JP2019205460A - 特定の代謝物の細胞内濃度がその野生型に比べて増加した細胞の同定方法であって、細胞の改変が組み換え操作によって達成される同定方法、ならびに特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞の製造方法、この代謝物の製造方法、およびそれに適した核酸 - Google Patents
特定の代謝物の細胞内濃度がその野生型に比べて増加した細胞の同定方法であって、細胞の改変が組み換え操作によって達成される同定方法、ならびに特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞の製造方法、この代謝物の製造方法、およびそれに適した核酸 Download PDFInfo
- Publication number
- JP2019205460A JP2019205460A JP2019142159A JP2019142159A JP2019205460A JP 2019205460 A JP2019205460 A JP 2019205460A JP 2019142159 A JP2019142159 A JP 2019142159A JP 2019142159 A JP2019142159 A JP 2019142159A JP 2019205460 A JP2019205460 A JP 2019205460A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- metabolite
- gene
- dna
- production
- 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
- 239000002207 metabolite Substances 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 76
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 72
- 230000001965 increasing effect Effects 0.000 title claims abstract description 37
- 230000003834 intracellular effect Effects 0.000 title claims abstract description 25
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 13
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 13
- 230000004048 modification Effects 0.000 title abstract description 3
- 238000012986 modification Methods 0.000 title abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 112
- 108010091086 Recombinases Proteins 0.000 claims abstract description 68
- 102000018120 Recombinases Human genes 0.000 claims abstract description 57
- 210000004027 cell Anatomy 0.000 claims description 139
- 108020004414 DNA Proteins 0.000 claims description 97
- 239000013612 plasmid Substances 0.000 claims description 59
- 230000035772 mutation Effects 0.000 claims description 48
- 230000006798 recombination Effects 0.000 claims description 45
- 238000005215 recombination Methods 0.000 claims description 45
- 210000000349 chromosome Anatomy 0.000 claims description 36
- 239000013598 vector Substances 0.000 claims description 33
- 235000018102 proteins Nutrition 0.000 claims description 24
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 239000006285 cell suspension Substances 0.000 claims description 23
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 21
- 150000001413 amino acids Chemical class 0.000 claims description 19
- 235000001014 amino acid Nutrition 0.000 claims description 17
- 239000004472 Lysine Substances 0.000 claims description 14
- 150000007524 organic acids Chemical class 0.000 claims description 10
- 239000004480 active ingredient Substances 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000011782 vitamin Substances 0.000 claims description 9
- 235000013343 vitamin Nutrition 0.000 claims description 9
- 229940088594 vitamin Drugs 0.000 claims description 9
- 229930003231 vitamin Natural products 0.000 claims description 9
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 8
- 229930195729 fatty acid Natural products 0.000 claims description 8
- 239000000194 fatty acid Substances 0.000 claims description 8
- 150000004665 fatty acids Chemical class 0.000 claims description 8
- 241000186216 Corynebacterium Species 0.000 claims description 7
- 235000019766 L-Lysine Nutrition 0.000 claims description 7
- 238000001917 fluorescence detection Methods 0.000 claims description 6
- 235000005985 organic acids Nutrition 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 5
- 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 claims description 4
- 241000588722 Escherichia Species 0.000 claims description 4
- 150000001720 carbohydrates Chemical class 0.000 claims description 4
- 235000014633 carbohydrates Nutrition 0.000 claims description 4
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 4
- 230000006696 biosynthetic metabolic pathway Effects 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000012239 gene modification Methods 0.000 claims description 2
- 230000005017 genetic modification Effects 0.000 claims description 2
- 235000013617 genetically modified food Nutrition 0.000 claims description 2
- 239000000047 product Substances 0.000 description 43
- 241000588724 Escherichia coli Species 0.000 description 40
- 238000012360 testing method Methods 0.000 description 33
- 239000002609 medium Substances 0.000 description 22
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 20
- 244000005700 microbiome Species 0.000 description 20
- 241000186226 Corynebacterium glutamicum Species 0.000 description 19
- 239000012634 fragment Substances 0.000 description 18
- 230000009466 transformation Effects 0.000 description 16
- 238000009396 hybridization Methods 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 12
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 12
- 229960000268 spectinomycin Drugs 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 230000010354 integration Effects 0.000 description 10
- 150000003384 small molecules Chemical class 0.000 description 10
- 102000053602 DNA Human genes 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000010367 cloning Methods 0.000 description 9
- 229930027917 kanamycin Natural products 0.000 description 9
- 229960000318 kanamycin Drugs 0.000 description 9
- 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 9
- 229930182823 kanamycin A Natural products 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 8
- 230000002759 chromosomal effect Effects 0.000 description 8
- 238000010276 construction Methods 0.000 description 8
- 238000004520 electroporation Methods 0.000 description 8
- 235000011187 glycerol Nutrition 0.000 description 8
- 101150043597 murE gene Proteins 0.000 description 8
- 238000005457 optimization Methods 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 235000018977 lysine Nutrition 0.000 description 7
- 230000000813 microbial effect Effects 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241001138501 Salmonella enterica Species 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 description 5
- 102000004594 DNA Polymerase I Human genes 0.000 description 5
- 108010017826 DNA Polymerase I Proteins 0.000 description 5
- 241000192132 Leuconostoc Species 0.000 description 5
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 5
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 230000002950 deficient Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 230000008929 regeneration Effects 0.000 description 5
- 238000011069 regeneration method Methods 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 229930101283 tetracycline Natural products 0.000 description 5
- 241000193403 Clostridium Species 0.000 description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 4
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 description 4
- JEVVKJMRZMXFBT-XWDZUXABSA-N Lycophyll Natural products OC/C(=C/CC/C(=C\C=C\C(=C/C=C/C(=C\C=C\C=C(/C=C/C=C(\C=C\C=C(/CC/C=C(/CO)\C)\C)/C)\C)/C)\C)/C)/C JEVVKJMRZMXFBT-XWDZUXABSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 241000589516 Pseudomonas Species 0.000 description 4
- 239000004098 Tetracycline Substances 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 description 4
- 229960004999 lycopene Drugs 0.000 description 4
- 235000012661 lycopene Nutrition 0.000 description 4
- 239000001751 lycopene Substances 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 229960002180 tetracycline Drugs 0.000 description 4
- 235000019364 tetracycline Nutrition 0.000 description 4
- 150000003522 tetracyclines Chemical class 0.000 description 4
- ZCIHMQAPACOQHT-ZGMPDRQDSA-N trans-isorenieratene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/c1c(C)ccc(C)c1C)C=CC=C(/C)C=Cc2c(C)ccc(C)c2C ZCIHMQAPACOQHT-ZGMPDRQDSA-N 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000589291 Acinetobacter Species 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 206010061765 Chromosomal mutation Diseases 0.000 description 3
- 241000427397 Corynebacterium aurimucosum Species 0.000 description 3
- 241000186227 Corynebacterium diphtheriae Species 0.000 description 3
- 101100316841 Escherichia phage lambda bet gene Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000605118 Thiobacillus Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 101150045500 galK gene Proteins 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 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 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- -1 linoleic acid, alcohols Chemical class 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 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
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000726110 Azoarcus Species 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
- 101100364969 Dictyostelium discoideum scai gene Proteins 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- 101000906736 Escherichia phage Mu DNA circularization protein N Proteins 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 101100364971 Mus musculus Scai gene Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241000520272 Pantoea Species 0.000 description 2
- 108091036333 Rapid DNA Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 241000863430 Shewanella Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 244000057717 Streptococcus lactis Species 0.000 description 2
- 235000014897 Streptococcus lactis Nutrition 0.000 description 2
- 101000764570 Streptomyces phage phiC31 Probable tape measure protein Proteins 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical group O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000012742 biochemical analysis Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- 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 2
- 239000013611 chromosomal DNA Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical group NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 235000013681 dietary sucrose Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000006481 glucose medium Substances 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 235000021109 kimchi Nutrition 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 101150035025 lysC gene Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000013586 microbial product Substances 0.000 description 2
- 238000007479 molecular analysis Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000010187 selection method Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 229960004793 sucrose Drugs 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- BWLBGMIXKSTLSX-UHFFFAOYSA-N 2-hydroxyisobutyric acid Chemical compound CC(C)(O)C(O)=O BWLBGMIXKSTLSX-UHFFFAOYSA-N 0.000 description 1
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 description 1
- 108010000700 Acetolactate synthase Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241000235349 Ascomycota Species 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 241000195597 Chlamydomonas reinhardtii Species 0.000 description 1
- 241001112695 Clostridiales Species 0.000 description 1
- 241000193401 Clostridium acetobutylicum Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 241001655326 Corynebacteriales Species 0.000 description 1
- 241001517047 Corynebacterium acetoacidophilum Species 0.000 description 1
- 241000186145 Corynebacterium ammoniagenes Species 0.000 description 1
- 241000807905 Corynebacterium glutamicum ATCC 14067 Species 0.000 description 1
- 241000133018 Corynebacterium melassecola Species 0.000 description 1
- 241000337023 Corynebacterium thermoaminogenes Species 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 101100226347 Escherichia phage lambda exo gene Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 102000048120 Galactokinases Human genes 0.000 description 1
- 108700023157 Galactokinases Proteins 0.000 description 1
- 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 1
- 108010020056 Hydrogenase Proteins 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- 241000144155 Microbacterium ammoniaphilum Species 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 241001208561 Mycobacterium virus Che9c Species 0.000 description 1
- 241000141172 Mycobacterium virus Halo Species 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 241001522316 Pyrrhula pyrrhula Species 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 241001223867 Shewanella oneidensis Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 101100309436 Streptococcus mutans serotype c (strain ATCC 700610 / UA159) ftf gene Proteins 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 241000235013 Yarrowia Species 0.000 description 1
- 241000235015 Yarrowia lipolytica Species 0.000 description 1
- 241000607477 Yersinia pseudotuberculosis Species 0.000 description 1
- 208000025087 Yersinia pseudotuberculosis infectious disease Diseases 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229930013930 alkaloid Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001195 anabolic effect Effects 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 1
- 229960004191 artemisinin Drugs 0.000 description 1
- 229930101531 artemisinin Natural products 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000037429 base substitution Effects 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 238000013452 biotechnological production Methods 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- FLKYBGKDCCEQQM-WYUVZMMLSA-M cefazolin sodium Chemical compound [Na+].S1C(C)=NN=C1SCC1=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)CN3N=NN=C3)[C@H]2SC1 FLKYBGKDCCEQQM-WYUVZMMLSA-M 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011437 continuous method Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 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
- 239000003480 eluent Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000015244 frankfurter Nutrition 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 230000008826 genomic mutation Effects 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 108010072136 iron hydrogenase Proteins 0.000 description 1
- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 125000000904 isoindolyl group Chemical class C=1(NC=C2C=CC=CC12)* 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 125000003977 lipoyl group Chemical group S1SC(C([H])([H])C(C(C(C(=O)[*])([H])[H])([H])[H])([H])[H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910001510 metal chloride Inorganic materials 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 210000001577 neostriatum Anatomy 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229930015704 phenylpropanoid Chemical class 0.000 description 1
- 125000001474 phenylpropanoid group Chemical class 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001522 polyglycol ester Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000007363 regulatory process Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 102220265074 rs1442801795 Human genes 0.000 description 1
- 101150025220 sacB gene Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- OFVLGDICTFRJMM-WESIUVDSSA-N tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
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.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
-
- 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
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
i)前記種類の細胞を含む細胞懸濁物を準備する段階、
ii)少なくとも一つの改変遺伝子G1〜Gnまたは少なくとも一つの変異M1〜Mmを含むDNAの添加下での組み換え操作によって細胞を遺伝的に改変して、中の細胞が特定の代謝物の細胞内濃度に関して異なる細胞懸濁物を得る段階、
iii)代謝物センサーを用いた蛍光検出によって、特定の代謝物の細胞内濃度が増加した個別の細胞を細胞懸濁物中から同定する段階
を含む方法を含む。
i)前記種類の細胞を含む細胞懸濁物を準備する段階;
ii)少なくとも一つの改変遺伝子G1〜Gnまたは少なくとも一つの変異M1〜Mmを含むDNAの添加下での組み換え操作によって細胞を遺伝的に改変して、中の細胞が特定の代謝物の細胞内濃度に関して異なる細胞懸濁物を得る段階;
iii)代謝物センサーを用いた蛍光検出によって、特定の代謝物の細胞内濃度が増加した個別の細胞を細胞懸濁物中から同定する段階;
iv)細胞懸濁物から同定後の細胞を分離する段階;
v)同定および分離後の細胞中から、特定の代謝物の細胞内濃度増加の原因である遺伝的に改変された遺伝子G1〜Gnまたは変異M1〜Mmを同定する段階;
vi)特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞であって、ゲノムが少なくとも一つの遺伝子G1〜Gnおよび/または少なくとも一つの変異M1〜Mmを含む産生細胞を製造する段階
を含む方法にも関する。
a)特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞を上記の方法によって製造する工程段階、
b)栄養素を含む培養培地中で、産生細胞が栄養素から特定の代謝物を産生する条件で細胞を培養する工程段階
を含む、代謝物の製造方法に関する。
National Center for Biotechnology Information(NCBI、Bethesda、Md.、USA)のデータベースにアクセス番号CAD61789.1で寄託されている、Escherichia coliのプロファージRac由来RecTの配列を用いて、プログラムBlast、BLAST2.2.27+による相同性検索を実施した(Wheeler、David; Bhagwat、Medha (2007).「Chapter 9、BLAST QuickStart」、Bergman、Nicholas H. Comparative Genomics Volumes 1 and 2. Methods in Molecular Biology. 395〜396頁、Totowa、NJ: Humana Press(非特許文献39))。相同性検索を、以下のコリネバクテリア種のゲノム中にコードされている全てのタンパク質に対して行った:C.accolens、C.ammoniagenes、C.amycolatum、C.aurimucosum、C.bovis、C.diphtheriae、C.efficiens、C.genitalium、C.glucuronolyticum、C.glutamicum、C.jeikeium、C.kroppenstedtii、C.lipophiloflavum、C.matruchotii、C.nuruki、C.pseudogenitalium、C.pseudotuberculosis、C.resistens、C.striatum、C.tuberculostearicum、C.ulcerans、C.urealyticumおよびC.variabile。
リコンビナーゼのクローニングは、発現ベクターpCLTON2(A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression. Lausberg F、Chattopadhyay AR、Heyer A、Eggeling L、Freudl R. Plasmid. 2012 68(2):142〜7頁(非特許文献32))、およびベクターpEKEx3(The E2 domain of OdhA of Corynebacterium glu−tamicum has succinyltransferase activity dependent on lipoyl residues of the acetyltransferase AceF. Hoffelder M、Raasch K、van Ooyen J、Eggeling L. J Bacteriol. 2010; 192(19):5203〜11頁(非特許文献40))に入れて行った。
Betをクローニングするために、QIAGEN Plasmid Plus Maxi Kit(注文番号12963)を用いてE.coliからベクターpSIM8(Rekombineering: in vivo genetic engineering in E. coli、S. enterica、and beyond. Sawitzke JA、Thomason LC、Costantino N、Bubunenko M、Datta S、Court DL. Methods Enzymol. 2007;421:171〜99頁(非特許文献8))を単離した。このプラスミドは、プライマー対bet−Fおよびbet−Rを用いたPCR増幅のためのテンプレートとして役立った。
bet−F aaggagatatagatATGAGTACTGCACTCGCAAC
bet−R TCATGCTGCCACCTTCTGCTC
Pcl_fw GTAACTATTGCCGATGATAAGCPcl_ rv−pEKEx2_fw CGGCGTTTCACTTCTGAGTTCGGC
recTのクローニングのために、QIAGEN Plasmid Plus Maxi Kit(注文番号12963)を用いて、E.coliからベクターpRAC3(Roles of RecJ、RecO、and RecR in RecET−mediated illegitimate recombination in Escherichia coli. Shiraishi K、Hanada K、Iwakura Y、Ikeda H、J Bacteriol. 2002 Sep;184(17):4715〜21頁(非特許文献41))を単離した。このプラスミドは、プライマー対recT−FおよびrecT−Rを用いたPCR増幅のためのテンプレートとして役立った。
recT−F aaggagatatagatATGACTAAGCAACCACCAATC
recT−R CGGTTATTCCTCTGAATTATCG
gp43のクローニングのために、遺伝子は、Eurofins−MWG−Operon(Anzingerstr.7a、85560 Ebersberg、Deutschland)で合成された。合成された断片の配列を配列番号10として挙げる。この断片を、1407bpの断片として制限酵素BglIIおよびEcoRIを用いて調製し、クレノウ断片で処理し、続いてFermentas製のT4ポリヌクレオチドキナーゼ(注文番号EK0031)でリン酸化した。この断片を例2a記載のように単離し、pCLTON2と連結し、それを用いてE.coli DH5を形質転換した。形質転換後の細胞を、100ng/mLスペクチノマイシン含有複合培地上で平板培養した。
gp61のクローニングのために、遺伝子は、Eurofins−MWG−Operon(Anzingerstr.7a、85560 Ebersberg、Deutschland)で合成された。合成された断片の配列を配列番号11として挙げる。この断片を、制限酵素BglIIおよびMunIを用いて1082bpとして調製し、クレノウ断片で処理し、続いてFermentas製のT4ポリヌクレオチドキナーゼ(注文番号EK0031)でリン酸化した。これを例2a記載のように単離し、pCLTON2と連結し、それを用いてE.coli DH5を形質転換した。形質転換後の細胞を、100ng/mLスペクチノマイシン含有複合培地上で平板培養した。
rCau(cauri_1962)のクローニングのために、遺伝子は、Eurofins−MWG−Operon(Anzingerstr.7a、85560 Ebersberg、Deutschland)で合成された。合成された断片の配列を配列番号1として挙げる。この断片を制限酵素BglIIおよびMunIを用いて839bpとして調製し、クレノウ断片で処理し、続いてFermentas製のT4ポリヌクレオチドキナーゼ(注文番号EK0031)でリン酸化した。これを例2a記載のように単離し、pCLTON2と連結し、それを用いてE.coli DH5を形質転換した。形質転換後の細胞を、100ng/mLスペクチノマイシン含有複合培地上で平板培養した。
recTをpEKEx3中にクローニングするために、PCR増幅用のテンプレートとして例2bからのpCLTON2−recTを使用した。この遺伝子を、プライマー対BglII−RBS−RecT−FおよびEcoRI−RecT−Rを用いて増幅した。
BglII−RBS−RecT−F gcagatctaaggagatatacatATGACTAAGCAACCACCAATCG
EcoRI−RecT−R gcgcgaattccaggCTGAATTATTCCTC
col−pEKEx3−F CGCCGACATCATAACGGTTCTGcol−pEKEx3−R TTATCAGACCGCTTCTGCGTTC
リコンビナーゼBetのクローニングのために、PCR増幅用のテンプレートとして例2eからのpCLTON2−rCauを使用した。この遺伝子を、プライマー対BglII−RBS−bet−FおよびEcoRI−bet−Rを用いて増幅した。
BglII−RBS−bet−F cggcagatctaaggagatatacatATGAGTACTGCACTCGCAAC
EcoRI−bet−R gcgcggaattCATGCTGCCACCTTCTGC
カナマイシン耐性付与遺伝子の非機能コピーをC.glutamicum ATCC13032の染色体中に組み込むために、まず、プライマー対ScaI−KanR−F/Kan(−)−L−RおよびMunI−R−R/Kan(−)−R−Fを用いて、ベクターpJC1(Cremer J、Treptow C、Eggeling L、Sahm H. Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. J Gen Microbiol. 1988;134(12):3221〜9頁(非特許文献42))と融合されるべき2種のPCR断片をテンプレートとして製造した。
ScaI−KanR−F CGAGTACTACAAACGCGGCCATAACKan(−)−L−R GTCGGAAGAGGCATAGAATTCCGTCAGCCAGTTTAG
Kan(−)−R−F GCTGACGGAATTCTATGCCTCTTCCGACCATC
MunI−R−R ATACAATTGAACAAAGCCGCCGTCC
colNCR−L2: CATTGGTCACCTTTGGCGTGTGG
colNCR−R2: AATCAATGAGCGCCGTGAAGAAGG
試験株の形質転換は、TauchらによってCorynebacterium diphtheriaeおよびC.glutamicumについて記載されたように行った(Efficient electrotransformation of Corynebacterium diphtheriae with a mini−replicon derived from the Corynebacterium glutamicum plasmid pGC1. Tauch A、Kirchner O、Loeffler B、Goetker S、Puehler A、Kalinowski J. Curr Microbiol. 2002 Nov;45(5):362〜367頁(非特許文献37))。この株をコンピテントにし、リコンビナーゼをコードするベクターそれぞれ0.5μgをエレクトロポレーションのために使用した。スペクチノマイシン耐性クローンは、スペクチノマイシン100μgを含有する複合培地ブレイン−ハート−インフュージョン−ソルビトール、BHIS(High efficiency electroporation of intact Corynebacterium glutamicum cells. Liebl W、Bayerl A、Schein B、Stillner U、Schleifer KH. FEMS Microbiol Lett. 1989 Dec;53(3):299〜303頁(非特許文献44))(BHIS−Spec100)で選択した。
ATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGC
を有するオリゴKan100*を使用した。このDNAは、Eurofins−MWG−Operon(Anzingerstr.7a、85560 Ebersberg、Deutschland)で合成された。
pEKEx3−recTを有する試験株をBHIS−Spec100 50ml中に接種し、三角フラスコに入れて130rpmおよび30℃で一晩培養した。翌朝、一晩インキュベーションされた培地10mlを、BHIS−Spec100 500ml+0.5mM IPTGに接種し、0、1、または4時間培養した。pCLTON2−recTを有する試験株をBHIS−Spec100 50ml中に接種し、三角フラスコに入れて130rpmおよび30℃で一晩培養した。翌朝、一晩インキュベーションされた培地10mlを、BHIS−Spec100 500ml+テトラサイクリン250ngに接種し、0、1、または4時間培養した。続いて培養物を氷上で30分間冷却し、10%グリセリン、1mMトリス(pH8)50mlで2回、続いて10%グリセリンで2回洗浄した。戻し工程で細胞ペレットを追加的に10%グリセリン1ml中に再懸濁し、それぞれ150μlに小分けし、液体窒素中で瞬間冷凍し、使用まで−75℃で保存した。使用するために、細胞を20分以内に氷の上で穏やかに解凍し、DNA 1μgと混合した。
lysC−100*: TCTTCAAGATCTCCATCGCGCGGCGGCCGTCGGAACGAGGGCAGGTGAAGATGATATCGGTGGTGCCGTCTTCTACAGAAGAGACGTTCTGCAGAACCAT
出発株由来で、murE変異によりリシン産生が増大した株を直接単離するために、pSenLysおよびpEKEx3−recTを有する例6記載の出発株C.glutamicum ATCC13032を使用した。個別のmurE DNAオリゴは、Eurofins−MWG−Operon(Anzingerstr.7a、85560 Ebersberg、Deutschland)によって合成された。以下のmurE配列を使用した:murEG81amb*、配列番号12;murEG81A*、配列番号13;murEG81C*、配列番号14;murE G81D*、配列番号15;murEG81E*、配列番号16;murEG81F*、配列番号17;murEG81H*、配列番号18;murEG81I*、配列番号19;murEG81K*、配列番号20;murEG81L*、配列番号21;murEG81M*、配列番号22;murEG81N*、配列番号23;murEG81P*、配列番号24;murEG81Q*、配列番号25;murEG81R*、配列番号26;murEG81S*、配列番号27;murEG81T*、配列番号28;murEG81V*、配列番号29;murEG81W*、配列番号30;murEG81Y*、配列番号31。
Claims (20)
- 野生型に存在しないリコンビナーゼをコードする遺伝子配列および追加的に代謝物センサーをコードする遺伝子配列を含む、その野生型に比べて遺伝的に改変された細胞。
- 代謝物センサーをコードする遺伝子配列が、アミノ酸、有機酸、脂肪酸、ビタミンまたは植物活性成分を認識するタンパク質をコードする配列であることを特徴とする、請求項1に記載の細胞。
- リコンビナーゼをコードする遺伝子配列が、細胞外から添加されたDNAを細胞固有のDNAと組み換えるタンパク質をコードする配列であることを特徴とする、請求項1または2に記載の細胞。
- リコンビナーゼをコードする遺伝子配列が、配列番号1によるDNAであることを特徴とする、請求項1〜3のいずれか一つに記載の細胞。
- Corynebacterium、EnterobakteriumまたはEscherichia属の細胞であることを特徴とする、請求項1〜4のいずれか一つに記載の細胞。
- 細胞懸濁物中の特定の代謝物の細胞内濃度がその野生型に比べて増加した細胞を同定するための方法であって、
i)請求項1〜4のいずれか一つに記載の細胞を含む細胞懸濁物を準備する工程段階;
ii)少なくとも一つの改変された遺伝子G1〜Gnまたは少なくとも一つの変異M1〜Mmを含むDANNの添加下での組み換え操作によって段階i)による細胞を遺伝的に改変して、中の細胞が特定の代謝物の細胞内濃度に関して異なる細胞懸濁物を得る工程段階;
iii)代謝物センサーを用いた蛍光検出によって、特定の代謝物の細胞内濃度が増加した個別の細胞を細胞懸濁物中から同定する工程段階、
を含む方法。 - 特定の代謝物の産生が最適化された、その野生型に比べて遺伝的に改変された細胞の製造方法であって:
i)請求項1〜4のいずれか一つに記載の細胞を含む細胞懸濁物を準備する段階;
ii)少なくとも一つの改変された遺伝子G1〜Gnまたは少なくとも一つの変異M1〜Mmを含むDNAの添加下での組み換え操作によって段階i)による細胞を遺伝的に改変して、中の細胞が特定の代謝物の細胞内濃度に関して異なる細胞懸濁物を得る段階;
iii)代謝物センサーを用いた蛍光検出によって、特定の代謝物の細胞内濃度が増加した個別の細胞を細胞懸濁物中から同定する段階;
iv)細胞懸濁物から同定後の細胞を分離する段階;
v)同定および分離後の細胞中から、特定の代謝物の細胞内濃度増加の原因である少なくとも一つの遺伝的に改変された遺伝子G1〜Gnまたは少なくとも一つの変異M1〜Mmを同定する段階;
vi)特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞であって、ゲノムが少なくとも一つの遺伝子G1〜Gmおよび/または少なくとも一つの変異M1〜Mmを含む産生細胞を製造する段階
を含む製造方法。 - 段階iii)による同定が、アミノ酸、有機酸、脂肪酸、ビタミン、炭水化物、または植物活性成分についての代謝物センサーによって行われることを特徴とする、請求項6または7に記載の方法。
- 段階ii)による細胞の遺伝的改変が、一つもしくは複数の改変された遺伝子G1〜Gnおよび/または一つもしくは複数の変異M1〜Mmを含む、細胞中に挿入された一つまたは複数のDNA分子を、染色体またはプラスミドとして存在する細胞固有のDNA中に組み込むリコンビナーゼによって行われることを特徴とする、請求項6〜8のいずれか一つに記載の方法。
- 請求項1〜4のいずれか一つに記載の、特定の代謝物の細胞内濃度がその野生型に比べて増加した同定用の細胞が、ベクター中に存在するリコンビナーゼ遺伝子を用いて形質転換されることを特徴とする、請求項6〜9のいずれか一つに記載の方法。
- リコンビナーゼ遺伝子として、配列番号1による遺伝子が使用されることを特徴とする、請求項9または10に記載の方法。
- 配列番号3〜9によるベクターが使用されることを特徴とする、請求項6〜11のいずれか一つに記載の方法。
- 少なくとも一つの改変された遺伝子G1〜Gnおよび/または少なくとも一つの変異M1〜Mmのために、代謝物の生合成経路由来の段階の一つをコードするDNAが使用されることを特徴とする、請求項7〜12のいずれか一つに記載の方法。
- 改変された遺伝子が、配列番号33〜44による配列の群からの少なくとも一つの要素であることを特徴とする、請求項13に記載の方法。
- a)特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された細胞を、請求項7〜14のいずれか一つに記載の方法によって製造する工程段階、
b)栄養素を含む培養培地中で、細胞が栄養素から特定の代謝物を産生する条件で細胞を培養する工程段階
を含む、代謝物の製造方法。 - 代謝物が、アミノ酸、有機酸、脂肪酸、ビタミン、炭水化物、または植物活性成分の群からの要素であることを特徴とする、請求項15に記載の方法。
- 代謝物がアミノ酸L−リシンであることを特徴とする、請求項15または16に記載の方法。
- 配列番号1によるDNAと70%〜100%の相同性を有するリコンビナーゼ遺伝子。
- 配列番号2によるタンパク質と80〜100%の相同性のリコンビナーゼ。
- 配列番号33〜44による核酸。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102012024435.5 | 2012-12-14 | ||
DE102012024435.5A DE102012024435A1 (de) | 2012-12-14 | 2012-12-14 | Verfahren zur Identifizierung einer Zelle mit gegenüber ihrem Wildtyp erhöhten intrazellulären Konzentration eines bestimmten Metaboliten, wobei die Veränderung der Zelle durch Rekombi-neering erreicht wird, sowie ein Verfahren zur Herstellung einer gegenüber ihrem Wildtyp genetisch veränderten Produktionszelle mit optimierter Produktion eines bestimmten Metaboliten, ein Verfahren zur Herstellung dieses Metaboliten, sowie dafür geeignete Nukleinsäuren |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2015546847A Division JP6609475B2 (ja) | 2012-12-14 | 2013-11-15 | 特定の代謝物の細胞内濃度がその野生型に比べて増加した細胞の同定方法であって、細胞の改変が組み換え操作によって達成される同定方法、ならびに特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞の製造方法、この代謝物の製造方法、およびそれに適した核酸 |
Publications (1)
Publication Number | Publication Date |
---|---|
JP2019205460A true JP2019205460A (ja) | 2019-12-05 |
Family
ID=49958130
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2015546847A Expired - Fee Related JP6609475B2 (ja) | 2012-12-14 | 2013-11-15 | 特定の代謝物の細胞内濃度がその野生型に比べて増加した細胞の同定方法であって、細胞の改変が組み換え操作によって達成される同定方法、ならびに特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞の製造方法、この代謝物の製造方法、およびそれに適した核酸 |
JP2019142159A Pending JP2019205460A (ja) | 2012-12-14 | 2019-08-01 | 特定の代謝物の細胞内濃度がその野生型に比べて増加した細胞の同定方法であって、細胞の改変が組み換え操作によって達成される同定方法、ならびに特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞の製造方法、この代謝物の製造方法、およびそれに適した核酸 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2015546847A Expired - Fee Related JP6609475B2 (ja) | 2012-12-14 | 2013-11-15 | 特定の代謝物の細胞内濃度がその野生型に比べて増加した細胞の同定方法であって、細胞の改変が組み換え操作によって達成される同定方法、ならびに特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞の製造方法、この代謝物の製造方法、およびそれに適した核酸 |
Country Status (9)
Country | Link |
---|---|
US (1) | US20160298094A1 (ja) |
EP (1) | EP2931918B1 (ja) |
JP (2) | JP6609475B2 (ja) |
CN (1) | CN104838016B (ja) |
DE (1) | DE102012024435A1 (ja) |
DK (1) | DK2931918T5 (ja) |
ES (1) | ES2643014T3 (ja) |
HU (1) | HUE037001T2 (ja) |
WO (1) | WO2014090208A2 (ja) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10271295B2 (en) | 2016-04-20 | 2019-04-23 | Convida Wireless, Llc | Downlink synchronization |
EP3446432A1 (en) | 2016-04-20 | 2019-02-27 | Convida Wireless, LLC | Configurable reference signals |
KR20180135479A (ko) | 2016-04-20 | 2018-12-20 | 콘비다 와이어리스, 엘엘씨 | 뉴 라디오에서의 물리 채널들 |
WO2017184842A1 (en) | 2016-04-20 | 2017-10-26 | Convida Wireless, Llc | System information provisioning and light weight connection signaling |
WO2017197125A1 (en) | 2016-05-11 | 2017-11-16 | Convida Wireless, Llc | New radio downlink control channel |
US10631319B2 (en) | 2016-06-15 | 2020-04-21 | Convida Wireless, Llc | Grant-less uplink transmission for new radio |
KR20190017994A (ko) | 2016-06-15 | 2019-02-20 | 콘비다 와이어리스, 엘엘씨 | 새로운 라디오를 위한 업로드 제어 시그널링 |
CN109644493A (zh) | 2016-06-15 | 2019-04-16 | 康维达无线有限责任公司 | 无许可操作 |
CN109845129B (zh) | 2016-08-11 | 2023-10-31 | 交互数字专利控股公司 | 针对新无线电在弹性帧结构中进行波束成形扫描和训练 |
WO2018097947A2 (en) | 2016-11-03 | 2018-05-31 | Convida Wireless, Llc | Reference signals and control channels in nr |
CN112753265A (zh) | 2018-09-27 | 2021-05-04 | 康维达无线有限责任公司 | 新无线电的未经许可的频谱中的子频带操作 |
Family Cites Families (42)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB1141107A (en) | 1966-01-26 | 1969-01-29 | Kyowa Hakko Kogyo Company Ltd | Process for producing sugars by fermentation |
DK173507B1 (da) | 1988-09-30 | 2001-01-15 | Hoffmann La Roche | Fremgangsmåde til fremstilling af 2-keto-L-gulonsyre |
US5976843A (en) | 1992-04-22 | 1999-11-02 | Ajinomoto Co., Inc. | Bacterial strain of Escherichia coli BKIIM B-3996 as the producer of L-threonine |
JP3165688B2 (ja) | 1990-04-03 | 2001-05-14 | 協和醗酵工業株式会社 | 発酵法によるd―イソクエン酸の製造法 |
JP3023615B2 (ja) | 1990-08-30 | 2000-03-21 | 協和醗酵工業株式会社 | 発酵法によるl―トリプトファンの製造法 |
DE4130867A1 (de) | 1991-09-17 | 1993-03-18 | Degussa | Verfahren zur fermentativen herstellung von aminosaeuren |
CA2097512A1 (en) | 1991-10-01 | 1993-04-02 | Yoshikuni Deguchi | Thermoplastic resin composition and production thereof |
FR2688515B1 (fr) | 1992-03-13 | 1995-03-31 | Institut Rech Agronomique | Plasmide thermosensible. |
US5322779A (en) | 1992-04-16 | 1994-06-21 | The Research And Development Institute, Inc. At Montana State University | Taxol production by taxomyces andreanae |
ES2149830T3 (es) | 1994-02-22 | 2000-11-16 | Biotechnolog Forschung Gmbh | Procedimiento de fermentacion continuo que es util para la produccion optima simultanea de acido propionico y vitamina b12. |
HU223706B1 (hu) | 1994-12-09 | 2004-12-28 | Ajinomoto Co., Inc. | Új lizin dekarboxiláz gén és eljárás L-lizin termelésére |
AU5265696A (en) | 1995-04-14 | 1996-10-30 | Novopharm Limited | Fermentation for taxol production |
GB2304718B (en) | 1995-09-05 | 2000-01-19 | Degussa | The production of tryptophan by the bacterium escherichia coli |
EP0861902A4 (en) | 1996-09-13 | 2001-07-25 | Kyowa Hakko Kogyo Kk | PROCESS FOR PRODUCING SUGAR NUCLEOTIDES AND COMPLEX CARBON HYDRATES |
US5990350A (en) | 1997-12-16 | 1999-11-23 | Archer Midland Company | Process for making granular L-lysine |
JP4445668B2 (ja) | 1998-04-02 | 2010-04-07 | エボニック デグサ ゲーエムベーハー | アースロバクターアウレッセンスからの組み換えl−n−カルバモイラーゼ、それによるl−アミノ酸の製造方法 |
TWI222464B (en) | 1999-02-08 | 2004-10-21 | Kyowa Hakko Kogyo Kk | Process for producing purine nucleotides |
US20050191732A1 (en) * | 1999-06-25 | 2005-09-01 | Basf Aktiengesellschaft | Corynebacterium glutamicum genes encoding proteins involved in homeostasis and adaptation |
ES2394877T3 (es) | 2000-08-14 | 2013-02-06 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Recombinación homóloga mejorada mediada por proteínas de recombinación de lambda |
DE10154175A1 (de) * | 2001-11-05 | 2003-05-15 | Basf Ag | Gene die für Homeostase-und Adaptions-Proteine codieren |
US7045338B2 (en) | 2002-03-08 | 2006-05-16 | E. I. Du Pont De Nemours And Company | Temperature sensitive mutant derivatives of the broad host range plasmid pBHR1 |
US7674621B2 (en) | 2004-05-21 | 2010-03-09 | The United States Of America As Represented By The Department Of Health And Human Services | Plasmids and phages for homologous recombination and methods of use |
EP1616963B1 (en) | 2004-06-25 | 2009-11-18 | Kyowa Hakko Bio Co., Ltd. | Process for producing dipeptides or dipeptide derivatives |
US7422889B2 (en) * | 2004-10-29 | 2008-09-09 | Stowers Institute For Medical Research | Dre recombinase and recombinase systems employing Dre recombinase |
BRPI0613662A2 (pt) | 2005-07-18 | 2017-05-09 | Basf Ag | microorganismo recombinante, e, método para produzir metionina |
US20080274526A1 (en) | 2007-05-02 | 2008-11-06 | Bramucci Michael G | Method for the production of isobutanol |
EP2386650B1 (en) | 2006-04-07 | 2013-07-03 | Evonik Degussa GmbH | Method for producing L-amino acids using the gap promoter |
DE102006025821A1 (de) | 2006-06-02 | 2007-12-06 | Degussa Gmbh | Ein Enzym zur Herstellung von Mehylmalonatsemialdehyd oder Malonatsemialdehyd |
DE102006032634A1 (de) | 2006-07-13 | 2008-01-17 | Evonik Degussa Gmbh | Verfahren zur Herstellung von L-Aminosäuren |
DE102006048882A1 (de) | 2006-10-17 | 2008-04-24 | Evonik Degussa Gmbh | Allele des rel-Gens aus coryneformen Bakterien |
RU2365622C2 (ru) | 2006-12-22 | 2009-08-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) | СПОСОБ ПРОДУКЦИИ ПУРИНОВЫХ НУКЛЕОЗИДОВ И НУКЛЕОТИДОВ МЕТОДОМ ФЕРМЕНТАЦИИ С ИСПОЛЬЗОВАНИЕМ БАКТЕРИЙ, ПРИНАДЛЕЖАЩИХ К РОДУ Escherichia ИЛИ Bacillus |
US20120165387A1 (en) * | 2007-08-28 | 2012-06-28 | Smolke Christina D | General composition framework for ligand-controlled RNA regulatory systems |
WO2009043372A1 (en) | 2007-10-02 | 2009-04-09 | Metabolic Explorer | Increasing methionine yield |
GB2455335A (en) | 2007-12-06 | 2009-06-10 | United Utilities Plc | Dewatering of Sludge by Fermentation |
WO2009088404A1 (en) | 2007-12-30 | 2009-07-16 | Amyris Biotechnologies, Inc. | Processes for the preparation of artemisinin an its precursors |
JP5395063B2 (ja) | 2008-04-25 | 2014-01-22 | 公益財団法人地球環境産業技術研究機構 | イソプロパノール生産能を有するコリネ型細菌の形質転換体 |
JP5170012B2 (ja) | 2009-06-26 | 2013-03-27 | 東レ株式会社 | カダベリン発酵コリネ型細菌を用いたポリアミドの製造方法 |
EP2327776A1 (en) | 2009-11-30 | 2011-06-01 | Institut National De La Recherche Agronomique | Method for the production of Very Long Chain Fatty Acids (VLCFA) by fermentation with a recombinant Yarrowia sp |
WO2011069105A2 (en) | 2009-12-04 | 2011-06-09 | Richard Allen Kohn | Process for producing fermentation products and fermentation medium compositions therefor |
DE102010019059A1 (de) | 2010-05-03 | 2011-11-03 | Forschungszentrum Jülich GmbH | Sensoren zur intrazellulären Metabolit-Detektion |
DE102012016716A1 (de) | 2012-08-22 | 2014-02-27 | Forschungszentrum Jülich GmbH | Verfahren zur Herstellung von Vektoren enthaltend ein für in seiner feedback-Inhibierung gemindertes oder ausgeschaltetes Enzym kodierendes Gen und deren Verwendung für die Herstellung von Aminosäuren und Nukleotiden |
DE102012017026A1 (de) | 2012-08-28 | 2014-03-06 | Forschungszentrum Jülich GmbH | Sensor für NADP(H) und Entwicklung von Alkoholdehydrogenasen |
-
2012
- 2012-12-14 DE DE102012024435.5A patent/DE102012024435A1/de not_active Withdrawn
-
2013
- 2013-11-15 EP EP13821429.1A patent/EP2931918B1/de not_active Not-in-force
- 2013-11-15 DK DK13821429.1T patent/DK2931918T5/en active
- 2013-11-15 HU HUE13821429A patent/HUE037001T2/hu unknown
- 2013-11-15 JP JP2015546847A patent/JP6609475B2/ja not_active Expired - Fee Related
- 2013-11-15 CN CN201380065322.8A patent/CN104838016B/zh not_active Expired - Fee Related
- 2013-11-15 US US14/651,502 patent/US20160298094A1/en not_active Abandoned
- 2013-11-15 ES ES13821429.1T patent/ES2643014T3/es active Active
- 2013-11-15 WO PCT/DE2013/000683 patent/WO2014090208A2/de active Application Filing
-
2019
- 2019-08-01 JP JP2019142159A patent/JP2019205460A/ja active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2014090208A2 (de) | 2014-06-19 |
ES2643014T3 (es) | 2017-11-21 |
DE102012024435A1 (de) | 2014-07-10 |
EP2931918B1 (de) | 2017-07-12 |
DK2931918T5 (en) | 2017-10-16 |
JP6609475B2 (ja) | 2019-11-20 |
CN104838016A (zh) | 2015-08-12 |
EP2931918A2 (de) | 2015-10-21 |
DK2931918T3 (en) | 2017-10-02 |
WO2014090208A3 (de) | 2014-09-04 |
JP2015536682A (ja) | 2015-12-24 |
CN104838016B (zh) | 2019-05-28 |
US20160298094A1 (en) | 2016-10-13 |
HUE037001T2 (hu) | 2018-08-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6609475B2 (ja) | 特定の代謝物の細胞内濃度がその野生型に比べて増加した細胞の同定方法であって、細胞の改変が組み換え操作によって達成される同定方法、ならびに特定の代謝物を最適に産生する、その野生型に比べて遺伝的に改変された産生細胞の製造方法、この代謝物の製造方法、およびそれに適した核酸 | |
Mahr et al. | Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum | |
Baumgart et al. | Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology | |
Müller et al. | Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity production from methanol | |
Schwentner et al. | Metabolic engineering to guide evolution–Creating a novel mode for L-valine production with Corynebacterium glutamicum | |
JP6359037B2 (ja) | L−バリン産生能が向上した菌株及びこれを用いたl−バリンの産生方法 | |
JP6648345B1 (ja) | 新規ポリペプチド及びこれを用いたimpの生産方法 | |
CN113201535B (zh) | 谷氨酸脱氢酶基因启动子的突变体及其应用 | |
CN106029879B (zh) | 具有提高的l-苏氨酸生产能力的微生物以及使用其生产l-苏氨酸的方法 | |
Tan et al. | Dynamic control of 4-hydroxyisoleucine biosynthesis by modified L-isoleucine biosensor in recombinant Corynebacterium glutamicum | |
US20110300588A1 (en) | Mutations and genetic targets for enhanced l-tyrosine production | |
Prell et al. | Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum | |
CN108350040A (zh) | 用于精细化学品的改进生产的重组微生物 | |
Choi et al. | Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum | |
CN107406864A (zh) | 用于使用缺乏丝氨酸降解途径的基因工程微生物生产l‑丝氨酸的方法 | |
JP2018528771A (ja) | L−リジン生産能を有するコリネバクテリウム属微生物及びそれを用いたl−リジン生産方法 | |
JP2018518977A (ja) | L−リジンを生産する微生物及びそれを用いたl−リジン生産方法 | |
JP2018523496A (ja) | L−リジン生産能を有するコリネバクテリウム属微生物及びそれを用いたl−リジン生産方法 | |
Srivastava et al. | Gene expression systems in corynebacteria | |
Stock et al. | Disruption and complementation of the selenocysteine biosynthesis pathway reveals a hierarchy of selenoprotein gene expression in the archaeon Methanococcus maripaludis | |
Su et al. | Improved ssDNA recombineering for rapid and efficient pathway engineering in Corynebacterium glutamicum | |
Gorshkova et al. | Mu-driven transposition of recombinant mini-Mu unit DNA in the Corynebacterium glutamicum chromosome | |
Kalinowski | The genomes of amino acid-producing corynebacteria | |
KR101411716B1 (ko) | 리보스위치를 이용한 라이신 고생산균 스크리닝 방법 | |
Bott et al. | Novel technologies for optimal strain breeding |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190828 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20190828 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200902 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20201124 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210225 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20210317 |