JP6495940B2 - L−リシン生産能を有する微生物及びそれを用いたl−リシンの生産方法 - Google Patents
L−リシン生産能を有する微生物及びそれを用いたl−リシンの生産方法 Download PDFInfo
- Publication number
- JP6495940B2 JP6495940B2 JP2016561009A JP2016561009A JP6495940B2 JP 6495940 B2 JP6495940 B2 JP 6495940B2 JP 2016561009 A JP2016561009 A JP 2016561009A JP 2016561009 A JP2016561009 A JP 2016561009A JP 6495940 B2 JP6495940 B2 JP 6495940B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- microorganism
- activity
- lysine
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 title claims description 109
- 244000005700 microbiome Species 0.000 title claims description 75
- 238000004519 manufacturing process Methods 0.000 title claims description 27
- 239000004472 Lysine Substances 0.000 title description 63
- 235000019766 L-Lysine Nutrition 0.000 title description 45
- 108090000623 proteins and genes Proteins 0.000 claims description 80
- 230000000694 effects Effects 0.000 claims description 55
- 108091033319 polynucleotide Proteins 0.000 claims description 46
- 102000040430 polynucleotide Human genes 0.000 claims description 46
- 239000002157 polynucleotide Substances 0.000 claims description 46
- 230000004952 protein activity Effects 0.000 claims description 42
- 102000004169 proteins and genes Human genes 0.000 claims description 42
- 230000001965 increasing effect Effects 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 35
- 241000186216 Corynebacterium Species 0.000 claims description 33
- 210000000349 chromosome Anatomy 0.000 claims description 33
- 108010092060 Acetate kinase Proteins 0.000 claims description 32
- 108700023175 Phosphate acetyltransferases Proteins 0.000 claims description 23
- 150000001413 amino acids Chemical group 0.000 claims description 18
- 108010049926 Acetate-CoA ligase Proteins 0.000 claims description 17
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 15
- 230000001105 regulatory effect Effects 0.000 claims description 10
- 230000035772 mutation Effects 0.000 claims description 8
- 102000008146 Acetate-CoA ligase Human genes 0.000 claims 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 253
- 239000013598 vector Substances 0.000 description 44
- 101150006213 ackA gene Proteins 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 29
- 235000018102 proteins Nutrition 0.000 description 26
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 24
- 230000014509 gene expression Effects 0.000 description 18
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 17
- 235000018977 lysine Nutrition 0.000 description 17
- 101150108780 pta gene Proteins 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 16
- 239000013612 plasmid Substances 0.000 description 14
- 102100035709 Acetyl-coenzyme A synthetase, cytoplasmic Human genes 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 230000004913 activation Effects 0.000 description 12
- 238000004925 denaturation Methods 0.000 description 12
- 230000036425 denaturation Effects 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- 230000037361 pathway Effects 0.000 description 12
- 238000006116 polymerization reaction Methods 0.000 description 12
- 108091008146 restriction endonucleases Proteins 0.000 description 11
- 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 10
- 239000008103 glucose Substances 0.000 description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 9
- 101100433987 Latilactobacillus sakei subsp. sakei (strain 23K) ackA1 gene Proteins 0.000 description 9
- 229910052799 carbon Inorganic materials 0.000 description 9
- 238000003780 insertion Methods 0.000 description 9
- 230000037431 insertion Effects 0.000 description 9
- 108700026244 Open Reading Frames Proteins 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 101150047711 acs gene Proteins 0.000 description 6
- 238000000137 annealing Methods 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 230000006801 homologous recombination Effects 0.000 description 6
- 238000002744 homologous recombination Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- -1 glucose Chemical class 0.000 description 5
- 230000009036 growth inhibition Effects 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 description 4
- 241000588722 Escherichia Species 0.000 description 4
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 4
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000002708 enhancing effect Effects 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 150000007524 organic acids Chemical class 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 4
- 150000003628 tricarboxylic acids Chemical class 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 102000000562 Monocarboxylic Acid Transporters Human genes 0.000 description 3
- 101710204259 Monocarboxylic acid transporter Proteins 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 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 3
- 239000004202 carbamide Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000013519 translation 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
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- 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 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-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
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 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
- 229930006000 Sucrose Natural products 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 2
- 229960002079 calcium pantothenate Drugs 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000013611 chromosomal DNA Substances 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 150000002762 monocarboxylic acid derivatives Chemical class 0.000 description 2
- 229960003966 nicotinamide Drugs 0.000 description 2
- 235000005152 nicotinamide Nutrition 0.000 description 2
- 239000011570 nicotinamide Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000013587 production medium Substances 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 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
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 101150025831 Ack gene 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
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 101100242035 Bacillus subtilis (strain 168) pdhA gene Proteins 0.000 description 1
- 101100350224 Bacillus subtilis (strain 168) pdhB gene Proteins 0.000 description 1
- 101100439426 Bradyrhizobium diazoefficiens (strain JCM 10833 / BCRC 13528 / IAM 13628 / NBRC 14792 / USDA 110) groEL4 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000186145 Corynebacterium ammoniagenes Species 0.000 description 1
- 101100236536 Corynebacterium glutamicum (strain ATCC 13032 / DSM 20300 / BCRC 11384 / JCM 1318 / LMG 3730 / NCIMB 10025) glcB gene Proteins 0.000 description 1
- 241000337023 Corynebacterium thermoaminogenes Species 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102100030801 Elongation factor 1-alpha 1 Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000901842 Escherichia coli W Species 0.000 description 1
- 241000488157 Escherichia sp. Species 0.000 description 1
- 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 1
- 101100123255 Komagataeibacter xylinus aceC gene Proteins 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 150000008545 L-lysines Chemical class 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
- 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
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 108010049977 Peptide Elongation Factor Tu Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 101100134871 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) aceE gene Proteins 0.000 description 1
- 101100406344 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) aceF gene Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 101150094017 aceA gene Proteins 0.000 description 1
- 101150036393 aceB gene Proteins 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001371 alpha-amino acids Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 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
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- SCJNCDSAIRBRIA-DOFZRALJSA-N arachidonyl-2'-chloroethylamide Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)NCCCl SCJNCDSAIRBRIA-DOFZRALJSA-N 0.000 description 1
- 101150070136 axeA gene Proteins 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
- 230000002457 bidirectional effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000013601 cosmid vector Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 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
- 239000003814 drug Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 235000013882 gravy Nutrition 0.000 description 1
- 101150077981 groEL gene Proteins 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 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
- 239000008101 lactose Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000037323 metabolic rate Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 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
- 230000017066 negative regulation of growth Effects 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
- 230000008520 organization Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001522 polyglycol ester Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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)
- C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
-
- 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.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01008—Phosphate acetyltransferase (2.3.1.8)
-
- 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)
- C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
- C12Y207/02001—Acetate kinase (2.7.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
[1]酢酸キナーゼ(Acetate kinase)タンパク質活性が内在的活性より強化された、L−リシン生産能を有するコリネバクテリウム属微生物。
[2]上記微生物は、さらに、
(i)ホスホトランスアセチラーゼ(Phosphotransacetylase)タンパク質活性が内在的活性より強化されるか、
(ii)アセチル−CoAシンテターゼ(Acetyl-CoA synthetase)タンパク質活性が導入もしくは強化されるか、又は
(iii)ホスホトランスアセチラーゼタンパク質活性が内在的活性より強化され、かつアセチル−CoAシンテターゼタンパク質活性が導入もしくは強化された、上記[1]に記載の微生物。
[3]上記酢酸キナーゼは、配列番号12のアミノ酸配列を有する、上記[1]に記載の微生物。
[4]上記ホスホトランスアセチラーゼは、配列番号13のアミノ酸配列を有する、上記[2]に記載の微生物。
[5]上記アセチル−CoAシンテターゼは、配列番号14のアミノ酸配列を有する、上記[2]に記載の微生物。
[6]上記微生物はコリネバクテリウム・グルタミカム(Corynebacterium glutamicum)である、上記[1]に記載の微生物。
[7](i)上記[1〜6のいずれかの微生物を培養して培養物を得るステップと、
(ii)上記培養物又は微生物からL−リシンを回収するステップとを含む、L−リシンの生産方法。
本発明者らは、発酵過程中に生成される酢酸又は新たな炭素源として提供される酢酸の効率的な利用のための微生物を開発すべく鋭意努力した結果、酢酸活性化経路における酢酸キナーゼ活性を強化した微生物の酢酸利用能とL−リシン生産能が増加したことを確認し、本発明を完成するに至った。
[実施例]
コリネバクテリウム染色体上にackA遺伝子とpta遺伝子をそれぞれ又は同時に追加導入するためのベクターを作製するために、トランスポゾンの遺伝子位置に標的遺伝子を挿入できるようにpDZベクターから考案されたpDZTNベクター(特許文献5)を基本ベクターとして用いた。2つの遺伝子は染色体上でpta−ackA形態のオペロンを形成しているので、追加導入するackA遺伝子の単独過剰発現のために、pta−ackAオペロンのプロモーター部位をackA遺伝子ORF(open-reading frame)の上流に作動可能に連結した。本実施例に用いたプライマー配列を下記表1に示す。
実施例1で作製した3種のベクターであるpDZTN−Ppta_ackA、pDZTN−pta、pDZTN−pta−ackAを電気パルス法によりコリネバクテリウム・グルタミカムKCCM11016P(上記微生物は、KFCC10881として公開されたが、ブダペスト条約上の国際寄託機関に再寄託され、KCCM11016Pの寄託番号が与えられた。特許文献2,6)に形質転換し、相同組換えにより染色体上に標的遺伝子が挿入された菌株をPCR法で選択的に分離し、KCCM11016P::Ppta_ackA(Tn)、KCCM11016P::pta(Tn)、KCCM11016P::pta−ackA(Tn)と命名した。
<種培地(pH7.0)>
ブドウ糖20g,(NH4)2SO4 10g,ペプトン10g,酵母抽出物5g,尿素1.5g,KH2PO44g,K2HPO4 8g,MgSO4・7H2O 0.5g,ビオチン100μg,チアミンHCl 1000μg,パントテン酸カルシウム2000μg,ニコチンアミド2000μg(蒸留水1リットル中)
<生産培地(pH7.0)>
ブドウ糖100g,酢酸アンモニウム7.1g(添加又は非添加),(NH4)2SO440g,大豆タンパク質2.5g,コーンスティープソリッド(corn steep solid)5g,尿素3g,KH2PO41g,MgSO4・7H2O 0.5g,ビオチン100μg,チアミン塩酸塩1000μg,パントテン酸カルシウム2000μg,ニコチンアミド3000μg,CaCO330g(蒸留水1リットル中)
ackA遺伝子を過剰発現させた場合の酢酸利用能及びその効果を再確認するために、既に報告されている強力なプロモーターであるlysCP1プロモーター(特許文献1)を用いて追加導入するackA遺伝子の発現を誘導した。本実施例で用いたプライマーを下記表2に示す。
実施例3で作製したコリネバクテリウム・グルタミカムKCCM11016P::lysCP1_ackA(Tn)菌株のリシン生産能を確認するために、対照区KCCM11016Pと共に実施例2と同様に培養し、培養終了後にHPLCを用いて分析したL−リシン濃度を下記表3に示す。
エシェリキア由来の酢酸活性化経路をコリネバクテリウムの染色体上に挿入するために、エシェリキア由来の酢酸活性化経路として知られるacs遺伝子(配列番号11)を得て、実施例1のpDZTnベクターを基本ベクターとして染色体挿入用ベクターを作製した。コリネバクテリウム由来pta−ackA遺伝子のプロモーターをacs遺伝子開始コドンの上流に作動可能に連結することにより発現を誘導した。本実施例で用いたプライマーを下記表4に示す。
ackA過剰発現菌株のリシン生産能増加効果を確認するために、コリネバクテリウム属由来の様々なリシン生産菌株を対象にL−リシン生産能を比較した。
Claims (7)
- 改変されていない微生物と比較してより強化されたL−リシン生産能を有するコリネバクテリウム属微生物であって、
その内在的活性に比較して酢酸キナーゼ(Acetate kinase)タンパク質活性がより強化されており、
前記酢酸キナーゼタンパク質活性が、前記タンパク質をコードするポリヌクレオチドのコピー数を増加させる方法、前記タンパク質をコードする遺伝子の調節配列における変異を導入する方法、前記タンパク質をコードする染色体上の遺伝子の調節配列を活性が強力な配列に置換する方法、前記タンパク質をコードする遺伝子を変異した遺伝子で置換して前記タンパク質の活性を増加させる方法、及び前記タンパク質活性が強化されるように前記タンパク質をコードする染色体上の遺伝子に変異を導入する方法からなる群から選択される少なくとも1つの方法によって強化される、微生物。 - 前記微生物は、さらに、
(i)ホスホトランスアセチラーゼ(Phosphotransacetylase)タンパク質活性が内在的活性より強化されているか、
(ii)アセチル−CoAシンテターゼ(Acetyl-CoA synthetase)タンパク質活性が導入もしくは強化されているか、又は
(iii)ホスホトランスアセチラーゼタンパク質活性が内在的活性より強化され、かつアセチル−CoAシンテターゼタンパク質活性が導入もしくは強化されている、請求項1に記載の微生物。 - 前記酢酸キナーゼは、配列番号12のアミノ酸配列を有する、請求項1に記載の微生物。
- 前記ホスホトランスアセチラーゼは、配列番号13のアミノ酸配列を有する、請求項2に記載の微生物。
- 前記アセチル−CoAシンテターゼは、配列番号14のアミノ酸配列を有する、請求項2に記載の微生物。
- 前記コリネバクテリウム属微生物はコリネバクテリウム・グルタミカム(Corynebacterium glutamicum)である、請求項1に記載の微生物。
- (i)請求項1〜6のいずれかに記載のコリネバクテリウム属微生物を培地中で培養するステップと、
(ii)前記培養された微生物又は培養された培地からL−リシンを回収するステップとを含む、L−リシンの生産方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2014-0042397 | 2014-04-09 | ||
KR1020140042397A KR101601404B1 (ko) | 2014-04-09 | 2014-04-09 | L-라이신 생산능을 갖는 미생물 및 이를 이용한 l-라이신 생산 방법 |
PCT/KR2015/003478 WO2015156583A1 (ko) | 2014-04-09 | 2015-04-07 | L-라이신 생산능을 갖는 미생물 및 이를 이용한 l-라이신 생산 방법 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2018193381A Division JP2019030316A (ja) | 2014-04-09 | 2018-10-12 | L−リシン生産能を有する微生物及びそれを用いたl−リシンの生産方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2017510280A JP2017510280A (ja) | 2017-04-13 |
JP6495940B2 true JP6495940B2 (ja) | 2019-04-03 |
Family
ID=54288096
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2016561009A Active JP6495940B2 (ja) | 2014-04-09 | 2015-04-07 | L−リシン生産能を有する微生物及びそれを用いたl−リシンの生産方法 |
JP2018193381A Withdrawn JP2019030316A (ja) | 2014-04-09 | 2018-10-12 | L−リシン生産能を有する微生物及びそれを用いたl−リシンの生産方法 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2018193381A Withdrawn JP2019030316A (ja) | 2014-04-09 | 2018-10-12 | L−リシン生産能を有する微生物及びそれを用いたl−リシンの生産方法 |
Country Status (12)
Country | Link |
---|---|
US (1) | US10179903B2 (ja) |
EP (1) | EP3130663B1 (ja) |
JP (2) | JP6495940B2 (ja) |
KR (1) | KR101601404B1 (ja) |
CN (1) | CN106574232B (ja) |
BR (1) | BR112016023337B1 (ja) |
ES (1) | ES2855048T3 (ja) |
HU (1) | HUE053437T2 (ja) |
MY (1) | MY195658A (ja) |
PL (1) | PL3130663T3 (ja) |
RU (1) | RU2694729C2 (ja) |
WO (1) | WO2015156583A1 (ja) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3613850A1 (en) * | 2018-08-22 | 2020-02-26 | Evonik Operations GmbH | Amino acid production |
EP3927772B1 (de) | 2019-02-20 | 2023-04-12 | Basf Se | Thermoplastische formmasse |
JP7500592B2 (ja) | 2019-02-25 | 2024-06-17 | ビーエーエスエフ ソシエタス・ヨーロピア | 熱可塑性成形組成物 |
KR102271333B1 (ko) * | 2021-01-29 | 2021-06-30 | 씨제이제일제당 (주) | 신규한 미코티온 리덕타제 변이체 및 이를 이용한 l-라이신 생산 방법 |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR940001307B1 (ko) | 1991-12-27 | 1994-02-19 | 제일제당 주식회사 | L-라이신을 생산하는 신규 미생물 |
KR0159812B1 (ko) | 1995-12-20 | 1998-11-16 | 손경식 | 코리네박테리움 글루타미컴 씨에이치 77 및 이 균주를 이용한 l-라이신의 제조 방법 |
KR100397322B1 (ko) | 2000-12-30 | 2003-09-06 | 씨제이 주식회사 | 엘-라이신의 제조방법 |
EP1664318B1 (en) * | 2004-01-30 | 2009-09-23 | Ajinomoto Co., Inc. | L-amino acid-producing microorganism and method for producing l-amino acid |
KR100789271B1 (ko) * | 2005-11-30 | 2008-01-02 | 씨제이 주식회사 | L-라이신 생산능이 향상된 코리네박테리움 속 미생물 및그를 이용하여 l-라이신을 생산하는 방법 |
JP5157180B2 (ja) * | 2006-01-27 | 2013-03-06 | 味の素株式会社 | L−アミノ酸の製造法 |
CN101374953B (zh) | 2006-01-27 | 2011-09-28 | 味之素株式会社 | 用于产生l-氨基酸的方法 |
JP2009060791A (ja) | 2006-03-30 | 2009-03-26 | Ajinomoto Co Inc | L−アミノ酸生産菌及びl−アミノ酸の製造法 |
JP5756259B2 (ja) | 2006-09-15 | 2015-07-29 | シージェイ チェイルジェダン コーポレイション | L−リジン生産能の向上したコリネバクテリウム属およびそれを用いたl−リジン生産方法 |
KR100838035B1 (ko) * | 2006-12-29 | 2008-06-12 | 씨제이제일제당 (주) | L-라이신 생산능이 향상된 코리네박테리움 속 미생물 및그를 이용한 l-라이신 생산 방법 |
KR100930203B1 (ko) | 2008-01-28 | 2009-12-07 | 씨제이제일제당 (주) | 개량된 프로모터 및 이를 이용한 l-라이신의 생산 방법 |
KR101126041B1 (ko) | 2008-04-10 | 2012-03-19 | 씨제이제일제당 (주) | 트랜스포존을 이용한 형질전환용 벡터, 상기 벡터로형질전환된 미생물 및 이를 이용한 l-라이신 생산방법 |
-
2014
- 2014-04-09 KR KR1020140042397A patent/KR101601404B1/ko active IP Right Grant
-
2015
- 2015-04-07 ES ES15776058T patent/ES2855048T3/es active Active
- 2015-04-07 EP EP15776058.8A patent/EP3130663B1/en active Active
- 2015-04-07 US US15/302,738 patent/US10179903B2/en active Active
- 2015-04-07 CN CN201580030811.9A patent/CN106574232B/zh active Active
- 2015-04-07 RU RU2016141594A patent/RU2694729C2/ru active
- 2015-04-07 MY MYPI2016703580A patent/MY195658A/en unknown
- 2015-04-07 BR BR112016023337-9A patent/BR112016023337B1/pt active IP Right Grant
- 2015-04-07 PL PL15776058T patent/PL3130663T3/pl unknown
- 2015-04-07 JP JP2016561009A patent/JP6495940B2/ja active Active
- 2015-04-07 HU HUE15776058A patent/HUE053437T2/hu unknown
- 2015-04-07 WO PCT/KR2015/003478 patent/WO2015156583A1/ko active Application Filing
-
2018
- 2018-10-12 JP JP2018193381A patent/JP2019030316A/ja not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
BR112016023337A2 (pt) | 2018-01-16 |
HUE053437T2 (hu) | 2021-06-28 |
US20180135031A1 (en) | 2018-05-17 |
RU2016141594A3 (ja) | 2018-05-10 |
EP3130663A1 (en) | 2017-02-15 |
KR20150117337A (ko) | 2015-10-20 |
WO2015156583A1 (ko) | 2015-10-15 |
PL3130663T3 (pl) | 2021-10-25 |
US10179903B2 (en) | 2019-01-15 |
ES2855048T3 (es) | 2021-09-23 |
RU2016141594A (ru) | 2018-05-10 |
CN106574232B (zh) | 2020-03-20 |
MY195658A (en) | 2023-02-03 |
BR112016023337B1 (pt) | 2023-10-17 |
EP3130663A4 (en) | 2017-08-30 |
KR101601404B1 (ko) | 2016-03-09 |
EP3130663B1 (en) | 2020-11-18 |
JP2019030316A (ja) | 2019-02-28 |
JP2017510280A (ja) | 2017-04-13 |
CN106574232A (zh) | 2017-04-19 |
RU2694729C2 (ru) | 2019-07-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
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 | |
KR101493585B1 (ko) | 퓨트레신 생산능이 향상된 재조합 미생물 및 이를 이용하여 퓨트레신을 생산하는 방법 | |
JP5945336B2 (ja) | キシロース利用能が付与されたコリネバクテリウム属微生物、及びそれを用いたl−リジンの生産方法 | |
CA3073180C (en) | Microorganism having increased glycine productivity and method for producing fermented composition using the same | |
JP6297134B2 (ja) | プトレシン生産性を有する微生物及びそれを用いたプトレシン生産方法 | |
JP6625133B2 (ja) | ピルビン酸デヒドロゲナーゼ変異体、それを含む微生物及びそれを用いるl−アミノ酸生産方法 | |
TWI632237B (zh) | 用於製造腐胺或鳥胺酸之微生物及使用該微生物製造腐胺或鳥胺酸之方法 | |
JP6286571B2 (ja) | L−リジン生産能が向上したコリネバクテリウム属微生物、及びそれを利用したl−リジン生産方法 | |
JP2019030316A (ja) | L−リシン生産能を有する微生物及びそれを用いたl−リシンの生産方法 | |
JP6291042B2 (ja) | L−アミノ酸の生産方法 | |
JP6465997B2 (ja) | グルコネートリプレッサー変異体、これを含むl−リシンを生産する微生物及びこれを利用したl−リシン生産方法 | |
JP6860565B2 (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 | |
JP6526058B2 (ja) | L−リジンを生産するコリネバクテリウム属微生物、及びそれを利用したl−リジンの生産方法 | |
JP6859437B2 (ja) | L‐リジンを生産するコリネバクテリウム属微生物及びそれを用いたl‐リジンの生産方法 | |
KR101564753B1 (ko) | 코리네형 세균 유래의 치환된 개시코돈을 포함하는 재조합 벡터, 형질전환된 숙주세포 및 이를 이용한 아미노산의 생산방법 | |
KR101493586B1 (ko) | 퓨트레신 생산능이 향상된 재조합 미생물 및 이를 이용하여 퓨트레신을 생산하는 방법 | |
RU2672323C2 (ru) | Микроорганизм для продуцирования диамина и способ получения диамина с его использованием | |
RU2696504C2 (ru) | Микроорганизм для продуцирования диамина и способ получения диамина с его использованием | |
JP6806895B2 (ja) | L−アルギニンを生産するコリネバクテリウム属微生物及びこれを用いたl−アルギニン生産方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20161005 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20161005 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20170704 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20170925 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20171129 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20171227 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20180612 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20181012 |
|
A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20181126 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20190205 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20190307 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6495940 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |