CN111433366A - 具有增加的甘氨酸生产能力的微生物和利用其生产发酵组合物的方法 - Google Patents
具有增加的甘氨酸生产能力的微生物和利用其生产发酵组合物的方法 Download PDFInfo
- Publication number
- CN111433366A CN111433366A CN201980004266.4A CN201980004266A CN111433366A CN 111433366 A CN111433366 A CN 111433366A CN 201980004266 A CN201980004266 A CN 201980004266A CN 111433366 A CN111433366 A CN 111433366A
- Authority
- CN
- China
- Prior art keywords
- hisg
- ala
- microorganism
- leu
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 title claims abstract description 117
- 239000004471 Glycine Substances 0.000 title claims abstract description 59
- 244000005700 microbiome Species 0.000 title claims abstract description 56
- 238000000855 fermentation Methods 0.000 title claims abstract description 41
- 230000004151 fermentation Effects 0.000 title claims abstract description 41
- 239000000203 mixture Substances 0.000 title claims abstract description 33
- 230000001965 increasing effect Effects 0.000 title claims abstract description 26
- 238000004519 manufacturing process Methods 0.000 title claims description 33
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 45
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 20
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 20
- 239000004220 glutamic acid Substances 0.000 claims abstract description 20
- 241000186216 Corynebacterium Species 0.000 claims abstract description 19
- 150000001413 amino acids Chemical group 0.000 claims description 50
- 235000001014 amino acid Nutrition 0.000 claims description 32
- 230000000694 effects Effects 0.000 claims description 25
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 19
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 16
- 108010058756 ATP phosphoribosyltransferase Proteins 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 12
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 claims description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 4
- 230000035772 mutation Effects 0.000 abstract description 41
- 108090000623 proteins and genes Proteins 0.000 description 37
- 229960002989 glutamic acid Drugs 0.000 description 36
- 101150032598 hisG gene Proteins 0.000 description 36
- 239000013598 vector Substances 0.000 description 36
- 229940024606 amino acid Drugs 0.000 description 30
- 239000002609 medium Substances 0.000 description 29
- 108020004414 DNA Proteins 0.000 description 22
- 108091033319 polynucleotide Proteins 0.000 description 22
- 102000040430 polynucleotide Human genes 0.000 description 22
- 239000002157 polynucleotide Substances 0.000 description 22
- 239000002773 nucleotide Substances 0.000 description 18
- 125000003729 nucleotide group Chemical group 0.000 description 18
- 108090000790 Enzymes Proteins 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 15
- 238000006467 substitution reaction Methods 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 13
- 239000000047 product Substances 0.000 description 10
- 230000008569 process Effects 0.000 description 9
- 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 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 210000000349 chromosome Anatomy 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 235000015097 nutrients Nutrition 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 235000000346 sugar Nutrition 0.000 description 8
- VIPDPMHGICREIS-GVXVVHGQSA-N Glu-Val-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O VIPDPMHGICREIS-GVXVVHGQSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 108010005233 alanylglutamic acid Proteins 0.000 description 6
- 108010038633 aspartylglutamate Proteins 0.000 description 6
- 108010047857 aspartylglycine Proteins 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000001802 infusion Methods 0.000 description 5
- 235000019640 taste Nutrition 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- 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 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 4
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 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 4
- 229960000344 thiamine hydrochloride Drugs 0.000 description 4
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 4
- 239000011747 thiamine hydrochloride Substances 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 3
- WGDNWOMKBUXFHR-BQBZGAKWSA-N Ala-Gly-Arg Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N WGDNWOMKBUXFHR-BQBZGAKWSA-N 0.000 description 3
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 3
- NIZKGBJVCMRDKO-KWQFWETISA-N Ala-Gly-Tyr Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NIZKGBJVCMRDKO-KWQFWETISA-N 0.000 description 3
- KTXKIYXZQFWJKB-VZFHVOOUSA-N Ala-Thr-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O KTXKIYXZQFWJKB-VZFHVOOUSA-N 0.000 description 3
- UISQLSIBJKEJSS-GUBZILKMSA-N Arg-Arg-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(O)=O UISQLSIBJKEJSS-GUBZILKMSA-N 0.000 description 3
- PQWTZSNVWSOFFK-FXQIFTODSA-N Arg-Asp-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)CN=C(N)N PQWTZSNVWSOFFK-FXQIFTODSA-N 0.000 description 3
- YQGZIRIYGHNSQO-ZPFDUUQYSA-N Arg-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N YQGZIRIYGHNSQO-ZPFDUUQYSA-N 0.000 description 3
- YKZJPIPFKGYHKY-DCAQKATOSA-N Arg-Leu-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O YKZJPIPFKGYHKY-DCAQKATOSA-N 0.000 description 3
- FVBZXNSRIDVYJS-AVGNSLFASA-N Arg-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N FVBZXNSRIDVYJS-AVGNSLFASA-N 0.000 description 3
- NVPHRWNWTKYIST-BPNCWPANSA-N Arg-Tyr-Ala Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 NVPHRWNWTKYIST-BPNCWPANSA-N 0.000 description 3
- CMLGVVWQQHUXOZ-GHCJXIJMSA-N Asn-Ala-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O CMLGVVWQQHUXOZ-GHCJXIJMSA-N 0.000 description 3
- MYRLSKYSMXNLLA-LAEOZQHASA-N Asn-Val-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O MYRLSKYSMXNLLA-LAEOZQHASA-N 0.000 description 3
- SBHUBSDEZQFJHJ-CIUDSAMLSA-N Asp-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O SBHUBSDEZQFJHJ-CIUDSAMLSA-N 0.000 description 3
- KHBLRHKVXICFMY-GUBZILKMSA-N Asp-Glu-Lys Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O KHBLRHKVXICFMY-GUBZILKMSA-N 0.000 description 3
- KTTCQQNRRLCIBC-GHCJXIJMSA-N Asp-Ile-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O KTTCQQNRRLCIBC-GHCJXIJMSA-N 0.000 description 3
- VNXQRBXEQXLERQ-CIUDSAMLSA-N Asp-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N VNXQRBXEQXLERQ-CIUDSAMLSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- NVEASDQHBRZPSU-BQBZGAKWSA-N Gln-Gln-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O NVEASDQHBRZPSU-BQBZGAKWSA-N 0.000 description 3
- AJDMYLOISOCHHC-YVNDNENWSA-N Gln-Gln-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AJDMYLOISOCHHC-YVNDNENWSA-N 0.000 description 3
- QBLMTCRYYTVUQY-GUBZILKMSA-N Gln-Leu-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O QBLMTCRYYTVUQY-GUBZILKMSA-N 0.000 description 3
- SZXSSXUNOALWCH-ACZMJKKPSA-N Glu-Ala-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O SZXSSXUNOALWCH-ACZMJKKPSA-N 0.000 description 3
- RLZBLVSJDFHDBL-KBIXCLLPSA-N Glu-Ala-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RLZBLVSJDFHDBL-KBIXCLLPSA-N 0.000 description 3
- QYPKJXSMLMREKF-BPUTZDHNSA-N Glu-Glu-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)O)N QYPKJXSMLMREKF-BPUTZDHNSA-N 0.000 description 3
- QXUPRMQJDWJDFR-NRPADANISA-N Glu-Val-Ser Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXUPRMQJDWJDFR-NRPADANISA-N 0.000 description 3
- JRDYDYXZKFNNRQ-XPUUQOCRSA-N Gly-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN JRDYDYXZKFNNRQ-XPUUQOCRSA-N 0.000 description 3
- KFMBRBPXHVMDFN-UWVGGRQHSA-N Gly-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCNC(N)=N KFMBRBPXHVMDFN-UWVGGRQHSA-N 0.000 description 3
- UESJMAMHDLEHGM-NHCYSSNCSA-N Gly-Ile-Leu Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O UESJMAMHDLEHGM-NHCYSSNCSA-N 0.000 description 3
- CLNSYANKYVMZNM-UWVGGRQHSA-N Gly-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N CLNSYANKYVMZNM-UWVGGRQHSA-N 0.000 description 3
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- QIVPRLJQQVXCIY-HGNGGELXSA-N His-Ala-Gln Chemical compound C[C@H](NC(=O)[C@@H](N)Cc1cnc[nH]1)C(=O)N[C@@H](CCC(N)=O)C(O)=O QIVPRLJQQVXCIY-HGNGGELXSA-N 0.000 description 3
- MKWSZEHGHSLNPF-NAKRPEOUSA-N Ile-Ala-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O)N MKWSZEHGHSLNPF-NAKRPEOUSA-N 0.000 description 3
- WRDTXMBPHMBGIB-STECZYCISA-N Ile-Tyr-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=C(O)C=C1 WRDTXMBPHMBGIB-STECZYCISA-N 0.000 description 3
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 3
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 3
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 3
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 3
- WSGXUIQTEZDVHJ-GARJFASQSA-N Leu-Ala-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O WSGXUIQTEZDVHJ-GARJFASQSA-N 0.000 description 3
- OXRLYTYUXAQTHP-YUMQZZPRSA-N Leu-Gly-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(O)=O OXRLYTYUXAQTHP-YUMQZZPRSA-N 0.000 description 3
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 3
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 3
- FBNPMTNBFFAMMH-AVGNSLFASA-N Leu-Val-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-AVGNSLFASA-N 0.000 description 3
- FBNPMTNBFFAMMH-UHFFFAOYSA-N Leu-Val-Arg Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(=O)NC(C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-UHFFFAOYSA-N 0.000 description 3
- MUXNCRWTWBMNHX-SRVKXCTJSA-N Lys-Leu-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O MUXNCRWTWBMNHX-SRVKXCTJSA-N 0.000 description 3
- WGBMNLCRYKSWAR-DCAQKATOSA-N Met-Asp-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN WGBMNLCRYKSWAR-DCAQKATOSA-N 0.000 description 3
- CHQWUYSNAOABIP-ZPFDUUQYSA-N Met-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCSC)N CHQWUYSNAOABIP-ZPFDUUQYSA-N 0.000 description 3
- KMSMNUFBNCHMII-IHRRRGAJSA-N Met-Leu-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN KMSMNUFBNCHMII-IHRRRGAJSA-N 0.000 description 3
- VYDLZDRMOFYOGV-TUAOUCFPSA-N Met-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCSC)N VYDLZDRMOFYOGV-TUAOUCFPSA-N 0.000 description 3
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 3
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 3
- ZLGQEBCCANLYRA-RYUDHWBXSA-N Phe-Gly-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O ZLGQEBCCANLYRA-RYUDHWBXSA-N 0.000 description 3
- BIYWZVCPZIFGPY-QWRGUYRKSA-N Phe-Gly-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CO)C(O)=O BIYWZVCPZIFGPY-QWRGUYRKSA-N 0.000 description 3
- CBENHWCORLVGEQ-HJOGWXRNSA-N Phe-Phe-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CBENHWCORLVGEQ-HJOGWXRNSA-N 0.000 description 3
- APKRGYLBSCWJJP-FXQIFTODSA-N Pro-Ala-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O APKRGYLBSCWJJP-FXQIFTODSA-N 0.000 description 3
- MTHRMUXESFIAMS-DCAQKATOSA-N Pro-Asn-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O MTHRMUXESFIAMS-DCAQKATOSA-N 0.000 description 3
- 108010003201 RGH 0205 Proteins 0.000 description 3
- SMIDBHKWSYUBRZ-ACZMJKKPSA-N Ser-Glu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O SMIDBHKWSYUBRZ-ACZMJKKPSA-N 0.000 description 3
- PVDTYLHUWAEYGY-CIUDSAMLSA-N Ser-Glu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PVDTYLHUWAEYGY-CIUDSAMLSA-N 0.000 description 3
- DJACUBDEDBZKLQ-KBIXCLLPSA-N Ser-Ile-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O DJACUBDEDBZKLQ-KBIXCLLPSA-N 0.000 description 3
- KCNSGAMPBPYUAI-CIUDSAMLSA-N Ser-Leu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O KCNSGAMPBPYUAI-CIUDSAMLSA-N 0.000 description 3
- XNCUYZKGQOCOQH-YUMQZZPRSA-N Ser-Leu-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O XNCUYZKGQOCOQH-YUMQZZPRSA-N 0.000 description 3
- FKYWFUYPVKLJLP-DCAQKATOSA-N Ser-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FKYWFUYPVKLJLP-DCAQKATOSA-N 0.000 description 3
- DYEGLQRVMBWQLD-IXOXFDKPSA-N Ser-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CO)N)O DYEGLQRVMBWQLD-IXOXFDKPSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- XSLXHSYIVPGEER-KZVJFYERSA-N Thr-Ala-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O XSLXHSYIVPGEER-KZVJFYERSA-N 0.000 description 3
- AMXMBCAXAZUCFA-RHYQMDGZSA-N Thr-Leu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMXMBCAXAZUCFA-RHYQMDGZSA-N 0.000 description 3
- MXDOAJQRJBMGMO-FJXKBIBVSA-N Thr-Pro-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O MXDOAJQRJBMGMO-FJXKBIBVSA-N 0.000 description 3
- UGFOSENEZHEQKX-PJODQICGSA-N Trp-Val-Ala Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)Cc1c[nH]c2ccccc12)C(=O)N[C@@H](C)C(O)=O UGFOSENEZHEQKX-PJODQICGSA-N 0.000 description 3
- PYJKETPLFITNKS-IHRRRGAJSA-N Tyr-Pro-Asn Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O PYJKETPLFITNKS-IHRRRGAJSA-N 0.000 description 3
- VDPRBUOZLIFUIM-GUBZILKMSA-N Val-Arg-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C(C)C)N VDPRBUOZLIFUIM-GUBZILKMSA-N 0.000 description 3
- DJQIUOKSNRBTSV-CYDGBPFRSA-N Val-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](C(C)C)N DJQIUOKSNRBTSV-CYDGBPFRSA-N 0.000 description 3
- BMOFUVHDBROBSE-DCAQKATOSA-N Val-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N BMOFUVHDBROBSE-DCAQKATOSA-N 0.000 description 3
- WMRWZYSRQUORHJ-YDHLFZDLSA-N Val-Phe-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N WMRWZYSRQUORHJ-YDHLFZDLSA-N 0.000 description 3
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 3
- DVLWZWNAQUBZBC-ZNSHCXBVSA-N Val-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N)O DVLWZWNAQUBZBC-ZNSHCXBVSA-N 0.000 description 3
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 3
- 239000002518 antifoaming agent Substances 0.000 description 3
- 108010008355 arginyl-glutamine Proteins 0.000 description 3
- 108010089975 arginyl-glycyl-aspartyl-serine Proteins 0.000 description 3
- 238000010923 batch production Methods 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 108010078144 glutaminyl-glycine Proteins 0.000 description 3
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 108010050343 histidyl-alanyl-glutamine Proteins 0.000 description 3
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 3
- 108010000761 leucylarginine Proteins 0.000 description 3
- 108010003700 lysyl aspartic acid Proteins 0.000 description 3
- 108010064235 lysylglycine Proteins 0.000 description 3
- 108010017391 lysylvaline Proteins 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 108010065135 phenylalanyl-phenylalanyl-phenylalanine Proteins 0.000 description 3
- 108010004914 prolylarginine Proteins 0.000 description 3
- 108010070643 prolylglutamic acid Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 108010005834 tyrosyl-alanyl-glycine Proteins 0.000 description 3
- IKKVASZHTMKJIR-ZKWXMUAHSA-N Ala-Asp-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O IKKVASZHTMKJIR-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
- ZVGRHIRJLWBWGJ-ACZMJKKPSA-N Asp-Ser-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZVGRHIRJLWBWGJ-ACZMJKKPSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000186146 Brevibacterium Species 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- 241000588698 Erwinia Species 0.000 description 2
- CLODWIOAKCSBAN-BQBZGAKWSA-N Gly-Arg-Asp Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O CLODWIOAKCSBAN-BQBZGAKWSA-N 0.000 description 2
- SCWYHUQOOFRVHP-MBLNEYKQSA-N Gly-Ile-Thr Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SCWYHUQOOFRVHP-MBLNEYKQSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 241000588748 Klebsiella Species 0.000 description 2
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 2
- ADJWHHZETYAAAX-SRVKXCTJSA-N Leu-Ser-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ADJWHHZETYAAAX-SRVKXCTJSA-N 0.000 description 2
- 241000520272 Pantoea Species 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- CXGLFEOYCJFKPR-RCWTZXSCSA-N Pro-Thr-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O CXGLFEOYCJFKPR-RCWTZXSCSA-N 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 241000319304 [Brevibacterium] flavum Species 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- FOCAUTSVDIKZOP-UHFFFAOYSA-N chloroacetic acid Chemical compound OC(=O)CCl FOCAUTSVDIKZOP-UHFFFAOYSA-N 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 235000015872 dietary supplement Nutrition 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
- 235000011194 food seasoning agent Nutrition 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 101150054929 hisE gene Proteins 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 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 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 235000019583 umami taste Nutrition 0.000 description 2
- 235000019607 umami taste sensations Nutrition 0.000 description 2
- 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
- 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
- 108091023037 Aptamer Proteins 0.000 description 1
- HKRXJBBCQBAGIM-FXQIFTODSA-N Arg-Asp-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N)CN=C(N)N HKRXJBBCQBAGIM-FXQIFTODSA-N 0.000 description 1
- JNNVNVRBYUJYGS-CIUDSAMLSA-N Asp-Leu-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O JNNVNVRBYUJYGS-CIUDSAMLSA-N 0.000 description 1
- 102100027708 Astrotactin-1 Human genes 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
- 239000002028 Biomass Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 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
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 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
- 241000588722 Escherichia Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 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
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- REJJNXODKSHOKA-ACZMJKKPSA-N Gln-Ala-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N REJJNXODKSHOKA-ACZMJKKPSA-N 0.000 description 1
- KGVHCTWYMPWEGN-FSPLSTOPSA-N Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CN KGVHCTWYMPWEGN-FSPLSTOPSA-N 0.000 description 1
- 101000936741 Homo sapiens Astrotactin-1 Proteins 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
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- -1 L-form amino acids Chemical class 0.000 description 1
- 229930195714 L-glutamate Natural products 0.000 description 1
- 125000003338 L-glutaminyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C(=O)N([H])[H] 0.000 description 1
- 125000000769 L-threonyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])[C@](O[H])(C([H])([H])[H])[H] 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
- RGUXWMDNCPMQFB-YUMQZZPRSA-N Leu-Ser-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RGUXWMDNCPMQFB-YUMQZZPRSA-N 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-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
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 235000019483 Peanut oil 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
- UAYHMOIGIQZLFR-NHCYSSNCSA-N Pro-Gln-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O UAYHMOIGIQZLFR-NHCYSSNCSA-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
- 238000003559 RNA-seq method Methods 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 108700026226 TATA Box Proteins 0.000 description 1
- KCRQEJSKXAIULJ-FJXKBIBVSA-N Thr-Gly-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCRQEJSKXAIULJ-FJXKBIBVSA-N 0.000 description 1
- 241000700605 Viruses Species 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
- 101150094017 aceA gene Proteins 0.000 description 1
- 101150036393 aceB gene Proteins 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 235000016127 added sugars Nutrition 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 150000001298 alcohols 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
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 229940069428 antacid Drugs 0.000 description 1
- 239000003159 antacid agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000013459 approach 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
- 239000003899 bactericide agent Substances 0.000 description 1
- 150000007514 bases Chemical class 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
- 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
- 230000004071 biological effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 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
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 235000005822 corn Nutrition 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
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 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
- 229940079920 digestives acid preparations Drugs 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 230000002964 excitative effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 101150077981 groEL gene Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000003978 infusion fluid Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000007483 microbial process Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 239000002858 neurotransmitter agent Substances 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
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 235000019629 palatability Nutrition 0.000 description 1
- 239000000312 peanut oil Substances 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
- 239000013612 plasmid Substances 0.000 description 1
- 229920001522 polyglycol ester Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000014347 soups Nutrition 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
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 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
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
- C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
- C12Y306/01031—Phosphoribosyl-ATP diphosphatase (3.6.1.31)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- 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
- 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.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1077—Pentosyltransferases (2.4.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
-
- 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
-
- 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
- C12P13/14—Glutamic acid; Glutamine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02017—ATP phosphoribosyltransferase (2.4.2.17)
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/55—Vector systems having a special element relevant for transcription from bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Seasonings (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本申请涉及具有增强的甘氨酸生产能力的微生物和利用其生产发酵组合物的方法,和更具体地,涉及将突变引入到HisG中而具有增加的甘氨酸生产能力的棒杆菌属的微生物,利用其制备包含甘氨酸和谷氨酸的发酵组合物的方法,以及发酵组合物。
Description
技术领域
本公开涉及具有增加的甘氨酸生产能力的微生物和利用该微生物生产发酵组合物的方法,和更具体地,涉及由于在HisG中引入突变而具有增加的甘氨酸生产能力的棒杆菌属(genus Corynebacterium)的微生物、利用棒杆菌属的微生物制备包含甘氨酸和谷氨酸的发酵组合物的方法、以及发酵组合物。
背景技术
L-氨基酸是蛋白质的基本结构单元,以及被用作如医药原料、食品添加剂、动物饲料、营养补充剂、农药、杀菌剂等的重要材料。其中,L-谷氨酸是通过发酵产生的代表性氨基酸,且具有特有的、独特的味道(鲜味(umami taste)),且因此是广泛应用于食品领域以及医药领域和其他动物饲料领域的重要氨基酸。进一步,甘氨酸由于其甜味,在食品工业中主要被用作增味剂,并与天然增味剂一起使用以增强味道。此外,甘氨酸还因其抗氧化活性、缓冲作用等而被使用,在医药方面,它被用于输液溶液、制酸剂、多氨基酸制剂、和营养补充剂中。
生产氨基酸的典型方法包括使用短杆菌属(genus Brevibacterium)或棒杆菌属的微生物(Amino Acid Fermentation,Gakkai Shuppan Center:195-215,1986)或使用大肠杆菌(Escherichia coli)或芽孢杆菌属(genus Bacillus)、链霉菌属(genusStreptomyces)、青霉菌属(genus Penicillum)、克雷伯氏菌属(genus Klebsiella)、欧文氏菌属(genus Erwinia)、泛菌属(genus Pantoea)等的微生物(美国专利号3,220,929和6,682,912)的发酵方法。另外,这种氨基酸也通过使用如一氯乙酸法、Strecker法等合成工艺的工业方法生产。
另外,为了有效地生产氨基酸,已经进行了各种研究;例如,努力开发用于有效生产氨基酸的微生物或发酵工艺技术。具体地,已经开发了目的物质的特异性接近方法(specific approaches),如增强编码在棒杆菌属的菌株中参与氨基酸生物合成的酶的基因的表达或缺失氨基酸生物合成所不必要的基因(韩国专利号10-0924065和1208480)。除了这些方法之外,还利用了移除不参与氨基酸生产的基因的方法和移除其生产氨基酸的具体功能未知的基因的方法。然而,对研究有效、高产率地生产氨基酸的方法的需求仍在增长。
发明内容
技术问题
本发明人致力于开发能够同时生产几种氨基酸的方法,且结果,他们确认当能够产生谷氨酸的微生物的HisG活性与其亲本菌株的HisG活性相比被增强时,能够在维持谷氨酸生产能力的同时提高甘氨酸生产能力,从而完成本公开。
技术方案
本公开的目的是提供具有增加的甘氨酸生产能力的棒杆菌属的微生物,其中ATP磷酸核糖基转移酶(HisG)的活性被增强。
本公开的另一个目的是提供制备包含甘氨酸和谷氨酸的发酵组合物的方法,其包括通过培养棒杆菌属的微生物进行发酵。
本公开的又一个目的是提供通过上述方法制备的发酵组合物。
有益效果
由于本公开的HisG突变可以被引入到微生物中并同时生产谷氨酸和甘氨酸,因此其可以有效地用于氨基酸的生产。另外,本公开可通过调节发酵产物中谷氨酸和甘氨酸的量来改善发酵产物的味道和适口性,用于发酵液的制备及其在调味产品中的应用。
具体实施方式
下面,将详细描述本公开。同时,本公开中公开的每个描述和实施方式可应用于其它描述和实施方式。也就是说,本公开中公开的各种要素的所有组合都落入本公开的范围内。进一步,下面公开的具体描述不应被解释为限制本公开的范围。
为了实现上述目的,本公开的一方面提供了具有增加的甘氨酸生产能力的棒杆菌属的微生物,其中ATP磷酸核糖基转移酶(HisG)的活性被增强。
具体地,可以提供具有增加的甘氨酸生产能力的微生物,其中,在ATP磷酸核糖基转移酶中,用组氨酸(H)取代SEQ ID NO:4的氨基酸序列的第233位氨基酸。
另外,具体地,可以提供具有增加的甘氨酸生产能力的微生物,其中,在ATP磷酸核糖基转移酶中,分别用组氨酸(H)和谷氨酰胺(Q)取代SEQ ID NO:4的氨基酸序列的第233和235位氨基酸。
如本文所用,术语“ATP磷酸核糖基转移酶”,也称为“HisG”,是指参与组氨酸合成途径的酶。组氨酸合成途径由总共9种酶组成(HisG-HisE-HisI-HisA-HisH-HisB-HisC-HisN-HisD),且HisG构成其第一步。
已知HisG参与组氨酸的产生,但尚不知道其与甘氨酸的产生的关系,并且是由本发明人首次鉴定的。更具体地,本发明人首次确认了通过增强HisG的活性可以增加甘氨酸生产的量。特别地,HisG经受产物组氨酸的反馈抑制,并且在本公开中,引入了突变(组氨酸反馈抑制被解除),结果,增加甘氨酸生产的量和维持谷氨酸量的效果由本发明人首次确定。
如本文所用,术语“HisG活性的增强”是指HisG酶的活性与棒杆菌属的微生物在其自然状态下所具有的内源活性相比被增加。提高HisG活性的方法的实例可以包括:(i)通过将含有编码HisG的核苷酸序列的多核苷酸进一步***到染色体中的方法,或通过将含有编码HisG的核苷酸序列的多核苷酸引入到载体***中的方法等来增加编码酶的核苷酸序列的拷贝数的方法;(ii)增强hisG基因启动子的方法(例如,用更强的启动子取代、在启动子上引入突变等);(iii)通过基因突变修饰为活性更强的酶的方法等。
具体地,在本公开中,可以用组氨酸取代SEQ ID NO:4的HisG氨基酸序列的第233位氨基酸(即,甘氨酸);或者在SEQ ID NO:4的HisG氨基酸序列中,可以用组氨酸取代第233位氨基酸(即,甘氨酸),且可以用谷氨酰胺取代第235位氨基酸(即,苏氨酸)。因此,包含上面描述的修饰的HisG的棒杆菌属的微生物可以显著增加甘氨酸生产能力,同时维持谷氨酸生产能力且不会对其产生任何不利影响。甘氨酸生产能力的增加可以意味着与具有不含本公开修饰的HisG(即,不具有上述突变的HisG)的微生物相比,甘氨酸生产能力增加了。
在另一个实施方式中,HisG酶的启动子可以经由突变或取代被修饰为比天然启动子更强的启动子。可以代替内源性酶启动子连接具有核苷酸取代突变的改良启动子或异源启动子,异源启动子的实例可以包括cj7启动子、lysCP1启动子、EF-Tu启动子、groEL启动子、aceA启动子、aceB启动子等,但异源启动子不限于此。
此外,由于hisG基因由hisE基因和操纵子组成,因此可以通过经由hisEG基因的启动子序列的突变或取代的hisG的过表达来增强hisG酶的活性。更具体地,可以使用通过在hisEG基因的启动子序列中的突变制备的比天然启动子强的启动子来增强HisG酶的活性,其中在SEQ ID NO:1的核苷酸序列中,用T取代第53和55个核苷酸;或者用T取代第53和55个核苷酸且用G取代第60个核苷酸。回顾关于谷氨酸棒杆菌(Corynebacterium glutamicum)的启动子序列研究的文献(Microb Biotechnol.2013Mar;6(2):103-117),可以通过RNA测序(RNA-seq)检测多个转录起始点(TSPs)和启动子的位置。因此,本发明人已经经由ATCC13869菌株的RNA-seq实验确认了hisEG基因的启动子序列,并且另外,已经尝试经由其天然启动子的突变来诱导hisEG基因的启动子序列的过表达。作为修饰天然启动子的方法,可以修饰来自谷氨酸棒杆菌启动子区的-35和-10位的核苷酸序列,使得修饰的启动子序列变得接近共有序列。特别地,当来自hisEG基因的启动子序列的-10区(TATA框)的序列被修饰为接近共有序列时,启动子可以被修饰为相比于天然启动子更强的启动子。
具体地,棒杆菌属的微生物中包括的ATP磷酸核糖基转移酶可以由SEQ ID NO:5或SEQ ID NO:6的氨基酸序列组成。
另外,本公开的氨基酸序列可以通过已知的诱变方法进行修饰,如定向进化、定点诱变等。
因此,ATP磷酸核糖基转移酶可以包括HisG,HisG包括与SEQ ID NO:5或SEQ IDNO:6的氨基酸序列具有至少60%、具体地至少70%、更具体地至少80%、和甚至更具体地至少83%、至少84%、至少88%、至少90%、至少93%、至少95%、或至少97%的同源性的核苷酸序列。显然,只要所得氨基酸序列具有与SEQ ID NO:5或SEQ ID NO:6的氨基酸序列实质上等同或对应的生物活性,具有这种同源性的任何氨基酸序列(其中部分序列被缺失、修饰、取代或添加)也在本公开的范围内。
特别地,术语“L-谷氨酸(L-glutamic acid或L-glutamate)”是指一种被分类为非必需氨基酸的氨基酸。已知L-谷氨酸是中枢神经***中最常见的兴奋性神经递质。此外,由于L-谷氨酸具有鲜味,因此由其开发了谷氨酸一钠(MSG),并被广泛用作增味剂。它一般通过生产L-谷氨酸的微生物的发酵产生。
此外,术语“甘氨酸”是指具有无色结晶形式和甜味的氨基酸。甘氨酸主要用作食品的增味剂,和在医药方面,其被用于输液溶液、制酸剂、多氨基酸制剂、营养补充剂。通常,甘氨酸通过诸如一氯乙酸法、Strecker法等工业合成方法制备。然而,由于在使用合成方法制备氨基酸时产生D-型和L-型氨基酸的混合物,因此存在有必要进行光学拆分的不便。因此,要求通过发酵法制备甘氨酸,该方法具有多种优点,即反应条件温和、可在短时间内大量生产、工艺环境友好、且所产物质是可生物降解的。
如本文所用,术语“同源性”可以表明与给定氨基酸序列或核苷酸序列的匹配程度,并且可以表示为百分比(%)。在本公开中,具有与给定氨基酸序列或核苷酸序列相同或相似的活性的同源序列表示为“%同源性”。对氨基酸序列或核苷酸序列的同源性可以通过例如算法BLAST(参见Karlin和Altschul,Pro.Natl.Acad.Sci.USA,90,5873(1993))或FASTA(参见Pearson,Methods Enzymol.,183,63,1990)来确定。基于该算法BLAST,已经开发了程序BLASTN和BLASTX(参见http://www.ncbi.nlm.nih.gov)。
如本文所用,术语“严格条件”是指允许多核苷酸之间特异性杂交的条件。在文献(例如,J.Sambrook等人)中具体描述了这种严格的条件。例如,严格条件可以包括具有高同源性(例如,60%或更高、具体地90%或更高、更具体地95%或更高、甚至更具体地97%或更高、和甚至更具体地99%或更高)的基因能彼此杂交,而具有比其同源性低的基因不能彼此杂交的条件;或常规Southern杂交的条件(即,对应于60℃、1×SSC、0.1%SDS,具体地在60℃、0.1×SSC、0.1%SDS;且更具体地在68℃、0.1×SSC、0.1%SDS的盐浓度和温度下洗涤一次、更具体地两次或三次的条件)。尽管根据杂交的严格程度,碱基之间的错配是可能的,但杂交要求两个核苷酸具有互补序列。术语“互补”用于描述能够相互杂交的核苷酸碱基之间的关系。例如,关于DNA,腺苷与胸腺嘧啶互补,胞嘧啶与鸟嘌呤互补。因此,本公开还可包括实质上相似的核苷酸序列以及与整个序列互补的分离的多核苷酸片段。
具体地,在上述条件下使用包括在55℃的Tm值下的杂交步骤的杂交条件,可以检测具有同源性的多核苷酸。另外,Tm值可以为60℃、63℃或65℃,但不限于此。本领域普通技术人员可以根据其目的适当地调整Tm值。杂交多核苷酸的适当严格程度取决于多核苷酸的长度和互补程度,这些变量在本领域中是公知的(参见Sambrook等人,同上,9.50-9.51,11.7-11.8)。
如本文所用,术语“微生物”包括所有野生型微生物和自然或人工遗传修饰的微生物,并且它可以是由于外源基因的***或内源性基因活性的加强或减弱而具有特定的减弱或加强机制的微生物。
在本公开中,微生物可以包含ATP磷酸核糖基转移酶。另外,ATP磷酸核糖基转移酶可以通过经由载体转化而引入到微生物中,但转化方法不限于此。此外,只要HisG能够在微生物中表达,编码HisG的基因是位于染色体上还是染色体外都无所谓。
如本文所用,术语“载体”是具有能够在适当宿主中表达目的基因的遗传物质的人工DNA分子,并且可以指包括编码HisG的基因的核苷酸序列的DNA构建体。
只要载体可以在宿主细胞中表达,本公开中使用的载体没有特别的限制,并且本领域中已知的任何载体都可以用于转化宿主细胞。常规载体的实例可包括天然或重组质粒、粘粒、病毒和噬菌体。
例如,作为噬菌体载体或粘粒载体,可以使用pWE15、M13、λLB3、λBL4、λIXII、λASHII、λAPII、λt10、λt11、Charon4A、Charon21A等;且作为质粒载体,可以使用基于pBR、pUC、pBluescriptII、pGEM、pTZ、pCL、pET等的质粒载体。
另外,编码本公开的HisG的多核苷酸可经由载体(用于在宿主细胞中进行染色体***)引入到宿主细胞的染色体中。例如,可以使用载体pECCG117、pDZ、pACYC177、pACYC184、pCL、pUC19、pBR322、pMW118、pCC1BAC、pCES208、pXMJ19等,但不限于这些。
另外,将多核苷酸***到染色体中可以通过本领域已知的任何方法来完成,例如通过同源重组。
由于可以通过诱导同源重组将本公开的载体***到染色体中,因此可以额外包括选择标记以确认基因是否成功***到染色体中。选择标记用于筛选被载体转化的细胞,换句话说,用于确定是否***了多核苷酸。可以使用提供可选择表型(如抗药性、辅源营养、对毒剂的抗性、或表面蛋白的表达)的标记。在用选择剂处理的环境中,只有表达选择标记的细胞才能存活,或者细胞表现出不同的表型,因此可以通过这种方法选择成功转化的细胞。
如本文所用,术语“转化”是指将包含多核苷酸或编码HisG的基因引入到宿主细胞中,以允许基因和HisG在宿主细胞中表达。此外,只要目的基因能够在宿主细胞中表达,转化基因是位于宿主细胞的染色体上还是染色体外都无所谓,且包括这两种情况。
转化方法可以包括将基因引入到细胞中的所有方法,并且可以根据宿主细胞通过选择本领域已知的合适的标准技术来进行。例如,合适的标准技术可选自电穿孔、磷酸钙(CaPO4)沉淀、氯化钙(CaCl2)沉淀、显微注射、聚乙二醇(PEG)技术、DEAE-葡聚糖技术、阳离子脂质体技术和乙酸锂-DMSO技术,但适合的标准技术不限于此。
在本公开中,微生物可以不受限制地是任何微生物,其中引入了本公开的HisG且因此增加了甘氨酸生产能力。
具体地,微生物可以是棒杆菌属的微生物;更具体地,是谷氨酸棒杆菌或黄色短杆菌(Brevibacterium flavum);和最具体地,是谷氨酸棒杆菌,但微生物不限于此。
本公开的另一方面提供了用于制备发酵组合物的方法,其包括通过在培养基中培养棒杆菌属的微生物进行发酵。
本公开的又一方面提供了通过上述方法制备的发酵组合物。
发酵组合物可以是其中甘氨酸的量增加的组合物。
微生物如上文所述。
如本文所用,术语“培养”是指在人工控制的环境条件下培养微生物。在本公开中,利用微生物生产目的物质的方法可以通过本领域中广泛已知的方法进行。具体地,培养可以在分批工艺中或连续工艺(例如分批补料工艺或重复分批补料工艺)中进行,但不限于此。用于培养的培养基必须满足所使用的特定菌株的要求。适合用于培养棒杆菌菌株的培养基是本领域已知的(例如,Manual of Methods for General Bacteriology by theAmerican Society for Bacteriology,Washington D.C.,USA,1981)。
可用于培养基的碳源可以是糖和碳水化合物(例如,葡萄糖、蔗糖、乳糖、果糖、麦芽糖、淀粉和纤维素);油和脂肪(例如,大豆油、葵花籽油、花生油、和椰子油);脂肪酸(例如,棕榈酸、硬脂酸、和亚油酸);醇(例如,甘油和乙醇);和有机酸(例如,乙酸)。这些物质可以单独使用或组合使用,但使用的方式不限于此。
可使用的氮源的实例包括蛋白胨、酵母提取物、肉汁、麦芽提取物、玉米浆、豆粕和尿素、或无机化合物(例如,硫酸铵、氯化铵、磷酸铵、碳酸铵和硝酸铵)。这些氮源也可以单独使用或组合使用,但使用的方式不限于此。
可用于培养基的磷源可包括磷酸氢二钾、磷酸二氢钾、或相应的含钠盐。另外,培养基可以含有细胞生长所必要的金属盐。最后,除了上述物质之外,还可以使用生长所必需的物质(例如,氨基酸和维生素)。进一步,可以使用适合于培养基的前体。上述原料可以以分批或连续的方式充分地供给到培养物中。
在微生物的培养期间,可通过适当的碱性化合物(例如,氢氧化钠、氢氧化钾或氨),或酸性化合物(例如,磷酸或硫酸)来调节培养物的pH。起泡可以通过抗起泡剂(例如,脂肪酸聚乙二醇酯)来调节。可通过引入氧气或含氧气体(例如,空气)来维持培养物的好氧条件。
培养物(培养基)的温度一般可以在20℃至45℃,具体地为25℃至40℃的范围。可以继续培养直到获得目的物质的期望的生产量,且具体地为10至160小时。
从培养物(培养基)中回收目的物质可以通过本领域已知的常规分离方法进行。对于分离方法,可以使用诸如离心、过滤、层析、结晶等方法。例如,可以将通过在低速下离心培养基以除去生物质而获得的上清液通过离子交换层析分离,但分离方法不限于此。在可取代的方法中,可以通过进行从培养产物(培养基)中分离和过滤细菌细胞的工艺,而不需要附加的纯化工艺来回收目的物质。在另一可取代方法中,回收步骤可进一步包括纯化工艺。
如本文所用,术语“发酵组合物”是指通过培养本公开的微生物而获得的组合物。此外,发酵组合物可以包括在培养微生物之后进行合适的后处理之后获得的液体或粉末形式的组合物。特别地,合适的后处理工艺可以包括例如培养微生物的工艺、移除细菌细胞的工艺、浓缩工艺、过滤工艺和混合载体的工艺,并且可以进一步包括干燥工艺。在一些情况下,后处理工艺可不包括纯化工艺。通过培养本发明的微生物而获得的发酵组合物含有增加的甘氨酸的量同时维持一定水平的谷氨酸生产量,因此使得其能够提供最佳味道。
另外,“发酵组合物”不排除含有液体或粉末形式的组合物的调味产品(例如,粉末汤产品、零食调味产品等)。此外,只要通过培养本公开的微生物获得的组合物被包含其中,“发酵组合物”不排除其中进一步包括通过非发酵工艺获得的物质和/或通过非自然工艺获得的其他物质的情况。
实施例
在下文,将结合所附示例性实施方式详细描述本公开。然而,本文公开的示例性实施方式仅用于说明性目的,而不应被解释为限制本公开的范围。
实施例1.为了增加甘氨酸生产能力在KFCC11074菌株中引入突变,和确认引入突变的KFCC11074中谷氨酸和甘氨酸的生产量
实施例1-1:引入突变的载体的制备
为了确认在能够生产谷氨酸的菌株中增强HisG活性对增加甘氨酸生产能力的影响,制备了在hisEG基因的启动子内诱导突变的菌株和诱导组氨酸反馈抑制解除突变的菌株,并确认了这些菌株的甘氨酸生产能力。
同时,hisE和hisG基因由操纵子组成,且这些基因参与组氨酸生物合成途径。特别地,由于HisG被产物组氨酸反馈抑制,因此尝试确认当引入反馈抑制解除突变以增加hisG基因的活性时,是否可以增加这些菌株的甘氨酸生产能力。因此,尝试将hisEG启动子突变和反馈抑制解除突变中的每一个引入到已知作为谷氨酸生产菌株的KFCC11074菌株(韩国专利号10-0292299)中。具体地,制备用于基因取代的载体,以用T取代SEQ ID NO:1的多核苷酸序列(其包括hisEG启动子)的第53和55个核苷酸;以及用T取代SEQ ID NO:1的多核苷酸序列的第53和55个核苷酸,且用G取代SEQ ID NO:1的多核苷酸序列的第60个核苷酸。
另外,制备用于基因取代的载体,以用组氨酸(His/H)取代SEQ ID NO:4的HisG的氨基酸序列的第233位氨基酸(即,甘氨酸(Gly/G)),以及分别用组氨酸(His/H)和谷氨酰胺(Gln/Q)取代SEQ ID NO:4的HisG的氨基酸序列的第233位氨基酸(即,甘氨酸(Gly/G))和第235位氨基酸(即,苏氨酸(Thr/T))。使用ATCC13869基因组DNA为模板,通过PCR获得用于制备各取代载体的基因片段。每个引物对是根据在美国国立卫生研究院GenBank(NationalInstitutes of Health GenBank,NIH GenBank)中登记的谷氨酸棒杆菌(ATCC13869)的基因和相邻核苷酸序列信息制备的。
为了制备用于hisEG启动子取代的载体,PCR按以下顺序进行:(1)在95℃下变性5分钟;(2)在95℃下变性30秒,在55℃下退火30秒,和在72℃下聚合1分钟,共30个循环;以及(3)在72℃下聚合5分钟。更具体地,获得了利用SEQ ID NO:7和SEQ ID NO:8的引物扩增的多核苷酸(500bp)和利用SEQ ID NO:9和SEQ ID NO:10的引物扩增的多核苷酸(500bp)。将获得的两个DNA片段利用输注酶(infusion enzyme)连接到已用限制性内切酶SalI消化的载体pDZ(韩国专利号10-0924065和国际专利公开号WO 2008-033001),从而制备了用于取代包括hisEG启动子的两个基因的单个载体,并且将该载体命名为“pDZ-hisEG-pro-2mt”。另外,获得了利用SEQ ID NO:7和SEQ ID NO:11的引物扩增的500bp多核苷酸和利用SEQ IDNO:10和SEQ ID NO:12的引物扩增的500bp多核苷酸。将获得的两个DNA片段利用输注酶连接到已用限制性内切酶SalI消化的载体pDZ(韩国专利号10-0924065和国际公开号WO2008-033001),从而制备了用于取代包括hisEG启动子的一个基因的单个载体,并且将该载体命名为“pDZ-hisE-Pro-3mt”。用于载体制备的引物序列的信息示于下表1中。
为了用H取代第233位氨基酸,以及分别用H和Q取代第233位氨基酸和第235位氨基酸,制备了用于基因取代的载体。具体地,PCR按以下顺序进行:(1)在95℃下变性5分钟;(2)在95℃下变性30秒,在55℃退火30秒,和在72℃下聚合1分钟,共30个循环;以及(3)在72℃下聚合5分钟。另外,获得了利用SEQ ID NO:13和SEQ ID NO:14的引物扩增的722bp多核苷酸和利用SEQ ID NO:15和SEQ ID NO:16的引物扩增的798bp多核苷酸。将获得的两个DNA片段利用输注酶连接到已用限制性内切酶SalI消化的载体pDZ(韩国专利号10-0924065和国际公开号WO 2008-033001),从而制备了包括含有HisG(G233H)突变的多核苷酸的用于基因取代的单个1.5kbp载体,并将该载体命名为“pDZ-hisG(G233H)”。另外,获得了利用SEQ IDNO:13和SEQ ID NO:17的引物扩增的722bp多核苷酸和利用SEQ ID NO:16和SEQ ID NO:18的引物扩增的798bp多核苷酸。将获得的两个DNA片段利用输注酶连接到已用限制性内切酶SalI消化的载体pDZ(韩国专利号10-0924065和国际公开号WO2008-033001),从而制备了包括含有HisG(G233H/T235Q)突变的多核苷酸的用于基因取代的单个1.5kbp载体,并将该载体命名为“pDZ-hisG(G233H/T235Q)”。用于载体制备的引物序列的信息示于下表1中。
[表1]
SEQ ID NO | 引物 | 序列(5′至3′) |
7 | hisEG-pro-2mt-AF | GATCCTCTAGAGTCGACTTCGACGAATCCCTCG |
8 | hisEG-pro-2mt-AR | CGGT<u>ACATTATA</u>CCACACAACAGTTATCAATG |
9 | hisEG-pro-2mt-BF | GTGG<u>TATAATGT</u>ACCGAGTGAAGACATTTGAC |
10 | hisEG-pro-2mt-BR | ATGCCTGCAGGTCGACTGATACCCAAATCGAG |
11 | hisEG-pro-3mt-AR | CGGT<u>CCATTATA</u>CCACACAACAGTTATCAATG |
12 | hisEG-pro-3mt-BF | GTGGTATAATGGACCGAGTGAAGACATTTGAC |
13 | hisG(G233H)-AF | GATCCTCTAGAGTCGACCCCAAACAAGGGCTCGC |
14 | hisG(G233H)-AR | CGTGCCAGTGGGGATACCGTTGGGTGGG |
15 | hisG(G233H)-BF | AACCCCAGGCCTATCCCACCCAACGGTATC |
16 | hisG(G233H)-BR | ATGCCTGCAGGTCGACGCAAGGTTGGCAACAAC |
17 | hisG(G233H/T235Q)-AR | CGTGCCAGTGGGGATACCTGTGGGTGGG |
18 | hisG(G233H/T235Q)-BF | AACCCCAGGCCTATCCCACCCACAGGTATC |
实施例1-2:引入突变的KFCC11074的制备以及谷氨酸和甘氨酸的生产量的确认
将实施例1-1中已经制备的用于hisEG启动子取代的载体(即,pDZ-hisEG-pro-2mt和pDZ-hisEG-pro-3mt)和用于基因取代的载体(即,pDZ-hisG(G233H)和pDZ-hisG(G233H/T235Q))各自通过电穿孔引入到KFCC11074菌株中,以制备“KFCC11074_Pro(2mt)_hisEG”、“KFCC11074_Pro(3mt)_hisEG”、“KFCC11074_hisG(G233H)”和“KFCC11074_hisG(G233H/T235Q)”,它们是分别引入突变的谷氨酸和甘氨酸生产菌株。
具体地,这些菌株是通过转化制备的(Appl.Microbiol.Biotechnol.,1999,52:541-545)。在含有卡那霉素(25mg/L)的琼脂营养培养基上选择通过同源序列的重组将载体***到染色体上的菌株。选择的初级菌株经受二次交换(crossover),并分别选择了引入了两个或三个目的突变的菌株。利用SEQ ID NO:7和SEQ ID NO:10的引物对和SEQ ID NO:13和SEQ ID NO:16的引物对分别进行PCR后,通过测序确认最终转化菌株的突变(取代)。
然后将选择的菌株KFCC11074_Pro(2mt)_hisEG、KFCC11074_Pro(3mt)_hisEG、KFCC11074_hisG(G233H)和KFCC11074_hisG(G233H/T235Q)平皿接种在营养培养基上,并在30℃下培养16小时。将已在121℃下高压灭菌15分钟的发酵培养基(25mL)分配到每个用于振荡的锥形瓶(Erlenmeyer flask)(250mL)中,并将在营养培养基中培养的各菌株接种到其上并培养48小时。培养条件设定为200rpm、37℃、和pH8.0。营养培养基和发酵培养基的组成如下。
营养培养基:
葡萄糖1%、肉汁0.5%、聚蛋白胨1%、氯化钠0.25%、酵母提取物0.5%、琼脂2%、尿素0.2%、pH 7.2
发酵培养基:
原糖6%、碳酸钙5%、硫酸铵2.25%、一磷酸钾0.1%、硫酸镁0.04%、硫酸铁(10mg/L)、生物素(0.3mg/L)、盐酸硫胺素(0.2mg/L)
完成培养后,通过利用HPLC的方法测定L-谷氨酸和甘氨酸的生产量,并且测定结果示于下表2中。
[表2]
菌株 | L-谷氨酸(g/L) | 甘氨酸(mg/L) |
KFCC11074 | 11.5 | 165 |
KFCC11074_Pro(2mt)_hisEG | 11.4 | 198 |
KFCC11074_Pro(3mt)_hisEG | 12.0 | 209 |
KFCC11074_hisG(G233H) | 11.8 | 210 |
KFCC11074_hisG(G233H/T235Q) | 12.3 | 433 |
如表2中所示,确认了引入突变的谷氨酸棒杆菌菌株KFCC11074_Pro(2mt)_hisEG、KFCC11074_Pro(3mt)_hisEG、KFCC11074_hisG(G233H)和KFCC11074_hisG(G233H/T235Q)各自生产的L-谷氨酸浓度与无突变的谷氨酸棒杆菌菌株KFCC11074生产的L-谷氨酸浓度相似。
另一方面,确认了菌株KFCC11074_Pro(2mt)_hisEG、KFCC11074_Pro(3mt)_hisEG、和KFCC11074_hisG(G233H)各自生产的甘氨酸浓度相对于菌株KFCC11074生产的甘氨酸浓度分别增加了33mg/L、44mg/L和45mg/L。特别地,KFCC11074_hisG(G233H/T235Q)菌株表现出显著增加了268mg/L的甘氨酸浓度。
即,确认了包括hisEG启动子突变和HisG反馈抑制解除突变的突变显著增加了甘氨酸生产能力,同时维持了微生物中的L-谷氨酸生产能力且对其没有显著影响。
实施例2.引入突变的ATCC13869中的谷氨酸和甘氨酸生产量的确认
为了确认上述突变是否即使在野生型谷氨酸棒杆菌ATCC13869菌株中也具有在不影响谷氨酸生产能力的情况下增加甘氨酸生产能力的效果,尝试制备引入突变的基于ATCC13869的菌株。
将实施例1-1中已经制备的用于hisEG启动子取代的载体(即,pDZ-hisEG-pro-2mt和pDZ-hisEG-pro-3mt)和用于hisG基因取代的载体(即,pDZ-hisG(G233H)和pDZ-hisG(G233H/T235Q))各自通过电穿孔引入到ATCC13869菌株中,以制备“ATCC13869_Pro(2mt)_hisEG”、“ATCC13869_Pro(3mt)_hisEG”、“ATCC13869_hisG(G233H)”和“ATCC13869_hisG(G233H/T235Q)”,它们是分别引入突变的谷氨酸和甘氨酸产生菌株。
具体地,这些菌株是通过转化制备的(Appl.Microbiol.Biotechnol.,1999,52:541-545)。在含有卡那霉素(25mg/L)的琼脂营养培养基上选择通过同源序列重组将载体***到染色体上的菌株。选择的初级菌株经受二次交换,并分别选择了引入了两个或三个目的突变的菌株。利用SEQ ID NO:7和SEQ ID NO:10的引物对和SEQ ID NO:13和SEQ ID NO:16的引物对中的一对进行PCR后,通过测序确认最终转化菌株的突变(取代)。
每个菌落在营养培养基中继代培养,然后在发酵培养基中培养5小时。然后,将25%吐温40加入到各培养基中,浓度为0.4%,以及将每个菌落再次培养32小时。
营养培养基:
葡萄糖1%、肉汁0.5%、聚蛋白胨1%、氯化钠0.25%、酵母提取物0.5%、琼脂2%、尿素0.2%、pH 7.2
发酵培养基:
原糖6%、碳酸钙5%、硫酸铵2.25%、一磷酸钾0.1%、硫酸镁0.04%、硫酸铁(10mg/L)、生物素(0.3mg/L)、盐酸硫胺素(0.2mg/L)
在上述条件下培养每个菌落,以及利用YSI测定L-谷氨酸浓度,且利用HPLC测定甘氨酸浓度。测得的L-谷氨酸和甘氨酸的浓度示于下表3中。
[表3]
菌株 | L-谷氨酸(g/L) | 甘氨酸(mg/L) |
ATCC13869 | 13.8 | 117 |
ATCC13869_Pro(2mt)_hisEG | 13.7 | 128 |
ATCC13869_Pro(3mt)_hisEG | 14.0 | 135 |
ATCC13869_hisG(G233H) | 13.5 | 144 |
ATCC13869_hisG(G233H/T235Q) | 13.7 | 306 |
如表3中所示,确认引入突变的谷氨酸棒杆菌菌株ATCC13869_Pro(2mt)_hisEG、ATCC13869_Pro(3mt)_hisEG、ATCC13869_hisG(G233H)和ATCC13869_hisG(G233H/T235Q)各自产生的L-谷氨酸浓度与谷氨酸棒杆菌菌株ATCC13869产生的L-谷氨酸浓度相似;然而,与谷氨酸棒杆菌菌株ATCC13869相比,谷氨酸棒杆菌菌株ATCC13869_Pro(2mt)_hisEG、ATCC13869_Pro(3mt)_hisEG、ATCC13869_hisG(G233H)和ATCC13869_hisG(G233H/T235Q)全部显示了甘氨酸浓度的增加。
即,确认了包括hisEG启动子突变和HisG反馈抑制解除突变的突变显著增加了甘氨酸生产能力,同时维持了微生物中的L-谷氨酸生产能力且对其没有显著影响。
同时,菌株ATCC13869_hisG(G233H)和ATCC13869_hisG(G233H/T235Q)于2019年3月14日以菌株名称“CA02-9216”和“CA02-9217”保藏在根据布达佩斯条约下的国际保藏机构韩国微生物保藏中心(KCCM),并被分配保藏号“KCCM12458P”和“KCCM12459P”。
实施例3.用于制备调味产品的发酵组合物的制备
如上所述,确认了HisG活性增强的菌株显示出甘氨酸生产能力的增加,同时对L-谷氨酸生产能力没有显示出显著的影响。因此,尝试利用HisG活性被增强的本公开的棒杆菌属的微生物制备发酵组合物。
例如,尝试使用基本上是公知调味料的谷氨酸作为活性成分来制备发酵组合物,并且控制发酵菌株和发酵过程以为了增加浓郁味道的构成的目的,增加调味料的其他副产物成分的比例。
尝试利用包括hisEG启动子突变和HisG反馈抑制解除突变两者的菌株在5升发酵罐中制备发酵组合物。
用于制备所用培养基的所有成分都是与食品级相对应的成分。
初级种子培养基的制备如下:
葡萄糖(1%)、酵母提取物(1%)、蛋白胨(1%)、硫酸铵(0.1%)、NaCl(0.25%)、KH2PO4(0.15%)、K2HPO4(0.15%)、pH 8.0
二级种子培养基的制备如下:
有机原糖(4.6%,纯度98.5%)、硫酸镁(0.05%)、酵母提取物(0.5%)、KH2PO4(0.2%)、硫酸铁(0.002%)、生物素(1mg/L)、盐酸硫胺素(2mg/L)、少量消泡剂、pH 7.2
发酵培养基的制备如下:
有机原糖(4%,纯度98.5%)、硫酸镁(0.03%)、酵母提取物(1%)、磷酸(0.22%)、KOH(0.4%)、生物素(0.2mg/L)、盐酸硫胺素(0.6mg/L)、硫酸锰(0.002%)、硫酸铁(0.002%)、硫酸锌(0.002%)、硫酸铜(0.006%)、少量消泡剂、pH 7.4
将初级种子培养基(50mL)分配到每个500mL锥形摇瓶中,在121℃下在压力下高压灭菌20分钟。然后,接种每种菌株,并以200rpm的转速,在30℃下振荡温育5至7小时。
在1.5L试验发酵罐中以0.25L的量制备二级种子培养基,在121℃下在压力下高压灭菌20分钟,并冷却。然后,接种初级种子培养基(50mL),并以900rpm的转速,在31.5℃下温育15小时。
在5L试验发酵罐中以0.25L的量制备发酵培养基,在121℃下在压力下高压灭菌20分钟,并冷却。然后,将二级种子培养基(0.26L)接种到其上,并以900rpm的转速,在30℃至34℃下温育。
在上述条件下培养的同时,在谷氨酸棒杆菌培养期间,使用28%氨水连续调节发酵培养物的pH,以使其在7.0至7.4的范围内。当培养物中残留糖的浓度在0.5%至1.5%的范围内时,频繁地添加灭菌的有机原糖以继续培养,直到添加的糖的总量变为发酵液量的30%至34%。
[表4]
结果,如上表4所示,确认虽然在两个菌株之间谷氨酸生产量没有显著差异,但是引入突变的谷氨酸棒杆菌KFCC11074_hisG(G233H/T235Q)_Pro(3mt)_hisEG菌株生产的发酵液中甘氨酸的量显著增加。
即使在利用3kL发酵罐制备发酵组合物的情况下,在两个菌株之间谷氨酸产量也没有显著差异。然而,尽管在两个菌株之间谷氨酸产量没有显著差异(64.2g/L vs 73g/L),但是引入突变的谷氨酸棒杆菌KFCC11074_hisG(G233H/T235Q)_Pro(3mt)_hisEG菌株与KFCC11074菌株相比,显示出甘氨酸量的显著增加(即0.2g/L vs 3.2g/L)。
根据以上所述,本公开所属领域的普通技术人员将能够理解,在不修改本公开的技术概念或本质特征的情况下,本公开可以以其它具体形式体现。就这一点而言,本文公开的示例性实施方式仅用于说明性目的,而不应被解释为限制本公开的范围。相反,本公开意在不仅覆盖示例性实施方式,而且还覆盖可包括在如由所附权利要求限定的本公开的精神和范围内的各种替代、修改、等同和其它实施方式。
<110> CJ第一制糖株式会社
<120> 具有增加的甘氨酸生产能力的微生物和利用其生产发酵组合物的方法
<130> OPA19064
<150> KR 10-2018-0035156
<151> 2018-03-27
<160> 18
<170> KoPatentIn 3.0
<210> 1
<211> 65
<212> DNA
<213> 谷氨酸棒杆菌
<400> 1
aattattcga ctaatatcct cccccaaaca cacattgata actgttgtgt ggaagaatgt 60
accga 65
<210> 2
<211> 65
<212> DNA
<213> 人工序列
<220>
<223> hisEG启动子
<400> 2
aattattcga ctaatatcct cccccaaaca cacattgata actgttgtgt ggtataatgt 60
accga 65
<210> 3
<211> 65
<212> DNA
<213> 人工序列
<220>
<223> hisEG启动子
<400> 3
aattattcga ctaatatcct cccccaaaca cacattgata actgttgtgt ggtataatgg 60
accga 65
<210> 4
<211> 281
<212> PRT
<213> 谷氨酸棒杆菌
<400> 4
Met Leu Lys Ile Ala Val Pro Asn Lys Gly Ser Leu Ser Glu Arg Ala
1 5 10 15
Met Glu Ile Leu Ala Glu Ala Gly Tyr Ala Gly Arg Gly Asp Ser Lys
20 25 30
Ser Leu Asn Val Phe Asp Glu Ala Asn Asn Val Glu Phe Phe Phe Leu
35 40 45
Arg Pro Lys Asp Ile Ala Ile Tyr Val Ala Gly Gly Gln Leu Asp Leu
50 55 60
Gly Ile Thr Gly Arg Asp Leu Ala Arg Asp Ser Gln Ala Asp Val His
65 70 75 80
Glu Val Leu Ser Leu Gly Phe Gly Ser Ser Thr Phe Arg Tyr Ala Ala
85 90 95
Pro Ala Asp Glu Glu Trp Ser Ile Glu Lys Leu Asp Gly Lys Arg Ile
100 105 110
Ala Thr Ser Tyr Pro Asn Leu Val Arg Asp Asp Leu Ala Ala Arg Gly
115 120 125
Leu Ser Ala Glu Val Leu Arg Leu Asp Gly Ala Val Glu Val Ser Ile
130 135 140
Lys Leu Gly Val Ala Asp Ala Ile Ala Asp Val Val Ser Thr Gly Arg
145 150 155 160
Thr Leu Arg Gln Gln Gly Leu Ala Pro Phe Gly Glu Val Leu Cys Thr
165 170 175
Ser Glu Ala Val Ile Val Gly Arg Lys Asp Glu Lys Val Thr Pro Glu
180 185 190
Gln Gln Ile Leu Leu Arg Arg Ile Gln Gly Ile Leu His Ala Gln Asn
195 200 205
Phe Leu Met Leu Asp Tyr Asn Val Asp Arg Asp Asn Leu Asp Ala Ala
210 215 220
Thr Ala Val Thr Pro Gly Leu Ser Gly Pro Thr Val Ser Pro Leu Ala
225 230 235 240
Arg Asp Asn Trp Val Ala Val Arg Ala Met Val Pro Arg Arg Ser Ala
245 250 255
Asn Ala Ile Met Asp Lys Leu Ala Gly Leu Gly Ala Glu Ala Ile Leu
260 265 270
Ala Ser Glu Ile Arg Ile Ala Arg Ile
275 280
<210> 5
<211> 281
<212> PRT
<213> 人工序列
<220>
<223> HisG(G233H)
<400> 5
Met Leu Lys Ile Ala Val Pro Asn Lys Gly Ser Leu Ser Glu Arg Ala
1 5 10 15
Met Glu Ile Leu Ala Glu Ala Gly Tyr Ala Gly Arg Gly Asp Ser Lys
20 25 30
Ser Leu Asn Val Phe Asp Glu Ala Asn Asn Val Glu Phe Phe Phe Leu
35 40 45
Arg Pro Lys Asp Ile Ala Ile Tyr Val Ala Gly Gly Gln Leu Asp Leu
50 55 60
Gly Ile Thr Gly Arg Asp Leu Ala Arg Asp Ser Gln Ala Asp Val His
65 70 75 80
Glu Val Leu Ser Leu Gly Phe Gly Ser Ser Thr Phe Arg Tyr Ala Ala
85 90 95
Pro Ala Asp Glu Glu Trp Ser Ile Glu Lys Leu Asp Gly Lys Arg Ile
100 105 110
Ala Thr Ser Tyr Pro Asn Leu Val Arg Asp Asp Leu Ala Ala Arg Gly
115 120 125
Leu Ser Ala Glu Val Leu Arg Leu Asp Gly Ala Val Glu Val Ser Ile
130 135 140
Lys Leu Gly Val Ala Asp Ala Ile Ala Asp Val Val Ser Thr Gly Arg
145 150 155 160
Thr Leu Arg Gln Gln Gly Leu Ala Pro Phe Gly Glu Val Leu Cys Thr
165 170 175
Ser Glu Ala Val Ile Val Gly Arg Lys Asp Glu Lys Val Thr Pro Glu
180 185 190
Gln Gln Ile Leu Leu Arg Arg Ile Gln Gly Ile Leu His Ala Gln Asn
195 200 205
Phe Leu Met Leu Asp Tyr Asn Val Asp Arg Asp Asn Leu Asp Ala Ala
210 215 220
Thr Ala Val Thr Pro Gly Leu Ser His Pro Thr Val Ser Pro Leu Ala
225 230 235 240
Arg Asp Asn Trp Val Ala Val Arg Ala Met Val Pro Arg Arg Ser Ala
245 250 255
Asn Ala Ile Met Asp Lys Leu Ala Gly Leu Gly Ala Glu Ala Ile Leu
260 265 270
Ala Ser Glu Ile Arg Ile Ala Arg Ile
275 280
<210> 6
<211> 281
<212> PRT
<213> 人工序列
<220>
<223> HisG(G233H/T235Q)
<400> 6
Met Leu Lys Ile Ala Val Pro Asn Lys Gly Ser Leu Ser Glu Arg Ala
1 5 10 15
Met Glu Ile Leu Ala Glu Ala Gly Tyr Ala Gly Arg Gly Asp Ser Lys
20 25 30
Ser Leu Asn Val Phe Asp Glu Ala Asn Asn Val Glu Phe Phe Phe Leu
35 40 45
Arg Pro Lys Asp Ile Ala Ile Tyr Val Ala Gly Gly Gln Leu Asp Leu
50 55 60
Gly Ile Thr Gly Arg Asp Leu Ala Arg Asp Ser Gln Ala Asp Val His
65 70 75 80
Glu Val Leu Ser Leu Gly Phe Gly Ser Ser Thr Phe Arg Tyr Ala Ala
85 90 95
Pro Ala Asp Glu Glu Trp Ser Ile Glu Lys Leu Asp Gly Lys Arg Ile
100 105 110
Ala Thr Ser Tyr Pro Asn Leu Val Arg Asp Asp Leu Ala Ala Arg Gly
115 120 125
Leu Ser Ala Glu Val Leu Arg Leu Asp Gly Ala Val Glu Val Ser Ile
130 135 140
Lys Leu Gly Val Ala Asp Ala Ile Ala Asp Val Val Ser Thr Gly Arg
145 150 155 160
Thr Leu Arg Gln Gln Gly Leu Ala Pro Phe Gly Glu Val Leu Cys Thr
165 170 175
Ser Glu Ala Val Ile Val Gly Arg Lys Asp Glu Lys Val Thr Pro Glu
180 185 190
Gln Gln Ile Leu Leu Arg Arg Ile Gln Gly Ile Leu His Ala Gln Asn
195 200 205
Phe Leu Met Leu Asp Tyr Asn Val Asp Arg Asp Asn Leu Asp Ala Ala
210 215 220
Thr Ala Val Thr Pro Gly Leu Ser His Pro Gln Val Ser Pro Leu Ala
225 230 235 240
Arg Asp Asn Trp Val Ala Val Arg Ala Met Val Pro Arg Arg Ser Ala
245 250 255
Asn Ala Ile Met Asp Lys Leu Ala Gly Leu Gly Ala Glu Ala Ile Leu
260 265 270
Ala Ser Glu Ile Arg Ile Ala Arg Ile
275 280
<210> 7
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> hisEG-pro-2mt-AF
<400> 7
gatcctctag agtcgacttc gacgaatccc tcg 33
<210> 8
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> hisEG-pro-2mt-AR
<400> 8
cggtacatta taccacacaa cagttatcaa tg 32
<210> 9
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> hisEG-pro-2mt-BF
<400> 9
gtggtataat gtaccgagtg aagacatttg ac 32
<210> 10
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> hisEG-pro-2mt-BR
<400> 10
atgcctgcag gtcgactgat acccaaatcg ag 32
<210> 11
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> hisEG-pro-3mt-AR
<400> 11
cggtccatta taccacacaa cagttatcaa tg 32
<210> 12
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> hisEG-pro-3mt-BF
<400> 12
gtggtataat ggaccgagtg aagacatttg ac 32
<210> 13
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> hisG(G233H)-AF
<400> 13
gatcctctag agtcgacccc aaacaagggc tcgc 34
<210> 14
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> hisG(G233H)-AR
<400> 14
cgtgccagtg gggataccgt tgggtggg 28
<210> 15
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> hisG(G233H)-BF
<400> 15
aaccccaggc ctatcccacc caacggtatc 30
<210> 16
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> hisG(G233H)-BR
<400> 16
atgcctgcag gtcgacgcaa ggttggcaac aac 33
<210> 17
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> hisG(G233H/T235Q)-AR
<400> 17
cgtgccagtg gggatacctg tgggtggg 28
<210> 18
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> hisG(G233H/T235Q)-BF
<400> 18
aaccccaggc ctatcccacc cacaggtatc 30
Claims (8)
1.具有增加的甘氨酸生产能力的棒杆菌属(genus Corynebacterium)的微生物,其中ATP磷酸核糖基转移酶(HisG)的活性被增强。
2.根据权利要求1所述的微生物,其中,在所述ATP磷酸核糖基转移酶中,用组氨酸(H)取代SEQ ID NO:4的氨基酸序列的第233位氨基酸。
3.根据权利要求1所述的微生物,其中,在所述ATP磷酸核糖基转移酶中,分别用组氨酸(H)和谷氨酰胺(Q)取代SEQ ID NO:4的氨基酸序列的第233和235位氨基酸。
4.根据权利要求2所述的微生物,其中所述ATP磷酸核糖基转移酶由SEQ ID NO:5的氨基酸序列组成。
5.根据权利要求3所述的微生物,其中所述ATP磷酸核糖基转移酶由SEQ ID NO:6的氨基酸序列组成。
6.根据权利要求1至5中任一项所述的微生物,其中所述棒杆菌属的微生物是谷氨酸棒杆菌(Corynebacterium glutamicum)。
7.制备包含甘氨酸和谷氨酸的发酵组合物的方法,所述方法包括通过在培养基中培养权利要求1至5中任一项所述的棒杆菌属的微生物进行发酵。
8.通过权利要求7所述的方法制备的发酵组合物。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR20180035156 | 2018-03-27 | ||
KR10-2018-0035156 | 2018-03-27 | ||
PCT/KR2019/003568 WO2019190193A1 (ko) | 2018-03-27 | 2019-03-27 | 글라이신 생산능이 증가된 미생물 및 이를 이용한 발효 조성물 생산 방법 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111433366A true CN111433366A (zh) | 2020-07-17 |
CN111433366B CN111433366B (zh) | 2024-01-09 |
Family
ID=68060246
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201980004245.2A Active CN111655860B (zh) | 2018-03-27 | 2019-03-27 | 新型启动子及利用其生产l-氨基酸的方法 |
CN201980004266.4A Active CN111433366B (zh) | 2018-03-27 | 2019-03-27 | 具有增加的甘氨酸生产能力的微生物和利用其生产发酵组合物的方法 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201980004245.2A Active CN111655860B (zh) | 2018-03-27 | 2019-03-27 | 新型启动子及利用其生产l-氨基酸的方法 |
Country Status (13)
Country | Link |
---|---|
US (2) | US11661616B2 (zh) |
EP (3) | EP4130281A1 (zh) |
JP (3) | JP7089582B2 (zh) |
KR (3) | KR102137806B1 (zh) |
CN (2) | CN111655860B (zh) |
AR (2) | AR115301A1 (zh) |
AU (2) | AU2019243241B2 (zh) |
BR (2) | BR112020003867B1 (zh) |
CA (2) | CA3073180C (zh) |
MY (1) | MY202021A (zh) |
SG (2) | SG11202001753TA (zh) |
TW (3) | TWI746953B (zh) |
WO (2) | WO2019190192A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115003806A (zh) * | 2021-01-26 | 2022-09-02 | Cj第一制糖株式会社 | 新atp磷酸核糖基转移酶变体及使用其生产l-缬氨酸的方法 |
CN116254242A (zh) * | 2022-12-21 | 2023-06-13 | 江南大学 | 一种atp磷酸核苷转移酶突变体及产l-组氨酸的谷氨酸棒杆菌 |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AR115301A1 (es) * | 2018-03-27 | 2020-12-23 | Cj Cheiljedang Corp | Microorganismo que tiene aumento de productividad de glicina y método para producir una composición fermentada usando el mismo |
KR20220094257A (ko) * | 2020-12-28 | 2022-07-06 | 대상 주식회사 | 히스티딘에 의한 피드백 억제가 감소된 atp-prt 변이체 및 이를 발현하는 히스티딘 생산 균주 |
KR102433200B1 (ko) * | 2020-12-28 | 2022-08-19 | 대상 주식회사 | 히스티딘에 의한 피드백 억제가 감소된 atp-prt 변이체 및 이를 발현하는 히스티딘 생산 균주 |
EP4105333A4 (en) * | 2021-05-07 | 2024-07-10 | Cj Cheiljedang Corp | NEW PROMOTER AND ITS USE |
KR102377745B1 (ko) * | 2021-05-12 | 2022-03-23 | 씨제이제일제당 주식회사 | 신규 프로모터 및 이의 용도 |
EP4381961A1 (en) * | 2021-08-02 | 2024-06-12 | Ajinomoto Co., Inc. | Method for improving flavor of food |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090104667A1 (en) * | 2006-04-28 | 2009-04-23 | Yoko Asakura | L-amino acid-producing microorganism and a method for producing an l-amino acid |
US20150284760A1 (en) * | 2012-08-22 | 2015-10-08 | Forschungszentrum Juelich Gmbh | Method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition and the use thereof for producing amino acids and nucleotides |
US20160222394A1 (en) * | 2013-10-23 | 2016-08-04 | Ajinomoto Co., Inc. | Method for Producing Target Substance |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3220929A (en) | 1964-02-10 | 1965-11-30 | Kyowa Hakko Kogyo Kk | Microbiological production of amino acid by reductive amination |
AU746542B2 (en) | 1998-03-18 | 2002-05-02 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
KR100292299B1 (ko) | 1999-03-22 | 2001-06-01 | 손경식 | 글루탐산 생산 미생물 및 이를 이용한 글루탐산 생산방법 |
US6984512B1 (en) * | 1999-08-02 | 2006-01-10 | Archer-Daniels-Midland Company | Bacterial strains, methods of preparing the same and use thereof in fermentation processes for L-lysine production |
KR100815041B1 (ko) * | 1999-08-02 | 2008-03-18 | 아처 다니엘 미드랜드 캄파니 | 아미노산 생산의 대사 공학 |
DE10044831A1 (de) * | 2000-03-01 | 2002-04-04 | Forschungszentrum Juelich Gmbh | Verbessertes Verfahren zur mikrobiellen Herstellung von L-Serin sowie ein dazu geeigneter genetisch veränderter Mikroorganismus |
WO2005010175A1 (en) | 2003-07-29 | 2005-02-03 | Ajinomoto Co., Inc. | Method for producing l-lysine or l-threonine using escherichia bacteria having attnuated malic enzyme activity |
US9109242B2 (en) | 2006-09-15 | 2015-08-18 | Cj Cheiljedang Corporation | Corynebacteria having enhanced L-lysine productivity and a method of producing L-lysine using the same |
KR101492134B1 (ko) | 2012-05-22 | 2015-02-12 | 백광산업 주식회사 | 코리네형 세균 유래 sod 유전자의 프로모터 변이체 및 상기 프로모터 변이체를 이용한 L-라이신의 생산 방법 |
CN104845923B (zh) | 2014-02-14 | 2018-03-23 | 中国科学院微生物研究所 | 生产l‑组氨酸的方法及其专用重组菌 |
KR20160072278A (ko) | 2014-12-12 | 2016-06-23 | 한국외국어대학교 연구산학협력단 | 합성 글루탐산 탈수소효소 프로모터를 이용한 목적단백질의 생산 방법 |
KR101904666B1 (ko) * | 2017-08-02 | 2018-11-29 | 씨제이제일제당 (주) | Atp 포스포리보실기 전이효소 변이체 및 이를 이용한 l-히스티딘 생산방법 |
AR115301A1 (es) * | 2018-03-27 | 2020-12-23 | Cj Cheiljedang Corp | Microorganismo que tiene aumento de productividad de glicina y método para producir una composición fermentada usando el mismo |
-
2019
- 2019-03-27 AR ARP190100764A patent/AR115301A1/es unknown
- 2019-03-27 BR BR112020003867-9A patent/BR112020003867B1/pt active IP Right Grant
- 2019-03-27 US US16/638,974 patent/US11661616B2/en active Active
- 2019-03-27 MY MYPI2020000969A patent/MY202021A/en unknown
- 2019-03-27 KR KR1020190035199A patent/KR102137806B1/ko active IP Right Grant
- 2019-03-27 WO PCT/KR2019/003565 patent/WO2019190192A1/ko unknown
- 2019-03-27 CN CN201980004245.2A patent/CN111655860B/zh active Active
- 2019-03-27 TW TW108110751A patent/TWI746953B/zh active
- 2019-03-27 TW TW108110750A patent/TWI716841B/zh active
- 2019-03-27 AR ARP190100765A patent/AR115302A1/es unknown
- 2019-03-27 AU AU2019243241A patent/AU2019243241B2/en active Active
- 2019-03-27 EP EP22181523.6A patent/EP4130281A1/en active Pending
- 2019-03-27 AU AU2019243242A patent/AU2019243242B2/en active Active
- 2019-03-27 WO PCT/KR2019/003568 patent/WO2019190193A1/ko unknown
- 2019-03-27 TW TW109131887A patent/TW202117002A/zh unknown
- 2019-03-27 EP EP19775192.8A patent/EP3778900A4/en active Pending
- 2019-03-27 EP EP19776661.1A patent/EP3778901A4/en active Pending
- 2019-03-27 CA CA3073180A patent/CA3073180C/en active Active
- 2019-03-27 JP JP2020512734A patent/JP7089582B2/ja active Active
- 2019-03-27 SG SG11202001753TA patent/SG11202001753TA/en unknown
- 2019-03-27 CN CN201980004266.4A patent/CN111433366B/zh active Active
- 2019-03-27 JP JP2020512742A patent/JP7125477B2/ja active Active
- 2019-03-27 KR KR1020190035198A patent/KR102028554B1/ko active IP Right Grant
- 2019-03-27 CA CA3073177A patent/CA3073177C/en active Active
- 2019-03-27 BR BR112020003467A patent/BR112020003467B8/pt active IP Right Grant
- 2019-03-27 US US16/639,364 patent/US10961554B2/en active Active
- 2019-03-27 SG SG11202001730TA patent/SG11202001730TA/en unknown
-
2020
- 2020-07-20 KR KR1020200089780A patent/KR102150041B1/ko active IP Right Grant
-
2022
- 2022-05-06 JP JP2022076489A patent/JP2022105171A/ja active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090104667A1 (en) * | 2006-04-28 | 2009-04-23 | Yoko Asakura | L-amino acid-producing microorganism and a method for producing an l-amino acid |
US20150284760A1 (en) * | 2012-08-22 | 2015-10-08 | Forschungszentrum Juelich Gmbh | Method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition and the use thereof for producing amino acids and nucleotides |
US20160222394A1 (en) * | 2013-10-23 | 2016-08-04 | Ajinomoto Co., Inc. | Method for Producing Target Substance |
Non-Patent Citations (1)
Title |
---|
GEORG SCHENDZIELORZ等: "Taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways" * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115003806A (zh) * | 2021-01-26 | 2022-09-02 | Cj第一制糖株式会社 | 新atp磷酸核糖基转移酶变体及使用其生产l-缬氨酸的方法 |
CN115003806B (zh) * | 2021-01-26 | 2023-09-08 | Cj第一制糖株式会社 | 新atp磷酸核糖基转移酶变体及使用其生产l-缬氨酸的方法 |
CN116254242A (zh) * | 2022-12-21 | 2023-06-13 | 江南大学 | 一种atp磷酸核苷转移酶突变体及产l-组氨酸的谷氨酸棒杆菌 |
CN116254242B (zh) * | 2022-12-21 | 2024-01-30 | 江南大学 | 一种atp磷酸核苷转移酶突变体及产l-组氨酸的谷氨酸棒杆菌 |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111433366B (zh) | 具有增加的甘氨酸生产能力的微生物和利用其生产发酵组合物的方法 | |
US8058036B2 (en) | Microorganism of Corynebacterium genus having enhanced L-lysine productivity and a method of producing L-lysine using the same | |
CN105392880B (zh) | 生产o-乙酰基高丝氨酸的微生物和用其生产o-乙酰基高丝氨酸的方法 | |
KR102132342B1 (ko) | 신규한 프로모터 및 이를 이용한 l-아미노산 생산 방법 | |
JP2001333769A (ja) | 析出を伴う発酵法によるl−グルタミン酸の製造法 | |
KR20130082124A (ko) | 자일로즈 이용능이 부여된 코리네박테리움 속 미생물 및 이를 이용한 l-라이신의 생산방법 | |
JP2021517802A5 (zh) | ||
CN117098842A (zh) | 具有增强的3-甲基-2-氧代丁酸羟甲基转移酶活性的微生物及其用途 | |
JP2007175016A (ja) | L−グルタミン酸生産菌及びl−グルタミン酸の製造方法 | |
CN113994003B (zh) | 新型启动子及其用途 | |
CN113994002B (zh) | 新型启动子及其用途 | |
RU2787592C1 (ru) | Новый промотор и его применение |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40025408 Country of ref document: HK |
|
GR01 | Patent grant | ||
GR01 | Patent grant |