CN1385537A - Process for producing adenosylmethionine by metabolic engineering bacteria - Google Patents
Process for producing adenosylmethionine by metabolic engineering bacteria Download PDFInfo
- Publication number
- CN1385537A CN1385537A CN 02112087 CN02112087A CN1385537A CN 1385537 A CN1385537 A CN 1385537A CN 02112087 CN02112087 CN 02112087 CN 02112087 A CN02112087 A CN 02112087A CN 1385537 A CN1385537 A CN 1385537A
- Authority
- CN
- China
- Prior art keywords
- sam
- synthetic enzyme
- expression plasmid
- cell
- yeast
- 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
- 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 title claims abstract description 88
- 241000894006 Bacteria Species 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims description 22
- 238000012269 metabolic engineering Methods 0.000 title claims description 5
- 230000008569 process Effects 0.000 title description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 66
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 25
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 14
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims abstract description 11
- 229930182817 methionine Natural products 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims description 33
- 102000004190 Enzymes Human genes 0.000 claims description 31
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 28
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 19
- 239000013613 expression plasmid Substances 0.000 claims description 15
- 235000011187 glycerol Nutrition 0.000 claims description 14
- 108010084455 Zeocin Proteins 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 claims description 8
- 230000033228 biological regulation Effects 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 3
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims 3
- LXJXRIRHZLFYRP-UHFFFAOYSA-N glyceraldehyde 3-phosphate Chemical compound O=CC(O)COP(O)(O)=O LXJXRIRHZLFYRP-UHFFFAOYSA-N 0.000 claims 2
- 238000000855 fermentation Methods 0.000 abstract description 22
- 230000004151 fermentation Effects 0.000 abstract description 22
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 229960004452 methionine Drugs 0.000 abstract description 9
- 102000003960 Ligases Human genes 0.000 abstract description 5
- 108090000364 Ligases Proteins 0.000 abstract description 5
- 239000001963 growth medium Substances 0.000 abstract description 3
- 101150028074 2 gene Proteins 0.000 abstract description 2
- 239000003755 preservative agent Substances 0.000 abstract 1
- 230000002335 preservative effect Effects 0.000 abstract 1
- 230000009897 systematic effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 28
- 230000008521 reorganization Effects 0.000 description 11
- 210000005253 yeast cell Anatomy 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000005611 electricity Effects 0.000 description 6
- 238000012262 fermentative production Methods 0.000 description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 238000010353 genetic engineering Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 4
- 101710194180 Alcohol oxidase 1 Proteins 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 4
- 239000008057 potassium phosphate buffer Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000000197 pyrolysis Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 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 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 3
- 229930195722 L-methionine Natural products 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 101150021948 SAM2 gene Proteins 0.000 description 3
- 229930003756 Vitamin B7 Natural products 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 235000011912 vitamin B7 Nutrition 0.000 description 3
- 239000011735 vitamin B7 Substances 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 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 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 150000001447 alkali salts Chemical class 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000013611 chromosomal DNA Substances 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-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
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 239000000592 Artificial Cell Substances 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 102100026115 S-adenosylmethionine synthase isoform type-1 Human genes 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 206010039361 Sacroiliitis Diseases 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 108010038083 amyloid fibril protein AS-SAM Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000002917 arthritic effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 208000024732 dysthymic disease Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000023266 generation of precursor metabolites and energy Effects 0.000 description 1
- 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 1
- 229960003180 glutathione Drugs 0.000 description 1
- 235000003969 glutathione Nutrition 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000005976 liver dysfunction Effects 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- QWVGKYWNOKOFNN-UHFFFAOYSA-N o-cresol Chemical compound CC1=CC=CC=C1O QWVGKYWNOKOFNN-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000005554 pickling Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 229940126532 prescription medicine Drugs 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a method for producing S-adenosyl-L-methionine, SAM by using simple, high-yield and low-cost fermentation, and is characterized by that the exogenous SAM synthetase 2 gene is placed under the control of constitutive GAP promotor, and integrated to methanol yeast to obtain gene engineering bacterium for high-producing SAM. In the simple fermentation culture medium with a certain quantity of externally-added methionine said gene engineering bacterium can be fermented to produce SAM. Said invention also provides a novel strain, the preservative number of said strain is CGMCC No.0746, and its systematic name is Pichia pastoris.
Description
Technical field
The present invention relates to the correlation technique of biotechnology, metabolic engineering and fermentation engineering, specifically, the present invention relates to utilize biotechnology to make up the methanol yeast genetic engineering bacterium, (S-adenosyl-L-methionine, SAM) synthetic metabolism stream comes high yield SAM to utilize this genetic engineering bacterium to strengthen adenosylmethionine.
Background technology
Adenosylmethionine is an intermediary metabolism substance important in the organism, participates in numerous biological respinses.SAM is as the metabolic main methyl donor of organism; participated in the synthetic of important physiologically active substances such as creatine, suprarenin, epiphysin and choline directly; ribose, the proteinic modification that methylates have also been participated in; also be active precursor such as sulfocompounds such as gsh, halfcystine, taurine and coenzyme As, most important to safeguarding HUMAN HEALTH.SAM becomes the clinical prescription medicine from the eighties, and Germany, Italy and Spain have the Duo Jia pharmaceutical factory to produce, and are used for the treatment of liver disorder, sacroiliitis and dysthymia disorders etc.1999, U.S. FDA approved SAMe went on the market as healthcare products, rapidly the U.S. become one of best-selling nutritious prod (poplar is waited quietly. pharmacy progress, 2001,25 (3): 164～167).China is populous, liver dysfunction, arthritic that significant proportion is wherein arranged, in addition, with the quickening pace of modern life with the increase of operating pressure, the patients with depression showed increased, advantages such as SAM effect and side effect in health care is little have huge market outlook, and market demand will increase constantly.
SAM can extract from microbial fermentation, and chemosynthesis or enzymatic are synthetic to be obtained.Chemical method is synthetic distinct disadvantage such as productive rate is low, racemism, and seldom by practical application (Matos J R et al.Biotechnol Appl Biochem, 1987,9:39～52).Although enzyme process synthesizes reaction product purity height, cost is too high, generally only be used for the synthetic isotope mark SAM (Reed B R et al.1986, EP0189322).Some microorganisms can be by cellular metabolism, cylinder accumulation SAM.Especially some bacterial strains of belonging to of yeast Saccharomyces, in substratum, there is a certain amount of methionine(Met), just can in cell, transform and accumulate SAM (Shiozaki S et al.Agric Biol Chem, 1984,48 (9): 2293～2300) of higher concentration.Therefore, by fermentation, zymetology conversion, the extraction purifying SAM of microorganism, be the major industry production approach of present SAM.
The production of SAM is always based on fermentation (Shiozaki S, Yamada H et al., US4562149,1985 of Saccharomyces Cerevisiae in S accharomyces cerevisiae; Shiozaki S, Yamada H et al.Arig Biol Chem, 1989,53:3269～3274).In early days, SAM produces bacterium and obtains by screening wild bacterium separation on a large scale, Shiozaki is by screening 300 multi-strain bacteria strains, be separated to a strain Saccharomyces sake K-6 bacterium (Shiozaki S et al., Agric BiolChem, 1984,48 (9): 2293～2300), SAM that can output 1.55g/L when the 10ml substratum ferments in a small amount; Further optimize culture condition, this bacterium is cultivated SAM output after 7 days up to 10.8g/L (Shiozaki S et al.Journal of Biotechnology, 1986,4:345～354) in the 10L fermentor tank.This is the production peak of being found on various patents and the periodical at present.Although SAM content is very high in the brewing yeast cell, but because brewing yeast cell can not be with high density growth, the thalline output is lower in the unit's of making fermentation volume, and, for fermentation in wild yeast saccharomyces cerevisiae obtains high SAM output, must prolong fermentation period, can reduce fermented quality on the one hand, increase fermentation costs on the other hand greatly.
The methanol yeast Pichia pastoris that develops rapidly at bioengineering field is used for the manufacture order cell protein the earliest in recent years, be compared to traditional yeast saccharomyces cerevisiae, Pichia pastoris has: (1) is the energy high-density growth in basic salt culture medium, and realizes high biological accumulation amount.Large scale fermentation can reach high cell density (dry cell weight of＞130g/L) and biological transformation ratio (12g stem cell/Lh).(2) have powerful and stringent controlled promotor, can accurately control expression of exogenous gene.(3) expression strain is more stable, and expression plasmid is difficult for losing.These characteristics, especially high-density growth make it possess with extremely low cost and produce mesostate in the cell.Yet the intracellular SAM content of wild-type methanol yeast is extremely low.The development of genetic engineering technique and the progress of study metabolic pathways make that on purpose engineered cells becomes possibility.
Thank in the network at the sulfo-of organism, the biosynthesizing of SAM is synthetic through SAM synthetic enzyme [EC 2.5.1.6] catalysis by substrate L-Met and ATP.The isozyme that two kinds of SAM synthetic enzyme are arranged in the S.cerevisiae cell is SAM synthetase 1 and SAM synthetic enzyme 2, and the consistence of both aminoacid sequences is up to 92%.The SAM synthetase 1 is responsible for the most of SAM in the synthetic cell, suppresses phenomenon but the SAM synthetase 1 shows stronger product, and in the presence of excessive Met, transcribing of it is suppressed; SAM synthetic enzyme 2 then shows different control methods, it transcribe the inhibition that is not subjected to Met, before cell grew into stationary phase, SAM synthetic enzyme 2 amounts of genes encoding increased (ThomasD et al.Mol Gen Genet with growth, 1991,226:224～232).We believe the methanol yeast Pichia pastoris genetic engineering bacterium of the SAM synthetic enzyme 2 that makes up recombinant expressed S.cerevisiae source, help fermentative production SAM.
In about the existing report of Pichia pastoris, when expressing exogenous protein, alcohol oxidase 1 (AOX1) promotor is to use at most, higher one of expression efficiency, numerous protein has obtained successful expression (Joan L C, James M C.FEMS Microbiology Reviews, 2000,24:45～66).
According to above-mentioned technical thought, among Yuan of Shanghai Inst. of Biochemistry, Chinese Academy of Sciences one, Li Dongyang and Ji Xinsong provide a kind of method of producing adenosylmethionine, this method has been applied for Chinese patent, and number of patent application is 01132379.5, and the applying date is November 30 calendar year 2001.This invention utilizes methanol yeast to be the host bacterium, by cloning SAM synthetic enzyme 2 genes of known yeast saccharomyces cerevisiae, this enzyme gene is fitted into the expression vector of methanol yeast, and place under the AOX1 promoter regulation, this plasmid is transformed into the methanol yeast cell through after the linearizing, has obtained transformant.The reorganization bacterium that this method utilization obtains is expressed through methanol induction and produces SAM earlier through growing in glycerine.
But the AOX1 expression system is owing to the toxicity of methyl alcohol pair cell and the glycerine reasons such as inhibition to methanol induction, must be earlier in fermentor tank through glycerine vegetative period (seeing the embodiment 6 of this application) of 1-2 days, limit the quantity of subsequently and add glycerine and make cell grow into proper density, just add methyl alcohol at last and begin abduction delivering, the whole cycle reaches 7-12 days, the method of this abduction delivering, the difficult control of condition on the one hand, microbiological contamination easily; The later stage fermentation mass ratio that ferments on the other hand is relatively poor, and production cost is corresponding to higher.The 8 days SAM output of fermenting reaches 1.72g/l.
And Glycerose 3-phosphate dehydrogenase (GAP) is although promotor just is in the news recently, but because its fermentation condition is fairly simple, need not to induce, can the constitutive expression foreign protein make advantages such as fermentation period is relatively shorter, more and more receive publicity, for large-scale industrial production, particularly favourable (Hans RWaterham et al.Gene, 1997,186:37～44).And the expression of exogenous gene under the GAP regulation and control, only need to make sole carbon source with cheap carbon sources such as glycerine, reduce production costs greatly.
The present invention be to Chinese Academy of Sciences's Shanghai biochemical research the improvement of 01132379.5 patent application, SAM synthetic enzyme 2 genes with Saccharomyces Cerevisiae in S accharomyces cerevisiae source place under the GAP promoter regulation first, integration is reconstituted in methanol yeast Pichia pastoris, with the genetic engineering bacterium fermentative production SAM of this constitutive expression.In the hope of providing a kind of not only high yield but also condition is simple, the method for the fermentative production SAM of the low and suitable industrial applications of cost.
Summary of the invention
The purpose of this invention is to provide a kind of simple, cycle weak point, high yield, low-cost fermentation production of adenosine methionine(Met) (S-adenosyl-L-methionine, method SAM).
The present invention designs two single-minded primers according to SAM synthetic enzyme 2 genes (login M23368) in the source of the S.cerevisiae among the GeneBank, and pcr amplification obtains this enzyme gene.SAM synthetic enzyme 2 genes that derive from yeast saccharomyces cerevisiae that the clone is obtained are fitted among the expression vector pGAPZ α A of methanol yeast, obtain expression plasmid pGAPZ α A-SAM2, make SAM synthetic enzyme 2 genes place under the control of promotor GAP.
The expression plasmid that more than obtains is through after the linearizing, and the method that transforms by protoplastis or electricity is transformed into methanol yeast Pichia pastoris host cell GS115.Promoter sequence in this expression plasmid or 3 ' end sequence by with Pichia pastoris chromosomal DNA homologous recombination, make SAM synthetic enzyme 2 gene integrations of external source go in the yeast chromosomal, and in reconstitution cell, stably obtain expressing.It is this that to be integrated in chromosomal recombinant expressed mode stronger than the stability of the plasmid expression form of duplicating separately, after even the experiment of Ohi shows this type of reorganization bacterium continuous passage through 83 generations, karyotype and expressing quantity all do not have obvious variation (H Ohi.yeast, 1998,14:895～903).
Because the expression plasmid that is integrated in the yeast contains the Zeocin resistant gene, make reorganization methanol yeast cell on the resistant panel that contains certain Zeocin concentration, to grow.We just utilize resistance screening to the positive colony cell very easily.
For filtering out the engineering bacteria of the present invention of high expression level, different strains to be cultivated in containing the fermention medium of an amount of methionine(Met), cell can utilize the methionine(Met) in the substratum to do substrate under the catalysis of SAM synthetic enzyme 2, synthetic and accumulation SAM in cell.Just screen according to expression amount and to obtain a plant height and produce bacterial strain.This bacterial strain has carried out preservation on June 10th, 2002 at China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.0746, classification called after Pichiapastoris.
This high yield engineering bacterium fermentation condition is optimized, and the result shows that carbon source, pH and dissolved oxygen are bigger to the cumulative effect of SAM.As carbon source, add a certain amount of methionine(Met) with glycerine, and add a certain amount of glycerine during the fermentation, the 4 days SAM output of fermenting reaches 2.2g/L, is compared to unloaded contrast bacterium output and has improved more than 100 times.
Utilize method fermentative production SAM of the present invention, output is higher than the report of be correlated with patent and document both at home and abroad.And this method fermentation condition is simple, high yield, cost are low, is suitable for suitability for industrialized production.
Effect of the present invention:
One aspect of the present invention has overcome in the method for existing production SAM, the very high but very low shortcoming of thalline output SAM in the unit fermentation volume of SAM content in the brewing yeast cell.That utilizes that methanol yeast has can be in basic salt culture medium middle-high density growth, and realize high biological accumulation amount, large scale fermentation can obtain the characteristics of high cell density and biological transformation ratio, SAM synthetic enzyme 2 genes that derive from yeast saccharomyces cerevisiae are placed under the regulation and control of promotor GAP, recombinant expressed in the methanol yeast cell, make in the cell synthetic and accumulate a large amount of SAM.
On the other hand, external source SAM synthetic enzyme 2 genes under the GAP regulation and control are constitutive expression in P.pastoris, do not need to induce, and, only use cheap glycerine as carbon source, expression condition is very simple, fermentation period is very short, stable yield, and this will reduce production costs greatly.The reorganization bacterium that we obtain, after having optimized fermentation condition, the 4 days SAM output of fermenting just reaches 2.2g/L.Volume productivity reaches 0.55g/L/d.Output and productive rate are higher than the report of relevant patent and document both at home and abroad.Application number is 01132379.5 patent application, ferments 8 days, and SAM output is 1.72g/L, and volume productivity is 0.22g/L/d.Therefore, utilize method fermentative production SAM of the present invention, output is higher, cost is lower, is more suitable for suitability for industrialized production.
Description of drawings
Fig. 1 is the synoptic diagram of recombinant expression plasmid.
Fig. 2 produces curve for the SAM of reorganization bacterium.
Embodiment
Experiment material and method:
1. bacterial strain and reagent
P.pastoris host bacterium GS115 (his4), expression plasmid of yeast pGAPZ α A are Invitrogen company product.Cloned plasmids pUCm-T carrier is preserved (as the T carrier) for this chamber.E.coli bacterial strain TG1 is in order to clone step.
Used T
4Dna ligase, Taq archaeal dna polymerase and restriction enzyme are all available from Takara company.Glue reclaims test kit available from Shanghai Zheng Gu biotech firm.Other all reagent are homemade or the import analytical reagent.
2. substratum
YPDS/Zeocin (10g/L yeast extract paste, 20g/L peptone, 20g/L glucose, 15g/L agar, 1mol/L sorbyl alcohol, 100mg/L Zeocin), YPD (10g/L yeast extract paste, the 20g/L peptone, 20g/L glucose), fermention medium A (10g/L yeast extract paste, the 10g/L peptone, 0.05mol/L potassium phosphate buffer pH6.0,13.4g/L YNB, 4 * 10
-4The g/L vitamin H, 30g/L glycerine), fermention medium B (10g/L yeast extract paste, 10g/L peptone, 0.05mol/L potassium phosphate buffer pH6.0,13.4g/L YNB, 4 * 10
-4The g/L vitamin H, 30g/L glycerine, 0.05mol/L L-methionine(Met))
3, conventional molecular biology operation
The extracting of S.cerevisiae genomic dna, gene clone are pressed the method (.1995 such as Ao Sibai, fine works molecular biology experiment guide, the third edition) of Ao Sibai etc. and are carried out.
The present invention can further illustrate by following embodiment:
The structure of the recombinant expressed SAM synthetic enzyme 2 plasmid pGAPZ α A-SAM2 of embodiment 1 methanol yeast
1, design of primers and PCR reaction
SAM synthetic enzyme 2 genes (accession number M23368) according to the source of the S.cerevisiae among the GeneBank, design two single-minded primers:
5’primer?TAT
TTCGAAACC
ATGGCCAAGAGCAAAACT
3’primer?GCGGCCGC
GAATTCAGCCTAGCATAAAGAAA
Comprise ribosome bind site at design gene 5 ' end primer, introduced a Nsp V restriction enzyme site simultaneously.Gene 3 ' end primer has comprised near the sequence the terminator codon TAG, and has introduced an EcoRI restriction enzyme site.
The total DNA of extracting S.cerevisiae genome karyomit(e) (.1995 such as Ao Sibai, fine works molecular biology experiment guide, the third edition), with the S.cerevisiae chromosomal DNA is template, add a certain amount of primer, Taq archaeal dna polymerase and dNTP mixture, carry out pcr amplification, PCR reaction conditions such as table 1:
Table 1
| Step | Temperature | Time | Cycle number |
| Initial sex change | ??94℃ | ??5min | ????1 |
| Sex change | ??94℃ | ??30sec | ????5 |
| Annealing | ??42℃ | ??30sec | |
| Extend | ??72℃ | ??3min | |
| Sex change | ??94℃ | ??30sec | ????30 |
| Annealing | ??55℃ | ??1min | |
| Extend | ??72℃ | ??3min | |
| Extend at last | ??72℃ | ??10min | ????1 |
2, the clone of PCR product
The PCR product adopts the Shanghai DNA of Zheng Gu biotech firm glue to reclaim the pcr amplified fragment that test kit reclaims the 1200bp size after 1% agarose electrophoresis.Be convenient later operation, with the PCR product subclone that reclaims to the pUCm-T carrier.The plasmid pUCm-SAM2 that obtains after with the two enzymes of Nsp V and EcoRI the SAM2 gene being cut out, is fitted into methanol yeast plasmid pGAPZ α A, obtains expression plasmid pGAPZ α A-SAM2.
The expression plasmid pGAPZ α A-SAM2 (seeing accompanying drawing 1) that obtains all can be obtained fragment about 1200bp through Nsp V and EcoRI double digestion and pcr amplification, consistent with theoretical SAM2 gene.Show after the order-checking that the SAM2 gene order of delivering among itself and the GeneBank is identical.
Embodiment 2 expresses the structure of the reconstitution cell of external source SAM synthetic enzyme 2
1, electricity transforms the preparation of methanol yeast cell
GS115 is in 50ml YPD substratum in inoculation, and 28～30 ℃ are cultured to OD
600Be 1.3～1.5,0～4 ℃, the centrifugal 10min of 4000rpm, bacterial sediment is respectively washed once with the freezing sorbyl alcohol of the freezing sterilized water of 50ml, the freezing sterilized water of 25ml and 2ml 1M respectively, wash equal 0～4 ℃ of back at every turn, the centrifugal 10min of 4000rpm also collects thalline, and is at last that the centrifugal somatic cells is resuspended with the freezing sorbyl alcohol of 100 μ l 1M, promptly obtains the electric shock competent cell.
2, recombinant expression plasmid pGAPZ α A-SAM2 electricity transformed competence colibacillus yeast cell
With the about 10 μ g of the recombinant expression plasmid pGAPZ α A-SAM2 Avr II linearization for enzyme restriction that builds, ethanol sedimentation reclaims linear DNA and is dissolved in the 10 μ l sterilized waters, simultaneously empty carrier pGAPZ α A plasmid is also reclaimed in contrast with identical linearization for enzyme restriction.Above-mentioned linearizing DNA is mixed with 80 μ l GS115 electricity transformant respectively, adopt Bio-Rad electricity conversion instrument GenePulser electric shock to transform, electric conversion condition is: voltage 1500V, electric capacity 25 μ F, resistance 200 Ω.Transform the sorbyl alcohol that adds the precooling of 1ml 1M ice bath in the cup to electricity immediately, and electric converted product transferred to respectively in the aseptic Eppendorf tube, 28～30 ℃ of insulation 1h, add the 0.5mlYPD substratum, 28～30 ℃ of insulation 1.5h, the centrifugal 5min of 6000rpm with 80 μ l supernatant re-suspended cells, coats the YPDS/Zeocin flat board.28～30 ℃ of cultivations are up to single bacterium colony occurring.
3, the multiple sieve of resistant strain
From the YPDS/Zeocin flat board that grows bacterium colony, to put successively with aseptic toothpick and to receive on the fresh YPDS/Zeocin flat board, 28～30 ℃ of cultivations are until single bacterium colony occurring.
SAM in the embodiment 3 in a small amount quick extracting cells
The centrifugal collection of the fermented liquid of known cell concn adds isopyknic 20% perchloric acid in 4 ℃ of extractings more than one hour, and 12000rpm with the membrane filtration of supernatant with 0.2 μ m, therefrom gets 20 μ l after centrifugal 5 minutes, the quantitative SAM of last sample HPLC.
High performance liquid chromatography (HPLC) standard measure of embodiment 4 SAM
Sulphur atom in the SAM molecule has positive charge, and this characteristic makes it to come analyzing and testing with ion exchange column.(4.6 * 250mm), moving phase is strong cation type ion exchange column Hypercil 10 SCX: 0.5M formic acid (pH4.0), flow velocity 2.0ml/min, the optical density(OD) of detection 254nm.Adopt external standard method, promptly the typical curve of doing according to the peak area of the SAM standard substance of different concns comes quantitatively.
The mensuration of embodiment 5 SAM synthetic enzyme vigor
In 10mL fermention medium A (10g/L yeast extract paste, 10g/L peptone, 0.05mol/L potassium phosphate buffer pH6.0,13.4g/L YNB, 4 * 10
-4The g/L vitamin H, 30g/L glycerine) cultivate after 5 days for 28～30 ℃ in, centrifugal collection thalline (4000rpm * 10min, 4 ℃), and use lysis buffer---50mM potassium phosphate buffer (pH7.4), 5% glycerine (V/V), the mercaptoethanol of 5mM and the EDTA of 1mM (methyl-phenoxide of adding 1mM and the PMSF of 1mM are as proteinase inhibitor) clean once.Add the long-pending pickling glass pearl of thalline monoploid and the lysis buffer of 4 times of volumes, handled lysing cell 10 minutes in 0～4 ℃ with ultrasonic wave (in maximum output, 50% work period).Granulated glass sphere and cell relic are through 0～4 ℃, and 10000rpm removed in centrifugal 15 minutes.Crude extract is through 0～4 ℃, and 12000rpm after centrifugal 30 minutes, keeps supernatant.
Set the reaction mixture of 1ml, wherein contain the L-methionine(Met) of 20mM, the ATP of 20mM, the reduced glutathion of 8mM, the MgCl of 20mM
2, the KCl of 100mM, the Tris-HCl of the pH7.4 of 150mM and an amount of cell pyrolysis liquid were 37 ℃ of incubations 60 minutes.The reaction that does not add the L-methionine(Met) is as blank.After reaction finished, 20% perchloric acid solution that adds 1ml stopped the centrifugal precipitation of removing.The gained supernatant is analyzed the content of SAM by HPLC.
Through 5 days cultivation, the enzyme activity of measuring the reorganization bacterium can reach 0.41 gram SAM/ hour/rise cell pyrolysis liquid, be compared to host bacterium GS115 less than 0.01 gram SAM/ hour/rise cell pyrolysis liquid, vigor has improved more than 40 times (table 2) at least.As seen, just because of external source SAM synthetic enzyme 2 expression of gene, improved reorganization bacterium SAM synthetic enzyme vigor.
The comparison of SAM output and SAM synthetic enzyme 2 vigor in table 2 reorganization bacterium and the wild mycetocyte
Wild bacterium reorganization bacterium SAM output (grams per liter)＜0.001 0.041SAM synthetic enzyme vigor (restrain SAM/ hour/rise cell pyrolysis liquid)＜0.01 0.41
Embodiment 6 reorganization bacterium fermentative production SAM
With the Pichia pastoris transformant inoculation 3ml YPD test tube that filters out, 28～30 ℃ of 300r/min overnight incubation contain in the 50mL centrifuge tube of 10mL fermention medium B with the access of 1% inoculum size.28～30 ℃ of 300r/min cultivate.Add glycerine to 0.1% since the 48th hour every 24h, cultured continuously 6 days.SAM is accumulation faster in the reorganization bacterium, and 96h can reach 2.2g/L, and decline is arranged subsequently slightly, is compared to the highest only 0.019g/L of unloaded contrast bacterium, and output has improved more than 100 times of (see figure 2).
Claims (4)
1. method of producing adenosylmethionine with metabolic engineering bacteria, the carrier of recombinant expressed external source SAM synthetic enzyme 2 transforms the back and obtains expression plasmid, expression plasmid is transformed into methanol yeast Pichia pastoris host cell GS115 later on through linearizing, utilize the Zeocin resistance screening positive colony of expression plasmid, in containing the fermention medium of methionine(Met), cultivate then, can generate adenosylmethionine, it is characterized in that SAM synthetic enzyme 2 genes of this external source place under the regulation and control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promotor.
2. the method for claim 1 is characterized in that, described expression vector is pGAPZ α A, obtains expression plasmid pGAPZ α A-SAM2.
3. method as claimed in claim 1 or 2 is characterized in that, in fermention medium, makes carbon source with glycerine.
4. a new metabolic engineering bacterial strain is characterized in that, has integrated SAM synthetic enzyme 2 genes in the yeast saccharomyces cerevisiae source under the GAP promoter regulation on the genomic dna of described bacterial strain, and its kind is Pichia pastoris, and preserving number is CGMCC No.0746.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021120870A CN1191369C (en) | 2002-06-14 | 2002-06-14 | Process for producing adenosylmethionine by metabolic engineering bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021120870A CN1191369C (en) | 2002-06-14 | 2002-06-14 | Process for producing adenosylmethionine by metabolic engineering bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1385537A true CN1385537A (en) | 2002-12-18 |
| CN1191369C CN1191369C (en) | 2005-03-02 |
Family
ID=4741889
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021120870A Expired - Fee Related CN1191369C (en) | 2002-06-14 | 2002-06-14 | Process for producing adenosylmethionine by metabolic engineering bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1191369C (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308457C (en) * | 2003-03-06 | 2007-04-04 | 电化学工业有限公司（国际） | Method for fermentatively producing s-adenosylmethionine |
| CN101012470B (en) * | 2007-01-25 | 2010-06-16 | 华东理工大学 | Method for zymolytic production of S-adenosine methionine |
| CN101392230B (en) * | 2008-01-07 | 2012-05-23 | 北京凯因科技股份有限公司 | Recombinant escherichia coli for expression of adenomethionine synthetase |
| CN102559726A (en) * | 2010-12-31 | 2012-07-11 | 华东理工大学 | Application of GAP (GTPase-Activating Protein) promoter library in regulation of metabolic pathway of S-adenosylmethionine |
| CN1916176B (en) * | 2006-09-11 | 2012-07-18 | 中国科学院上海生命科学研究院 | Method for synthesizing adenosyl methionine through extraneous enzyme catalysis |
| CN101676384B (en) * | 2008-09-16 | 2013-02-06 | 浙江升华拜克生物股份有限公司 | Method for simultaneously producing glutathione and S-adenosyl methionine at high yield |
| CN109957556A (en) * | 2019-03-15 | 2019-07-02 | 自然资源部第三海洋研究所 | A kind of preparation method of S-adenosylmethionine |
| CN115058350A (en) * | 2022-04-28 | 2022-09-16 | 广西大学 | Method for improving S-adenosylmethionine yield by introducing potassium ion transporter |
-
2002
- 2002-06-14 CN CNB021120870A patent/CN1191369C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308457C (en) * | 2003-03-06 | 2007-04-04 | 电化学工业有限公司（国际） | Method for fermentatively producing s-adenosylmethionine |
| CN1916176B (en) * | 2006-09-11 | 2012-07-18 | 中国科学院上海生命科学研究院 | Method for synthesizing adenosyl methionine through extraneous enzyme catalysis |
| CN101012470B (en) * | 2007-01-25 | 2010-06-16 | 华东理工大学 | Method for zymolytic production of S-adenosine methionine |
| CN101392230B (en) * | 2008-01-07 | 2012-05-23 | 北京凯因科技股份有限公司 | Recombinant escherichia coli for expression of adenomethionine synthetase |
| CN101676384B (en) * | 2008-09-16 | 2013-02-06 | 浙江升华拜克生物股份有限公司 | Method for simultaneously producing glutathione and S-adenosyl methionine at high yield |
| CN102559726A (en) * | 2010-12-31 | 2012-07-11 | 华东理工大学 | Application of GAP (GTPase-Activating Protein) promoter library in regulation of metabolic pathway of S-adenosylmethionine |
| CN102559726B (en) * | 2010-12-31 | 2013-10-09 | 华东理工大学 | Application of GAP (GTPase-Activating Protein) promoter library in regulation of metabolic pathway of S-adenosylmethionine |
| CN109957556A (en) * | 2019-03-15 | 2019-07-02 | 自然资源部第三海洋研究所 | A kind of preparation method of S-adenosylmethionine |
| CN109957556B (en) * | 2019-03-15 | 2021-06-04 | 自然资源部第三海洋研究所 | Preparation method of S-adenosylmethionine |
| CN115058350A (en) * | 2022-04-28 | 2022-09-16 | 广西大学 | Method for improving S-adenosylmethionine yield by introducing potassium ion transporter |
| CN115058350B (en) * | 2022-04-28 | 2023-12-01 | 湖南成大生物科技有限公司 | Method for improving S-adenosylmethionine yield by introducing potassium ion transporter |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1191369C (en) | 2005-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101173308A (en) | Method for ferment for producing adenomethionine with genetic engineering bacterium | |
| CN104946576A (en) | Escherichia coli gene engineering strain and construction method thereof, and application of strain in pyruvic acid production | |
| EP2738266B1 (en) | Fermentation process for producing chemicals | |
| CN1191369C (en) | Process for producing adenosylmethionine by metabolic engineering bacteria | |
| CN102933715A (en) | Novel method for producing 3-hydroxypropionic acid from glycerol | |
| CN101613707B (en) | Method for producing glutathione by use of metabolic engineering bacteria | |
| CN110628653A (en) | Proliferation medium of kluyveromyces marxianus and preparation method thereof | |
| Khattab et al. | A novel production method for high-fructose glucose syrup from sucrose-containing biomass by a newly isolated strain of osmotolerant Meyerozyma guilliermondii | |
| CN114317307B (en) | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof | |
| Nie et al. | A novel strategy on the high-cell-density cultivation of Candida utilis for the enhanced production of glutathione | |
| CN112940952B (en) | High-yield ethyl caproate saccharomycete and application thereof | |
| CN115838645A (en) | Yeast strain for high yield of orotic acid and application thereof | |
| CN109929853B (en) | Application of thermophilic bacteria source heat shock protein gene | |
| ES2776361T3 (en) | Strains of microorganism for the production of 2,3-butanediol | |
| CN1160467C (en) | Method of producing adenosylmethionine | |
| CN1916176B (en) | Method for synthesizing adenosyl methionine through extraneous enzyme catalysis | |
| WO2020187388A1 (en) | Lactobacillus diolivorans biotransformation process | |
| CN115093977B (en) | Brevibacterium pullulans strain EP01 for producing fumaric acid and use method thereof | |
| CN115058350B (en) | Method for improving S-adenosylmethionine yield by introducing potassium ion transporter | |
| CN106636251B (en) | Method for high yield of D-ribose by using bacillus subtilis engineering bacteria | |
| CN114634902B (en) | Culture medium and culture method for producing psicose epimerase and application of culture medium and culture method | |
| CN110713940B (en) | High-yield heavy oil aureobasidium pullulans strain and construction method and application thereof | |
| Zhao et al. | Enhanced D-ribose production by genetic modification and medium optimization in Bacillus subtilis 168 | |
| JP3007615B1 (en) | Fermentation method for xylitol production using Candida tropicalis | |
| CN116083379A (en) | Transporter variant and application thereof in R-pantoic acid preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: SHANGHAI INST. OF LIFE SCIENCE, CAS Free format text: FORMER OWNER: INSTITUTE OF BIOCHEMISTRY AND CELL BIOLOGY, SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES Effective date: 20090619 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20090619 Address after: No. 320, Yueyang Road, Shanghai Patentee after: Shanghai Institute of life Sciences, Chinese Academy of Sciences Address before: No. 320, Yueyang Road, Shanghai Patentee before: Inst. of Biochemistry and Cell Biology, Shanghai Research Academy of Life Scienc |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20050302 Termination date: 20160614 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |