CN116463310A - 一种粉防己4’-氧甲基转移酶4’-omt及应用 - Google Patents
一种粉防己4’-氧甲基转移酶4’-omt及应用 Download PDFInfo
- Publication number
- CN116463310A CN116463310A CN202211411600.8A CN202211411600A CN116463310A CN 116463310 A CN116463310 A CN 116463310A CN 202211411600 A CN202211411600 A CN 202211411600A CN 116463310 A CN116463310 A CN 116463310A
- Authority
- CN
- China
- Prior art keywords
- oxymethyl
- transferase
- omt
- tetrandra
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000992 Transferases Proteins 0.000 title claims abstract description 49
- 102000004357 Transferases Human genes 0.000 title claims abstract description 39
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 229930015408 benzyl-isoquinoline alkaloid Natural products 0.000 claims abstract description 12
- 150000005516 benzylisoquinolines Chemical class 0.000 claims abstract description 12
- 239000013612 plasmid Substances 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 19
- WVTKBKWTSCPRNU-KYJUHHDHSA-N (+)-Tetrandrine Chemical compound C([C@H]1C=2C=C(C(=CC=2CCN1C)OC)O1)C(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2C[C@@H]2N(C)CCC3=CC(OC)=C(OC)C1=C23 WVTKBKWTSCPRNU-KYJUHHDHSA-N 0.000 claims description 16
- KBMSVODXFLAQNJ-ZEBDJQDSSA-N Linderane Natural products O=C1O[C@@H]2[C@@H]3O[C@]13CC/C=C(/C)\Cc1occ(C)c21 KBMSVODXFLAQNJ-ZEBDJQDSSA-N 0.000 claims description 11
- 238000006555 catalytic reaction Methods 0.000 claims description 8
- WVTKBKWTSCPRNU-UHFFFAOYSA-N rac-Tetrandrin Natural products O1C(C(=CC=2CCN3C)OC)=CC=2C3CC(C=C2)=CC=C2OC(=C2)C(OC)=CC=C2CC2N(C)CCC3=CC(OC)=C(OC)C1=C23 WVTKBKWTSCPRNU-UHFFFAOYSA-N 0.000 claims description 8
- 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 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 229960001570 ademetionine Drugs 0.000 claims description 4
- -1 hydroxy-azamethyl Chemical group 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 3
- 150000001413 amino acids Chemical group 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- KBMSVODXFLAQNJ-DXGHHDSJSA-N Linderane Chemical compound C1=2C(C)=COC=2C\C(C)=C\CC[C@@]23C(=O)O[C@@H]1[C@@H]2O3 KBMSVODXFLAQNJ-DXGHHDSJSA-N 0.000 claims description 2
- 239000002585 base Substances 0.000 claims description 2
- 235000013399 edible fruits Nutrition 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- JSGZKHJWRITPII-UHFFFAOYSA-N oxoniumylideneazanide Chemical compound O[N] JSGZKHJWRITPII-UHFFFAOYSA-N 0.000 claims description 2
- 241000146029 Lindera aggregata Species 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 14
- 108090000790 Enzymes Proteins 0.000 abstract description 8
- 241000196324 Embryophyta Species 0.000 abstract description 7
- 230000014509 gene expression Effects 0.000 abstract description 7
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 230000003197 catalytic effect Effects 0.000 abstract description 5
- 241001369613 Stephania tetrandra Species 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 230000001276 controlling effect Effects 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 230000006798 recombination Effects 0.000 abstract 1
- 238000005215 recombination Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 10
- 239000000872 buffer Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- INVGWHRKADIJHF-UHFFFAOYSA-N Sanguinarin Chemical compound C1=C2OCOC2=CC2=C3[N+](C)=CC4=C(OCO5)C5=CC=C4C3=CC=C21 INVGWHRKADIJHF-UHFFFAOYSA-N 0.000 description 4
- OROGSEYTTFOCAN-DNJOTXNNSA-N codeine Chemical compound C([C@H]1[C@H](N(CC[C@@]112)C)C3)=C[C@H](O)[C@@H]1OC1=C2C3=CC=C1OC OROGSEYTTFOCAN-DNJOTXNNSA-N 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 4
- XQYZDYMELSJDRZ-UHFFFAOYSA-N papaverine Chemical compound C1=C(OC)C(OC)=CC=C1CC1=NC=CC2=CC(OC)=C(OC)C=C12 XQYZDYMELSJDRZ-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 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 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000009465 prokaryotic expression Effects 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- IPRPPFIAVHPVJH-UHFFFAOYSA-N (4-hydroxyphenyl)acetaldehyde Chemical compound OC1=CC=C(CC=O)C=C1 IPRPPFIAVHPVJH-UHFFFAOYSA-N 0.000 description 2
- 229930008281 A03AD01 - Papaverine Natural products 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- FCEXWTOTHXCQCQ-UHFFFAOYSA-N Ethoxydihydrosanguinarine Natural products C12=CC=C3OCOC3=C2C(OCC)N(C)C(C2=C3)=C1C=CC2=CC1=C3OCO1 FCEXWTOTHXCQCQ-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 229960004126 codeine Drugs 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- OROGSEYTTFOCAN-UHFFFAOYSA-N hydrocodone Natural products C1C(N(CCC234)C)C2C=CC(O)C3OC2=C4C1=CC=C2OC OROGSEYTTFOCAN-UHFFFAOYSA-N 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229960005181 morphine Drugs 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229960001789 papaverine Drugs 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 229940084560 sanguinarine Drugs 0.000 description 2
- YZRQUTZNTDAYPJ-UHFFFAOYSA-N sanguinarine pseudobase Natural products C1=C2OCOC2=CC2=C3N(C)C(O)C4=C(OCO5)C5=CC=C4C3=CC=C21 YZRQUTZNTDAYPJ-UHFFFAOYSA-N 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- AKNNEGZIBPJZJG-MSOLQXFVSA-N (-)-noscapine Chemical compound CN1CCC2=CC=3OCOC=3C(OC)=C2[C@@H]1[C@@H]1C2=CC=C(OC)C(OC)=C2C(=O)O1 AKNNEGZIBPJZJG-MSOLQXFVSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241001127714 Amomum Species 0.000 description 1
- 241001164374 Calyx Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- DITOENWBJBNZSL-UHFFFAOYSA-N O-methyl-hippeastrine Natural products C1=C2C3C4N(C)CCC4=CC(OC)C3OC(=O)C2=CC2=C1OCO2 DITOENWBJBNZSL-UHFFFAOYSA-N 0.000 description 1
- 235000008753 Papaver somniferum Nutrition 0.000 description 1
- 240000001090 Papaver somniferum Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- AKNNEGZIBPJZJG-UHFFFAOYSA-N alpha-noscapine Natural products CN1CCC2=CC=3OCOC=3C(OC)=C2C1C1C2=CC=C(OC)C(OC)=C2C(=O)O1 AKNNEGZIBPJZJG-UHFFFAOYSA-N 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin 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=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 230000003288 anthiarrhythmic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- IZTUINVRJSCOIR-UHFFFAOYSA-N benzylisoquinoline Chemical compound N=1C=CC2=CC=CC=C2C=1CC1=CC=CC=C1 IZTUINVRJSCOIR-UHFFFAOYSA-N 0.000 description 1
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 description 1
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 description 1
- 229940093265 berberine Drugs 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- QORVDGQLPPAFRS-XPSHAMGMSA-N galantamine hydrobromide Chemical compound Br.O1C(=C23)C(OC)=CC=C2CN(C)CC[C@]23[C@@H]1C[C@@H](O)C=C2 QORVDGQLPPAFRS-XPSHAMGMSA-N 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229930013397 isoquinoline alkaloid Natural products 0.000 description 1
- 150000002537 isoquinolines Chemical class 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- PLPRGLOFPNJOTN-UHFFFAOYSA-N narcotine Natural products COc1ccc2C(OC(=O)c2c1OC)C3Cc4c(CN3C)cc5OCOc5c4OC PLPRGLOFPNJOTN-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229960004708 noscapine Drugs 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 230000002048 spasmolytic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- SPOMEWBVWWDQBC-UHFFFAOYSA-K tripotassium;dihydrogen phosphate;hydrogen phosphate Chemical compound [K+].[K+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O SPOMEWBVWWDQBC-UHFFFAOYSA-K 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y201/00—Transferases transferring one-carbon groups (2.1)
- C12Y201/01—Methyltransferases (2.1.1)
- C12Y201/01116—3'-Hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase (2.1.1.116)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了一种粉防己4’‑氧甲基转移酶4’‑OMT及应用，本发明从药用植物粉防己中分离和鉴定了一条新的4’‑氧甲基转移酶，再利用大肠杆菌对该基因进行异源重组表达蛋白，该药用植物中关键酶基因参与调控苄基异喹啉生物碱的合成，奠定了一定的研究基础。相比于其他植物来源的4’‑氧甲基转移酶，本发明的4’‑氧甲基转移酶St4’‑OMT具有更高的底物特异性，催化效率更高，因此具有一定的应用价值。
Description
技术领域
本发明涉及一种粉防己4’-氧甲基转移酶4’-OMT的克隆表达及应用，并公开了其核苷酸序列、氨基酸序列、其酶学性质，属于生物工程技术领域。
背景技术
苄基异喹啉生物碱具有结构和活性多样性，是天然产物领域研究的热点之一。药理学研究表明苄基异喹啉大多活性优异，如罂粟中得到的罂粟碱有解痉作用；粉防己中的汉防己甲素具有防治肝纤维化及抗埃博拉病毒的活性；莲子心中的莲心碱具有广谱的抗心律失常作用。此外，镇痛药吗啡和可待因、抗菌活性的小糵碱和血根碱、止咳药诺斯卡品等已成为临床一线使用的药物。目前药用苄基异喹啉生物碱的获取方式以植物提取为主，但存在苄基异喹啉生物碱含量低、提取废物污染环境、成本高等问题。
虽然结构复杂多样，但苄基异喹啉生物碱生物合成途径的起始步骤却十分相似。以酪氨酸的代谢产物多巴胺和4-羟基-苯乙醛为底物，经去甲乌药碱合酶、去甲乌药碱6-氧甲基转移酶、乌药碱-氮-甲基转移酶、氮甲基乌药碱3’-羟化酶和4’-氧甲基转移酶等多个酶依次催化合成苄基异喹啉类生物碱的共同中间体——牛心果碱((S)-reticuline)。随后通过异构、偶联、重排、甲基化、去甲基化等反应，形成不同骨架类型的苄基异喹啉类生物碱，如吗啡、可待因、小檗碱、血根碱以及罂粟碱等。
4’-氧甲基转移酶(3′-hydroxy-N-methylcoclaurine 4’-O-methyltransferase，EC2.1.1.116,4’-OMT)可以催化3’羟基N甲基乌药碱形成牛心果碱，是苄基异喹啉生物碱合成过程中的关键限速酶。因此，挖掘催化活性高，底物特异性强的4’-氧甲基转移酶是提高苄基异喹啉生物碱含量的迫切现实需要。
发明内容
本发明目的在于从药用植物粉防己中分离和鉴定了一条新的4’-氧甲基转移酶基因，并将其命名为St4’-OMT，利用大肠杆菌对该基因进行异源重组表达蛋白，并公开了其酶学性质及其应用。
本发明采用的技术方案是：
一种粉防己4’-氧甲基转移酶4’-OMT，具有如SEQ ID NO.2所示的氨基酸序列。
所述的粉防己4’-氧甲基转移酶4’-OMT的编码基因，具有如SEQ ID NO.1所示的核苷酸序列。
一种粉防己4’-氧甲基转移酶重组质粒，所述重组质粒中包括所述的粉防己4’-氧甲基转移酶的编码基因。
进一步地，所述重组质粒中的原核表达载体为pET28a。所述重组质粒pET28a-St4’-OMT的序列如SEQ ID NO.4所示，其中原核表达载体pET28a的序列如SEQ ID NO.3所示。其中，构建重组质粒pET28a-St4’-OMT时，先提取粉防己RNA，并将其反转录获得全基因组cDNA。设计4’-氧甲基转移酶基因St4’-OMT的特异性引物，扩增得到4’-氧甲基转移酶基因St4’-OMT全长编码框序列，将扩增得到的4’-氧甲基转移酶基因St4’-OMT全长编码框序列与双酶切后的pET28a质粒片段进行同源重组连接从而构建得到重组表达质粒pET28a-St4’-OMT。
进一步地，4’-氧甲基转移酶基因St4’-OMT的特异性引物如SEQ ID NO.5、SEQ IDNO.6所示。
一种粉防己4’-氧甲基转移酶重组菌株，所述重组菌株包含所述重组质粒的表达载体。
进一步地，通过如下方法构建：
通过将4’-氧甲基转移酶基因St4’-OMT构建至原核表达载体pET28a中得到重组质粒pET28a-St4’-OMT，将阳性重组质粒pET28a-St4’-OMT转入BL21(DE3)感受态细胞中组成重组菌株。重组菌株在诱导剂IPTG的诱导下表达出可溶性蛋白-4’-氧甲基转移酶St4’-OMT。
一种所述粉防己4’-氧甲基转移酶或所述粉防己4’-氧甲基转移酶重组菌株在苄基异喹啉生物碱催化反应中的应用。
所述应用具体为：催化3’羟基-氮甲基乌药碱反应合成牛心果碱。
进一步地，反应体系中还包含辅因子S-腺苷甲硫氨酸，浓度优选为0.1mmol·L-1。
进一步地，所述催化反应的温度为16～45℃，pH为4.0～10.0。
进一步地，所述催化反应的最适温度为30℃，最适pH为8.0。
进一步地，所述粉防己4’-氧甲基转移酶重组菌株催化3’羟基-氮甲基乌药碱反应合成牛心果碱之前，所述粉防己4’-氧甲基转移酶重组菌株在IPTG溶液条件下进行诱导表达。IPTG溶液的浓度优选为0.4mmol·L-1。
本发明的有益效果是：从药用植物粉防己中鉴定出一条新的4’-氧甲基转移酶St4’-OMT，为进一步解析该药用植物中关键酶基因参与调控苄基异喹啉生物碱的合成奠定了一定的研究基础。相比于其他植物来源的4’-氧甲基转移酶，本发明的4’-氧甲基转移酶St4’-OMT具有更高的底物特异性，催化效率更高，因此具有一定的应用价值。
附图说明
图1：pET28a-St4’-OMT的重组载体谱；
图2：SDS-PAGE分析重组工程菌E.coil BL21 IPTG诱导表达情况结果图：M:Marker；1:含pET28a的菌株胞内可溶表达；2：含pET28a-St4’-OMT菌株沉淀表达；3：含pET28a-St4’-OMT菌株胞内可溶表达；
图3：SDS-PAGE分析重组工程菌E.coil BL21胞内可溶蛋白的纯化情况结果图：M：Marker；1:流穿液；2：Elution buffer第一次洗脱；3：Elution buffer第二次洗脱；4：Elution buffer第三次洗脱；5：Elution buffer第四次洗脱。
图4：A:标准品的HPLC分析图(峰1：3’羟基-氮甲基乌药碱；峰2：牛心果碱。)；B:纯化后的pET28a-St4’-OMT蛋白(灭活)催化反应产物的HPLC分析图；C：纯化后pET28a-St4’-OMT蛋白(未灭活)催化反应产物的HPLC分析图；
图5：不同温度对4’-氧甲基转移酶活性的影响结果图；
图6：不同pH对4’-氧甲基转移酶活性的影响，图中，实心方块对应pH 4-6,实心圆对应pH 6-8,实心三角对应pH 8-10；
图7：以3’羟基N甲基乌药碱为底物研究4’-氧甲基转移酶的催化动力学参数拟合结果图；
图8：以不同异喹啉生物碱为底物研究4’-氧甲基转移酶的底物特异性对比图。
具体实施方式
实施例1
本实施例为本发明所述的4’-氧甲基转移酶基因的克隆及大肠杆菌工程菌的构建
粉防己全植株总RNA提取及反转录
1.将粉防己全植株洗净放入液氮速冻，使用已经处理过的研钵对其进行充分研磨，取50-100mg粉末进行RNA提取。利用RNA提取试剂盒和反转录试剂盒得到全植株cDNA备用。
2.大肠杆菌感受态的制备
从LB平板上分别挑取大肠杆菌JM109和BL21(DE3)单菌落，接种于20mL LB培养基中，37℃振荡培养过夜至对数生长中后期。然后将菌液按照1:50(v/v)的比例接种于50mLLB培养基中，37℃震荡培养3h左右至OD600＝0.5得到培养菌液。4℃5000r/min离心5min，收集菌体加入5mL冰上预冷的Solution A(感受态试剂盒)，轻轻晃动离心管使菌体充分悬浮。4℃5000r/min离心5min，收集菌体加入5mL冰上预冷的SolutionB，轻轻晃动离心管使菌体充分悬浮，按照每管100μL进行分装。制作完成得到JM109和BL21感受态细胞。本感受态细胞可存储于-80℃冰箱用于后续DNA的转化实验。
3.PCR扩增4’-氧甲基转移酶基因
1)引物的设计
根据本实验室粉防己转录组数据库中注释的4’-氧甲基转移酶基因St4’-OMT的序列设计引物，引物直接带有同源臂(下划线标示)，具体引物设计为：
F：GCAAATGGGTCGCGGATCCATGGAAGAAGCTATGGATGTCCA(SEQ ID NO.5)
R：CGAGTGCGGCCGCAAGCTTCTAAGGAAAGACCTCAATGACTGATT(SEQ ID NO.6)
2)PCR扩增
扩增反应体系为：模板1μL，上下游引物各1μL，dNTP Mix 4μL，5×primeSTARBuffer 10μL，灭菌的双蒸水32.5μL，primeSTAR DNA聚合酶0.5μL。
PCR反应的条件：98℃30s，56℃30s，72℃2min，35个循环，得到PCR产物最后保存于4℃中。
PCR产物经过胶回收试剂盒回收后送浙江尚亚生物有限公司测序，测序所得到4’-氧甲基转移酶基因St4’-OMT基因序列如SEQ ID NO.1所示。
3)双酶切和连接
利用限制性内切酶BamH I与Hind III对pET28a质粒(序列如SEQ ID NO.3所示)进行双酶切，酶切体系为10×Fast Digest Buffer 5μL，pET28a质粒30μL，限制性内切酶BamHI与Hind III各2.5μL，灭菌蒸馏水10μL。上述反应在37℃反应1h后采用胶回收试剂盒进行胶回收，然后与PCR回收产物进行连接。
连接体系为：MultiS 1μL，5×CE MultiS Buffer 2μL，pET28a胶回收产物1μL，PCR回收产物1μL，灭菌蒸馏水5μL。
上述反应在37℃反应30min后冰浴5min，准备转化。
4)转化
1.取1管前述制备的JM109感受态置于冰上融化。
2.将连接反应液全部转入JM109感受态中，并吹打均匀。
3.冰浴30min后，在42℃水浴中热激90s，然后冰浴2min。
4.加入600μL LB培养基，在37℃条件下培养45min。
5.5000r/min离心2min，弃500μL上清，剩余菌体和培养液吹打均匀，涂布含有卡那霉素抗性的平板。
6.37℃过夜倒置培养。
7.挑选5个单克隆重组质粒进行菌落PCR阳性鉴定，并将阳性克隆送浙江尚亚生物进行测序。测序正确的阳性重组质粒命名为pET28a-St4’-OMT(如SEQ ID NO.4所示)。其图谱如图1所示。
8.将阳性重组质粒转入BL21(DE3)感受态得到重组菌株备用。
实施例2
本实施例为本发明所述的重组菌株的诱导表达
1.取100μL实施例1制得的重组菌株加入20mL LB培养基，加入20μL卡那霉素溶液(终浓度为100μg/ml)，以空载菌株(pET28a质粒转入BL21(DE3))作为对照，37℃，220rpm，过夜培养得到种子菌液。
2.取上一步骤种子菌液200μL转接加入含20mL LB培养基的三角瓶，加入20μL卡那霉素溶液(终浓度为100μg/ml)，培养至OD600＝0.5。
3.每个三角瓶中加入50μL的IPTG溶液(终浓度为0.4mmol·L-1)，25℃，220rpm，过夜诱导培养。
4.分别取步骤3中诱导的空质粒菌液和带目的基因的重组质粒菌液1mL，8000rpm离心5min，弃上清，再分别加入1mL磷酸缓冲液(pH＝8.0)重悬菌体，超声破碎5min。
5.4℃下，8000rpm离心5min，取上清跑蛋白胶验证，如图2所示，重组蛋白可溶性表达。
6.蛋白纯化及酶活性鉴定
通过上述方法，将剩余的pET28a-St4’-OMT发酵液进行菌体富集破碎得到粗酶液。通过AKTA pure蛋白纯化系统进行Ni-柱纯化，用含300mM的高浓度咪唑缓冲液梯度洗脱目的蛋白，如图3所示，将吸附在柱子上的蛋白洗脱下来得到4’-氧甲基转移酶备用。
实施例3
取20mmol·L-1磷酸二氢钾和磷酸氢二钾缓冲液88μL(pH＝8.0)，加入10μL纯酶(终浓度为10μg·ml-1)，30℃预热5min，加入1μL 3’羟基N甲基乌药碱(终浓度为2mM)，加入1μLS-腺苷甲硫氨酸(终浓度为0.1mmol·L-1)反应15min，加入50μL甲醇终止反应，通过HPLC检测在波长为280nm处的特征峰。HPLC结果如图4所示，本发明所得到的纯酶St4’-OMT可以催化3’羟基N甲基乌药碱进行甲基化反应。
实施例4-7是对纯化得到的蛋白进行酶学性质研究。其反应体系为100μL反应体系，其中底物母液浓度是10mmol·L-1，辅因子为S-腺苷甲硫氨酸，4’-氧甲基转移酶浓度0.63mg·ml-1，反应15min后加入50μL甲醇终止反应，并进行HPLC定量分析。
实施例4
本实施例为本发明所述的研究4’-氧甲基转移酶酶促反应的最适温度值。以3’羟基N甲基乌药碱为底物，在pH＝8.0、辅因子浓度为0.1mmol·L-1的条件下，分别测定不同的温度值(16、20、25、30、40、45、50℃)条件下的牛心果碱的产量。如图5所示，可知4’-氧甲基转移酶酶促反应的最适温度值为30℃。
实施例5
本实施例为本发明所述的研究4’-氧甲基转移酶酶促反应的最适pH值。以3’羟基N甲基乌药碱为底物，在30℃、辅因子浓度为0.1mmol·L-1的条件下，分别测定不同的pH(20mmol·L-1Acetic acid-Sodium acetate，pH 4.0-6.0；20mmol·L-1KH2PO4-K2HPO4，pH6.0-8.0；20mmol·L-1Tris-HCl，pH 8.0-10.0)条件下的牛心果碱的产量。如图6所示，可知4’-氧甲基转移酶酶促反应的最适pH值为8.0。
实施例6
本实施例为本发明所述的以3’羟基N甲基乌药碱为底物研究4’-氧甲基转移酶的催化动力学参数。
在最适反应pH和反应温度下测定该4’-氧甲基转移酶催化不同浓度的3’羟基N甲基乌药碱时的反应速率，反应体系中含0.1mmol·L-1辅因子S-腺苷甲硫氨酸，参照Linewear-Burk双倒数作图法，计算Vmax和Km值，进而算出kcat/Km值，如图7所示，Km＝36.1317μM，Vmax＝0.01263μM·min-1·mg-1，kcat＝0.33S-1，kcat/Km＝0.0092μM-1·S-1。
实施例7
本实施例为本发明所述的以3’羟基N甲基乌药碱、去甲乌药碱、乌药碱、N甲基乌药碱和防己乙素为底物研究4’-氧甲基转移酶的底物特异性。如图8所示，结果表明该4’-氧甲基转移酶只对3’羟基N甲基乌药碱的催化，具有优异的底物特异性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干优化改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (9)
1.一种粉防己4’-氧甲基转移酶4’-OMT，其特征在于，具有如SEQ ID NO.2所示的氨基酸序列。
2.权利要求1所述的粉防己4’-氧甲基转移酶4’-OMT的编码基因，其特征在于，具有如SEQ ID NO.1所示的核苷酸序列。
3.一种粉防己4’-氧甲基转移酶重组质粒，其特征在于，所述重组质粒中包括权利要求2所述的粉防己4’-氧甲基转移酶的编码基因。
4.一种粉防己4’-氧甲基转移酶重组菌株，其特征在于，所述重组菌株包含权利要求3所述重组质粒的表达载体。
5.权利要求1所述粉防己4’-氧甲基转移酶或权利要求4所述粉防己4’-氧甲基转移酶重组菌株在苄基异喹啉生物碱催化反应中的应用。
6.根据权利要求5所述的应用，其特征在于，所述应用具体为：催化3’羟基-氮甲基乌药碱反应合成牛心果碱。
7.根据权利要求6所述的应用，其特征在于，反应体系中还包含辅因子S-腺苷甲硫氨酸。
8.根据权利要求6所述的应用，其特征在于，催化反应的温度为16～45℃，pH为4.0～10.0。
9.根据权利要求6所述的应用，其特征在于，所述粉防己4’-氧甲基转移酶重组菌株催化3’羟基-氮甲基乌药碱反应合成牛心果碱之前，所述粉防己4’-氧甲基转移酶重组菌株在IPTG溶液条件下进行诱导表达。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211411600.8A CN116463310A (zh) | 2022-11-11 | 2022-11-11 | 一种粉防己4’-氧甲基转移酶4’-omt及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211411600.8A CN116463310A (zh) | 2022-11-11 | 2022-11-11 | 一种粉防己4’-氧甲基转移酶4’-omt及应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116463310A true CN116463310A (zh) | 2023-07-21 |
Family
ID=87174117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211411600.8A Pending CN116463310A (zh) | 2022-11-11 | 2022-11-11 | 一种粉防己4’-氧甲基转移酶4’-omt及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116463310A (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3033347A1 (en) * | 2013-08-16 | 2016-06-22 | Epimeron Inc. | Compositions and methods for making noscapine and synthesis intermediates thereof |
| US20180163212A1 (en) * | 2015-05-04 | 2018-06-14 | The Board Of Trustees Of The Leland Stanford Junior University | Benzylisoquinoline alkaloid (bia) precursor producing microbes, and methods of making and using the same |
| US20190078100A1 (en) * | 2017-08-03 | 2019-03-14 | Antheia, Inc. | Engineered benzylisoquinoline alkaloid epimerases and methods of producing benzylisoquinoline alkaloids |
| CN111235081A (zh) * | 2020-01-20 | 2020-06-05 | 赣南师范大学 | 一种生物合成牛心果碱的微生物及其方法 |
| CN112996902A (zh) * | 2018-10-30 | 2021-06-18 | 国立大学法人神户大学 | 用于产生苄基异喹啉生物碱(bia)的重组宿主细胞和苄基异喹啉生物碱(bia)的新的制造方法 |
-
2022
- 2022-11-11 CN CN202211411600.8A patent/CN116463310A/zh active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3033347A1 (en) * | 2013-08-16 | 2016-06-22 | Epimeron Inc. | Compositions and methods for making noscapine and synthesis intermediates thereof |
| US20180163212A1 (en) * | 2015-05-04 | 2018-06-14 | The Board Of Trustees Of The Leland Stanford Junior University | Benzylisoquinoline alkaloid (bia) precursor producing microbes, and methods of making and using the same |
| US20190078100A1 (en) * | 2017-08-03 | 2019-03-14 | Antheia, Inc. | Engineered benzylisoquinoline alkaloid epimerases and methods of producing benzylisoquinoline alkaloids |
| CN112996902A (zh) * | 2018-10-30 | 2021-06-18 | 国立大学法人神户大学 | 用于产生苄基异喹啉生物碱(bia)的重组宿主细胞和苄基异喹啉生物碱(bia)的新的制造方法 |
| CN111235081A (zh) * | 2020-01-20 | 2020-06-05 | 赣南师范大学 | 一种生物合成牛心果碱的微生物及其方法 |
Non-Patent Citations (5)
| Title |
|---|
| LI KL 等: "Transcriptome Analysis of Stephania tetrandra and Characterization of Norcoclaurine-6-O-Methyltransferase Involved in Benzylisoquinoline Alkaloid Biosynthesis", FRONTIERS IN PLANT SCIENCE, vol. 13, 31 March 2022 (2022-03-31), pages 1 - 13 * |
| LI QS 等: "Characterization of O-methyltransferases involved in the biosynthesis of tetrandrine in Stephania tetrandra", JOURNAL OF PLANT PHYSIOLOGY, vol. 250, 12 May 2020 (2020-05-12), pages 1 - 7, XP086185427, DOI: 10.1016/j.jplph.2020.153181 * |
| YANGYANG ZHANG 等: "Comparative Transcriptome Analysis Reveals Candidate Genes Involved in Isoquinoline Alkaloid Biosynthesis in Stephania tetrandra", PLANTA MEDICA, vol. 86, no. 17, 5 August 2020 (2020-08-05), pages 1258 - 1268 * |
| 刘金凤 等: "苄基异喹啉类生物碱生物合成与代谢工程研究进展", 基因组学与应用生物学, vol. 35, no. 8, 30 August 2016 (2016-08-30), pages 2194 - 2200 * |
| 李琦爽: "粉防己碱生物合成途径候选功能基因筛选及去甲乌药碱-6-O-甲基转移酶的功能研究", 中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑, no. 2, 15 February 2021 (2021-02-15), pages 047 - 490 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109609474B (zh) | 一种氨基酸脱氢酶突变体及其在合成l-草铵膦中的应用 | |
| CN108060092B (zh) | 一种重组菌及其用途 | |
| CN115927139A (zh) | 用于制备(r)-网状番荔枝碱及其前体的组合物和方法 | |
| CN108753669B (zh) | 一种腺嘌呤生产菌株及其构建方法和应用 | |
| WO2023208146A1 (zh) | 生物合成麦角硫因的方法及载体 | |
| CN104480127B (zh) | 超嗜热糖苷酶突变体及其在人参皂苷ck制备中的应用 | |
| WO2024045796A1 (zh) | 一种溶剂耐受性提高的环糊精葡萄糖基转移酶及其制备 | |
| CN113088501B (zh) | 一种用于生产l-草铵膦的谷氨酸脱氢酶突变体及l-草铵膦生产方法 | |
| CN111154665B (zh) | 一株重组解脂耶罗维亚酵母及其构建方法和应用 | |
| CN105255934A (zh) | 一种高效联产α-氨基丁酸及葡萄糖酸的策略 | |
| CN116463310A (zh) | 一种粉防己4’-氧甲基转移酶4’-omt及应用 | |
| CN115109769B (zh) | 一种l-苏氨酸醛缩酶突变体以及在l-丝氨酸合成中的应用 | |
| CN116536285A (zh) | 一种粉防己乌药碱氮甲基转移酶cnmt及应用 | |
| CN115960879A (zh) | 一种d-阿洛酮糖3-差向异构酶突变体文库的高通量筛选方法及获得的突变体 | |
| CN115873836A (zh) | 一种橙花叔醇合成酶及应用 | |
| CN114990043A (zh) | 代谢赖氨酸的工程菌及其构建方法与应用 | |
| CN116790545A (zh) | 用于重楼皂苷生物合成的糖基转移酶PpUGT2 | |
| CN109609522B (zh) | 博落回二氢苯并菲啶氧化酶基因优化序列及其应用 | |
| CN112410353B (zh) | 一种fkbS基因、含其的基因工程菌及其制备方法和用途 | |
| CN109762801B (zh) | 卤醇脱卤酶突变体及其在合成手性药物中间体中的应用 | |
| CN110846289A (zh) | 一种鲍氏不动杆菌黄嘌呤脱氢酶突变体及其应用 | |
| CN111808836A (zh) | 耐热的i型普鲁兰酶的突变体酶及其制备方法与应用 | |
| CN105018504A (zh) | 一种改造后的荧光素酶基因、含该基因的重组载体、工程菌及其构建方法和发酵方法 | |
| CN115261343B (zh) | 一种黑曲霉酯合成酶An3131、编码基因及其应用 | |
| CN115261396B (zh) | 一种黑曲霉酯合成酶An605、编码基因及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |