JP2017514480A - 高麗人参由来の糖転移酵素を用いた新規なジンセノサイド糖転移方法 - Google Patents
高麗人参由来の糖転移酵素を用いた新規なジンセノサイド糖転移方法 Download PDFInfo
- Publication number
- JP2017514480A JP2017514480A JP2016565151A JP2016565151A JP2017514480A JP 2017514480 A JP2017514480 A JP 2017514480A JP 2016565151 A JP2016565151 A JP 2016565151A JP 2016565151 A JP2016565151 A JP 2016565151A JP 2017514480 A JP2017514480 A JP 2017514480A
- Authority
- JP
- Japan
- Prior art keywords
- ginsenoside
- protein
- glycosyltransferase
- udp
- ppd
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229930182494 ginsenoside Natural products 0.000 title claims abstract description 140
- 229940089161 ginsenoside Drugs 0.000 title claims abstract description 128
- 238000000034 method Methods 0.000 title claims abstract description 27
- 235000008434 ginseng Nutrition 0.000 title claims description 45
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 title claims description 36
- 235000003140 Panax quinquefolius Nutrition 0.000 title claims description 36
- 102000051366 Glycosyltransferases Human genes 0.000 title claims description 17
- 108700023372 Glycosyltransferases Proteins 0.000 title claims description 17
- 238000012546 transfer Methods 0.000 title description 12
- 125000003147 glycosyl group Chemical group 0.000 title description 3
- 241000208340 Araliaceae Species 0.000 title 1
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 116
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 104
- PYXFVCFISTUSOO-HKUCOEKDSA-N (20S)-protopanaxadiol Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@H]([C@@](C)(O)CCC=C(C)C)[C@H]4[C@H](O)C[C@@H]3[C@]21C PYXFVCFISTUSOO-HKUCOEKDSA-N 0.000 claims abstract description 90
- PYXFVCFISTUSOO-UHFFFAOYSA-N betulafolienetriol Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC(C(C)(O)CCC=C(C)C)C4C(O)CC3C21C PYXFVCFISTUSOO-UHFFFAOYSA-N 0.000 claims abstract description 90
- SWQINCWATANGKN-UHFFFAOYSA-N protopanaxadiol Natural products CC(CCC=C(C)C)C1CCC2(C)C1C(O)CC1C3(C)CCC(O)C(C)(C)C3CCC21C SWQINCWATANGKN-UHFFFAOYSA-N 0.000 claims abstract description 90
- BBEUDPAEKGPXDG-UHFFFAOYSA-N protopanaxatriol Natural products CC(CCC=C(C)C)C1CCC2(C)C1C(O)CC3C4(C)CCC(O)C(C)(C)C4C(O)CC23C BBEUDPAEKGPXDG-UHFFFAOYSA-N 0.000 claims abstract description 75
- SHCBCKBYTHZQGZ-DLHMIPLTSA-N protopanaxatriol Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2[C@@H](O)C[C@@]3(C)[C@]4(C)CC[C@H]([C@](C)(O)CCC=C(C)C)[C@H]4[C@H](O)C[C@@H]3[C@]21C SHCBCKBYTHZQGZ-DLHMIPLTSA-N 0.000 claims abstract description 74
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 58
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 58
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 43
- 230000000694 effects Effects 0.000 claims abstract description 33
- 101000932831 Panax ginseng Protopanaxadiol 6-hydroxylase Proteins 0.000 claims abstract description 27
- 230000006098 transglycosylation Effects 0.000 claims abstract description 12
- 238000005918 transglycosylation reaction Methods 0.000 claims abstract description 12
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims abstract 4
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 claims abstract 2
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims abstract 2
- 229940045145 uridine Drugs 0.000 claims abstract 2
- 108091033319 polynucleotide Proteins 0.000 claims description 62
- 102000040430 polynucleotide Human genes 0.000 claims description 62
- 239000002157 polynucleotide Substances 0.000 claims description 62
- 239000013604 expression vector Substances 0.000 claims description 54
- 150000001413 amino acids Chemical group 0.000 claims description 51
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 50
- FVIZARNDLVOMSU-UHFFFAOYSA-N ginsenoside K Natural products C1CC(C2(CCC3C(C)(C)C(O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O FVIZARNDLVOMSU-UHFFFAOYSA-N 0.000 claims description 46
- 239000013598 vector Substances 0.000 claims description 46
- 240000004371 Panax ginseng Species 0.000 claims description 45
- 101000932810 Panax ginseng Dammarenediol 12-hydroxylase Proteins 0.000 claims description 44
- 108030005934 Dammarenediol II synthases Proteins 0.000 claims description 35
- 238000004519 manufacturing process Methods 0.000 claims description 31
- 235000000346 sugar Nutrition 0.000 claims description 23
- 102000004286 Hydroxymethylglutaryl CoA Reductases Human genes 0.000 claims description 20
- 108090000895 Hydroxymethylglutaryl CoA Reductases Proteins 0.000 claims description 20
- XCCTYIAWTASOJW-XVFCMESISA-N Uridine-5'-Diphosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-XVFCMESISA-N 0.000 claims description 18
- 108010045510 NADPH-Ferrihemoprotein Reductase Proteins 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- -1 tHMGR) Proteins 0.000 claims description 12
- 235000002789 Panax ginseng Nutrition 0.000 claims description 11
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 10
- 241000219194 Arabidopsis Species 0.000 claims description 9
- 239000012634 fragment Substances 0.000 claims description 9
- ZUXNULGHCOXCFL-UHFFFAOYSA-N 2-(4-tert-butyl-2,6-dimethylphenyl)acetonitrile Chemical compound CC1=CC(C(C)(C)C)=CC(C)=C1CC#N ZUXNULGHCOXCFL-UHFFFAOYSA-N 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 230000001747 exhibiting effect Effects 0.000 claims description 5
- 239000004480 active ingredient Substances 0.000 claims description 3
- 101000841399 Arabidopsis thaliana ERBB-3 BINDING PROTEIN 1 Proteins 0.000 claims 4
- 102000018832 Cytochromes Human genes 0.000 claims 4
- 108010052832 Cytochromes Proteins 0.000 claims 4
- 102000004316 Oxidoreductases Human genes 0.000 claims 4
- 108090000854 Oxidoreductases Proteins 0.000 claims 4
- 101000761643 Panax ginseng UDP-glucosyltransferase 74AE2 Proteins 0.000 claims 2
- 239000002253 acid Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 57
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 45
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 45
- 239000002773 nucleotide Substances 0.000 description 42
- 125000003729 nucleotide group Chemical group 0.000 description 42
- 238000004128 high performance liquid chromatography Methods 0.000 description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 30
- 230000014759 maintenance of location Effects 0.000 description 26
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 25
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 24
- GEWDNTWNSAZUDX-WQMVXFAESA-N (-)-methyl jasmonate Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-WQMVXFAESA-N 0.000 description 19
- 101150107463 ERG7 gene Proteins 0.000 description 19
- 238000000855 fermentation Methods 0.000 description 19
- 230000004151 fermentation Effects 0.000 description 19
- GEWDNTWNSAZUDX-UHFFFAOYSA-N methyl 7-epi-jasmonate Natural products CCC=CCC1C(CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-UHFFFAOYSA-N 0.000 description 19
- 230000015572 biosynthetic process Effects 0.000 description 17
- 230000004071 biological effect Effects 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- FBFMBWCLBGQEBU-RXMALORBSA-N (2s,3r,4s,5s,6r)-2-[(2r,3r,4s,5s,6r)-2-[[(3s,5r,6s,8r,9r,10r,12r,13r,14r,17s)-3,12-dihydroxy-4,4,8,10,14-pentamethyl-17-[(2s)-6-methyl-2-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhept-5-en-2-yl]-2,3,5,6,7,9,11,12,13,15,16,17-dodecah Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O FBFMBWCLBGQEBU-RXMALORBSA-N 0.000 description 12
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 12
- 239000008186 active pharmaceutical agent Substances 0.000 description 12
- SWIROVJVGRGSPO-JBVRGBGGSA-N ginsenoside F2 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O SWIROVJVGRGSPO-JBVRGBGGSA-N 0.000 description 12
- 229930182817 methionine Natural products 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 229920002469 poly(p-dioxane) polymer Polymers 0.000 description 12
- FBFMBWCLBGQEBU-GYMUUCMZSA-N 20-gluco-ginsenoside-Rf Natural products O([C@](CC/C=C(\C)/C)(C)[C@@H]1[C@H]2[C@H](O)C[C@H]3[C@](C)([C@]2(C)CC1)C[C@H](O[C@@H]1[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O2)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1C(C)(C)[C@@H](O)CC[C@]31C)[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 FBFMBWCLBGQEBU-GYMUUCMZSA-N 0.000 description 11
- HYPFYJBWSTXDAS-UHFFFAOYSA-N Ginsenoside Rd Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C4CCC5C(C)(C)C(CCC5(C)C4CC(O)C23C)OC6OC(CO)C(O)C(O)C6OC7OC(CO)C(O)C(O)C7O)C HYPFYJBWSTXDAS-UHFFFAOYSA-N 0.000 description 11
- XNGXWSFSJIQMNC-FIYORUNESA-N ginsenoside F1 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@H](O)[C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O XNGXWSFSJIQMNC-FIYORUNESA-N 0.000 description 11
- UOJAEODBOCLNBU-UHFFFAOYSA-N vinaginsenoside R4 Natural products C1CC(C2(CC(O)C3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O UOJAEODBOCLNBU-UHFFFAOYSA-N 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 238000004809 thin layer chromatography Methods 0.000 description 10
- BGHNZAWRRWLKPO-UHFFFAOYSA-N Ginsenoside F1 Natural products CC(=C)CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5C(O)CC34C BGHNZAWRRWLKPO-UHFFFAOYSA-N 0.000 description 9
- SWIROVJVGRGSPO-UHFFFAOYSA-N Ginsenoside F2 Natural products C1CC(C2(CCC3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O SWIROVJVGRGSPO-UHFFFAOYSA-N 0.000 description 9
- AVTXSAWPGCSYFO-UHFFFAOYSA-N Ginsenoside Ia Natural products C1CC(C2(CC(O)C3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O AVTXSAWPGCSYFO-UHFFFAOYSA-N 0.000 description 9
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 9
- 230000009466 transformation Effects 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 8
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 8
- 230000003828 downregulation Effects 0.000 description 8
- 238000000527 sonication Methods 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 210000005253 yeast cell Anatomy 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 7
- 238000011426 transformation method Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 241000196324 Embryophyta Species 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
- 230000001851 biosynthetic effect Effects 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 239000002207 metabolite Substances 0.000 description 6
- 150000007523 nucleic acids Chemical group 0.000 description 6
- CBIDRCWHNCKSTO-UHFFFAOYSA-N prenyl diphosphate Chemical compound CC(C)=CCO[P@](O)(=O)OP(O)(O)=O CBIDRCWHNCKSTO-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- FVIZARNDLVOMSU-IRFFNABBSA-N ginsenoside C-K Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O FVIZARNDLVOMSU-IRFFNABBSA-N 0.000 description 5
- 230000007704 transition Effects 0.000 description 5
- 244000000626 Daucus carota Species 0.000 description 4
- 235000002767 Daucus carota Nutrition 0.000 description 4
- 101150050575 URA3 gene Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- QQGWBRJQPRTJDA-UHFFFAOYSA-N [Li].CC(O)=O Chemical compound [Li].CC(O)=O QQGWBRJQPRTJDA-UHFFFAOYSA-N 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 150000001875 compounds Chemical group 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 4
- 229930182490 saponin Natural products 0.000 description 4
- 150000007949 saponins Chemical class 0.000 description 4
- 150000003648 triterpenes Chemical class 0.000 description 4
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- 108700010070 Codon Usage Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 102100022346 Serine/threonine-protein phosphatase 5 Human genes 0.000 description 3
- 101710129069 Serine/threonine-protein phosphatase 5 Proteins 0.000 description 3
- 101710199542 Serine/threonine-protein phosphatase T Proteins 0.000 description 3
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 229920000470 poly(p-phenylene terephthalate) polymer Polymers 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229940031439 squalene Drugs 0.000 description 3
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- CKUVNOCSBYYHIS-IRFFNABBSA-N (20S)-ginsenoside Rh2 Chemical compound O([C@H]1CC[C@]2(C)[C@H]3C[C@@H](O)[C@H]4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@@H]4[C@@](C)(O)CCC=C(C)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O CKUVNOCSBYYHIS-IRFFNABBSA-N 0.000 description 2
- 101710165761 (2E,6E)-farnesyl diphosphate synthase Proteins 0.000 description 2
- QYIMSPSDBYKPPY-RSKUXYSASA-N (S)-2,3-epoxysqualene Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C=C(/C)CC\C=C(/C)CC[C@@H]1OC1(C)C QYIMSPSDBYKPPY-RSKUXYSASA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- 102100028626 4-hydroxyphenylpyruvate dioxygenase Human genes 0.000 description 2
- SEHFUALWMUWDKS-UHFFFAOYSA-N 5-fluoroorotic acid Chemical compound OC(=O)C=1NC(=O)NC(=O)C=1F SEHFUALWMUWDKS-UHFFFAOYSA-N 0.000 description 2
- 101100262420 Arabidopsis thaliana UGT78D2 gene Proteins 0.000 description 2
- 101100371461 Arabidopsis thaliana UGT89C1 gene Proteins 0.000 description 2
- 101710095468 Cyclase Proteins 0.000 description 2
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 2
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101710156207 Farnesyl diphosphate synthase Proteins 0.000 description 2
- 102100035111 Farnesyl pyrophosphate synthase Human genes 0.000 description 2
- 101710125754 Farnesyl pyrophosphate synthase Proteins 0.000 description 2
- 101710089428 Farnesyl pyrophosphate synthase erg20 Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 108010065958 Isopentenyl-diphosphate Delta-isomerase Proteins 0.000 description 2
- 102100027665 Isopentenyl-diphosphate Delta-isomerase 1 Human genes 0.000 description 2
- 101150068888 MET3 gene Proteins 0.000 description 2
- 101000997933 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (2E,6E)-farnesyl diphosphate synthase Proteins 0.000 description 2
- 101001015102 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) Dimethylallyltranstransferase Proteins 0.000 description 2
- 101100022915 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cys-11 gene Proteins 0.000 description 2
- 241001527087 Panax vietnamensis Species 0.000 description 2
- 101710150389 Probable farnesyl diphosphate synthase Proteins 0.000 description 2
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 101100022918 Schizosaccharomyces pombe (strain 972 / ATCC 24843) sua1 gene Proteins 0.000 description 2
- 102000005782 Squalene Monooxygenase Human genes 0.000 description 2
- 108020003891 Squalene monooxygenase Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- NLHQJXWYMZLQJY-TXNIMPHESA-N dammarenediol-II Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@H]([C@@](C)(O)CCC=C(C)C)[C@H]4CC[C@@H]3[C@]21C NLHQJXWYMZLQJY-TXNIMPHESA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- CJFGBCWGOQRURQ-UHFFFAOYSA-N ginsenoside Mc Natural products C1CC(C2(CCC3C(C)(C)C(O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC(C(C(O)C1O)O)OC1COC1OC(CO)C(O)C1O CJFGBCWGOQRURQ-UHFFFAOYSA-N 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000000348 glycosyl donor Substances 0.000 description 2
- 230000033444 hydroxylation Effects 0.000 description 2
- 238000005805 hydroxylation reaction Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 2
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 description 2
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 2
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 150000002772 monosaccharides Chemical group 0.000 description 2
- 238000002552 multiple reaction monitoring Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229910021654 trace metal Inorganic materials 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- RWXIFXNRCLMQCD-JBVRGBGGSA-N (20S)-ginsenoside Rg3 Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1CC[C@]2(C)[C@H]3C[C@@H](O)[C@H]4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@@H]4[C@@](C)(O)CCC=C(C)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RWXIFXNRCLMQCD-JBVRGBGGSA-N 0.000 description 1
- RAQNTCRNSXYLAH-RFCGZQMISA-N (20S)-ginsenoside Rh1 Chemical compound O([C@@H]1[C@H]2C(C)(C)[C@@H](O)CC[C@]2(C)[C@H]2C[C@@H](O)[C@H]3[C@@]([C@@]2(C1)C)(C)CC[C@@H]3[C@@](C)(O)CCC=C(C)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RAQNTCRNSXYLAH-RFCGZQMISA-N 0.000 description 1
- 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
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 description 1
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 description 1
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 244000260524 Chrysanthemum balsamita Species 0.000 description 1
- 235000005633 Chrysanthemum balsamita Nutrition 0.000 description 1
- 241000951471 Citrus junos Species 0.000 description 1
- RKWHWFONKJEUEF-GQUPQBGVSA-O Cyanidin 3-O-glucoside Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 RKWHWFONKJEUEF-GQUPQBGVSA-O 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 1
- NLHQJXWYMZLQJY-UHFFFAOYSA-N Dammarendiol Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC(C(C)(O)CCC=C(C)C)C4CCC3C21C NLHQJXWYMZLQJY-UHFFFAOYSA-N 0.000 description 1
- 240000007190 Daucus pusillus Species 0.000 description 1
- 235000002196 Daucus pusillus Nutrition 0.000 description 1
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical group OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108010022535 Farnesyl-Diphosphate Farnesyltransferase Proteins 0.000 description 1
- 101150066516 GST gene Proteins 0.000 description 1
- 241000702463 Geminiviridae Species 0.000 description 1
- AGBCLJAHARWNLA-DQUQINEDSA-N Ginsenoside RG2 Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@@](C)(O)CCC=C(C)C)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O AGBCLJAHARWNLA-DQUQINEDSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101150020741 Hpgds gene Proteins 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 230000004989 O-glycosylation Effects 0.000 description 1
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 description 1
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000180649 Panax notoginseng Species 0.000 description 1
- 235000017726 Panax vietnamensis Nutrition 0.000 description 1
- 240000003296 Petasites japonicus Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 1
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 244000000231 Sesamum indicum Species 0.000 description 1
- 235000003434 Sesamum indicum Nutrition 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 102100037997 Squalene synthase Human genes 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 241000219793 Trifolium Species 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- DRDCJEIZVLVWNC-SLBWPEPYSA-N UDP-beta-L-rhamnose Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 DRDCJEIZVLVWNC-SLBWPEPYSA-N 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- YTMNONATNXDQJF-UBNZBFALSA-N chrysanthemin Chemical compound [Cl-].O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC=C(O)C(O)=C1 YTMNONATNXDQJF-UBNZBFALSA-N 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- OORMXZNMRWBSTK-UHFFFAOYSA-N dammaran Natural products C1CCC(C)(C)C2CCC3(C)C4(C)CCC(C(C)CCCC(C)C)C4CCC3C21C OORMXZNMRWBSTK-UHFFFAOYSA-N 0.000 description 1
- OORMXZNMRWBSTK-LGFJJATJSA-N dammarane Chemical compound C1CCC(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@H]([C@H](C)CCCC(C)C)[C@H]4CC[C@@H]3[C@]21C OORMXZNMRWBSTK-LGFJJATJSA-N 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 description 1
- 150000007946 flavonol Chemical class 0.000 description 1
- 235000011957 flavonols Nutrition 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000002546 full scan Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 229940107131 ginseng root Drugs 0.000 description 1
- GZYPWOGIYAIIPV-JBDTYSNRSA-N ginsenoside Rb1 Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GZYPWOGIYAIIPV-JBDTYSNRSA-N 0.000 description 1
- PWAOOJDMFUQOKB-WCZZMFLVSA-N ginsenoside Re Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@](C)(CCC=C(C)C)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O PWAOOJDMFUQOKB-WCZZMFLVSA-N 0.000 description 1
- UZIOUZHBUYLDHW-XUBRWZAZSA-N ginsenoside Rf Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H]2C(C)(C)[C@@H](O)CC[C@]2(C)[C@H]2C[C@@H](O)[C@H]3[C@@]([C@@]2(C1)C)(C)CC[C@@H]3[C@@](C)(O)CCC=C(C)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UZIOUZHBUYLDHW-XUBRWZAZSA-N 0.000 description 1
- YURJSTAIMNSZAE-HHNZYBFYSA-N ginsenoside Rg1 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O YURJSTAIMNSZAE-HHNZYBFYSA-N 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 239000000937 glycosyl acceptor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 235000015092 herbal tea Nutrition 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000000640 hydroxylating effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 235000020344 instant tea Nutrition 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
- 235000008777 kaempferol Nutrition 0.000 description 1
- 238000001972 liquid chromatography-electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229940100243 oleanolic acid Drugs 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000003863 physical function Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical group N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 235000015961 tonic Nutrition 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
-
- 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.)
-
- 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.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- 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
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01017—Glucuronosyltransferase (2.4.1.17)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Mycology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
Abstract
Description
高麗人参cDNAからPgUGT71A1−F
(5’-AGGCAGGATCCATGAAGTCAGAATTGATATTCTTGCCCGCCCCGGC-3’;配列番号17)、PgUGT71A1−R
(5’-AGGCATCTCGAGTCACATAATTTTCTCAAATAGTTTGGCCAATGAAT-3’;配列番号18)のプライマーとポリメラーゼを用いてPCRにより遺伝子を増幅し、制限酵素であるBamHI及びXhoIを用いて遺伝子の末端を切り取った。その後、前記遺伝子をpGEX−4T1ベクターにクローニングして発現ベクターを構築し、これを大腸菌BL21(DE3)−RIL菌株に形質転換してPgUGT71A1発現菌株を得た。
前記菌株はIPTGでタンパク質発現を誘導した後、生じたタンパク質をセファロース−4Bレジンを用いてPgUGT71A1酵素を精製した。
精製されたPgUGT71A1(30μg)、ジンセノサイド化合物(5mM)及びUDP−グルコース(50mM)を含む反応緩衝液(10mM PBS緩衝液、pH7)で糖転移酵素分析を行った。この分析には、4つの異なる種類のジンセノサイド、すなわち、プロトパナキサジオール(PPD)、プロトパナキサトリオール(PPT)、Rh2及びRg3を使用して本発明の酵素と反応させた。前記ジンセノサイドの構造については、図1に示した。
トータルRNAは、スペクトルプラントトータルRNAキット(Spectrum plant total RNA kit、Sigma-Aldrich)を使用し、15か月の高麗人参の葉又は根から分離した。200μMのジャスモン酸メチル(MeJA)を5日間毎日高麗人参の葉に噴霧し、6日目にサンプルを採取した。1μgのトータルRNAをcDNA合成に使用した。
異なる遺伝子の発現レベルは、表1に示したプライマーセットを使用し、定量的RT−PCR を行って確認し、チューブリンの発現レベルで標準化した。
MeJAを処理した高麗人参の葉と根、及びMeJAを処理していない高麗人参の葉と根からRNAを抽出し、TruSeq RNAライブラリキットを使用して1μgのトータルRNAからcDNAライブラリーを構築した。polyA−selected RNA抽出、RNA断片化 及びランダムヘキサマープライム逆転写に続いて、100 ntのペアエンドシーケンシングはイルミナHiSeq2000を用いて行った。
実施例5.1:ERG7ダウンレギュレーションカセット の構築
ERG7ダウンレギュレーション菌株(S. cerevisiaeMET3p-ERG7)を構築するために、ERG7ダウンレギュレーションカセットを構築した。MET3プロポーター、ERG7遺伝子及びCYC1ターミネータを含むERG7ダウンレギュレーションカセットのために、pRS306プラスミドをプラットフォームとして使用した。ERG7遺伝子を阻害するために、メチオニン供給により転写レベルを抑制することができるMET3プロモーター(MET3p)を選択した。MET3pはS. cerevisiaeCEN.PK2−1DのゲノムDNAから次のプライマーセットを用いてPCRにより増幅した:
pRS306−F(SacI)に対するMET3p及びpRS306−B(XbaI)に対するMET3p(表2)。
pRS306−F(SpeI)に対するERG7及びpRS306−B(XhoI)に対するERG7(表3)。
pRS306−F(XhoI)に対するCYC1ターミネータ及びpRS306−B(KpnI)に対するCYC1ターミネータ(表4)。
構築されたERG7ダウンレギュレーションカセットをリチウム酢酸方法(リチウム酢酸、一本鎖キャリアDNA、ポリエチレングリコール方法による酵母の形質転換)によりS. cerevisiaeCEN.PK2−1Dゲノムに挿入した。形質転換体はSD−Leu−Trp−Hisプレート上で選択した。選択された形質転換体をERG7ノックダウンコンフォーム−F及びERG7ノックダウンコンフォーム−Bプライマー(表6)を用いてPCRによりカセット挿入を確認するためのゲノムDNA分離のために5ml YPD培地で培養した。その後、URA3マーカーを回収するため5−FOA(5-Fluoroorotic acid)プレートから酵母を選択し、さらに分析するために、使用した。
ジンセノサイドの新規合成はバイオリアクターによりジンセノサイド生合成遺伝子を有しているS. cerevisiaeを培養して測定した。非特許文献4により説明されたメチオニンを含む培地を発酵のために使用した。前記バッチ培養培地は、グルコース20g/L、(NH4)2SO4 15g/L、KH2PO4 8g/L、ZnSO4・7H2O 0.72g/L、MgSO4・7H2O 6.15g/L、ビタミン溶液12mL/L、メチオニン0.3g/L及び微量金属溶液 10 mL/Lを含む。培地の微量金属溶液は、EDTA 15g/L、ZnSO4・7H2O 10.2g/L、MnCl2・4H2O 0.50g/L、無水CuSo4 0.5g/L、CoCl2・6H2O 0.86g/L、Na2MO4・2H2O 0.56g/L、CaCl2・2H2O 3.84g/L及びFeSO4・7H2O 5.12g/Lを含む。ビタミン溶液は、ビオチン0.05g/L、パントテン酸カルシウム1g/L、ナイアシン1g/L、イノシトール25g/L、チアミンHCL 1g/L、ピリドキソールHCL 1g/L及び4−アミノ安息香酸0.2g/Lを含む。流入溶液は、グルコース578g/L、KH2PO4 9g/L、K2SO4 3.5g/L、Na2SO4 0.28g/L、MgSO4・7H2O 5.12g/L、及びメチオニン1 mLを含む。流加培養式発酵は30℃の1.5−Lバイオリアクター(Biotron、韓国)により1L/分の気流及び400rpmで攪拌して行った。pH−stat feeding戦略はグルコース(> pH 5.51)を注入及び5%のNH4OH(<pH 5.4)を追加することにより、流加培養式発酵に使用した。
HPLC分析はODS(2)カラム(Phenomenex、CA、米国)を使用し、次のように1 mL/分の流速で行った:0分、68%の水及び32%のアセトニトリル;8分、35%の水及び65%のアセトニトリル;12分、0%の水及び100%のアセトニトリル;20分、0%の水及び100%のアセトニトリル;20.1分、68%の水及び32%のアセトニトリル;及び28分、68%の水及び32%のアセトニトリル。ジンセノサイドはUV−検出器(Agilent Technologies、CA、米国)を用いて203nmの波長で観察した。
実施例1において同定したPgUGT71A1の基質特異性及び位置特異性下記のように確認した。
伝統的に薬用の目的で使用されてきた高麗人参の根に本発明のPgUGT71A1が主に発現されているかどうかを調査した。また、3つの異なるジンセノサイド生合成遺伝子であるダンマレンジオールIIシンターゼ(dammarenediol-II synthase、PgDS)、プロトパナキサジオール合成酵素(protopanaxadiol synthase、PgPPDS)及びプロトパナキサトリオール合成酵素(protopanaxatriol synthase、PgPPTS)を用いて、本発明のPgUGT71A1の器官特異的発現パターンを測定した。
C-K(compound K)の生産のために、pRS424−DS、pRS426−tHMG1、pRS425−PPD及びPgUGT71A1を含むpRS423−C-Kをを S. cerevisiaeMET3p−ERG7に導入して、C-Kの生合成経路を有するS. cerevisiaeC-K菌株を製造した(図6)。
ジンセノサイドF1の生産のために、pRS424−DS、pRS426−tHMG1、pRSPPD、及びPgUGT71A1とPgPPTSを含むpRS423−F1をS. cerevisiae MET3p−ERG7に導入して、ジンセノサイドF1の生合成経路を有するS. cerevisiae F1菌株を製造(図8)。
ジンセノサイドF2の生産のために、pRS424−DS、pRS426−tHMG1、pRSPPD、及びPgUGT74A1とPgUGT71A1を含むpRS423をS. cerevisiae MET3p−ERG7に導入して、ジンセノサイドF2の生合成経路を有するS. cerevisiaeF2菌株を製造した(図10)。
ジンセノサイドRdの生産のために、pRS424−DS、pRS426−tHMG1、pRS425−PPD、及びPgUGT74A1、PgUGT94B1及びPgUGT71A1を含むpRS423−RdをS. cerevisiaeMET3p−ERG7に導入して、ジンセノサイドRdの生合成経路を有するS. cerevisiaeRd菌株を製造した(図12)。
Claims (25)
- 20位の炭素にヒドロキシル基を有するプロトパナキサジオール(protopanaxadiol、PPD)又はプロトパナキサトリオール(protopanaxatriol、PPT)系のジンセノサイドを、
ウリジン二リン酸(UDP)−糖転移酵素(uridine diphosphate glycosyltransferase)タンパク質;前記タンパク質をコードするポリヌクレオチドを含むベクター、又は前記ベクターの一部が導入され、前記タンパク質の活性を示す形質転換細胞;前記形質転換細胞が含まれた生物体;又は前記形質転換細胞の培養物で処理する段階を含む、
20位の炭素のヒドロキシル基が糖転移された、PPD系又はPPT系のジンセノサイドを製造する方法。 - 前記20位の炭素にヒドロキシル基を有するPPD又はPPT系のジンセノサイドは、PPD、Rh2、Rg3及びPPTからなる群から選択される一つ以上である、請求項1に記載のジンセノサイドを製造する方法。
- 前記方法は、PPDのC-K(compound K)への転換、Rh2のF2への転換、Rg3のRdへの転換及びPPTのF1への転換からなる群から選択された一つ以上の転換段階を含む、請求項1に記載の方法。
- 前記UDP−糖転移酵素タンパク質は、配列番号1のアミノ酸配列で定義される、請求項1に記載のジンセノサイドを製造する方法。
- 20位の炭素にヒドロキシル基を有するプロトパナキサジオール (protopanaxadiol、PPD)又はプロトパナキサトリオール (protopanaxatriol、PPT)系のジンセノサイドのヒドロキシル基に対して糖転移活性を有するウリジン二リン酸(UDP)−糖転移酵素(uridine diphosphate glycosyltransferase)タンパク質;前記タンパク質をコードするポリヌクレオチドを含むベクター、又は前記ベクターの一部が導入され、前記タンパク質の活性を示す形質転換細胞;前記形質転換細胞が含まれた生物体;及び前記形質転換細胞の培養物からなる群から選択される一つ以上を有効成分として含む、
20位の炭素に位置するヒドロキシル基が糖転移された、PPD又はPPT系のジンセノサイドの製造用組成物。 - 前記20位の炭素にヒドロキシル基を有するPPD又はPPT系のジンセノサイドは、PPD、Rh2、Rg3及びPPTからなる群から選択される一つ以上である、請求項5に記載のジンセノサイドの製造用組成物。
- 前記UDP−糖転移酵素は、PPDをC−K(compound K)に、Rh2をF2に、Rg3をRdに、又はPPTをF1に転換する、請求項5に記載の組成物。
- 前記UDP−糖転移酵素タンパク質は、配列番号1のアミノ酸配列で定義される、請求項5に記載のジンセノサイドの製造用組成物。
- 配列番号1のアミノ酸配列で定義される、分離されたウリジン二リン酸(UDP)−糖転移酵素(Uridine diphosphate-glycosyltransferase)タンパク質(PgUGT71A1)。
- 前記タンパク質は、プロトパナキサジオール (protopanaxadiol、PPD)又はプロトパナキサトリオール(protopanaxatriol、PPT)系のジンセノサイドの20位の炭素に位置したヒドロキシル基に対して糖転移活性を有する、請求項9に記載のウリジン二リン酸(UDP)−糖転移酵素(uridine diphosphate glycosyltransferase)タンパク質。
- 前記タンパク質は、PPDをC−K(compound K)に、Rh2をF2に、Rg3をRdに、又はPPTをF1に転換する、請求項9に記載のウリジン二リン酸(UDP)−糖転移酵素(uridine diphosphate glycosyltransferase)タンパク質。
- 請求項9に記載タンパク質をコードするポリヌクレオチド。
- 請求項12に記載ポリヌクレオチドを含む発現ベクター。
- 前記ベクターは、ダンマレンジオールIIシンターゼ(dammarenediol-IIsynthase、DS)、トランケーとされたHMG-CoAレダクターゼ(truncated HMG-CoA reductase、tHMGR)、プロトパナキサジオール合成酵素 (protopanaxadiol synthase、PPDS)及びシロイヌナズナシトクロムp450jレダクターゼ(Arabidopsis thaliana cytochrome p450 reductase、AtCPR)タンパク質のそれぞれをコードするポリヌクレオチドをさらに含む、請求項13に記載の発現ベクター。
- 前記ベクターは、ダンマレンジオールIIシンターゼ (dammarenediol-IIsynthase、DS)、トランケーとされたHMG-CoAレダクターゼ (truncated HMG-CoA reductase、tHMGR)、プロトパナキサジオール合成酵素 (protopanaxadiol synthase、PPDS)、シロイヌナズナシトクロムp450jレダクターゼ (Arabidopsis thaliana cytochrome p450 reductase、AtCPR)及びプロトパナキサトリオール合成酵素(protopanaxatriol synthase、PPTS)タンパク質のそれぞれをコードポリヌクレオチドをさらに含む、請求項13に記載の発現ベクター。
- 前記ベクターは、ダンマレンジオールIIシンターゼ(dammarenediol-IIsynthase、DS )、トランケーとされたHMG-CoAレダクターゼ (truncated HMG-CoA reductase、tHMGR)、プロトパナキサジオール合成酵素 (protopanaxadiol synthase、PPDS)、シロイヌナズナシトクロムp450jレダクターゼ(Arabidopsis thaliana cytochrome p450 reductase、AtCPR)及び高麗人参UDP-糖転移酵素74A1(panax ginseng UDP-glycosyltransferase 74A1、PgUGT74A1)タンパク質のそれぞれをコードするポリヌクレオチドをさらに含む、請求項13に記載の発現ベクター。
- 前記ベクターは、ダンマレンジオールIIシンターゼ (dammarenediol-IIsynthase、DS)、トランケーとされたHMG-CoAレダクターゼ (truncated HMG-CoA reductase、tHMGR)、プロトパナキサジオール合成酵素 (protopanaxadiol synthase、PPDS)、シロイヌナズナシトクロムp450jレダクターゼ (Arabidopsis thaliana cytochrome p450 reductase、AtCPR)、 高麗人参UDP-糖転移酵素74A1(panax ginseng UDP-glycosyltransferase、PgUGT74A1)及び 高麗人参UDP-糖転移酵素94B1(panax ginseng UDP-glycosyltransferase 94B1、PgUGT94B1)タンパク質のそれぞれをコードするポリヌクレオチドをさらに含む、請求項13に記載の発現ベクター。
- 請求項14に記載発現ベクター又はそのフラグメントを含むC−K(compound K)生産用形質転換細胞。
- 請求項15に記載発現ベクター又はそのフラグメントを含むF1生産用形質転換細胞。
- 請求項16に記載発現ベクター又はそのフラグメントを含むF2生産用形質転換細胞。
- 請求項17に記載発現ベクター又はそのフラグメントを含むRd生産用形質転換細胞。
- 前記形質転換細胞は酵母である、請求項18に記載の形質転換細胞。
- 前記形質転換細胞は酵母である、請求項19に記載の形質転換細胞。
- 前記形質転換細胞は酵母である、請求項20に記載の形質転換細胞。
- 前記形質転換細胞は酵母である、請求項21に記載の形質転換細胞。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2014-0052728 | 2014-04-30 | ||
KR20140052728 | 2014-04-30 | ||
PCT/KR2015/004399 WO2015167282A1 (en) | 2014-04-30 | 2015-04-30 | A novel method for glycosylation of ginsenoside using a glycosyltransferase derived from panax ginseng |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2017514480A true JP2017514480A (ja) | 2017-06-08 |
JP6526716B2 JP6526716B2 (ja) | 2019-06-05 |
Family
ID=54358919
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2016565151A Expired - Fee Related JP6526716B2 (ja) | 2014-04-30 | 2015-04-30 | 高麗人参由来の糖転移酵素を用いた新規なジンセノサイド糖転移方法 |
Country Status (6)
Country | Link |
---|---|
US (1) | US10174355B2 (ja) |
EP (1) | EP3137612A4 (ja) |
JP (1) | JP6526716B2 (ja) |
KR (1) | KR101788608B1 (ja) |
CN (1) | CN106459987B (ja) |
WO (1) | WO2015167282A1 (ja) |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106544310B (zh) * | 2016-09-27 | 2019-07-16 | 南开大学 | 一种绞股蓝糖基转移酶的工程菌及其构建方法与应用 |
CN106701647B (zh) * | 2016-09-27 | 2019-12-24 | 南开大学 | 绞股蓝糖基转移酶在合成稀有人参皂苷中的应用 |
CN108330111B (zh) * | 2017-01-20 | 2023-01-31 | 生合万物(苏州)生物科技有限公司 | 细胞色素p450突变蛋白及其应用 |
CN108728423B (zh) * | 2017-04-13 | 2021-12-07 | 中国医学科学院药物研究所 | 枯草芽孢杆菌糖基转移酶及其应用 |
KR101957910B1 (ko) * | 2017-05-02 | 2019-03-15 | 재단법인 지능형 바이오 시스템 설계 및 합성 연구단 | 효모의 세포 소기관(organelle) 개량을 통한 진세노사이드 생산 증대 |
CN108866020A (zh) * | 2017-05-16 | 2018-11-23 | 中国科学院上海生命科学研究院 | 糖基转移酶、突变体及其应用 |
CN109666714A (zh) * | 2017-10-15 | 2019-04-23 | 中国医学科学院药物研究所 | 一种制备12β-O-Glc-PPD和12β-O-Glc-PPT的方法 |
CN110438099B (zh) * | 2018-05-04 | 2022-04-15 | 中国科学院天津工业生物技术研究所 | 糖基转移酶及其相关材料在构建产人参皂苷Rb1和Rg1的工程菌中的应用 |
CN109234347B (zh) * | 2018-09-30 | 2022-03-01 | 长春中医药大学 | 一种转化原人参三醇型皂苷生成c25-oh衍生物的方法 |
CN109295027B (zh) * | 2018-11-06 | 2020-12-29 | 浙江华睿生物技术有限公司 | 一种糖基转移酶突变体 |
CN111471704B (zh) * | 2019-01-23 | 2024-02-06 | 中国医学科学院药物研究所 | 生产稀有人参皂苷20S-O-Glc-DM的重组菌及其应用 |
CN114045270B (zh) * | 2020-02-17 | 2024-06-04 | 中国科学院天津工业生物技术研究所 | 一种生产人参皂苷Rg1的方法及其专用的工程菌 |
CN113444703B (zh) * | 2020-03-26 | 2023-09-01 | 生合万物(苏州)生物科技有限公司 | 催化糖链延伸的糖基转移酶突变体及其应用 |
CN114507646B (zh) * | 2020-11-17 | 2023-11-10 | 生合万物(上海)生物科技有限公司 | 细胞色素p450突变体蛋白及其应用 |
CN113493795B (zh) * | 2021-08-03 | 2022-10-28 | 昆明理工大学 | 一种人参皂苷Rh2的制备方法 |
CN113549649B (zh) * | 2021-08-05 | 2022-07-01 | 昆明理工大学 | 一种人参皂苷f1的制备方法 |
WO2023039518A1 (en) * | 2021-09-09 | 2023-03-16 | Manus Bio Inc. | Enzymes, host cells, and methods for biosynthesis of dammarenediol and derivatives |
CN115725620B (zh) * | 2022-09-12 | 2023-09-15 | 昆明理工大学 | 一种在三七细胞中合成竹节参皂苷的方法 |
KR20240070299A (ko) * | 2022-11-14 | 2024-05-21 | 주식회사 메디카코리아 | 발효를 통해 생산된 컴파운드 k의 효율적인 정제방법 |
CN116396876B (zh) * | 2022-11-17 | 2024-06-21 | 云南农业大学 | 一种生产人参皂苷Rd的酿酒酵母工程菌及其构建方法 |
CN117187297B (zh) * | 2023-09-07 | 2024-08-23 | 昆明理工大学 | 一种在珠子参细胞中合成人参皂苷ck的方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014051215A1 (en) * | 2012-09-27 | 2014-04-03 | Korea Advanced Institute Of Science And Technology | Novel udp-glycosyltransferase derived from ginseng and use thereof |
WO2014051214A1 (en) * | 2012-09-27 | 2014-04-03 | Korea Advanced Institute Of Science And Technology | Novel udp-glycosyltransferase derived from ginseng and use thereof |
JP2016504023A (ja) * | 2012-12-06 | 2016-02-12 | 上海 インスティテューツ フォー バイオロジカル サイエンシーズ、チャイニーズ アカデミー オブ サイエンシーズShanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | 一連の糖転移酵素およびその応用 |
-
2015
- 2015-04-30 WO PCT/KR2015/004399 patent/WO2015167282A1/en active Application Filing
- 2015-04-30 JP JP2016565151A patent/JP6526716B2/ja not_active Expired - Fee Related
- 2015-04-30 KR KR1020150061691A patent/KR101788608B1/ko active IP Right Grant
- 2015-04-30 EP EP15785502.4A patent/EP3137612A4/en not_active Withdrawn
- 2015-04-30 CN CN201580023380.3A patent/CN106459987B/zh not_active Expired - Fee Related
- 2015-04-30 US US15/307,582 patent/US10174355B2/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014051215A1 (en) * | 2012-09-27 | 2014-04-03 | Korea Advanced Institute Of Science And Technology | Novel udp-glycosyltransferase derived from ginseng and use thereof |
WO2014051214A1 (en) * | 2012-09-27 | 2014-04-03 | Korea Advanced Institute Of Science And Technology | Novel udp-glycosyltransferase derived from ginseng and use thereof |
JP2016504023A (ja) * | 2012-12-06 | 2016-02-12 | 上海 インスティテューツ フォー バイオロジカル サイエンシーズ、チャイニーズ アカデミー オブ サイエンシーズShanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | 一連の糖転移酵素およびその応用 |
Non-Patent Citations (3)
Title |
---|
"Panax ginseng UGT1 mRNA, complete cds.", GENBANK[ONLINE], ACCESSION NO. KF377585, JPN6017042594, 5 July 2014 (2014-07-05), ISSN: 0003852075 * |
CELL RESEARCH, vol. 24, JPN6017042596, 7 March 2014 (2014-03-07), pages 770 - 773, ISSN: 0003852074 * |
METABOLIC ENGINEERING, vol. 20, JPN6017042595, November 2013 (2013-11-01), pages 146 - 156, ISSN: 0003852076 * |
Also Published As
Publication number | Publication date |
---|---|
EP3137612A4 (en) | 2018-01-24 |
JP6526716B2 (ja) | 2019-06-05 |
KR20150125902A (ko) | 2015-11-10 |
CN106459987A (zh) | 2017-02-22 |
CN106459987B (zh) | 2019-12-03 |
US10174355B2 (en) | 2019-01-08 |
WO2015167282A1 (en) | 2015-11-05 |
US20170121750A1 (en) | 2017-05-04 |
EP3137612A1 (en) | 2017-03-08 |
KR101788608B1 (ko) | 2017-10-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6526716B2 (ja) | 高麗人参由来の糖転移酵素を用いた新規なジンセノサイド糖転移方法 | |
US20230203458A1 (en) | Group of udp-glycosyltransferase for catalyzing carbohydrate chain elongation and application thereof | |
CN107058446B (zh) | 一组糖基转移酶及其应用 | |
JP6072180B2 (ja) | アマチャヅル由来の新規な糖転移酵素及びその用途 | |
De Costa et al. | Molecular cloning of an ester-forming triterpenoid: UDP-glucose 28-O-glucosyltransferase involved in saponin biosynthesis from the medicinal plant Centella asiatica | |
Dong et al. | Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast | |
EP2900812B1 (en) | Novel udp-glycosyltransferase derived from ginseng and use thereof | |
US12084688B2 (en) | Glucuronosyltransferase, gene encoding same and use thereof | |
EP3033347B1 (en) | Methods for making noscapine and synthesis intermediates thereof | |
CN115094046A (zh) | 一组糖基转移酶及其应用 | |
KR101662268B1 (ko) | 감초속 식물 유래 트리테르펜 산화 효소, 그것을 코딩하는 유전자 및 그의 이용법 | |
KR20160039867A (ko) | 올레아난계 진세노사이드 생합성 촉진용 조성물 | |
WO2023006109A1 (zh) | 鼠李糖高度专一的糖基转移酶及其应用 | |
JP5526323B2 (ja) | カンゾウ属植物由来トリテルペン酸化酵素、それをコードする遺伝子およびその利用 | |
CN114829577A (zh) | 葡萄糖醛酸转移酶、编码该酶的基因及其利用方法 | |
CN117062914A (zh) | 方法和组合物 | |
KR20170127675A (ko) | 형질전환 담배세포의 세포 현탁 배양을 이용하는 프로토파낙사디올의 생산방법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20171023 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20171107 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20180207 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20180806 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20190422 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20190508 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6526716 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
LAPS | Cancellation because of no payment of annual fees |