldiphosphate),再继续将牻牛儿基牻牛儿基焦磷酸转化为紫杉二烯(taxadiene)的微生物。pFZ131与pXC02相比,采用更高拷贝的pBBR322复制子,其产量高于低拷贝表达的pXC02,这表明为了得到较高紫杉二烯产量需要增加GGPPS和TS的表达量。
实施例5紫杉二烯的GC-MS定量方法
首先将紫杉二烯溶于正己烷中,配成1g/L的溶液,向正己烷中加入内标物β-石竹烯(β-caryophyllene)使内标物的浓度也为1g/L,再用正己烷进行逐级稀释,配成一系列浓度分别为500ppm、200ppm、100ppm、10ppm的溶液。将溶液转入有内置管的样品瓶中,进行进行GC-MS(气相色谱-质谱联用仪)检测。使用柱子为HP-5MS,离子源为CI源,氦气流速1mL/min,进样量1μL,程序升温程序为:100℃1min,以10℃/min升温至220℃保持8min。以浓度为横坐标,峰面积为纵坐标,制作紫杉二烯(图9A)和内标物(图9B)的标准曲线,根据同浓度时峰面积之比计算出紫杉二烯对于内标物的相对响应因子Rf。最后使用内标法对T4菌株发酵产物中的紫杉二烯进行定量,获得的最高产量为1.84mg/L(图10)。
计算得相对响应因子公式为Rf=(A1/C1)/(AIS/CIS)=0.4776
其中A1:紫杉二烯峰面积;AIS:内标物峰面积;C1:紫杉二烯浓度;CIS:内标物浓度。
待测样品中紫杉二烯含量的计算公式:C=(A*CIS)/(AIS*Rf)
其中A:待测样品紫杉二烯峰面积;AIS:内标物峰面积;C:待测样品中紫杉二烯含量;CIS:内标物浓度。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
SEQUENCELISTING
<110>武汉大学
<120>一种生产紫杉二烯的系统和微生物及其应用
<130>1
<160>37
<170>PatentInversion3.5
<210>1
<211>919
<212>DNA
<213>ArtificialSequence
<220>
<223>经过密码子优化的GGPPS序列
<400>1
gtcgacagaaggagatatacatatgtttgatttcaatgaatatatgaaaagtaaggctgt60
tgcggtagacgcggctctggataaagcgattccgctggaatatcccgagaagattcacga120
atcgatgcgctactccctgttagcaggagggaaacgcgttcgtccggcattatgcatcgc180
ggcctgtgaactcgtcggcggttcacaggacttagcaatgccaactgcttgcgcaatgga240
aatgattcacacaatgagcctgattcatgatgatttgccttgcatggacaacgatgactt300
tcggcgcggtaaacctactaatcataaggtttttggcgaagatactgcagtgctggcggg360
cgatgcgctgctgtcgtttgccttcgaacatatcgccgtcgcgacctcgaaaaccgtccc420
gtcggaccgtacgcttcgcgtgatttccgagctgggaaagaccatcggctctcaaggact480
cgtgggtggtcaggtagttgatatcacgtctgagggtgacgcgaacgtggacctgaaaac540
cctggagtggatccatattcacaaaacggccgtgctgctggaatgtagcgtggtgtcagg600
ggggatcttggggggcgccacggaggatgaaatcgcgcgtattcgtcgttatgcccgctg660
tgttggactgttatttcaggtggtggatgacatcctggatgtcacaaaatccagcgaaga720
gcttggcaagaccgcgggcaaagaccttctgacggataaggctacatacccgaaattgat780
gggcttggagaaagccaaggagttcgcagctgaacttgccacgcgggcgaaggaagaact840
ctcttctttcgatcaaatcaaagccgcgccactgctgggcctcgccgattacattgcgtt900
tcgtcagaactgagcatgc919
<210>2
<211>2421
<212>DNA
<213>ArtificialSequence
<220>
<223>经过密码子优化的TS序列
<400>2
gagctcatgtctagctctacgggtacgtctaaagtcgtgagtgaaacctcatcgacgatc60
gtggacgatattccacgcttgtcggcgaactatcatggagatctgtggcatcataacgtc120
attcagacattggaaaccccgtttcgcgaaagtagcacctaccaggaacgggcagatgaa180
ttagtcgtgaaaatcaaagatatgtttaatgcattaggagatggagacatctcgcccagc240
gcatatgatacggcgtgggtggctcggttggccacgattagctccgatggcagtgaaaag300
ccgcgtttcccgcaggcgctgaactgggtgtttaataatcaattgcaggatggcagctgg360
ggcattgaatctcactttagcctctgtgaccggttactcaacacgacaaactccgtaatt420
gcgttgtcagtttggaaaacgggccatagccaggttcaacagggcgcggaatttatcgct480
gaaaatctgcgcctgctgaacgaggaggacgaactgtcacccgattttcagattattttt540
ccggctttactccagaaagccaaagccttaggcatcaacctgccatatgatctgccgttc600
atcaagtatctgtctactacccgcgaagcccgtctcactgacgtctctgcggcggcggac660
aatattccagcgaacatgctgaacgcactggaagggctggaagaggttatcgactggaat720
aaaatcatgcgcttccaaagcaaggacggtagcttcttaagcagcccagcatctactgct780
tgtgttctgatgaataccggagacgaaaagtgctttacgtttctgaacaatctgctggac840
aaatttgggggttgtgttccttgtatgtattccattgatctgttggaacgtctgtcgctg900
gtcgataacattgaacacttaggtatcggccgccacttcaaacaagaaatcaagggggcg960
ttggattatgtataccgtcattggagcgagcgtggtattggttgggggcgcgatagcttg1020
gtacctgatctgaacaccactgctttgggactgcgcactcttcgtatgcacggatacaac1080
gttagttccgatgtcctcaataatttcaaggacgagaacggccgttttttcagctcggcc1140
ggtcagacgcatgttgaactgcggtccgtagtcaatctctttcgcgctagtgatctggcc1200
ttccccgacgagcgcgctatggacgatgcacggaagtttgccgagccgtatctccgcgaa1260
gccctggccaccaaaatttcaaccaacaccaagcttttcaaagaaattgagtatgtagta1320
gagtatccgtggcatatgtctattccgcgcctggaagcccgctcgtatatcgattcttac1380
gatgacaattatgtgtggcaacgcaaaacactgtaccgtatgcccagcctgtcaaatagt1440
aagtgtctggagctggcgaaactggatttcaacattgtgcaatccctgcaccaagaagag1500
ctgaaattactgactcgctggtggaaggaatccggcatggcagacatcaattttacgcgt1560
caccgtgttgcagaggtgtacttctcctcggcgacctttgagccggagtattcggccaca1620
cgtattgcatttaccaagattggctgccttcaggtgctttttgacgatatggcggatatt1680
tttgcgacacttgatgagcttaaatcatttaccgaaggcgtgaagcgttgggatacctct1740
ctgttgcatgaaatccccgaatgtatgcagacctgcttcaaagtttggttcaaactgatg1800
gaagaagtgaacaacgacgtcgtgaaagttcagggtcgtgatatgttagcacacatccgc1860
aagccgtgggaactctatttcaattgctatgtgcaggagcgtgaatggttagaagcgggc1920
tacattcctaccttcgaagagtacttaaaaacctatgccatttccgtcggtttaggcccg1980
tgcactctgcagcctatcttgctgatgggtgagctggtaaaggatgatgtggtggaaaaa2040
gttcactacccgtcgaatatgtttgaactggtaagtctgagttggcgtctgacaaacgac2100
accaaaacgtaccaggcagaaaaggcacgtgggcaacaggcaagcggtatcgcgtgttat2160
atgaaggataatccgggcgctactgaggaagatgccattaagcatatctgccgtgttgtg2220
gatcgcgctcttaaagaagcgtcattcgaatattttaaacctagtaatgatattccgatg2280
ggttgtaagtcattcattttcaatcttcgcctgtgcgtgcaaattttttacaaatttatt2340
gacggctacggaatcgccaacgaagaaatcaaagactatattcgtaaagtttacatcgat2400
ccaatccaggtctaagtcgac2421
<210>3
<211>4515
<212>DNA
<213>ArtificialSequence
<220>
<223>pMH1序列(不含骨架载体序列)
<400>3
gcgcgtaatacgactcactatagggcgaattggagctccaccgcggtggcggccgctcta60
gaactagtggatccttaggatttaatgcaggtgacggacccatctttcaaacgatttata120
tcagtggcgtccaaattgttaggttttgttggttcagcaggtttcctgttgtgggtcata180
tgactttgaaccaaatggccggctgctagggcagcacataaggataattcacctgccaag240
acggcacaggcaactattcttgctaattgacgtgcgttggtaccaggagcggtagcatgc300
gggcctcttacacctaataagtccaacatggcaccttgtggttctagaacagtaccacca360
ccgatggtacctacttcgatggatggcatggatacggaaattctcaaatcaccgtccact420
tctttcatcaatgttatacagttggaactttcaacattttgtgcaggatcttgtcctaat480
gccaagaaaacagctgtcactaaattagctgcatgtgcgttaaatccaccaacagaccca540
gccattgcagatccaaccaaattcttagcaatgttcaactcaaccaatgcggaaacatca600
ctttttaacacttttctgacaacatcaccaggaatagtagcttctgcgacgacactctta660
ccacgaccttcgatccagttgatggcagctggtttttttgtcggtacagtagttaccaga720
aacggagacaacctccatatcttcccagccatactcttctaccatttgctttaatgagta780
ttcgacacctttagaaatcatattcatacccattgcgtcaccagtagttgttctaaatct840
catgaagagtaaatctcctgctagacaagtttgaatatgttgcagacgtgcaaatcttga900
tgtagagttaaaagcttttttaattgcgttttgtccctcttctgagtctaaccatatctt960
acaggcaccagatcttttcaaagttgggaaacggactactgggcctcttgtcataccatc1020
cttagttaaaacagttgttgcaccaccgccagcattgattgccttacagccacgcatggc1080
agaagctaccaaacaaccctctgtagttgccattggtatatgataagatgtaccatcgat1140
aaccaaggggcctataacaccaacgggcaaaggcatgtaacctataacattttcacaaca1200
agcgccaaatacgcggtcgtagtcataatttttatatggtaaacgatcagatgctaatac1260
aggagcttctgccaaaattgaaagagccttcctacgtaccgcaaccgctctcgtagtatc1320
acctaattttttctccaaagcgtacaaaggtaacttaccgtgaataaccaaggcagcgac1380
ctctttgttcttcaattgttttgtatttccactacttaataatgcttctaattcttctaa1440
aggacgtattttcttatccaagctttcaatatcgcgggaatcatcttcctcactagatga1500
tgaaggtcctgatgagctcgattgcgcagatgataaacttttgactttcgatccagaaat1560
gactgttttattggttaaaacgaattcggatccgcgacccatttgctgtccaccagtcat1620
gctagccatatggctgccgcgcggcaccaggccgctgctgtgatgatgatgatgatggct1680
gctgcccatagtgtaatcctccttattttttaacatcgtaagatcttctaaatttgtcat1740
cgatgttggtcaagtagtaaacaccactttgcaaatgctcaatggaaccttgaggtttga1800
agttcttcttcaaatgggcattttctctcaattcgatggcagcttcgtaatcctttggag1860
tttcggtgattctcttggctaatttgttagtaatatctaattccttgataatatgttgga1920
cgtcaccaacaattttgcaagaatatagagatgcagctaaaccggaaccgtaagaaaata1980
aaccaacacgcttgccttgtaagtcgtcagatccaacatagtttaatagagatgcaaagg2040
cggcataaacagatgcggtgtacatgttacctgtgtttgttggaacaatcaaagattggg2100
caactctctctttgtggaatggcttagcaacattaacaaaagttttttcaatgttcttat2160
cggttaaagattcgtcataatcgcgagtagctaattcggcgtcaacttctgggaacaatt2220
gaggattggctctgaaatcgttatatagtaatctaccgtatgattttgtgaccaatttac2280
aggttggaacatggaaaacgttgtagtcgaaatatttcaaaacgttcaaagcatccgaac2340
cagcgggatcgctaaccaaccctttagaaatagccttcttggaataactcttgtaaactt2400
gatcaagagccttgacgtaacaagttaatgaaaaatgaccatcgacgtaaggatattcgc2460
tggtgaaatctggcttgtaaaaatcgtaggcgtgttccatgtaagaagctcttacagagt2520
caaatacaattggagcatcaggaccgatccacatagcaacagtaccggcaccaccggttg2580
gtcttgcggcacccttatcgtagatggcaatatcaccgcaaactacaatggcgtctctac2640
catcccatgcgttagattcaatccagttcaaagagttgaacaacgcgttggtaccaccgt2700
aacaggcattaagcgtgtcaataccttcgacgtcagtgttttcaccaaacaattgcatca2760
agacagacttgacagacttggacttgtcaatcagagtttcagtaccgacttctaatctac2820
caattttgttggtgtcgatgttgtaactcttgatcaacttagacaaaacagttagggaca2880
tcgagtagatatcttctctgtcattgacaaaagacatgttggtttggcccagaccaattg2940
tgtatttaccttgagaaacgccatcaaatttctctagctcagattggttgacacattgag3000
ttgggatgtaaatttggatacctttaataccgacattttgaggtctggttttttgttcag3060
cggtcttttgtttttttagttcagtcatttgcaagtttgtattgtgtaattgttgttgct3120
tttgcggcctaagtcttcctttaataccacaccaacaaagtttagttgagagtttcattt3180
agctgtcctccttaattcaaccgttcaatcaccatcgcaattccctgaccgccgccaatg3240
cacagtgttgccagccccagcgttttatcgcgtgcctgcatggcatgtaatagtgtgacc3300
agaatacgagcaccactggcaccgataggatgcccgagcgcgatggccccgccgttgaca3360
ttcactttctcagaatcaaagcccaggtttttcccaacggcaaggaactgtgcagcaaat3420
gcttcattagcctcaatgagatcaatatccgccagttgcagccccgccagttgtaacgct3480
ttttgcgtggcaggtactggccccatacccatcaatgcggggggcacgccaccgctggca3540
taacttttaatgcgagccaggggggtaaggcctgctgccagcgccgcagattcttccata3600
atcaccagagcggcagcaccgtcgttaataccagacgcgttcccagcggtgactgttcct3660
gctttatcgaaggccgggcgcaatgcacctaacgcttcagccgttgaattcgctttcggg3720
aattcgtcttgactgaagacgaaggttttctttcgagtgacaacatttaccgggacgatt3780
tcggctgtaaaagcaccggactcaattgcggctgccgctttacgctgtgaatgtagcgcc3840
agttcatcctgcatttcacgggtaattccgtactctttagccacgttttcggcggtaatc3900
cccatatgataaccatgggtggcgcacatcaggccatcgcgcaggattacgtcataaacc3960
tgtccgtctccaagacgataaccagagcgtgcttttgcatcgagtaagtagggggctaaa4020
ctcatattttccataccccccgccacaatgctctgcgcctgacctgcctgaatggcctgg4080
gcggcaagcgccacacttttaagacccgaaccacatactttattgaccgtgaatccgcac4140
accgtttctgccagcccgctttttaacagtgcctgacgcgccggattttgccccagcccg4200
gcttgtaacacgttacccataatcacttcatcaacgtgttgtgaatcgatttttgcacgt4260
tcaatggcggctttaattactgtcgcccccaggtcgatggcgctggtggaagcgagtgaa4320
ccgttaaaactaccgatagcagtacgtaccgcactgacgatgacacaatttttcatttta4380
tattcctcctagtcgactctagaggatccccgggctgcaggaattcgatatcaagcttat4440
cgataccgtcgacctcgagggggggcccggtacccagcttttgttccctttagtgagggt4500
taattgcgcgctggg4515
<210>4
<211>4667
<212>DNA
<213>ArtificialSequence
<220>
<223>pFZ81序列(不含骨架载体序列)
<400>4
cgcgcgtaatacgactcactatagggcgaattggagctcttatttaagctgggtaaatgc60
agataatcgttttctggcttcgcgatttgtcgcctgcatcaccatccacggactgaacgc120
ccacggcgtggcatcaataccgtgtaatacatctgctaaatcacaccattgataatccat180
cacttcatcatcattgatctgtaacgcactagtggtgcgtgcggcaaataccggacacac240
ttcattttccacaatgccactcggatcggtggcgcggtagcgaaagtcaggatagataga300
ttcaggaggcgtaatttccacgccaagctcataacggcaacggcggatcactgcgtcttc360
gttgctttctcccagttgtgggtgcccacaaaccgagttagtccacacgccaggccatgc420
ttttttgctcagtgcgcggcgggtaactaataattgtcctttggcattaaacagccaact480
ggagaacgcgagatgtaagcgggtgtctgccgtgtgtgcggcatacttttccagcgtacc540
cgtgggaactccctgtgcattcaataaaatgacgtgttccgtttgcatatggctgccgcg600
cggcaccaggccgctgctgtgatgatgatgatgatggctgctgcccatatagtaatcctc660
ctcccgggctgcagttattcctttggtagaccagtctttgcgtcaatcaaagattcgttt720
gtttcttgtgggcctgaaccgacttgagttaaaatcactctggcaacatccttttgcaac780
tcaagatccaattcacgtgcagtaaagttagatgattcaaattgatggttgaaagcctca840
agctgctcagtagtaaatttcttgtcccatccaggaacagagccaaacaatttatagata900
aatgcaaagagtttcgactcattttcagctaagtagtacaacacagcatttggacctgca960
tcaaacgtgtatgcaacgattgtttctccgtaaaactgattaatggtgtggcaccaactg1020
atgatacgcttggaagtgtcattcatgtagaatattggagggaaagagtccaaacatgtg1080
gcatggaaagagttggaatccatcattgtttcctttgcaaaggtggcgaaatctttttca1140
acaatggctttacgcatgacttcaaatctctttggtacgacatgttcaattctttcttta1200
aatagttcggaggttgccacggtcaattgcataccctgagtggaactcacatccttttta1260
atatcgctgacaactaggacacaagctttcatctgaggccagtcagagctgtctgcgatt1320
tgtactgccatggaatcatgaccatcttcagcttttcccatttcccaggccacgtatccg1380
ccaaacaacgatctacaagctgaaccagacccctttcttgctattctagatatttctgaa1440
gttgactgtggtaattggtataacttagcaattgcagagaccaatgcagcaaagccagca1500
gcggaggaagctaaaccagctgctgtaggaaagttattttcggagacaatgtggagtttc1560
cattgagataatgtgggcaatgaggcgtccttcgattccatttcctttcttaattggcgt1620
aggtcgcgcagacaattttgagttctttcattgtcgatgctgtgtggttctccatttaac1680
cacaaagtgtcgcgttcaaactcaggtgcagtagccgcagaggtcaacgttctgaggtca1740
tcttgcgataaagtcactgatatggacgaattggtgggcagattcaacttcgtgtccctt1800
ttcccccaatacttaagggttgcgatgttgacgggtgcggtaacggatgctgtgtaaacg1860
gtcatgagtattacctcctatttatcaagataagtttccggatctttttctttcctaaca1920
ccccagtcagcctgagttacatccagccattgaaccttagaaaatcttttgtcattagcg1980
gtttgagccctaagatcaacatcttgcttagtaatcactgcaatggcgtcataaccacca2040
gcaccaggtattaagcaagtaagaactccttttaaggtctggcaatcatccaataagcta2100
gtttgtacgggaggttcgatatcggcaccagattctttagttatttttctaaaggaacgt2160
ctaattgtggcaactgcatctctaacttctgtgatttcaggatacttttgacaggtacag2220
tcattcctctcaagagactcaaatatctgatcgctgtaatcgtcatgagtctcgtgtaag2280
cgatctagtttagatagtccatccataaatctagaatttgcatgatcgagttctgtatat2340
attttcaagctttctggcatatgcgaatcataccaattttttaccttctggaccagtttt2400
actgtttctgaaccattcttaatatcgcccatccataaagttaatcccgaaggtaaatgg2460
ttacttttaatcgtaatattccagtcttcttcatcaaccaaatgcgccagtttactgccg2520
taagtagcacttccaatatctggcaaattagagattaatgcgggtgggaatcttctatat2580
ctgatagatccatatgctgccgccgctacatcaaacccgcttccaattttaccctgagct2640
tgacaatgagcaacttgtgctaaattatgaataacttctctatatttgtctacattattt2700
tccaggtccgatacaaaaaaggaggccaaagctgtagttaaaactgtgactaaacctgcc2760
gaggagcccagccctgttttgggaacttcttcaattctgtgcgaatgaaaactcaatctt2820
ctgttgccacgatgttcggtaacgctatcctcctgagaatggtaggcatcatcagagaaa2880
atatcaataacgaacaagtttctattgcagtagtcgtccatgttaggtttaaagtagcta2940
aatacgttagcgataactttttcaatgaaagggttcttagatccgcctatcgaaacagga3000
atgaagccacttttaggacttatatggtacagccactccccatctttaaattgtttactt3060
ttcacacgcacttcaaacttatcagacccttgcaatgaaccgtaaggatgggctacagca3120
tgcattcttgccgataatccgactacaaatgcttcatattttgtatctaaaactaaatat3180
ccaccagctagtaacgctttccctggggcactgaaggctctcaactctgacatttgatct3240
gcctcctatgaagtccatggtaaattcgtgtttcctggcaataatagatcgtcaatttgt3300
tgctttgtggtagttttattttcaaataattggaatactagggatttgattttaggatct3360
ttattcaaattttttgcgcttaacaaacagcagccagtcccacccaagtctgtttcaaat3420
gtctcgtaactaaaatcatcttgcaatttctttttgaaactgtcaatttgctcttgagta3480
atgtctcttcgtaacaaagtcaaagagcaaccgccgccaccagcaccggtaagttttgtg3540
gagccaattctcaaatcatcgctcagatttttaataagttctaatccaggatgagaaaca3600
ccgattgagacaagcagtccatgatttattcttatcaattccaatagttgttcatacagt3660
tcattattagtttctacggcctcgtcatcggtgcctttacatttacttaactttgtcatg3720
atctctaagccttgtagggcacattcacccatggcatctagaattggcttcataacttca3780
ggaaatttctcggtgaccaacacacgaacgcgagcaacaagatcttttgtagaccttgga3840
attctagtataggttaggatcattggaatggctgggaaatcatctaagaacttaaaattg3900
tttgtatttattgttccattatgtgagtctttttcaaatagcagggcattaccataagtg3960
gccacagcgttatctattcctgaaggggtaccgtgaatacacttttcacctatgaaggcc4020
cattgattcactatatgcttatcgttttctgacagcttttccaagtcattagatcctatt4080
aacccccccaagtaggccatagctaaggccagtgatacagaaatagaggcgcttgagccc4140
aacccagcaccgatgggtaaagtagactttaaagaaaacttaatattcttggcatggggg4200
cataggcaaacaaacatatacaggaaacaaaacgctgcatggtagtggaaggattcggat4260
agttgagctaacaacggatccaaaagactaacgagttcctgagacaagccatcggtggct4320
tgttgagccttggccaatttttgggagtttacttgatcctcggtgatggcattgaaatca4380
ttgatggaccacttatgattaaagctaatgtccgggaagtccaattcaatagtatctggt4440
gcagatgactcgcttattagcaggtaggttctcaacgcagacacactagcagcgacggca4500
ggcttgttgtacacagcagagtgttcaccaaaaataataacctttcccggtgcagaagtt4560
aagaacggtaatgacatggttaattcctcctactgcaggaattcgatatcaagcttatcg4620
ataccgtcgacctcgagggggggcccggtacccagcttttgttcccc4667
<210>5
<211>3315
<212>DNA
<213>ArtificialSequence
<220>
<223>pXC02序列(不含骨架载体序列)
<400>5
atgtctagctctacgggtacgtctaaagtcgtgagtgaaacctcatcgacgatcgtggac60
gatattccacgcttgtcggcgaactatcatggagatctgtggcatcataacgtcattcag120
acattggaaaccccgtttcgcgaaagtagcacctaccaggaacgggcagatgaattagtc180
gtgaaaatcaaagatatgtttaatgcattaggagatggagacatctcgcccagcgcatat240
gatacggcgtgggtggctcggttggccacgattagctccgatggcagtgaaaagccgcgt300
ttcccgcaggcgctgaactgggtgtttaataatcaattgcaggatggcagctggggcatt360
gaatctcactttagcctctgtgaccggttactcaacacgacaaactccgtaattgcgttg420
tcagtttggaaaacgggccatagccaggttcaacagggcgcggaatttatcgctgaaaat480
ctgcgcctgctgaacgaggaggacgaactgtcacccgattttcagattatttttccggct540
ttactccagaaagccaaagccttaggcatcaacctgccatatgatctgccgttcatcaag600
tatctgtctactacccgcgaagcccgtctcactgacgtctctgcggcggcggacaatatt660
ccagcgaacatgctgaacgcactggaagggctggaagaggttatcgactggaataaaatc720
atgcgcttccaaagcaaggacggtagcttcttaagcagcccagcatctactgcttgtgtt780
ctgatgaataccggagacgaaaagtgctttacgtttctgaacaatctgctggacaaattt840
gggggttgtgttccttgtatgtattccattgatctgttggaacgtctgtcgctggtcgat900
aacattgaacacttaggtatcggccgccacttcaaacaagaaatcaagggggcgttggat960
tatgtataccgtcattggagcgagcgtggtattggttgggggcgcgatagcttggtacct1020
gatctgaacaccactgctttgggactgcgcactcttcgtatgcacggatacaacgttagt1080
tccgatgtcctcaataatttcaaggacgagaacggccgttttttcagctcggccggtcag1140
acgcatgttgaactgcggtccgtagtcaatctctttcgcgctagtgatctggccttcccc1200
gacgagcgcgctatggacgatgcacggaagtttgccgagccgtatctccgcgaagccctg1260
gccaccaaaatttcaaccaacaccaagcttttcaaagaaattgagtatgtagtagagtat1320
ccgtggcatatgtctattccgcgcctggaagcccgctcgtatatcgattcttacgatgac1380
aattatgtgtggcaacgcaaaacactgtaccgtatgcccagcctgtcaaatagtaagtgt1440
ctggagctggcgaaactggatttcaacattgtgcaatccctgcaccaagaagagctgaaa1500
ttactgactcgctggtggaaggaatccggcatggcagacatcaattttacgcgtcaccgt1560
gttgcagaggtgtacttctcctcggcgacctttgagccggagtattcggccacacgtatt1620
gcatttaccaagattggctgccttcaggtgctttttgacgatatggcggatatttttgcg1680
acacttgatgagcttaaatcatttaccgaaggcgtgaagcgttgggatacctctctgttg1740
catgaaatccccgaatgtatgcagacctgcttcaaagtttggttcaaactgatggaagaa1800
gtgaacaacgacgtcgtgaaagttcagggtcgtgatatgttagcacacatccgcaagccg1860
tgggaactctatttcaattgctatgtgcaggagcgtgaatggttagaagcgggctacatt1920
cctaccttcgaagagtacttaaaaacctatgccatttccgtcggtttaggcccgtgcact1980
ctgcagcctatcttgctgatgggtgagctggtaaaggatgatgtggtggaaaaagttcac2040
tacccgtcgaatatgtttgaactggtaagtctgagttggcgtctgacaaacgacaccaaa2100
acgtaccaggcagaaaaggcacgtgggcaacaggcaagcggtatcgcgtgttatatgaag2160
gataatccgggcgctactgaggaagatgccattaagcatatctgccgtgttgtggatcgc2220
gctcttaaagaagcgtcattcgaatattttaaacctagtaatgatattccgatgggttgt2280
aagtcattcattttcaatcttcgcctgtgcgtgcaaattttttacaaatttattgacggc2340
tacggaatcgccaacgaagaaatcaaagactatattcgtaaagtttacatcgatccaatc2400
caggtctaagaaggagatatacatatgtttgatttcaatgaatatatgaaaagtaaggct2460
gttgcggtagacgcggctctggataaagcgattccgctggaatatcccgagaagattcac2520
gaatcgatgcgctactccctgttagcaggagggaaacgcgttcgtccggcattatgcatc2580
gcggcctgtgaactcgtcggcggttcacaggacttagcaatgccaactgcttgcgcaatg2640
gaaatgattcacacaatgagcctgattcatgatgatttgccttgcatggacaacgatgac2700
tttcggcgcggtaaacctactaatcataaggtttttggcgaagatactgcagtgctggcg2760
ggcgatgcgctgctgtcgtttgccttcgaacatatcgccgtcgcgacctcgaaaaccgtc2820
ccgtcggaccgtacgcttcgcgtgatttccgagctgggaaagaccatcggctctcaagga2880
ctcgtgggtggtcaggtagttgatatcacgtctgagggtgacgcgaacgtggacctgaaa2940
accctggagtggatccatattcacaaaacggccgtgctgctggaatgtagcgtggtgtca3000
ggggggatcttggggggcgccacggaggatgaaatcgcgcgtattcgtcgttatgcccgc3060
tgtgttggactgttatttcaggtggtggatgacatcctggatgtcacaaaatccagcgaa3120
gagcttggcaagaccgcgggcaaagaccttctgacggataaggctacatacccgaaattg3180
atgggcttggagaaagccaaggagttcgcagctgaacttgccacgcgggcgaaggaagaa3240
ctctcttctttcgatcaaatcaaagccgcgccactgctgggcctcgccgattacattgcg3300
tttcgtcagaactga3315
<210>6
<211>50
<212>DNA
<213>ArtificialSequence
<220>
<223>P15A-f
<400>6
gcattgccgccgtgggtttctcgagcggggcggagatttcctggaagatg50
<210>7
<211>52
<212>DNA
<213>ArtificialSequence
<220>
<223>P15A-r
<400>7
ggggccactttttgccggagctcgagaaatattttatctgattaataagatg52
<210>8
<211>52
<212>DNA
<213>ArtificialSequence
<220>
<223>pBBR1MCS1-p15A-f
<400>8
catcttattaatcagataaaatatttctcgagctccggcaaaaagtggcccc52
<210>9
<211>50
<212>DNA
<213>ArtificialSequence
<220>
<223>pBBR1MCS1-p15A-r
<400>9
catcttccaggaaatctccgccccgctcgagaaacccacggcggcaatgc50
<210>10
<211>55
<212>DNA
<213>ArtificialSequence
<220>
<223>pBBR1MCS1-f
<400>10
tttgaaagatgggtccgtcacctgcattaaatcctaaggatccactagttctaga55
<210>11
<211>59
<212>DNA
<213>ArtificialSequence
<220>
<223>pBBR1MCS1-r
<400>11
ttttatattcctcctagtcgactctagaggatccccgggctgcaggaattcgatatcaa59
<210>12
<211>59
<212>DNA
<213>ArtificialSequence
<220>
<223>atoB-f
<400>12
cccggggatcctctagagtcgactaggaggaatataaaatgaaaaattgtgtcatcgtc59
<210>13
<211>49
<212>DNA
<213>ArtificialSequence
<220>
<223>atoB-r
<400>13
gttgagagtttcatttagctgtcctccttaattcaaccgttcaatcacc49
<210>14
<211>46
<212>DNA
<213>ArtificialSequence
<220>
<223>ERG13-f
<400>14
acggttgaattaaggaggacagctaaatgaaactctcaactaaact46
<210>15
<211>49
<212>DNA
<213>ArtificialSequence
<220>
<223>ERG13-r
<400>15
tggctgctgcccatagtgtaatcctccttattttttaacatcgtaagat49
<210>16
<211>47
<212>DNA
<213>ArtificialSequence
<220>
<223>tHMG1-f
<400>16
atgttaaaaaataaggaggattacactatgggcagcagccatcatca47
<210>17
<211>23
<212>DNA
<213>ArtificialSequence
<220>
<223>tHMG1-r
<400>17
ttaggatttaatgcaggtgacgg23
<210>18
<211>59
<212>DNA
<213>ArtificialSequence
<220>
<223>pBBR1MCS2-f
<400>18
cagaaaacgattatctgcatttacccagcttaaataagagctccaattcgccctatagt59
<210>19
<211>55
<212>DNA
<213>ArtificialSequence
<220>
<223>pBBR1MCS2-r
<400>19
ttaagaacggtaatgacatggttaattcctcctactgcaggaattcgatatcaag55
<210>20
<211>42
<212>DNA
<213>ArtificialSequence
<220>
<223>ERG12-f
<400>20
ctgcagtaggaggaattaaccatgtcattaccgttcttaact42
<210>21
<211>44
<212>DNA
<213>ArtificialSequence
<220>
<223>ERG12-r
<400>21
ctcaactctgacatttgatctgcctcctatgaagtccatggtaa44
<210>22
<211>51
<212>DNA
<213>ArtificialSequence
<220>
<223>ERG8-f
<400>22
ttaccatggacttcataggaggcagatcaaatgtcagagttgagagccttc51
<210>23
<211>53
<212>DNA
<213>ArtificialSequence
<220>
<223>ERG8-r
<400>23
gatgctgtgtaaacggtcatgagtattacctcctatttatcaagataagtttc53
<210>24
<211>50
<212>DNA
<213>ArtificialSequence
<220>
<223>MVD1-f
<400>24
atcttgataaataggaggtaatactcatgaccgtttacacagcatccgtt50
<210>25
<211>55
<212>DNA
<213>ArtificialSequence
<220>
<223>MVD1-r
<400>25
tgcccatatagtaatcctcctcccgggctgcagttattcctttggtagaccagtc55
<210>26
<211>51
<212>DNA
<213>ArtificialSequence
<220>
<223>Idi-f
<400>26
ggaataactgcagcccgggaggaggattactatatgggcagcagccatcat51
<210>27
<211>24
<212>DNA
<213>ArtificialSequence
<220>
<223>Idi-r
<400>27
ttatttaagctgggtaaatgcaga24
<210>28
<211>40
<212>DNA
<213>ArtificialSequence
<220>
<223>TS-duet-f
<400>28
ctttaagaaggagatataccatgtctagctctacgggtac40
<210>29
<211>41
<212>DNA
<213>ArtificialSequence
<220>
<223>TS-duet-r
<400>29
acatatgtatatctccttctttagacctggattggatcgat41
<210>30
<211>41
<212>DNA
<213>ArtificialSequence
<220>
<223>GS-duet-f
<400>30
atcgatccaatccaggtctaaagaaggagatatacatatgt41
<210>31
<211>39
<212>DNA
<213>ArtificialSequence
<220>
<223>GS-duet-r
<400>31
cggccgcaagcttgtcgactcagttctgacgaaacgcaa39
<210>32
<211>39
<212>DNA
<213>ArtificialSequence
<220>
<223>duet-TG-f
<400>32
ttgcgtttcgtcagaactgagtcgacaagcttgcggccg39
<210>33
<211>40
<212>DNA
<213>ArtificialSequence
<220>
<223>duet-TG-r
<400>33
gtacccgtagagctagacatggtatatctccttcttaaag40
<210>34
<211>41
<212>DNA
<213>ArtificialSequence
<220>
<223>duet-pSC101-f
<400>34
gtttgcgggcagcaaaacccggcagcagtcgcttcacgttc41
<210>35
<211>41
<212>DNA
<213>ArtificialSequence
<220>
<223>duet-pSC101-r
<400>35
atccgtcagtatcagcataagctacggggtctgacgctcag41
<210>36
<211>41
<212>DNA
<213>ArtificialSequence
<220>
<223>pSC101-f
<400>36
ctgagcgtcagaccccgtagcttatgctgatactgacggat41
<210>37
<211>41
<212>DNA
<213>ArtificialSequence
<220>
<223>pSC101-r
<400>37
gaacgtgaagcgactgctgccgggttttgctgcccgcaaac41
Claims (2)
1.一种生产紫杉二烯的微生物,其特征在于:所述的微生物为含有质粒pMH1、质粒pFZ81和质粒pXC02的大肠杆菌;所述的质粒pMH1启动子为lac启动子,复制子为p15A复制子,包含atoB、erg13和thmg1基因;所述的质粒pFZ81启动子为lac启动子,复制子为pBBR1MCS复制子,包含erg12、erg8、mvd1和idi基因;所述的质粒pXC02启动子为T7强启动子,复制子为pSC101低拷贝复制子,包含ggpps和ts基因;或为含有质粒pMH1、质粒pFZ81和质粒pFZ131的大肠杆菌;所述的质粒pFZ131启动子为T7强启动子,复制子为pBBR322高拷贝复制子,包含ggpps和ts基因,所述的ggpps和ts基因的序列分别如SEQIDNO.1和2所示。
2.权利要求1所述的微生物在生产紫杉二烯中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310595448.8A CN103571835B (zh) | 2013-11-22 | 2013-11-22 | 一种生产紫杉二烯的系统和微生物及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310595448.8A CN103571835B (zh) | 2013-11-22 | 2013-11-22 | 一种生产紫杉二烯的系统和微生物及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103571835A CN103571835A (zh) | 2014-02-12 |
CN103571835B true CN103571835B (zh) | 2016-01-20 |
Family
ID=50044547
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201310595448.8A Active CN103571835B (zh) | 2013-11-22 | 2013-11-22 | 一种生产紫杉二烯的系统和微生物及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103571835B (zh) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105255921B (zh) * | 2014-06-30 | 2019-05-31 | 中国科学院上海生命科学研究院 | 异源合成广霍香醇的方法和微生物 |
CN105802866A (zh) * | 2014-12-30 | 2016-07-27 | 天津大学 | 一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法 |
CN107674890A (zh) * | 2016-08-02 | 2018-02-09 | 武汉臻智生物科技有限公司 | 制备紫杉醇的方法 |
CN107674881A (zh) * | 2016-08-02 | 2018-02-09 | 武汉臻智生物科技有限公司 | 紫杉二烯过表达载体及其应用 |
CN106754896B (zh) * | 2017-03-13 | 2019-09-17 | 中国科学院武汉病毒研究所 | 一种杆状病毒基因组的合成方法及其在重组杆状病毒构建中的应用 |
CN106906201B (zh) * | 2017-04-10 | 2020-03-10 | 武汉大学 | 一种生产橙花叔醇的萜类合酶及其应用 |
CN110904148A (zh) * | 2019-11-19 | 2020-03-24 | 湖北大学 | 一种合成紫杉二烯的植物表达载体、菌株dzggppsts和盾叶薯蓣及其制备方法 |
CN115976075B (zh) * | 2023-01-09 | 2025-01-28 | 深圳大学 | 一种利用莱茵衣藻叶绿体异源合成紫杉二烯的方法及应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101484584A (zh) * | 2006-05-26 | 2009-07-15 | 阿米瑞斯生物技术公司 | 类异戊二烯的生产 |
CN102812129A (zh) * | 2009-11-10 | 2012-12-05 | 麻省理工学院 | 用于从类异戊二烯途径生产化学和医药产品的微生物工程 |
-
2013
- 2013-11-22 CN CN201310595448.8A patent/CN103571835B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101484584A (zh) * | 2006-05-26 | 2009-07-15 | 阿米瑞斯生物技术公司 | 类异戊二烯的生产 |
CN102812129A (zh) * | 2009-11-10 | 2012-12-05 | 麻省理工学院 | 用于从类异戊二烯途径生产化学和医药产品的微生物工程 |
Also Published As
Publication number | Publication date |
---|---|
CN103571835A (zh) | 2014-02-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103571835B (zh) | 一种生产紫杉二烯的系统和微生物及其应用 | |
Shi et al. | Current progress in the study on biosynthesis and regulation of ganoderic acids | |
Shankar Naik | Developments in taxol production through endophytic fungal biotechnology: a review | |
Zhang et al. | Microbial production of sabinene—a new terpene-based precursor of advanced biofuel | |
CA2780415C (en) | Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway | |
Hu et al. | Screening and modification of (+)-germacrene A synthase for the production of the anti-tumor drug (−)-β-elemene in engineered Saccharomyces cerevisiae | |
Yang et al. | Biosynthesis of β-caryophyllene, a novel terpene-based high-density biofuel precursor, using engineered Escherichia coli | |
Luo et al. | Characterization of a sesquiterpene cyclase from the glandular trichomes of Leucosceptrum canum for sole production of cedrol in Escherichia coli and Nicotiana benthamiana | |
CN103695441A (zh) | 一个参与丹参酮生物合成的cyp450基因及其编码产物与应用 | |
Zhang et al. | Molecular cloning and characterization of a cytochrome P450 taxoid 9a-hydroxylase in Ginkgo biloba cells | |
CN111004763A (zh) | 一种生产β-石竹烯的工程菌及其构建方法与应用 | |
CN105255921B (zh) | 异源合成广霍香醇的方法和微生物 | |
Carruthers et al. | Diversifying isoprenoid platforms via atypical carbon substrates and non-model microorganisms | |
Huang et al. | Side products of recombinant amorpha-4, 11-diene synthase and their effect on microbial artemisinin production | |
CN111286482A (zh) | 一种快速产香叶醇的大肠杆菌工程菌及其构建方法和应用 | |
CN106987578B (zh) | 一种生产koraiol的萜类合酶及其应用 | |
CN110317765A (zh) | 一种高产香叶醇葡萄糖苷的大肠杆菌表达菌株及其应用 | |
CN111394290A (zh) | 产长叶烯的基因工程菌及其构建方法与应用 | |
CN104789512A (zh) | 异戊二烯的生产菌以及生产异戊二烯的方法 | |
CN107083378A (zh) | 一种生产Longiborneol的萜类合酶及其应用 | |
CN111607546A (zh) | 一种高产法尼烯的基因工程菌及其构建方法与应用 | |
JP6856553B2 (ja) | マノオールの製造 | |
Wang et al. | Tandem expression of Ganoderma sinense sesquiterpene synthase and IDI promotes the production of gleenol in E. coli | |
US10337031B2 (en) | Production of fragrant compounds | |
JP2023539092A (ja) | 酸素化ジテルペノイド化合物の生成 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant |