CN114085800A - 新型异丙基苹果酸合酶变异体及使用其生产l-亮氨酸的方法 - Google Patents
新型异丙基苹果酸合酶变异体及使用其生产l-亮氨酸的方法 Download PDFInfo
- Publication number
- CN114085800A CN114085800A CN202111316466.9A CN202111316466A CN114085800A CN 114085800 A CN114085800 A CN 114085800A CN 202111316466 A CN202111316466 A CN 202111316466A CN 114085800 A CN114085800 A CN 114085800A
- Authority
- CN
- China
- Prior art keywords
- val
- ala
- glu
- asp
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 title claims abstract description 166
- 229960003136 leucine Drugs 0.000 title claims abstract description 90
- 239000004395 L-leucine Substances 0.000 title claims abstract description 75
- 235000019454 L-leucine Nutrition 0.000 title claims abstract description 75
- KPULXFNPTWGJQH-UHFFFAOYSA-N 3-hydroxy-4-oxo-4-propan-2-yloxybutanoic acid Chemical compound CC(C)OC(=O)C(O)CC(O)=O KPULXFNPTWGJQH-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 238000004519 manufacturing process Methods 0.000 title claims description 23
- 229920001184 polypeptide Polymers 0.000 claims abstract description 62
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 62
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 62
- 230000000694 effects Effects 0.000 claims abstract description 46
- 244000005700 microbiome Species 0.000 claims abstract description 40
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 24
- 239000002157 polynucleotide Substances 0.000 claims abstract description 24
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 62
- 239000013598 vector Substances 0.000 claims description 47
- 150000001413 amino acids Chemical group 0.000 claims description 22
- 241000186216 Corynebacterium Species 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 15
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 14
- 230000014509 gene expression Effects 0.000 claims description 13
- 239000004471 Glycine Substances 0.000 claims description 10
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 8
- 235000003704 aspartic acid Nutrition 0.000 claims description 8
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 5
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 claims description 4
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims 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 48
- 101150087199 leuA gene Proteins 0.000 description 43
- 108020004414 DNA Proteins 0.000 description 39
- 108090000623 proteins and genes Proteins 0.000 description 36
- DRCOAZZDQRCGGP-GHCJXIJMSA-N Asp-Ser-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DRCOAZZDQRCGGP-GHCJXIJMSA-N 0.000 description 32
- MVJRBCJCRYGCKV-GVXVVHGQSA-N Leu-Val-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MVJRBCJCRYGCKV-GVXVVHGQSA-N 0.000 description 32
- XABXVVSWUVCZST-GVXVVHGQSA-N Lys-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN XABXVVSWUVCZST-GVXVVHGQSA-N 0.000 description 32
- JLFKWDAZBRYCGX-ZKWXMUAHSA-N Val-Asn-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N JLFKWDAZBRYCGX-ZKWXMUAHSA-N 0.000 description 32
- 108010005233 alanylglutamic acid Proteins 0.000 description 32
- 125000003275 alpha amino acid group Chemical group 0.000 description 32
- 108010068380 arginylarginine Proteins 0.000 description 32
- 108010092854 aspartyllysine Proteins 0.000 description 32
- 108010015792 glycyllysine Proteins 0.000 description 32
- 108010012581 phenylalanylglutamate Proteins 0.000 description 32
- 108010026333 seryl-proline Proteins 0.000 description 32
- 108010071207 serylmethionine Proteins 0.000 description 32
- 229940024606 amino acid Drugs 0.000 description 31
- 235000001014 amino acid Nutrition 0.000 description 31
- 125000003729 nucleotide group Chemical group 0.000 description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 19
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 18
- UGLPMYSCWHTZQU-AUTRQRHGSA-N Ala-Ala-Tyr Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 UGLPMYSCWHTZQU-AUTRQRHGSA-N 0.000 description 16
- SVBXIUDNTRTKHE-CIUDSAMLSA-N Ala-Arg-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O SVBXIUDNTRTKHE-CIUDSAMLSA-N 0.000 description 16
- YAXNATKKPOWVCP-ZLUOBGJFSA-N Ala-Asn-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O YAXNATKKPOWVCP-ZLUOBGJFSA-N 0.000 description 16
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 16
- YCRAFFCYWOUEOF-DLOVCJGASA-N Ala-Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 YCRAFFCYWOUEOF-DLOVCJGASA-N 0.000 description 16
- IPZQNYYAYVRKKK-FXQIFTODSA-N Ala-Pro-Ala Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IPZQNYYAYVRKKK-FXQIFTODSA-N 0.000 description 16
- LSMDIAAALJJLRO-XQXXSGGOSA-N Ala-Thr-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O LSMDIAAALJJLRO-XQXXSGGOSA-N 0.000 description 16
- XMIAMUXIMWREBJ-HERUPUMHSA-N Ala-Trp-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(=O)N)C(=O)O)N XMIAMUXIMWREBJ-HERUPUMHSA-N 0.000 description 16
- XSLGWYYNOSUMRM-ZKWXMUAHSA-N Ala-Val-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XSLGWYYNOSUMRM-ZKWXMUAHSA-N 0.000 description 16
- BVLPIIBTWIYOML-ZKWXMUAHSA-N Ala-Val-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O BVLPIIBTWIYOML-ZKWXMUAHSA-N 0.000 description 16
- YYOVLDPHIJAOSY-DCAQKATOSA-N Arg-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N YYOVLDPHIJAOSY-DCAQKATOSA-N 0.000 description 16
- RVDVDRUZWZIBJQ-CIUDSAMLSA-N Arg-Asn-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O RVDVDRUZWZIBJQ-CIUDSAMLSA-N 0.000 description 16
- KMSHNDWHPWXPEC-BQBZGAKWSA-N Arg-Asp-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KMSHNDWHPWXPEC-BQBZGAKWSA-N 0.000 description 16
- YSUVMPICYVWRBX-VEVYYDQMSA-N Arg-Asp-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YSUVMPICYVWRBX-VEVYYDQMSA-N 0.000 description 16
- KRQSPVKUISQQFS-FJXKBIBVSA-N Arg-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N KRQSPVKUISQQFS-FJXKBIBVSA-N 0.000 description 16
- PAPSMOYMQDWIOR-AVGNSLFASA-N Arg-Lys-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PAPSMOYMQDWIOR-AVGNSLFASA-N 0.000 description 16
- AFNHFVVOJZBIJD-GUBZILKMSA-N Arg-Met-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O AFNHFVVOJZBIJD-GUBZILKMSA-N 0.000 description 16
- ASQKVGRCKOFKIU-KZVJFYERSA-N Arg-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ASQKVGRCKOFKIU-KZVJFYERSA-N 0.000 description 16
- MOGMYRUNTKYZFB-UNQGMJICSA-N Arg-Thr-Phe Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MOGMYRUNTKYZFB-UNQGMJICSA-N 0.000 description 16
- ULBHWNVWSCJLCO-NHCYSSNCSA-N Arg-Val-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N ULBHWNVWSCJLCO-NHCYSSNCSA-N 0.000 description 16
- SUMJNGAMIQSNGX-TUAOUCFPSA-N Arg-Val-Pro Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N1CCC[C@@H]1C(O)=O SUMJNGAMIQSNGX-TUAOUCFPSA-N 0.000 description 16
- PDQBXRSOSCTGKY-ACZMJKKPSA-N Asn-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N PDQBXRSOSCTGKY-ACZMJKKPSA-N 0.000 description 16
- DNYRZPOWBTYFAF-IHRRRGAJSA-N Asn-Arg-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)N)N)O DNYRZPOWBTYFAF-IHRRRGAJSA-N 0.000 description 16
- KXEGPPNPXOKKHK-ZLUOBGJFSA-N Asn-Asp-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O KXEGPPNPXOKKHK-ZLUOBGJFSA-N 0.000 description 16
- FAEFJTCTNZTPHX-ACZMJKKPSA-N Asn-Gln-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O FAEFJTCTNZTPHX-ACZMJKKPSA-N 0.000 description 16
- SJPZTWAYTJPPBI-GUBZILKMSA-N Asn-Gln-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)N)N SJPZTWAYTJPPBI-GUBZILKMSA-N 0.000 description 16
- FLJVGAFLZVBBNG-BPUTZDHNSA-N Asn-Trp-Arg Chemical compound N[C@@H](CC(=O)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(=N)N)C(=O)O FLJVGAFLZVBBNG-BPUTZDHNSA-N 0.000 description 16
- XOQYDFCQPWAMSA-KKHAAJSZSA-N Asn-Val-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XOQYDFCQPWAMSA-KKHAAJSZSA-N 0.000 description 16
- WQAOZCVOOYUWKG-LSJOCFKGSA-N Asn-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CC(=O)N)N WQAOZCVOOYUWKG-LSJOCFKGSA-N 0.000 description 16
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 description 16
- XEDQMTWEYFBOIK-ACZMJKKPSA-N Asp-Ala-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XEDQMTWEYFBOIK-ACZMJKKPSA-N 0.000 description 16
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 description 16
- NYLBGYLHBDFRHL-VEVYYDQMSA-N Asp-Arg-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NYLBGYLHBDFRHL-VEVYYDQMSA-N 0.000 description 16
- PYXXJFRXIYAESU-PCBIJLKTSA-N Asp-Ile-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PYXXJFRXIYAESU-PCBIJLKTSA-N 0.000 description 16
- WOPJVEMFXYHZEE-SRVKXCTJSA-N Asp-Phe-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O WOPJVEMFXYHZEE-SRVKXCTJSA-N 0.000 description 16
- KNDCWFXCFKSEBM-AVGNSLFASA-N Asp-Tyr-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O KNDCWFXCFKSEBM-AVGNSLFASA-N 0.000 description 16
- PLOKOIJSGCISHE-BYULHYEWSA-N Asp-Val-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PLOKOIJSGCISHE-BYULHYEWSA-N 0.000 description 16
- RKXVTTIQNKPCHU-KKHAAJSZSA-N Asp-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O RKXVTTIQNKPCHU-KKHAAJSZSA-N 0.000 description 16
- CYTSBCIIEHUPDU-ACZMJKKPSA-N Gln-Asp-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O CYTSBCIIEHUPDU-ACZMJKKPSA-N 0.000 description 16
- OFPWCBGRYAOLMU-AVGNSLFASA-N Gln-Asp-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O OFPWCBGRYAOLMU-AVGNSLFASA-N 0.000 description 16
- JHPFPROFOAJRFN-IHRRRGAJSA-N Gln-Glu-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O JHPFPROFOAJRFN-IHRRRGAJSA-N 0.000 description 16
- OSCLNNWLKKIQJM-WDSKDSINSA-N Gln-Ser-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O OSCLNNWLKKIQJM-WDSKDSINSA-N 0.000 description 16
- YADSXULAFMJZRL-QEJZJMRPSA-N Gln-Trp-Cys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)N)N YADSXULAFMJZRL-QEJZJMRPSA-N 0.000 description 16
- UBRQJXFDVZNYJP-AVGNSLFASA-N Gln-Tyr-Ser Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O UBRQJXFDVZNYJP-AVGNSLFASA-N 0.000 description 16
- UTKICHUQEQBDGC-ACZMJKKPSA-N Glu-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N UTKICHUQEQBDGC-ACZMJKKPSA-N 0.000 description 16
- PBEQPAZRHDVJQI-SRVKXCTJSA-N Glu-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)O)N PBEQPAZRHDVJQI-SRVKXCTJSA-N 0.000 description 16
- RDPOETHPAQEGDP-ACZMJKKPSA-N Glu-Asp-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RDPOETHPAQEGDP-ACZMJKKPSA-N 0.000 description 16
- IESFZVCAVACGPH-PEFMBERDSA-N Glu-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O IESFZVCAVACGPH-PEFMBERDSA-N 0.000 description 16
- PVBBEKPHARMPHX-DCAQKATOSA-N Glu-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCC(O)=O PVBBEKPHARMPHX-DCAQKATOSA-N 0.000 description 16
- AIGROOHQXCACHL-WDSKDSINSA-N Glu-Gly-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O AIGROOHQXCACHL-WDSKDSINSA-N 0.000 description 16
- VGBSZQSKQRMLHD-MNXVOIDGSA-N Glu-Leu-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VGBSZQSKQRMLHD-MNXVOIDGSA-N 0.000 description 16
- FGSGPLRPQCZBSQ-AVGNSLFASA-N Glu-Phe-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O FGSGPLRPQCZBSQ-AVGNSLFASA-N 0.000 description 16
- BPCLDCNZBUYGOD-BPUTZDHNSA-N Glu-Trp-Glu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 BPCLDCNZBUYGOD-BPUTZDHNSA-N 0.000 description 16
- ZSIDREAPEPAPKL-XIRDDKMYSA-N Glu-Trp-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCC(=O)O)N ZSIDREAPEPAPKL-XIRDDKMYSA-N 0.000 description 16
- HVKAAUOFFTUSAA-XDTLVQLUSA-N Glu-Tyr-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O HVKAAUOFFTUSAA-XDTLVQLUSA-N 0.000 description 16
- LZEUDRYSAZAJIO-AUTRQRHGSA-N Glu-Val-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LZEUDRYSAZAJIO-AUTRQRHGSA-N 0.000 description 16
- HQTDNEZTGZUWSY-XVKPBYJWSA-N Glu-Val-Gly Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)NCC(O)=O HQTDNEZTGZUWSY-XVKPBYJWSA-N 0.000 description 16
- 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 16
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 16
- FZQLXNIMCPJVJE-YUMQZZPRSA-N Gly-Asp-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O FZQLXNIMCPJVJE-YUMQZZPRSA-N 0.000 description 16
- IXKRSKPKSLXIHN-YUMQZZPRSA-N Gly-Cys-Leu Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O IXKRSKPKSLXIHN-YUMQZZPRSA-N 0.000 description 16
- PABFFPWEJMEVEC-JGVFFNPUSA-N Gly-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)CN)C(=O)O PABFFPWEJMEVEC-JGVFFNPUSA-N 0.000 description 16
- DHDOADIPGZTAHT-YUMQZZPRSA-N Gly-Glu-Arg Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DHDOADIPGZTAHT-YUMQZZPRSA-N 0.000 description 16
- GDOZQTNZPCUARW-YFKPBYRVSA-N Gly-Gly-Glu Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O GDOZQTNZPCUARW-YFKPBYRVSA-N 0.000 description 16
- SWQALSGKVLYKDT-ZKWXMUAHSA-N Gly-Ile-Ala Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SWQALSGKVLYKDT-ZKWXMUAHSA-N 0.000 description 16
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 16
- UTYGDAHJBBDPBA-BYULHYEWSA-N Gly-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN UTYGDAHJBBDPBA-BYULHYEWSA-N 0.000 description 16
- YTSVAIMKVLZUDU-YUMQZZPRSA-N Gly-Leu-Asp Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O YTSVAIMKVLZUDU-YUMQZZPRSA-N 0.000 description 16
- HFPVRZWORNJRRC-UWVGGRQHSA-N Gly-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN HFPVRZWORNJRRC-UWVGGRQHSA-N 0.000 description 16
- MKIAPEZXQDILRR-YUMQZZPRSA-N Gly-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN MKIAPEZXQDILRR-YUMQZZPRSA-N 0.000 description 16
- VNNRLUNBJSWZPF-ZKWXMUAHSA-N Gly-Ser-Ile Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VNNRLUNBJSWZPF-ZKWXMUAHSA-N 0.000 description 16
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 16
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 16
- CIWILNZNBPIHEU-DCAQKATOSA-N His-Arg-Asn Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O CIWILNZNBPIHEU-DCAQKATOSA-N 0.000 description 16
- OMNVOTCFQQLEQU-CIUDSAMLSA-N His-Asn-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N OMNVOTCFQQLEQU-CIUDSAMLSA-N 0.000 description 16
- NOQPTNXSGNPJNS-YUMQZZPRSA-N His-Asn-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O NOQPTNXSGNPJNS-YUMQZZPRSA-N 0.000 description 16
- VBOFRJNDIOPNDO-YUMQZZPRSA-N His-Gly-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N VBOFRJNDIOPNDO-YUMQZZPRSA-N 0.000 description 16
- VAXBXNPRXPHGHG-BJDJZHNGSA-N Ile-Ala-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)O)N VAXBXNPRXPHGHG-BJDJZHNGSA-N 0.000 description 16
- ASCFJMSGKUIRDU-ZPFDUUQYSA-N Ile-Arg-Gln Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O ASCFJMSGKUIRDU-ZPFDUUQYSA-N 0.000 description 16
- NULSANWBUWLTKN-NAKRPEOUSA-N Ile-Arg-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N NULSANWBUWLTKN-NAKRPEOUSA-N 0.000 description 16
- XENGULNPUDGALZ-ZPFDUUQYSA-N Ile-Asn-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N XENGULNPUDGALZ-ZPFDUUQYSA-N 0.000 description 16
- DVRDRICMWUSCBN-UKJIMTQDSA-N Ile-Gln-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N DVRDRICMWUSCBN-UKJIMTQDSA-N 0.000 description 16
- SJLVSMMIFYTSGY-GRLWGSQLSA-N Ile-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N SJLVSMMIFYTSGY-GRLWGSQLSA-N 0.000 description 16
- GVKKVHNRTUFCCE-BJDJZHNGSA-N Ile-Leu-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)O)N GVKKVHNRTUFCCE-BJDJZHNGSA-N 0.000 description 16
- ZUPJCJINYQISSN-XUXIUFHCSA-N Ile-Met-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)O)N ZUPJCJINYQISSN-XUXIUFHCSA-N 0.000 description 16
- OWSWUWDMSNXTNE-GMOBBJLQSA-N Ile-Pro-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N OWSWUWDMSNXTNE-GMOBBJLQSA-N 0.000 description 16
- CNMOKANDJMLAIF-CIQUZCHMSA-N Ile-Thr-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O CNMOKANDJMLAIF-CIQUZCHMSA-N 0.000 description 16
- ANTFEOSJMAUGIB-KNZXXDILSA-N Ile-Thr-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N ANTFEOSJMAUGIB-KNZXXDILSA-N 0.000 description 16
- 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 16
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 16
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 16
- IGUOAYLTQJLPPD-DCAQKATOSA-N Leu-Asn-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IGUOAYLTQJLPPD-DCAQKATOSA-N 0.000 description 16
- VPKIQULSKFVCSM-SRVKXCTJSA-N Leu-Gln-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VPKIQULSKFVCSM-SRVKXCTJSA-N 0.000 description 16
- RSFGIMMPWAXNML-MNXVOIDGSA-N Leu-Gln-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RSFGIMMPWAXNML-MNXVOIDGSA-N 0.000 description 16
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 16
- WRLPVDVHNWSSCL-MELADBBJSA-N Leu-His-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@@H]2C(=O)O)N WRLPVDVHNWSSCL-MELADBBJSA-N 0.000 description 16
- AVEGDIAXTDVBJS-XUXIUFHCSA-N Leu-Ile-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AVEGDIAXTDVBJS-XUXIUFHCSA-N 0.000 description 16
- KOSWSHVQIVTVQF-ZPFDUUQYSA-N Leu-Ile-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O KOSWSHVQIVTVQF-ZPFDUUQYSA-N 0.000 description 16
- HNDWYLYAYNBWMP-AJNGGQMLSA-N Leu-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N HNDWYLYAYNBWMP-AJNGGQMLSA-N 0.000 description 16
- ZRHDPZAAWLXXIR-SRVKXCTJSA-N Leu-Lys-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O ZRHDPZAAWLXXIR-SRVKXCTJSA-N 0.000 description 16
- YUTNOGOMBNYPFH-XUXIUFHCSA-N Leu-Pro-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YUTNOGOMBNYPFH-XUXIUFHCSA-N 0.000 description 16
- WQWZXKWOEVSGQM-DCAQKATOSA-N Lys-Ala-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN WQWZXKWOEVSGQM-DCAQKATOSA-N 0.000 description 16
- CLBGMWIYPYAZPR-AVGNSLFASA-N Lys-Arg-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O CLBGMWIYPYAZPR-AVGNSLFASA-N 0.000 description 16
- LZWNAOIMTLNMDW-NHCYSSNCSA-N Lys-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N LZWNAOIMTLNMDW-NHCYSSNCSA-N 0.000 description 16
- SSJBMGCZZXCGJJ-DCAQKATOSA-N Lys-Asp-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O SSJBMGCZZXCGJJ-DCAQKATOSA-N 0.000 description 16
- GKFNXYMAMKJSKD-NHCYSSNCSA-N Lys-Asp-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O GKFNXYMAMKJSKD-NHCYSSNCSA-N 0.000 description 16
- WTZUSCUIVPVCRH-SRVKXCTJSA-N Lys-Gln-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WTZUSCUIVPVCRH-SRVKXCTJSA-N 0.000 description 16
- LPAJOCKCPRZEAG-MNXVOIDGSA-N Lys-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCCCN LPAJOCKCPRZEAG-MNXVOIDGSA-N 0.000 description 16
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 16
- QBEPTBMRQALPEV-MNXVOIDGSA-N Lys-Ile-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN QBEPTBMRQALPEV-MNXVOIDGSA-N 0.000 description 16
- GAHJXEMYXKLZRQ-AJNGGQMLSA-N Lys-Lys-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GAHJXEMYXKLZRQ-AJNGGQMLSA-N 0.000 description 16
- YRAWWKUTNBILNT-FXQIFTODSA-N Met-Ala-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YRAWWKUTNBILNT-FXQIFTODSA-N 0.000 description 16
- DNDVVILEHVMWIS-LPEHRKFASA-N Met-Asp-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N DNDVVILEHVMWIS-LPEHRKFASA-N 0.000 description 16
- PHWSCIFNNLLUFJ-NHCYSSNCSA-N Met-Gln-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCSC)N PHWSCIFNNLLUFJ-NHCYSSNCSA-N 0.000 description 16
- LQMHZERGCQJKAH-STQMWFEESA-N Met-Gly-Phe Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 LQMHZERGCQJKAH-STQMWFEESA-N 0.000 description 16
- FBLBCGLSRXBANI-KKUMJFAQSA-N Met-Phe-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FBLBCGLSRXBANI-KKUMJFAQSA-N 0.000 description 16
- MQASRXPTQJJNFM-JYJNAYRXSA-N Met-Pro-Phe Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MQASRXPTQJJNFM-JYJNAYRXSA-N 0.000 description 16
- BJPQKNHZHUCQNQ-SRVKXCTJSA-N Met-Pro-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCSC)N BJPQKNHZHUCQNQ-SRVKXCTJSA-N 0.000 description 16
- PHURAEXVWLDIGT-LPEHRKFASA-N Met-Ser-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N PHURAEXVWLDIGT-LPEHRKFASA-N 0.000 description 16
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 16
- 108010066427 N-valyltryptophan Proteins 0.000 description 16
- WPTYDQPGBMDUBI-QWRGUYRKSA-N Phe-Gly-Asn Chemical compound N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O WPTYDQPGBMDUBI-QWRGUYRKSA-N 0.000 description 16
- CWFGECHCRMGPPT-MXAVVETBSA-N Phe-Ile-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O CWFGECHCRMGPPT-MXAVVETBSA-N 0.000 description 16
- NJJBATPLUQHRBM-IHRRRGAJSA-N Phe-Pro-Ser Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CO)C(=O)O NJJBATPLUQHRBM-IHRRRGAJSA-N 0.000 description 16
- RAGOJJCBGXARPO-XVSYOHENSA-N Phe-Thr-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 RAGOJJCBGXARPO-XVSYOHENSA-N 0.000 description 16
- BSKMOCNNLNDIMU-CDMKHQONSA-N Phe-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O BSKMOCNNLNDIMU-CDMKHQONSA-N 0.000 description 16
- CYQQWUPHIZVCNY-GUBZILKMSA-N Pro-Arg-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O CYQQWUPHIZVCNY-GUBZILKMSA-N 0.000 description 16
- SFECXGVELZFBFJ-VEVYYDQMSA-N Pro-Asp-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SFECXGVELZFBFJ-VEVYYDQMSA-N 0.000 description 16
- PULPZRAHVFBVTO-DCAQKATOSA-N Pro-Glu-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PULPZRAHVFBVTO-DCAQKATOSA-N 0.000 description 16
- LXVLKXPFIDDHJG-CIUDSAMLSA-N Pro-Glu-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O LXVLKXPFIDDHJG-CIUDSAMLSA-N 0.000 description 16
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 16
- ZLXKLMHAMDENIO-DCAQKATOSA-N Pro-Lys-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLXKLMHAMDENIO-DCAQKATOSA-N 0.000 description 16
- ZJXXCGZFYQQETF-CYDGBPFRSA-N Pro-Met-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@@H]1CCCN1 ZJXXCGZFYQQETF-CYDGBPFRSA-N 0.000 description 16
- WLJYLAQSUSIQNH-GUBZILKMSA-N Pro-Met-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@@H]1CCCN1 WLJYLAQSUSIQNH-GUBZILKMSA-N 0.000 description 16
- KWMZPPWYBVZIER-XGEHTFHBSA-N Pro-Ser-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWMZPPWYBVZIER-XGEHTFHBSA-N 0.000 description 16
- LZHHZYDPMZEMRX-STQMWFEESA-N Pro-Tyr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O LZHHZYDPMZEMRX-STQMWFEESA-N 0.000 description 16
- IMNVAOPEMFDAQD-NHCYSSNCSA-N Pro-Val-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IMNVAOPEMFDAQD-NHCYSSNCSA-N 0.000 description 16
- UCTIUWKCVNGEFH-OBJOEFQTSA-N Pro-Val-Gly-Pro Chemical compound N([C@@H](C(C)C)C(=O)NCC(=O)N1[C@@H](CCC1)C(O)=O)C(=O)[C@@H]1CCCN1 UCTIUWKCVNGEFH-OBJOEFQTSA-N 0.000 description 16
- KJMOINFQVCCSDX-XKBZYTNZSA-N Ser-Gln-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KJMOINFQVCCSDX-XKBZYTNZSA-N 0.000 description 16
- KCGIREHVWRXNDH-GARJFASQSA-N Ser-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N KCGIREHVWRXNDH-GARJFASQSA-N 0.000 description 16
- PYTKULIABVRXSC-BWBBJGPYSA-N Ser-Ser-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PYTKULIABVRXSC-BWBBJGPYSA-N 0.000 description 16
- NOWXWJLVGTVJKM-PBCZWWQYSA-N Thr-Asp-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O NOWXWJLVGTVJKM-PBCZWWQYSA-N 0.000 description 16
- CQNFRKAKGDSJFR-NUMRIWBASA-N Thr-Glu-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O CQNFRKAKGDSJFR-NUMRIWBASA-N 0.000 description 16
- BNGDYRRHRGOPHX-IFFSRLJSSA-N Thr-Glu-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O BNGDYRRHRGOPHX-IFFSRLJSSA-N 0.000 description 16
- XFTYVCHLARBHBQ-FOHZUACHSA-N Thr-Gly-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O XFTYVCHLARBHBQ-FOHZUACHSA-N 0.000 description 16
- XKWABWFMQXMUMT-HJGDQZAQSA-N Thr-Pro-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XKWABWFMQXMUMT-HJGDQZAQSA-N 0.000 description 16
- NQQMWWVVGIXUOX-SVSWQMSJSA-N Thr-Ser-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NQQMWWVVGIXUOX-SVSWQMSJSA-N 0.000 description 16
- LXXCHJKHJYRMIY-FQPOAREZSA-N Thr-Tyr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O LXXCHJKHJYRMIY-FQPOAREZSA-N 0.000 description 16
- OGOYMQWIWHGTGH-KZVJFYERSA-N Thr-Val-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O OGOYMQWIWHGTGH-KZVJFYERSA-N 0.000 description 16
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 16
- KPMIQCXJDVKWKO-IFFSRLJSSA-N Thr-Val-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O KPMIQCXJDVKWKO-IFFSRLJSSA-N 0.000 description 16
- VYVBSMCZNHOZGD-RCWTZXSCSA-N Thr-Val-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O VYVBSMCZNHOZGD-RCWTZXSCSA-N 0.000 description 16
- WSGPBCAGEGHKQJ-BBRMVZONSA-N Trp-Gly-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC1=CNC2=CC=CC=C21)N WSGPBCAGEGHKQJ-BBRMVZONSA-N 0.000 description 16
- GQNCRIFNDVFRNF-BPUTZDHNSA-N Trp-Pro-Asp Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O GQNCRIFNDVFRNF-BPUTZDHNSA-N 0.000 description 16
- SCCKSNREWHMKOJ-SRVKXCTJSA-N Tyr-Asn-Ser Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O SCCKSNREWHMKOJ-SRVKXCTJSA-N 0.000 description 16
- XBWKCYFGRXKWGO-SRVKXCTJSA-N Tyr-Cys-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(O)=O XBWKCYFGRXKWGO-SRVKXCTJSA-N 0.000 description 16
- KSCVLGXNQXKUAR-JYJNAYRXSA-N Tyr-Leu-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O KSCVLGXNQXKUAR-JYJNAYRXSA-N 0.000 description 16
- 108010064997 VPY tripeptide Proteins 0.000 description 16
- ZLFHAAGHGQBQQN-AEJSXWLSSA-N Val-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N ZLFHAAGHGQBQQN-AEJSXWLSSA-N 0.000 description 16
- ZLFHAAGHGQBQQN-GUBZILKMSA-N Val-Ala-Pro Natural products CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O ZLFHAAGHGQBQQN-GUBZILKMSA-N 0.000 description 16
- LABUITCFCAABSV-BPNCWPANSA-N Val-Ala-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 LABUITCFCAABSV-BPNCWPANSA-N 0.000 description 16
- LABUITCFCAABSV-UHFFFAOYSA-N Val-Ala-Tyr Natural products CC(C)C(N)C(=O)NC(C)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LABUITCFCAABSV-UHFFFAOYSA-N 0.000 description 16
- KKHRWGYHBZORMQ-NHCYSSNCSA-N Val-Arg-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKHRWGYHBZORMQ-NHCYSSNCSA-N 0.000 description 16
- OGNMURQZFMHFFD-NHCYSSNCSA-N Val-Asn-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N OGNMURQZFMHFFD-NHCYSSNCSA-N 0.000 description 16
- LHADRQBREKTRLR-DCAQKATOSA-N Val-Cys-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N LHADRQBREKTRLR-DCAQKATOSA-N 0.000 description 16
- VCAWFLIWYNMHQP-UKJIMTQDSA-N Val-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N VCAWFLIWYNMHQP-UKJIMTQDSA-N 0.000 description 16
- MHAHQDBEIDPFQS-NHCYSSNCSA-N Val-Glu-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)C(C)C MHAHQDBEIDPFQS-NHCYSSNCSA-N 0.000 description 16
- UEHRGZCNLSWGHK-DLOVCJGASA-N Val-Glu-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UEHRGZCNLSWGHK-DLOVCJGASA-N 0.000 description 16
- JTWIMNMUYLQNPI-WPRPVWTQSA-N Val-Gly-Arg Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N JTWIMNMUYLQNPI-WPRPVWTQSA-N 0.000 description 16
- ZTKGDWOUYRRAOQ-ULQDDVLXSA-N Val-His-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N ZTKGDWOUYRRAOQ-ULQDDVLXSA-N 0.000 description 16
- KDKLLPMFFGYQJD-CYDGBPFRSA-N Val-Ile-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N KDKLLPMFFGYQJD-CYDGBPFRSA-N 0.000 description 16
- YMTOEGGOCHVGEH-IHRRRGAJSA-N Val-Lys-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O YMTOEGGOCHVGEH-IHRRRGAJSA-N 0.000 description 16
- YKNOJPJWNVHORX-UNQGMJICSA-N Val-Phe-Thr Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=CC=C1 YKNOJPJWNVHORX-UNQGMJICSA-N 0.000 description 16
- QWCZXKIFPWPQHR-JYJNAYRXSA-N Val-Pro-Tyr Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QWCZXKIFPWPQHR-JYJNAYRXSA-N 0.000 description 16
- OFTXTCGQJXTNQS-XGEHTFHBSA-N Val-Thr-Ser Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N)O OFTXTCGQJXTNQS-XGEHTFHBSA-N 0.000 description 16
- QPJSIBAOZBVELU-BPNCWPANSA-N Val-Tyr-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N QPJSIBAOZBVELU-BPNCWPANSA-N 0.000 description 16
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 16
- 108010045350 alanyl-tyrosyl-alanine Proteins 0.000 description 16
- 108010044940 alanylglutamine Proteins 0.000 description 16
- 108010011559 alanylphenylalanine Proteins 0.000 description 16
- 108010087924 alanylproline Proteins 0.000 description 16
- 108010062796 arginyllysine Proteins 0.000 description 16
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 16
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 16
- 108010093581 aspartyl-proline Proteins 0.000 description 16
- 108010047857 aspartylglycine Proteins 0.000 description 16
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 16
- 239000012634 fragment Substances 0.000 description 16
- 108010080575 glutamyl-aspartyl-alanine Proteins 0.000 description 16
- 108010008237 glutamyl-valyl-glycine Proteins 0.000 description 16
- 108010049041 glutamylalanine Proteins 0.000 description 16
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 16
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 16
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 16
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 16
- 108010036413 histidylglycine Proteins 0.000 description 16
- 108010085325 histidylproline Proteins 0.000 description 16
- 230000005764 inhibitory process Effects 0.000 description 16
- 108010083708 leucyl-aspartyl-valine Proteins 0.000 description 16
- 108010000761 leucylarginine Proteins 0.000 description 16
- 108010057821 leucylproline Proteins 0.000 description 16
- 108010064235 lysylglycine Proteins 0.000 description 16
- 108010034507 methionyltryptophan Proteins 0.000 description 16
- 108010070643 prolylglutamic acid Proteins 0.000 description 16
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 16
- 108010027345 wheylin-1 peptide Proteins 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 15
- 239000002773 nucleotide Substances 0.000 description 15
- 239000004475 Arginine Substances 0.000 description 13
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 13
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 13
- 108010040030 histidinoalanine Proteins 0.000 description 12
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 10
- 230000002018 overexpression Effects 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 8
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 8
- 230000003115 biocidal effect Effects 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108091008146 restriction endonucleases Proteins 0.000 description 7
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 235000004279 alanine Nutrition 0.000 description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000000137 annealing Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 229910052697 platinum Inorganic materials 0.000 description 5
- 238000006116 polymerization reaction Methods 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 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 4
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 4
- JAMAWBXXKFGFGX-KZVJFYERSA-N Ala-Arg-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JAMAWBXXKFGFGX-KZVJFYERSA-N 0.000 description 4
- SFNFGFDRYJKZKN-XQXXSGGOSA-N Ala-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C)N)O SFNFGFDRYJKZKN-XQXXSGGOSA-N 0.000 description 4
- NJWJSLCQEDMGNC-MBLNEYKQSA-N Ala-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](C)N)O NJWJSLCQEDMGNC-MBLNEYKQSA-N 0.000 description 4
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH 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)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 4
- 241000588722 Escherichia Species 0.000 description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 4
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 4
- 239000013611 chromosomal DNA Substances 0.000 description 4
- 229940093530 coenzyme a Drugs 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 108010050343 histidyl-alanyl-glutamine Proteins 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000013587 production medium Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000004474 valine Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 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 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 3
- 239000005516 coenzyme A Substances 0.000 description 3
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 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 description 3
- 239000006166 lysate Substances 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- 101710102786 ATP-dependent leucine adenylase Proteins 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
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 2
- 241000186146 Brevibacterium Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 241000807905 Corynebacterium glutamicum ATCC 14067 Species 0.000 description 2
- 241000588698 Erwinia Species 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- 239000004472 Lysine Substances 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
- 229930193140 Neomycin Natural products 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
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 241000588768 Providencia Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 241000607720 Serratia Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 239000004098 Tetracycline Substances 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
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 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
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 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
- 235000005822 corn Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229910052564 epsomite Inorganic materials 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- -1 glycine amino acids Chemical class 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 229960000318 kanamycin Drugs 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
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229960004927 neomycin Drugs 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
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 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
- 229960000344 thiamine hydrochloride Drugs 0.000 description 2
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 2
- 239000011747 thiamine hydrochloride Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- 238000005406 washing Methods 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
- HWKRAUXFMLQKLS-UHFFFAOYSA-N 2-oxidanylidenepropanoic acid Chemical compound CC(=O)C(O)=O.CC(=O)C(O)=O HWKRAUXFMLQKLS-UHFFFAOYSA-N 0.000 description 1
- QHKABHOOEWYVLI-UHFFFAOYSA-N 3-methyl-2-oxobutanoic acid Chemical compound CC(C)C(=O)C(O)=O QHKABHOOEWYVLI-UHFFFAOYSA-N 0.000 description 1
- VPEKFOQZFCDXQN-UHFFFAOYSA-N 3-methyl-2-oxobutanoic acid Chemical compound CC(C)C(=O)C(O)=O.CC(C)C(=O)C(O)=O VPEKFOQZFCDXQN-UHFFFAOYSA-N 0.000 description 1
- YQKRCNKNISSARS-UHFFFAOYSA-N 4-methyl-2-oxopentanoic acid Chemical compound CC(C)CC(=O)C(O)=O.CC(C)CC(=O)C(O)=O YQKRCNKNISSARS-UHFFFAOYSA-N 0.000 description 1
- QTXZASLUYMRUAN-QLQASOTGSA-N Acetyl coenzyme A (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.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 QTXZASLUYMRUAN-QLQASOTGSA-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
- 238000009010 Bradford assay Methods 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 229920002307 Dextran Polymers 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
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 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
- 108020005350 Initiator Codon Proteins 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
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 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
- 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
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 240000007019 Oxalis corniculata Species 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 208000005074 Retroviridae Infections Diseases 0.000 description 1
- 238000002105 Southern blotting Methods 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
- 229930006000 Sucrose Natural products 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
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 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
- 239000000908 ammonium hydroxide Substances 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
- 239000003674 animal food additive Substances 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 239000011324 bead Substances 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
- 150000005693 branched-chain amino acids Chemical class 0.000 description 1
- 229910000019 calcium carbonate 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
- 235000013877 carbamide 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
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 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
- 238000005119 centrifugation Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000000975 co-precipitation 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
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004590 computer program Methods 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
- 230000007850 degeneration Effects 0.000 description 1
- 230000008021 deposition 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
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000000284 extract 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
- 239000006260 foam Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000008571 general function Effects 0.000 description 1
- 239000011521 glass Substances 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
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003317 industrial substance Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 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
- BRHPBVXVOVMTIQ-ZLELNMGESA-N l-leucine l-leucine Chemical compound CC(C)C[C@H](N)C(O)=O.CC(C)C[C@H](N)C(O)=O BRHPBVXVOVMTIQ-ZLELNMGESA-N 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
- 239000012139 lysis buffer Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000009126 molecular therapy Methods 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
- 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
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 239000008363 phosphate buffer 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
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229940001941 soy protein Drugs 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
- 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
- 230000005030 transcription termination Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000013519 translation Methods 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
- 239000003981 vehicle Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 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
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
- 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
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- 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
-
- 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)
-
- 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
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
- C12Y203/03013—2-Isopropylmalate synthase (2.3.3.13)
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本公开涉及具有异丙基苹果酸合酶活性的新型修饰多肽、编码该多肽的多核苷酸、包含该多肽的微生物和通过培养该微生物生产L‑亮氨酸的方法。
Description
分案信息
本申请是2017年10月20日递交的申请号为201780001521.0、发明名称为“新型异丙基苹果酸合酶变异体及使用其生产L-亮氨酸的方法”的发明专利申请的分案申请。
技术领域
本公开涉及具有异丙基苹果酸合酶活性的新型修饰多肽、编码该多肽的多核苷酸、包含该多肽的微生物和通过培养该微生物生产L-亮氨酸的方法。
背景技术
L-亮氨酸(L-Leucine)是一种必需氨基酸，其价格昂贵并广泛用于药物、食品、饲料添加剂、工业化学品等。此外，主要使用微生物来生产L-亮氨酸。包括L-亮氨酸在内的支链氨基酸的发酵主要通过埃希氏杆菌属的微生物(a microorganism of the genusEscherichia)或棒状杆菌属的微生物(a microorganism of the genusCorynebacterium)经过若干步骤来进行，已知的是由丙酮酸(pyruvic acid)来生物合成2-酮异己酸(2-ketoisocaproate)而作为前体(precursor)(韩国专利号10-0220018和10-0438146)。
作为参与亮氨酸生物合成的酶，异丙基苹果酸合酶(Isopropylmalate synthase，以下称为“IPMS”)是亮氨酸生物合成中第一步骤的酶，其将缬氨酸生物合成途径中产生的2-酮异戊酸(2-ketoisovalerate)转化为异丙基苹果酸，使得亮氨酸代替缬氨酸进行生物合成，因此IPMS是亮氨酸生物合成过程中重要的酶。然而，IPMS受到最终产物L-亮氨酸或其衍生物的反馈抑制。因此，尽管存在与释放反馈抑制而生产高浓度亮氨酸的IPMS变异体相关的各种现有技术(美国专利公开申请号2005-0079641和美国专利号6403342)，但仍在继续研究以发现更好的变异体。
在这些情况下，本发明人尝试开发一种可用于以高浓度生产L-亮氨酸的IPMS变异体，并由此开发出了一种新型IPMS变异体。据证实，该变异体释放出由L-亮氨酸(为最终产物)引起的反馈抑制并增强了其活性，使得该变异体能够通过含有其的微生物以高产率生产L-亮氨酸，从而实现本公开。
发明内容
技术问题
本公开的一个目的是提供具有异丙基苹果酸合酶活性的新型修饰多肽。
本公开的另一个目的是提供编码所述修饰多肽的多核苷酸。
本公开的又一个目的是提供含有所述多肽的生产L-亮氨酸的棒状杆菌(Corynebacterium)属的微生物。
本公开的又一个目的是提供通过在培养基中培养所述微生物来生产L-亮氨酸的方法。
技术方案
为了实现上述目的，本公开的一方面提供了具有异丙基苹果酸合酶活性的新型修饰多肽。该新型修饰多肽可以是如下具有异丙基苹果酸合酶活性的修饰多肽：其中由氨基酸序列SEQ ID NO：1组成的多肽的N端的558位的精氨酸被非精氨酸的氨基酸残基置换，或者由氨基酸序列SEQ ID NO：1组成的多肽的N端的561位的甘氨酸被非甘氨酸的氨基酸残基置换。本公开的修饰多肽不仅比具有异丙基苹果酸合酶活性的多肽SEQ ID NO：1具有更高的活性，而且还具有释放L-亮氨酸反馈抑制的特征。
如本文所用，术语“异丙基苹果酸合酶”是指通过与乙酰辅酶A(acetyl-CoA)反应来将2-酮异戊酸转化成异丙基苹果酸(L-亮氨酸的前体)的酶。可以包括本公开的异丙基苹果酸合酶，只要该酶具有转化活性即可，而不论微生物的来源。具体地，异丙基苹果酸合酶可以是源自棒状杆菌(Corynebacterium)属的微生物的酶。更具体地，异丙基苹果酸合酶可以是源自谷氨酸棒状杆菌(Corynebacterium glutamicum)的异丙基苹果酸合酶，具体地，可以包括氨基酸序列SEQ ID NO：1，但不限于此。此外，异丙基苹果酸合酶可以包括与氨基酸序列SEQ ID NO：1具有至少80％、90％、95％、96％、97％、98％或99％同源性的多肽。例如，显而易见的是，具有这种同源性并表现出对应于异丙基苹果酸合酶效果的氨基酸序列可以包括在本公开的范围之内，即使其氨基酸序列中的某些序列缺失、被修饰、被置换或被添加。
如本文所用，术语“异丙基苹果酸合酶活性的增加”是指异丙基苹果酸转化活性的增加。因此，与具有异丙基苹果酸合酶活性的多肽SEQ ID NO：1相比，本公开的修饰多肽具有更高水平的异丙基苹果酸转化活性。异丙基苹果酸转化活性可以通过测量产生的异丙基苹果酸水平而被直接证实，或者可以通过测量所产生的辅酶A(CoA)的水平来间接证实。如本文所用，术语“活性的增加”可与“增强的活性”结合使用。此外，异丙基苹果酸是L-亮氨酸的前体，因此与具有异丙基苹果酸合酶活性的多肽SEQ ID NO：1相比，使用本公开的修饰多肽产生了更高水平的L-亮氨酸。
此外，与具有异丙基苹果酸合酶活性的多肽SEQ ID NO：1不同，本公开的修饰多肽的特征在于释放了作为最终产物的L-亮氨酸或其衍生物的反馈抑制。如本文所用，术语“反馈抑制”是指在酶体系中，最终产物在酶体系的早期状态下的反应抑制。对于本公开的目的，反馈抑制可以是L-亮氨酸或其衍生物抑制异丙基苹果酸合酶(其介导生物合成途径的第一步骤)活性的反馈抑制，但不限于此。因此，当释放出对异丙基苹果酸合酶的反馈抑制时，与不释放反馈抑制的情况相比，可以增加L-亮氨酸的生产率。
如本文所用，术语“修饰”、“修饰的”或“变异体”是指在一种稳定表型中显示出遗传性或非遗传性交替的培养物或个体。具体地，术语“变异体”可以意指：与野生型相比，由于与源于谷氨酸棒状杆菌(Corynebacterium glutamicum)的异丙基苹果酸合酶对应的氨基酸序列被修饰而有效增加其活性的变异体；释放L-亮氨酸或其衍生物的反馈抑制的变异体；或释放反馈抑制和增加活性二者的变异体。
具体地，具有异丙基苹果酸合酶活性的本公开的修饰多肽可以是如下具有异丙基苹果酸合酶活性的修饰多肽：其中由氨基酸序列SEQ ID NO：1组成的多肽的N端的558位的氨基酸-精氨酸-被非精氨酸的氨基酸残基置换，或者由氨基酸序列SEQ ID NO：1组成的多肽的N端的561位的氨基酸残基-甘氨酸-被非甘氨酸的氨基酸残基置换。非精氨酸的氨基酸可以包括丙氨酸、亮氨酸、异亮氨酸、缬氨酸、脯氨酸、苯丙氨酸、色氨酸、甲硫氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、天冬酰胺、谷氨酰胺、赖氨酸、组氨酸、天冬氨酸和谷氨酸；且非甘氨酸的氨基酸可以包括丙氨酸、亮氨酸、异亮氨酸、缬氨酸、脯氨酸、苯丙氨酸、色氨酸、甲硫氨酸、精氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、天冬酰胺、谷氨酰胺、赖氨酸、组氨酸、天冬氨酸和谷氨酸；但所述氨基酸不限于此。更具体地，所述修饰多肽可以是如下修饰多肽：其中由氨基酸序列SEQ ID NO：1组成的多肽的N端的558位的氨基酸-精氨酸-被组氨酸、丙氨酸或谷氨酰胺置换，或者由氨基酸序列SEQ ID NO：1组成的多肽的N端的561位的氨基酸残基-甘氨酸-被天冬氨酸、精氨酸或酪氨酸置换，但不限于此。此外，所述修饰多肽可以是如下多肽：其中558位的精氨酸被组氨酸、丙氨酸或谷氨酰胺置换；并且561位的甘氨酸被天冬氨酸、精氨酸或酪氨酸置换，但不限于此。最具体地，所述修饰多肽可以包括SEQID NO：21至SEQ ID NO：35中任一个的氨基酸序列。
另外，所述修饰多肽可以包括与SEQ ID NO：21至SEQ ID NO：35中任一个的氨基酸序列具有至少80％、90％、95％、96％、97％、98％或99％同源性的多肽。例如，显而易见的是，具有如下氨基酸序列的酶变异体也应属于本公开的范围：其中一些序列缺失、被修饰、被置换或被添加，而与558位和/或561位的氨基酸序列对应的经修饰的氨基酸序列是固定的；只要氨基酸序列具有上述同源性并且展示与异丙基苹果酸合酶对应的效果即可。另一方面，作为特定修饰位置的558位和561位是指基于氨基酸序列SEQ ID NO：1中的N端确定的位置，因此，通过考虑向SEQ ID NO：1的N端上添加或从其缺失的氨基酸数量来确定这些位置的这一事实对于本领域普通技术人员是显而易见的，因此也属于本公开的范围。例如，leuA(编码异丙基苹果酸合酶的基因)通过由616个氨基酸组成的SEQ ID NO：1来表示。然而，在一些参考文献中，转译起始密码子表示leuA基因序列下游的35个氨基酸，即由581个氨基酸组成的基因。在这种情况下，第558位氨基酸被解释为第523位氨基酸，第561位氨基酸被解释为第526位氨基酸，从而包括在本公开的范围之内。
如本文所用，术语“同源性”是指两个多核苷酸或多肽半部(polypeptidemoieties)之间的同一性百分比。从一个半部到另一半部的序列之间的同源性可以通过本领域已知的技术来确定。例如，可以使用易于访问的计算机程序(例如：BLAST 2.0)通过直接排列序列信息(即诸如两个多核苷酸分子或两个多肽分子的得分、同一性、相似性等的参数)来确定同源性。另外，多核苷酸之间的同源性可以通过以下方式来确定：在同源区域之间形成稳定双链(double-strand)的条件下使多核苷酸杂交，用单链特异性核酸酶分解，然后确定所分解的片段的大小。
本公开的另一方面提供了编码修饰多肽的多核苷酸。
所述多核苷酸可以是编码如下具有异丙基苹果酸合酶活性的修饰多肽的多核苷酸：其中由氨基酸序列SEQ ID NO：1组成的多肽的N端的558位的氨基酸-精氨酸-被非精氨酸的其它氨基酸残基置换，或者由氨基酸序列SEQ ID NO：1组成的多肽的N端的561位的氨基酸残基-甘氨酸-被非甘氨酸的其它氨基酸残基置换。具体地，可以包括(但不限于)编码以下多肽的多核苷酸：包括氨基酸序列SEQ ID NO：21至35并具有异丙基苹果酸合酶活性的多肽；与上述多肽具有至少80％、90％、95％、96％、97％、98％或99％同源性的修饰多肽；或编码具有异丙基苹果酸合酶活性的修饰多肽，其中缺失、修饰、置换或添加了一些序列，而作为上述多肽中的特定修饰位置的558和/或561位的经修饰的氨基酸序列是固定的。或者，可以包括(但不限于)通过在严格条件(stringent conditions)下使上述全部或部分核苷酸序列的互补序列杂交而从已知基因序列(例如，编码具有异丙基苹果酸合酶活性的蛋白质的序列)制得的探针。
如本文所用，术语“严格条件”是指如下条件：在该条件下，形成所谓的杂交体而不形成非特异性杂交体(non-specific hybrids)。这些条件的示例包括如下条件：在该条件下，具有高度同源性的基因(如同源性为80％或以上、特别为90％或以上、更特别为95％或以上、再特别为97％或以上、最特别为99％或以上的基因)彼此杂交，而具有低度同源性的基因彼此不杂交；或者在该条件下，在60℃、1×SSC和0.1％SDS的温度和盐浓度下(特别为60℃、0.1×SSC和0.1％SDS，更特别为68℃、0.1×SSC和0.1％SDS，这些是普通Southern杂交的洗涤条件)洗涤基因1次(特别为2次和3次)(Sambrook等人，《分子克隆：实验室手册》第3版，冷泉港实验出版社，冷泉港，纽约(2001)(Sambrook et al.,Molecular Cloning:ALaboratory Manual,3rd Ed.,Cold Spring Harbor Laboratory Press,Cold SpringHarbor,N.Y.(2001)))。
杂交中使用的探针可以是核苷酸序列的互补序列的一部分。可以使用基于已知序列制得的寡核苷酸作为引物和含有该核苷酸序列的基因片段作为模板，通过PCR来构建此类探针。例如，可以使用长度为约300bp的基因片段作为探针。更具体地，在使用具有一定(约300bp)长度的探针的情况下，杂交的洗涤条件可建议为50℃、2×SSC和0.1％SDS。
另一方面，多核苷酸可以是具有SEQ ID NO：36至SEQ ID NO：50中任一个的核苷酸序列的多核苷酸，并且显而易见的是，多核苷酸还包括可通过密码子简(codondegeneracy)被转译成修饰多肽的多核苷酸。
本公开的又一方面是提供生产L-亮氨酸的棒状杆菌(Corynebacterium)属的微生物，其含有所述修饰多肽。
在本公开中，所述微生物可以包括通过转化而人工产生的微生物或天然存在的微生物中的所有微生物。
如本文所用，术语“转化”是指将基因引入宿主细胞进行表达。在本公开中，转化方法包括将基因引入细胞并可通过选择本领域已知的合适标准技术实施的任何方法。转化方法的示例是电穿孔法、磷酸钙共沉淀法、逆转录病毒感染法、显微注射法、DEAE-葡聚糖法、阳离子脂质体法、热休克法等，但不限于此。
待转化的基因可以包括插入宿主细胞的染色体中的形式和位于染色体外部的形式二者，只要其可以在宿主细胞中表达即可。此外，该基因包括作为能够编码多肽的多核苷酸的DNA和RNA，并且可以使用可引入宿主细胞并在其中表达的任何基因而无限制。例如，可以将基因以表达盒的形式引入宿主细胞，表达盒是含有自身表达所需的所有要素的多核苷酸构建体。表达盒通常包括可操作地连接到基因的启动子、转录终止信号、核糖体结合位点以及转译终止信号。表达盒可以是自我复制的表达载体(vector)的形式。此外，基因可以是引入宿主细胞本身或呈多核苷酸构建体形式(即载体的形式)的基因，并且可操作地连接至要求在宿主细胞中表达的序列。
如本文所用，术语“载体”是指用于将核苷酸克隆和/或转移至宿主细胞的任何运载体。载体可以是复制子(replicon)，以允许复制与其它DNA片段结合的片段。“复制子”是指进行自我复制直到DNA体内复制的任何遗传单位(例如质粒、噬菌体、粘粒、染色体和病毒)，即可通过自我调节进行复制。术语“载体”可以包括用于在体外、离体或体内将核苷酸引入宿主细胞的病毒运载体和非病毒运载体，并且还可以包括微球形DNA。例如，载体可以是不含细菌DNA序列的质粒。已经去除了富含CpG区域的细菌DNA序列，以减少转基因表达的消音(silencing)并促进质粒DNA载体的更连续表达(例如，Ehrhardt,A.等人(2003)《人类基因治疗》10:215-25；Yet,N.S.(2002)《分子治疗》5:731-38；Chen,Z.Y.等人(2004)《基因治疗》11：856-64(Ehrhardt,A.et al.(2003)Hum Gene Ther 10:215-25；Yet,N.S.(2002)MoI Ther 5:731-38；Chen,Z.Y.et al.(2004)Gene Ther 11:856-64))。术语“载体”也可以包括转座子(transposon)，如睡美人转座子(Izsvak等人，《分子生物学期刊》302：93-102(2000)(Izsvak et al.J.Mol.Biol.302:93-102(2000)))或人工染色体。通常使用的载体的示例可以是天然或重组质粒、粘粒、病毒和细菌噬菌体。例如，作为噬菌体载体或粘粒载体，可以使用pWE15、M13、λMBL3、λMBL4、λIXII、λASHII、λAPII、λt10、λt11、Charon4A、Charon21A等。此外，作为质粒载体，可以使用pDZ型、pBR型、pUC型、pBluescriptII型、pGEM型、pTZ型、pCL型、pET型等。具体地，可以使用pECCG117载体。本公开中可以使用的载体没有特别限制，可以使用已知的表达/置换型载体。
此外，载体可以是重组载体，该重组载体可进一步包括各种抗生素抗性基因。
如本文所用，术语“抗生素抗性基因”是指具有抗生素抗性的基因，并且包含该基因的细胞甚至能够存活在用相应的抗生素处理过的环境中。因此，抗生素抗性基因可以有效地用作微生物(如大肠杆菌等)中大规模生产质粒的筛选标记。在本发明中，由于抗生素抗性基因不是显著影响表达效率的因素(表达效率通过作为本发明关键特征的载体的成分的最佳组合而获得)，因此任何常见的抗生素抗性基因都可以用作筛选标记而不受限制。具体地，可以使用抗氨苄青霉素、抗四环素、抗卡那霉素、抗氯霉素、抗链霉素以及抗新霉素的抗性基因(the resistance genes against ampicilin,tetracyclin,kanamycin,chloramphenicol,streptomycin,or neomycin can be used)。
如本文所用，术语“可操作地连接”是指用于核苷酸表达的调节序列与编码靶蛋白的核苷酸序列的可操作连接而用于实现其一般功能，从而影响编码核苷酸序列的表达。可以使用本领域已知的基因重组技术与载体可操作地连接，并且可以使用本领域已知的限制酶和连接酶进行位点特异性DNA的分裂(cleavage)和连结(ligation)。
如本文所用，术语“载体引入(转化)的宿主细胞”是指用具有编码一种或多种靶蛋白的基因的载体转化的细胞。宿主细胞可以包括原核微生物和真核微生物中的任意一种，只要微生物包括通过引入上述载体能够产生异丙基苹果酸合酶的修饰多肽即可。例如，可以包括属于埃希氏杆菌(Escherichia)属、欧文氏菌(Erwinia)属、沙雷氏菌(Serratia)属、普罗威登氏菌(Providencia)属、棒状杆菌(Corynebacterium)属以及短杆菌(Brevibacterium)属的微生物菌株。棒状杆菌(Corynebacterium)属的微生物的示例可以是谷氨酸棒状杆菌(Corynebacterium glutamicum)，但不限于此。
生产L-亮氨酸的棒状杆菌属微生物(The microorganism of the genusCorynebacterium producing L-leucine)(能够表达具有异丙基苹果酸合酶活性的修饰多肽)包括除引入载体之外的能够通过各种已知方法表达修饰多肽的所有微生物。
本公开的另一方面提供了生产L-亮氨酸的方法，该方法包括：(a)培养产生L-亮氨酸的棒状杆菌(Corynebacterium)属的微生物；和(b)从培养的微生物或培养基中回收L-亮氨酸。
如本文所用，术语“培养”是指在适当控制的环境条件下培养微生物。本公开的培养过程可以根据本领域已知的合适培养基和培养条件来实施。根据将要选择的菌株，本领域普通技术人员可以容易地调整和使用这种培养过程。具体地，培养可以是间歇型(batchtype)、连续型和补料分批型(fed-batch type)，但不限于此。
培养基中含有的碳源可以包括：糖和碳水化合物，如葡萄糖、蔗糖、乳糖、果糖、麦芽糖、淀粉和纤维素；油和脂肪，如大豆油、葵花油、蓖麻油、椰子油等；脂肪酸，如棕榈酸、硬脂酸和亚油酸；醇类，如甘油和乙醇；以及有机酸，如乙酸。这些材料可以单独使用或组合使用，但不限于此。培养基中含有的氮源可以包括：有机氮源，如蛋白胨，酵母膏、肉汁、麦芽膏、玉米浆和大豆；以及无机氮源，如尿素、硫酸铵、氯化铵、磷酸铵、碳酸铵和硝酸铵。这些氮源可以单独使用或组合使用，但不限于此。培养基中含有的磷源可以包括磷酸二氢钾、磷酸氢二钾及相应的含钠盐，但不限于此。此外，可以含有金属盐，如硫酸镁或硫酸铁。此外，可以含有氨基酸、维生素、合适的前体等。这些培养基或前体可以以分批培养过程或连续培养过程添加到培养物中，但不限于此。
可以在培养期间通过添加适当的化合物(如氢氧化铵、氢氧化钾、氨、磷酸和硫酸)来调节培养物的pH，并且可以在培养期间通过使用消泡剂(如脂肪酸聚乙二醇酯)来抑制泡沫的产生。为了维持培养的有氧条件，可以将氧气或含氧气体注入到培养物中。为了保持厌氧和微氧条件，可以不注入气体或者也可以注入氮气、氢气或二氧化碳。培养的温度可以为27℃至37℃，特别为30℃至35℃，但不限于此。培养时间可以是连续的，只要回收了所需量的有用物料即可，并且优选为10至100小时，但培养时间不限于此。
回收在本公开的培养步骤中所生产的L-亮氨酸的步骤可以根据培养方法使用本领域已知的合适方法从微生物或培养基中收集所需的L-亮氨酸。例如，可以使用离心、过滤、阴离子交换层析、结晶、HPLC方法等，并且可以使用本领域已知的合适方法从培养基或微生物中回收所需的L-亮氨酸。另外，上述回收步骤可以包括纯化过程。
有益效果
与野生型相比，具有异丙基苹果酸合酶活性的新型修饰多肽是活性增加并释放L-亮氨酸反馈抑制的多肽，从而可以使用这种修饰多肽以高产率生产L-亮氨酸。
具体实施方式
在下文中，将通过所附示例性实施例来详细描述本公开。然而，本文公开的示例性实施例仅用于说明性的目的，不应被解释为限制本公开的范围。
示例1：确认KCCM11661P的leuA核苷酸序列、生产亮氨酸的微生物
在121℃下，在15分钟内将谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067接种到具有下述成分的种子培养基中，培养13小时，然后回收25mL培养基。用100mM柠檬酸盐缓冲液洗涤所回收的培养基，并用N-甲基-N'-硝基-N-亚硝基胍(NTG)处理30分钟，最终浓度为400μg/mL。之后，用100mM磷酸盐缓冲液洗涤所得物。在具有下述成分的基本培养基上涂抹菌株的结果是：NTG处理过的菌株的死亡率确定为99.6％。为了获得耐正亮氨酸(norleucine，NL)的变异体，将NTG处理过的菌株涂抹在最终浓度为20mM、40mM和50mM的基本培养基上，在30℃下培养5天，然后获得耐NL的变异体。
＜种子培养基＞
葡萄糖(20g)，蛋白胨(10g)，酵母膏(5g)，尿素(1.5g)，KH2PO4(4g)，K2HPO4(8g)，MgSO4·7H2O(0.5g)，生物素(100μg)，盐酸硫胺素(1000μg)，泛酸钙(2000μg)，烟酰胺(2000μg，按1升蒸馏水计)，pH 7.0
＜生产培养基＞
葡萄糖(100g)，(NH4)2SO4(40g)，大豆蛋白(2.5g)，玉米浆固体(5g)，尿素(3g)，KH2PO4(1g)，MgSO4·7H2O(0.5g),生物素(100μg)，盐酸硫胺素(1000μg)，泛酸钙(2000μg)，烟酰胺(3000μg)，CaCO3(30g，按1升蒸馏水计)，pH 7.0
通过上述方法获得的变异体被命名为谷氨酸棒状杆菌KCJ-24(Corynebacteriumglutamicum KCJ-24)和谷氨酸棒状杆菌KCJ-28(Corynebacterium glutamicum KCJ-28)，并且根据布达佩斯条约，于2015年1月22日将其保藏于国际保藏机构-韩国微生物培养中心，因此谷氨酸棒状杆菌KCJ-24(Corynebacterium glutamicum KCJ-24)和谷氨酸棒状杆菌KCJ-28(Corynebacterium glutamicum KCJ-28)分别以保藏号KCCM11661P和KCCM11662P保藏。谷氨酸棒状杆菌KCJ-24(Corynebacterium glutamicum KCJ-24)和谷氨酸棒状杆菌KCJ-28(Corynebacterium glutamicum KCJ-28)分别生产浓度为2.7g/L和3.1g/L的L-亮氨酸。因此，证实了由变异体生产的L-亮氨酸的生产率比野生型高10倍。
另外，尝试证实在变异体KCCM11661P中是否发生编码异丙基苹果酸合酶(IPMS)的leuA的变异。通过参照基因库的WP_003863358.1，确认了野生型leuA的氨基酸序列(SEQ IDNO：1)。使用聚合酶链反应(以下称为“PCR”)方法对变异体的染色体DNA进行扩增。尽管已知leuA基因由616个氨基酸组成，但在一些参考文献中公开了leuA基因序列下游的35个氨基酸被表示为转译起始密码子，因此leuA基因由581个氨基酸组成。在这种情况下，表示相应氨基酸的变异的位置编号可能改变。因此，在leuA基因被认为是由581个氨基酸组成的情况下，在括号中另外表示变异位置。
具体地，使用变异体的染色体DNA作为模板并使用引物SEQ ID NO：3和4在以下条件下进行PCR：在94℃下变性1分钟；在58℃下退火30秒；以及使用Taq DNA聚合酶在72℃下聚合2分钟。共重复上述PCR 28次，以扩增约2700个碱基对(base pairs)的片段。使用相同的引物来分析片段的核苷酸序列，结果证实，KCCM11661P中leuA的第1673位核苷酸G被A置换。该结果表明，第558位(或第523位；以下只表示为第558位)氨基酸-精氨酸-被组氨酸置换。此外，还证实，第1682和1683位核苷酸的GC被AT置换。该结果也意味着第561位(或第526位，以下只表示为第561位)氨基酸-甘氨酸-被天冬氨酸置换。
示例2：生产IPMS变异体的置换型载体
为了生产含有示例1中确认的经修饰的核苷酸序列的载体，使用上述变异体的染色体DNA作为模板并使用引物SEQ ID NO：5和6在以下条件下进行PCR：在94℃下变性1分钟；在58℃下退火30秒；以及使用Pfu DNA聚合酶在72℃下聚合1分钟。共重复25次上述PCR，用SalI和XbaI限制酶位点来扩增约1460个碱基对的片段。用限制酶SalI和XbaI处理扩增的片段，然后通过与用相同的酶处理的载体pDZ(韩国专利号10-0924065和国际专利公开号2008-033001)连结来制备pDZ-leuA(R558H，G561D)。另外，为了制备每个变异体的载体，使用ATCC14067作为模板，然后分别使用引物5和7以及引物8和6来扩增2个片段。使用制备的两个片段作为模板在以下条件下进行PCR：在94℃下变性1分钟；在58℃下退火30秒；以及使用Pfu DNA聚合酶在72℃下聚合1分钟。共重复25次上述PCR，用SalI和XbaI限制酶位点来扩增约1460个碱基对的片段。用限制酶SalI和XbaI处理扩增的片段，然后通过与用相同的酶处理的pDZ连结来制备pDZ-leuA(R558H)。通过用与上述相同的方法，使用引物5和9以及引物10和6来制备pDZ-leuA(G561D)。
示例3：生产IPMS变异体的置换型菌株
使用谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067作为亲本菌株，以制备含有在上述修饰菌株中发现的leuA修饰的核苷酸序列的菌株。
用示例2中制备的载体pDZ-leuA(R558H)、pDZ-leuA(G561D)和pDZ-leuA(R558H，G561D)通过电穿孔技术转化谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067。通过二次交换制备的每个菌株被命名为14067::leuA(R558H)、14067::leuA(G561D)和14067::leuA(R558H，G561D)。为了确认leuA的核苷酸是否被置换，使用引物SEQ ID NO：3和4在以下条件下进行PCR：在94℃下变性1分钟；在58℃下退火30秒；以及使用Taq DNA聚合酶在72℃下聚合2分钟。共重复上述PCR 28次，以扩增约2700个碱基对的片段。之后，通过用相同的引物分析核苷酸序列来确认leuA的核苷酸的置换。
将用载体pDZ-leuA(R558H，G561D)转化的菌株14067::leuA(R558H，G561D)命名为KCJ-0148，并且于2016年1月25日将其保藏于韩国微生物培养中心，由此该菌株以保藏号KCCM11811P保藏。
示例4：在IPMS变异体的置换型菌株中生产L-亮氨酸
为了从示例3制备的谷氨酸棒状杆菌(Corynebacterium glutamicum)14067::leuA(R558H)、14067::leuA(G561D)和14067::leuA(R558H，G561D)来生产L-亮氨酸，采用以下方式进行培养。
将亲本菌株谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067与制备的谷氨酸棒状杆菌(Corynebacterium glutamicum)14067::leuA(R558H)、14067::leuA(G561D)和14067::leuA(R558H，G561D)菌株中的每一种的白金环(platinum loop)接种到含有生产培养基(25mL)的挡板三角烧瓶(250mL)中。之后，在30℃的振荡水浴中，以200rpm的速率培育60小时来生产L-亮氨酸。
培育完成后，通过高效液相色谱法测定产生的L-亮氨酸的量。对于每种实验菌株，培养基中L-亮氨酸的浓度示于下表1中。
表1IPMS变异体的置换型菌株中的L-亮氨酸生产
| 菌株 | L-亮氨酸浓度(g/L) |
| ATCC14067 | 0.1 |
| 14067::leuA(R558H) | 1.2 |
| 14067::leuA(G561D) | 1.6 |
| 14067::leuA(R558H，G561D) | 2.5 |
如上表1所示，证实了与亲本菌株谷氨酸棒状杆菌(Corynebacteriumglutamicum)ATCC14067相比，具有leuA基因的R558H、G561D或R558H/G561D变异的L-亮氨酸生产菌株-谷氨酸棒状杆菌(Corynebacterium glutamicum)14067::leuA(R558H)、14067::leuA(G561D)和14067::leuA(R558H，G561D)的L-亮氨酸生产率增加到约12至25倍。
示例5：生产IPMS变异体的过表达载体(overexpressing vector)
为了生产含有在示例1中确认的经修饰的核苷酸序列的表达载体，使用ATCC14067和示例3中制备的3种变异体的染色体DNA作为模板，并使用引物SEQ ID NO：11和12在以下条件下进行PCR：在94℃下变性1分钟；在58℃下退火30秒；以及使用Pfu DNA聚合酶在72℃下聚合1分钟。共重复上述PCR25次，用NdeI和XbaI限制酶位点来扩增约2050个碱基对的片段。用限制酶NdeI和XbaI处理扩增的片段，然后使用p117_PCJ7(其中PCJ7启动子被插入到用相同的酶处理的载体pECCG117中)(《生物技术学报》第13卷，第10期，第721-726页(1991)(Biotechnology letters Vol.13,No.10,p.721-726(1991)))通过连结来制备表达载体p117_PCJ7-leuA(WT)、p117_PCJ7-leuA(R558H)、p117_PCJ7-leuA(G561D)和p117_PCJ7-leuA(R558H，G561D)。PCJ7启动子是增强基因表达的启动子，并且在韩国专利号10-0620092和国际专利公开号2006-065095中是公知的。
示例6：生产由IPMS变异体的过表达载体转化的菌株
为了生产用含有示例5中制备的经leuA修饰的核苷酸序列的过表达载体转化的菌株，使用了野生型谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067作为亲本菌株以及亮氨酸生产菌株KCCM11661P和KCCM11662P。
通过电穿孔技术，用示例5中制备的各个载体p117_PCJ7-leuA(WT)、p117_PCJ7-leuA(R558H)、p117_PCJ7-leuA(G561D)和p117_PCJ7-leuA(R558H，G561D)来转化谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067、KCCM11661P和KCCM11662P。由此生产了14067::p117_PCJ7-leuA(WT)、14067::p117_PCJ7-leuA(R558H)、14067::p117_PCJ7-leuA(G561D)、14067::p117_PCJ7-leuA(R558H，G561D)；KCCM11661P::p117_PCJ7-leuA(WT)、KCCM11661P::p117_PCJ7-leuA(R558H)、KCCM11661P::p117_PCJ7-leuA(G561D)、KCCM11661P::p117_PCJ7-leuA(R558H，G561D)；以及KCCM11662P::p117_PCJ7-leuA(WT)、KCCM11662P::p117_PCJ7-leuA(R558H)、KCCM11662P::p117_PCJ7-leuA(G561D)、KCCM11662P::p117_PCJ7-leuA(R558H，G561D)。
示例7：在用IPMS变异体的过表达载体转化的菌株中生产L-亮氨酸
为了从L-亮氨酸生产菌株、示例6中生产的谷氨酸棒状杆菌(Corynebacteriumglutamicum)14067::p117_PCJ7-leuA(WT)、14067::p117_PCJ7-leuA(R558H)、14067::p117_PCJ7-leuA(G561D)、14067::p117_PCJ7-leuA(R558H，G561D)；KCCM11661P::p117_PCJ7-leuA(WT)、KCCM11661P::117PCJ7-leuA(R558H)、KCCM11661P::p117_PCJ7-leuA(G561D)、KCCM11661P::p117_PCJ7-leuA(R558H，G561D)；以及KCCM11662P::p117_PCJ7-leuA(WT)、KCCM11662P::p117_PCJ7-leuA(R558H)、KCCM11662P::p117_PCJ7-leuA(G561D)、KCCM11662P::p117_PCJ7-leuA(R558H，G561D)中生产L-亮氨酸，采用以下方式进行培养。
将亲本菌株-谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067、KCCM11661P和KCCM11662P与示例6中生产的菌株中的每一种的白金环接种到含有生产培养基(25mL)的挡板三角烧瓶(250mL)中。之后，在30℃的振荡水浴中，以200rpm的速率培育60个小时来生产L-亮氨酸。
培育完成后，通过高效液相色谱法测定产生的L-亮氨酸的量。对于每个实验菌株，培养基中L-亮氨酸的浓度示于下表2中。
表2在过表达IPMS变异体菌株中生产L-亮氨酸
如上表2所示，证实了与亲本菌株ATCC14067相比，用包含在菌株ATCC14067中的leuA基因变异的过表达载体转化的L-亮氨酸生产菌株、14067::p117_PCJ7-leuA(R558H)、14067::p117_PCJ7-leuA(G561D)和14067::p117_PCJ7的L-亮氨酸生产增加到45至98倍(enhanced 45-to 98-fold)；与亲本菌株KCCM11661P相比，用包含在菌株KCCM11661P中的leuA基因变异的过表达载体转化的L-亮氨酸生产菌株、KCCM11661P::p117_PCJ7-leuA(R558H)、KCCM11661P::p117_PCJ7-leuA(G561D)和KCCM11661P::p117_PCJ7-leuA(R558H，G561D)的L-亮氨酸生产增加到2.3至4.5倍(enhanced 2.3-to 4.5-fold)；以及与亲本菌株KCCM11662P相比，用包含在菌株KCKCM11662P中的leuA基因的变异的过表达载体转化的L-亮氨酸生产菌株、KCCM11662P::p117_PCJ7-leuA(R558H)、KCCM11662P::p117_PCJ7-leuA(G561D)和KCCM11662P::p117_PCJ7-leuA(R558H，G561D)的L-亮氨酸生产率增加到2至4.2倍(enhanced 2-to 4.2-fold)。
示例8：在用leuA过表达载体转化的菌株中测定异丙基苹果酸合酶活性
为了测定示例6中生产的以下L-亮氨酸生产菌株中的异丙基苹果酸合酶活性(谷氨酸棒状杆菌(Corynebacterium glutamicum)14067::p117_PCJ7-leuA(WT)、14067::p117_PCJ7-leuA(R558H)、14067::p117_PCJ7-leuA(G561D)和14067::p117_PCJ7-leuA(R558H，G561D))，采用以下方式进行实验。
将上述4个菌株中的每一种的白金环接种到含有种子培养基(25mL)的挡板三角烧瓶(250mL)中。之后，将所得物在30℃的振荡水浴中以200rpm的速率培育16小时。培育完成后，将培养基离心以弃去上清液，洗涤沉淀物(pellet)并与裂解缓冲液混合，然后用玻珠均质器粉碎细胞。根据Bradford测定法对存在于裂解物中的蛋白质进行定量，并且通过测量使用含有蛋白质的裂解物(100μg/mL)时产生的辅酶A(CoA)来测定异丙基苹果酸合酶活性。每种菌株中的异丙基苹果酸合酶活性的测定结果示于下表3中。
表3
| 菌株 | 相对IPMS活性(％) |
| 14067::p117_PCJ7-leuA(WT) | 100 |
| 14067::p117_PCJ7-leuA(R558H) | 105 |
| 14067::p117_PCJ7-leuA(G561D) | 130 |
| 14067::p117_PCJ7-leuA(R558H，G561D) | 328 |
为了确认酶中亮氨酸反馈抑制的释放程度，通过在添加亮氨酸(3g/L)的条件下测量使用含有蛋白质(100μg/mL)的裂解物时产生的辅酶A来测定异丙基苹果酸合酶活性。每种菌株中的异丙基苹果酸合酶活性的测量结果示于下表4中。
表4
如上表3和表4所示，证实了与对照组谷氨酸棒状杆菌(Corynebacteriumglutamicum)14067::p117_PCJ7-leuA(WT)相比，用表达IPMS变异体的载体转化的L-亮氨酸生产菌株-谷氨酸棒状杆菌(Corynebacterium glutamicum)14067::p117_PCJ7-leuA(R558H)、14067::p117_PCJ7-leuA(G561D)和14067::p117_PCJ7-leuA(R558H，G561D)的异丙基苹果酸合酶活性分别增加1.05倍、1.3倍和3.2倍。此外，即使在添加亮氨酸(2g/L)的情况下，L-亮氨酸生产菌株的IPMS活性仍分别保持在61％、70％和89％，这证实了释放出了亮氨酸的反馈抑制。
示例9：生产用于改进异丙基苹果酸合酶(IPMS)变异体的载体
在示例4、7和8中，由于证实了异丙基苹果酸合酶的氨基酸序列(SEQ ID NO：1)中的第558位和561位氨基酸是IPMS变异体酶的活性的重要位点，因此尝试证实当用变异体中的氨基酸之外的氨基酸置换时，酶活性是否增加或反馈抑制是否进一步释放。因此，尝试制备被其它能够引起结构变异的氨基酸基团的氨基酸置换的变异体。
制备其中第558位氨基酸-精氨酸-被丙氨酸(Ala)或谷氨酰胺(Gln)置换的变异体。使用定点突变方法(a site-directed mutagenesis method)，并且通过使用载体p117_PCJ7-leuA(R558H)作为模板，使用引物SEQ ID NO：13和14及引物对SEQ ID NO：15和16来制备其中第558位氨基酸被丙氨酸(Ala)置换的载体p117_PCJ7-leuA(R558A)和其中第558位氨基酸被谷氨酰胺(Gln)置换的载体p117_PCJ7-leuA(R558Q)。
制备其中第561位氨基酸-甘氨酸-被精氨酸(Arg)或酪氨酸(Tyr)置换的变异体。使用定点突变方法，并且通过使用p117_PCJ7-leuA(G561D)作为模板，使用引物SEQ ID NO：17和18及引物对SEQ ID NO：19和20来获得其中第561位氨基酸被精氨酸(Arg)置换的载体p117_PCJ7-leuA(G561R)和其中第561位氨基酸被酪氨酸(Tyr)置换的载体p117_PCJ7-leuA(G561Y)。
示例10：生产其中引入异丙基苹果酸修饰变异体的菌株
为了制备用含有示例9中制备的leuA修饰的核苷酸序列的表达载体来转化的菌株，使用野生型谷氨酸棒状杆菌(wild-type Corynebacterium glutamicum)ATCC14067作为亲本菌株。
通过电穿孔技术，将示例9中制备的各个载体p117_PCJ7-leuA(R558A)、p117_PCJ7-leuA(R558Q)、p117_PCJ7-leuA(G561R)和p117_PCJ7-leuA(G561Y)转化到谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067中，以制备14067::p117_PCJ7-leuA(R558A)、14067::p117_PCJ7-leuA(R558Q)、14067::p117_PCJ7-leuA(G561R)和14067::p117_PCJ7-leuA(G561Y)。
示例11：在引入异丙基苹果酸合酶修饰的变异体的菌株中生产L-亮氨酸
为了从示例10中制备的以下L-亮氨酸生产菌株生产L-亮氨酸：谷氨酸棒状杆菌(Corynebacterium glutamicum)14067::p117_PCJ7-leuA(R558A)、14067::p117_PCJ7-leuA(R558Q)、14067::p117_PCJ7-leuA(G561R)和14067::p117_PCJ7-leuA(G561Y)，采用以下方式进行培养。
将亲本菌株、谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC14067和上述4个菌株中的每一种的白金环接种到含有生产培养基(25mL)的挡板三角烧瓶(250mL)中。之后，在30℃的振荡水浴中，以200rpm的速率培育60小时来生产L-亮氨酸。
培育完成后，通过高效液相色谱法测定产生的L-亮氨酸的量。对于每个实验菌株，培养基中L-亮氨酸的浓度示于下表5中。
表5过表达IPMS变异体的菌株中的L-亮氨酸生产
如上表5所示，证实了与亲本菌株-谷氨酸棒状杆菌(Corynebacteriumglutamicum)ATCC14067相比，L-亮氨酸生产菌株-谷氨酸棒状杆菌(Corynebacteriumglutamicum)14067::p117_PCJ7-leuA(R558A)和14067::p117_PCJ7-leuA(R558Q)的L-亮氨酸生产率增加到32至38倍。
此外，还证实了与亲本菌株-谷氨酸棒杆状菌(Corynebacterium glutamicum)ATCC14067相比，L-亮氨酸生产菌株-谷氨酸棒状杆菌(Corynebacterium glutamicum)14067::p117_PCJ7-leuA(G561R)和14067::p117_PCJ7-leuA(G561Y)的L-亮氨酸生产率增加到约36至40倍。
根据上述结果，证实了异丙基苹果酸合酶的氨基酸序列(SEQ ID NO：1)中第558和561位氨基酸是IPMS变异体酶活性的重要位点，并且即使野生型IPMS蛋白质的第558位和第561位氨基酸分别被组氨酸和天冬氨酸置换，具有这种修饰的菌株中的L-亮氨酸生产率仍显著增加。
虽然已经参考具体的示例性实施例描述了本公开，但是本公开所属领域的技术人员将会理解，本公开可以以其它的具体形式来实现而不脱离本公开的技术宗旨或基本特征。因此，上述实施例被认为是各方面的说明而非限制性的。此外，本公开的范围由所附权利要求书限定，而不是由具体的实施方式来限定，并且应当理解，从本公开及其等同物的含义和范围衍生出的所有修改或变化都包括在所附权利要求书的范围内。
<110> CJ第一制糖株式会社（CJ CheilJedang Corporation）
<120> 新型异丙基苹果酸合酶变异体及使用其生产L-亮氨酸的方法
<130> ZF1780109C-D1
<150> KR 10-2016-0181343
<151> 2016-12-28
<160> 50
<170> KoPatentIn 3.0
<210> 1
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> leuA AA
<400> 1
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 2
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> leuA NT
<400> 2
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680
ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 3
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> leuA sequencing-1
<400> 3
agaaggacaa agcactcatc c 21
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> leuA sequencing-2
<400> 4
tcgccctagt attctggcgt 20
<210> 5
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> leuA sub F
<400> 5
acgcgtcgac ttgaccctca gctggacttc 30
<210> 6
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> leuA sub R
<400> 6
gctctagaaa ttccctgtcg gtgaagca 28
<210> 7
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> leuA 588 mut P-2
<400> 7
atcgtcgccc gaggtgtggg cgtgctggtt gta 33
<210> 8
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> leuA 588 mut P-3
<400> 8
tacaaccagc acgcccacac ctcgggcgac gat 33
<210> 9
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> leuA 561 mut P-2
<400> 9
tgcttctgca tcgtcatccg aggtgcgggc gtgct 35
<210> 10
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> leuA 561 mut P-3
<400> 10
acgcccgcac ctcggatgac gatgcagaag ca 32
<210> 11
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> leuA over F
<400> 11
ggaattttcc atatgtctcc taacgatgca ttc 33
<210> 12
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> leuA over R
<400> 12
gctctagatc gccctagtat tctggcgt 28
<210> 13
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> R558A SD F
<400> 13
aggaatacaa ccagcacgcc gcaacctcgg gcgacgatgc aga 43
<210> 14
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> R558A SD R
<400> 14
tctgcatcgt cgcccgaggt tgcggcgtgc tggttgtatt cct 43
<210> 15
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> R558Q SD F
<400> 15
aggaatacaa ccagcacgcc cagacctcgg gcgacgatgc aga 43
<210> 16
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> R558Q SD R
<400> 16
tctgcatcgt cgcccgaggt ctgggcgtgc tggttgtatt cct 43
<210> 17
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> G561R SD F
<400> 17
accagcacgc ccgcacctcg cgcgacgatg cagaagcagc cgc 43
<210> 18
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> G561R SD R
<400> 18
gcggctgctt ctgcatcgtc gcgcgaggtg cgggcgtgct ggt 43
<210> 19
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> G561Y SD F
<400> 19
accagcacgc ccgcacctcg tacgacgatg cagaagcagc cgc 43
<210> 20
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> G561Y SD R
<400> 20
gcggctgctt ctgcatcgtc gtacgaggtg cgggcgtgct ggt 43
<210> 21
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558H AA
<400> 21
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala His Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 22
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558A AA
<400> 22
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Ala Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 23
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558Q AA
<400> 23
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Gln Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 24
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> G561D AA
<400> 24
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Asp Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 25
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> G561R AA
<400> 25
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Arg Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 26
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> G561Y AA
<400> 26
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Tyr Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 27
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558H, G561D AA
<400> 27
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala His Thr Ser
545 550 555 560
Asp Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 28
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558H, G561R AA
<400> 28
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala His Thr Ser
545 550 555 560
Arg Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 29
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558H, G561Y AA
<400> 29
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala His Thr Ser
545 550 555 560
Tyr Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 30
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558A, G561D AA
<400> 30
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Ala Thr Ser
545 550 555 560
Asp Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 31
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558A, G561R AA
<400> 31
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Ala Thr Ser
545 550 555 560
Arg Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 32
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558A, G561Y AA
<400> 32
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Ala Thr Ser
545 550 555 560
Tyr Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 33
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558Q, G561D AA
<400> 33
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Gln Thr Ser
545 550 555 560
Asp Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 34
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558Q, G561R AA
<400> 34
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Gln Thr Ser
545 550 555 560
Arg Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 35
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> R558Q, G561Y AA
<400> 35
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Gln Thr Ser
545 550 555 560
Tyr Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 36
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558H NT
<400> 36
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccacacctcg 1680
ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 37
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558A NT
<400> 37
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc cgcaacctcg 1680
ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 38
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558Q NT
<400> 38
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccagacctcg 1680
ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 39
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> G561D NT
<400> 39
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680
gatgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 40
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> G561R NT
<400> 40
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680
cgcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 41
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> G561Y NT
<400> 41
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680
tacgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 42
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558H, G561D NT
<400> 42
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccacacctcg 1680
gatgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 43
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558H, G561R NT
<400> 43
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccacacctcg 1680
cgcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 44
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558H, G561Y NT
<400> 44
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccacacctcg 1680
tacgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 45
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558A, G561D NT
<400> 45
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc cgcaacctcg 1680
gatgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 46
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558A, G561R NT
<400> 46
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccagacctcg 1680
cgcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 47
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558A, G561Y NT
<400> 47
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc cgcaacctcg 1680
tacgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 48
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558Q, G561D NT
<400> 48
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccagacctcg 1680
gatgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 49
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558Q, G561R NT
<400> 49
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccagacctcg 1680
cgcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 50
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> R558Q, G561Y NT
<400> 50
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccagacctcg 1680
tacgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
Claims (7)
1.一种用于生产L-亮氨酸的属于棒状杆菌属的修饰微生物，其包含具有异丙基苹果酸合酶活性的修饰多肽，
其中由氨基酸序列SEQ ID NO：1组成的多肽的N端的561位的甘氨酸被所述修饰多肽中的天冬氨酸置换，并且
其中所述修饰微生物具有对正亮氨酸或L-亮氨酸的抗性。
2.根据权利要求1所述的修饰微生物，其中编码所述修饰多肽的多核苷酸的天然启动子被与所述天然启动子相比增强所述多核苷酸的表达的启动子置换。
3.根据权利要求2所述的修饰微生物，其中与所述天然启动子相比增强所述多核苷酸的表达的所述启动子是PCJ7启动子。
4.根据权利要求1所述的修饰微生物，其中属于棒状杆菌属的所述修饰微生物源自谷氨酸棒状杆菌。
5.一种生产L-亮氨酸的棒状杆菌属的微生物，其由载体转化，所述载体包含：i)编码具有异丙基苹果酸合酶活性的修饰多肽的多核苷酸；和ii)增强与所述多核苷酸相关的L-亮氨酸生产率的启动子，
其中由氨基酸序列SEQ ID NO：1组成的多肽的N端的561位的甘氨酸被所述修饰多肽中的天冬氨酸置换，
其中编码所述修饰多肽的所述多核苷酸与所述载体中的所述启动子可操作地连接，并且
其中所述启动子与天然启动子相比增强L-亮氨酸生产率，所述天然启动子在棒状杆菌属的非转化微生物中表达编码具有异丙基苹果酸合酶活性的非修饰多肽的多核苷酸。
6.根据权利要求5所述的微生物，其中增强L-亮氨酸生产率的所述启动子是PCJ7启动子。
7.一种生产L-亮氨酸的方法，包括：
(a)在培养基中培养根据权利要求1至6中任一项所述的微生物，以生产L-亮氨酸；和
(b)从培养的微生物或所述培养基回收L-亮氨酸。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20160181343 | 2016-12-28 | ||
| KR10-2016-0181343 | 2016-12-28 | ||
| CN201780001521.0A CN108884449B (zh) | 2016-12-28 | 2017-10-20 | 新型异丙基苹果酸合酶变异体及使用其生产l-亮氨酸的方法 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201780001521.0A Division CN108884449B (zh) | 2016-12-28 | 2017-10-20 | 新型异丙基苹果酸合酶变异体及使用其生产l-亮氨酸的方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114085800A true CN114085800A (zh) | 2022-02-25 |
Family
ID=62710309
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201780001521.0A Active CN108884449B (zh) | 2016-12-28 | 2017-10-20 | 新型异丙基苹果酸合酶变异体及使用其生产l-亮氨酸的方法 |
| CN202111316466.9A Pending CN114085800A (zh) | 2016-12-28 | 2017-10-20 | 新型异丙基苹果酸合酶变异体及使用其生产l-亮氨酸的方法 |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201780001521.0A Active CN108884449B (zh) | 2016-12-28 | 2017-10-20 | 新型异丙基苹果酸合酶变异体及使用其生产l-亮氨酸的方法 |
Country Status (12)
| Country | Link |
|---|---|
| US (2) | US11104924B2 (zh) |
| EP (2) | EP3564366B1 (zh) |
| JP (2) | JP6848067B2 (zh) |
| KR (2) | KR102055874B1 (zh) |
| CN (2) | CN108884449B (zh) |
| CA (2) | CA3095659C (zh) |
| ES (2) | ES2896257T3 (zh) |
| HK (1) | HK1258202A1 (zh) |
| HU (2) | HUE060328T2 (zh) |
| MX (2) | MX2019007782A (zh) |
| PL (2) | PL3564366T3 (zh) |
| WO (1) | WO2018124440A2 (zh) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3095659C (en) * | 2016-12-28 | 2022-12-06 | Cj Cheiljedang Corporation | Isopropylmalate synthase variant and a method of producing l-leucine using the same |
| CN109456987B (zh) * | 2018-10-26 | 2021-05-25 | 天津科技大学 | 高产l-亮氨酸的相关基因及工程菌构建方法与应用 |
| KR102134418B1 (ko) * | 2019-06-17 | 2020-07-16 | 씨제이제일제당 주식회사 | L-타이로신을 생산하는 미생물 및 이를 이용한 l-타이로신 생산 방법 |
| CN110540976B (zh) * | 2019-08-29 | 2020-07-24 | 天津科技大学 | 一种异丙基苹果酸合成酶及其应用 |
| WO2021037190A1 (zh) * | 2019-08-29 | 2021-03-04 | 天津科技大学 | 一种2-异丙基苹果酸合成酶及其工程菌与应用 |
| KR102153534B1 (ko) | 2019-09-02 | 2020-09-09 | 씨제이제일제당 주식회사 | 신규한 프로모터 및 이를 이용한 아미노산 생산 방법 |
| KR102143964B1 (ko) * | 2019-12-06 | 2020-08-12 | 씨제이제일제당 주식회사 | 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 |
| KR102207867B1 (ko) | 2020-01-21 | 2021-01-26 | 씨제이제일제당 주식회사 | Nadp 의존적 글리세르알데하이드-3-포스페이트 디하이드로지나제를 포함하는 미생물을 이용하여 ｌ-아미노산을 생산하는 방법 |
| KR102285951B1 (ko) * | 2020-01-30 | 2021-08-04 | 씨제이제일제당 주식회사 | 시트레이트 신타아제의 활성이 약화된 신규한 변이형 폴리펩티드 및 이를 이용한 l-아미노산 생산 방법 |
| KR102360900B1 (ko) | 2020-05-20 | 2022-02-09 | 씨제이제일제당 (주) | 신규한 폴리펩티드 및 이를 이용한 l-류신의 생산 방법 |
| KR102495918B1 (ko) | 2021-01-26 | 2023-02-06 | 씨제이제일제당 주식회사 | aroG 알돌라아제 (Phospho-2-dehydro-3-deoxyheptonate aldolase) 변이체 및 이를 이용한 분지쇄 아미노산 생산 방법 |
| KR102527096B1 (ko) | 2021-02-01 | 2023-04-28 | 씨제이제일제당 주식회사 | 프리페네이트 탈수 효소 (Prephenate dehydratase) 변이체 및 이를 이용한 분지쇄 아미노산 생산 방법 |
| KR102527102B1 (ko) * | 2021-03-05 | 2023-04-28 | 씨제이제일제당 주식회사 | 이소프로필말레이트 신타제 변이체 및 이를 이용한 l-류신의 생산 방법 |
| JP2024075803A (ja) * | 2021-03-29 | 2024-06-05 | ＧｒｅｅｎＥａｒｔｈＩｎｓｔｉｔｕｔｅ株式会社 | 改変型α－イソプロピルマレートシンターゼ |
| KR102281370B1 (ko) * | 2021-04-07 | 2021-07-22 | 씨제이제일제당 (주) | 신규한 2-이소프로필말레이트합성효소 변이체 및 이를 이용한 l-발린 생산 방법 |
| KR20240139382A (ko) | 2023-03-14 | 2024-09-23 | 씨제이제일제당 (주) | 여과 유속이 개선된 아미노산의 제조방법 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1222576A (zh) * | 1997-10-29 | 1999-07-14 | 味之素株式会社 | 生产l－亮氨酸的方法 |
| JP2000116393A (ja) * | 1998-10-16 | 2000-04-25 | Ajinomoto Co Inc | Ｌ−ロイシンの製造法 |
| CN1280135A (zh) * | 1999-07-09 | 2001-01-17 | 味之素株式会社 | L－亮氨酸的生产方法及有关dna和微生物 |
| US20030167513A1 (en) * | 2001-11-30 | 2003-09-04 | Mourad George S. | Selection and use of isopropylmalate synthase (IPMS) mutants desensitized in L-leucine negative feedback control |
| CN101087880A (zh) * | 2004-12-16 | 2007-12-12 | Cj株式会社 | 来自棒杆菌属细菌的新的启动子核酸、包含该启动子的表达盒和包含该表达盒的载体、包含该载体的宿主细胞和使用该细胞表达基因的方法 |
| CN104302765A (zh) * | 2012-04-27 | 2015-01-21 | 赢创工业集团股份有限公司 | 反馈抗性α-异丙基苹果酸合酶 |
| CN104480058A (zh) * | 2014-12-30 | 2015-04-01 | 福建师范大学 | 一株高产l-亮氨酸工程菌及其应用 |
| CN106190921A (zh) * | 2016-08-08 | 2016-12-07 | 天津科技大学 | 一种谷氨酸棒状杆菌与应用 |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100438146B1 (ko) | 1996-11-28 | 2004-11-03 | 씨제이 주식회사 | L-루이신생산미생물인코리네박테리움글루타미컴ch25 |
| KR100220018B1 (ko) | 1997-06-25 | 1999-10-01 | 손 경 식 | L-루이신을 생산하는 신규한 미생물 코리네박테리움 글루타미컴(Corynebacterium glutamicum) CH45 |
| KR20060065095A (ko) | 2004-12-09 | 2006-06-14 | 엘지전자 주식회사 | 부품 냉각수단을 포함하는 휴대형 통신 단말기 및 냉각방법 |
| KR100924065B1 (ko) | 2006-09-15 | 2009-10-27 | 씨제이제일제당 (주) | Ｌ－라이신 생산능이 향상된 코리네박테리아 및 그를 이용한 ｌ－라이신 생산 방법 |
| KR20080033001A (ko) | 2006-10-12 | 2008-04-16 | 삼성전자주식회사 | 백라이트 유닛 및 이를 구비하는 액정 표시 장치 |
| KR101119593B1 (ko) * | 2009-08-14 | 2012-03-06 | 한국과학기술원 | Ｌ-루신 생성능이 개선된 변이 미생물 및 이를 이용한 ｌ-루신의 제조방법 |
| TWI510621B (zh) * | 2013-07-08 | 2015-12-01 | Cj Cheiljedang Corp | 具有增加之腐胺生產力之重組微生物及使用該微生物之製造腐胺之方法 |
| KR101632642B1 (ko) * | 2015-01-29 | 2016-06-22 | 씨제이제일제당 주식회사 | 신규한 프로모터 및 그의 용도 |
| KR101796830B1 (ko) * | 2015-08-25 | 2017-11-13 | 씨제이제일제당 (주) | Ｌ-류신 생산능을 가지는 미생물 및 이를 이용한 ｌ-류신의 제조방법 |
| CA3095659C (en) * | 2016-12-28 | 2022-12-06 | Cj Cheiljedang Corporation | Isopropylmalate synthase variant and a method of producing l-leucine using the same |
| CN106754807B (zh) * | 2016-12-29 | 2020-10-30 | 廊坊梅花生物技术开发有限公司 | 生产l-亮氨酸菌株和生产l-亮氨酸的方法 |
-
2017
- 2017-10-20 CA CA3095659A patent/CA3095659C/en active Active
- 2017-10-20 MX MX2019007782A patent/MX2019007782A/es unknown
- 2017-10-20 EP EP17889165.1A patent/EP3564366B1/en active Active
- 2017-10-20 CN CN201780001521.0A patent/CN108884449B/zh active Active
- 2017-10-20 PL PL17889165T patent/PL3564366T3/pl unknown
- 2017-10-20 EP EP21154505.8A patent/EP3858987B1/en active Active
- 2017-10-20 US US16/473,941 patent/US11104924B2/en active Active
- 2017-10-20 HU HUE21154505A patent/HUE060328T2/hu unknown
- 2017-10-20 PL PL21154505.8T patent/PL3858987T3/pl unknown
- 2017-10-20 ES ES17889165T patent/ES2896257T3/es active Active
- 2017-10-20 KR KR1020170136244A patent/KR102055874B1/ko active IP Right Grant
- 2017-10-20 ES ES21154505T patent/ES2932086T3/es active Active
- 2017-10-20 WO PCT/KR2017/011622 patent/WO2018124440A2/ko active Application Filing
- 2017-10-20 CA CA3048802A patent/CA3048802C/en active Active
- 2017-10-20 JP JP2019535387A patent/JP6848067B2/ja active Active
- 2017-10-20 HU HUE17889165A patent/HUE056931T2/hu unknown
- 2017-10-20 CN CN202111316466.9A patent/CN114085800A/zh active Pending
-
2019
- 2019-01-14 HK HK19100559.2A patent/HK1258202A1/zh unknown
- 2019-05-20 KR KR1020190058985A patent/KR102094875B1/ko active IP Right Grant
- 2019-06-27 MX MX2023009109A patent/MX2023009109A/es unknown
-
2020
- 2020-09-23 JP JP2020158999A patent/JP7217250B2/ja active Active
-
2021
- 2021-01-25 US US17/156,998 patent/US11905539B2/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1222576A (zh) * | 1997-10-29 | 1999-07-14 | 味之素株式会社 | 生产l－亮氨酸的方法 |
| JP2000116393A (ja) * | 1998-10-16 | 2000-04-25 | Ajinomoto Co Inc | Ｌ−ロイシンの製造法 |
| CN1280135A (zh) * | 1999-07-09 | 2001-01-17 | 味之素株式会社 | L－亮氨酸的生产方法及有关dna和微生物 |
| US20030167513A1 (en) * | 2001-11-30 | 2003-09-04 | Mourad George S. | Selection and use of isopropylmalate synthase (IPMS) mutants desensitized in L-leucine negative feedback control |
| CN101087880A (zh) * | 2004-12-16 | 2007-12-12 | Cj株式会社 | 来自棒杆菌属细菌的新的启动子核酸、包含该启动子的表达盒和包含该表达盒的载体、包含该载体的宿主细胞和使用该细胞表达基因的方法 |
| CN104302765A (zh) * | 2012-04-27 | 2015-01-21 | 赢创工业集团股份有限公司 | 反馈抗性α-异丙基苹果酸合酶 |
| CN104480058A (zh) * | 2014-12-30 | 2015-04-01 | 福建师范大学 | 一株高产l-亮氨酸工程菌及其应用 |
| CN106190921A (zh) * | 2016-08-08 | 2016-12-07 | 天津科技大学 | 一种谷氨酸棒状杆菌与应用 |
Non-Patent Citations (4)
| Title |
|---|
| GUI,Y.等: "登录号：ALP49010.1", pages 1 - 616 * |
| MICHAEL VOGT等: "Pushing product formation to its limit: Metabolic engineering of Corynebacterium glutamicum for l-leucine overproduction", vol. 22, pages 40 - 52, XP055111480, DOI: 10.1016/j.ymben.2013.12.001 * |
| YONGLI GUI等: "Complete genome sequence of Corynebacterium glutamicum CP, a Chinese l-leucine producing strain", vol. 220, pages 64 - 65 * |
| 张跃等: "L-亮氨酸生物合成关键酶基因的克隆和表达", vol. 34, no. 11, pages 170 - 173 * |
Also Published As
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108884449B (zh) | 新型异丙基苹果酸合酶变异体及使用其生产l-亮氨酸的方法 | |
| KR101904666B1 (ko) | Atp 포스포리보실기 전이효소 변이체 및 이를 이용한 l-히스티딘 생산방법 | |
| US20220098569A1 (en) | Novel adenylosuccinate synthetase and method for producing purine nucleotides using the same | |
| EP3508580A1 (en) | Novel promoter and use thereof | |
| KR102434925B1 (ko) | 3-메틸-2-옥소뷰타노에이트 하이드록시 메틸트랜스퍼라아제의 활성이 강화된 미생물, 및 이의 용도 | |
| KR102589135B1 (ko) | 3-메틸-2-옥소뷰타노에이트 하이드록시 메틸트랜스퍼라아제의 활성이 강화된 미생물, 및 이의 용도 | |
| EP4056675A1 (en) | Mutant of corynebacterium glutamicum with enhanced l-lysine productivity and method for preparing l-lysine using the same | |
| CN115052976B (zh) | 新型乙酰羟酸合酶变体和包括其的微生物 | |
| KR102360900B1 (ko) | 신규한 폴리펩티드 및 이를 이용한 l-류신의 생산 방법 | |
| RU2811433C1 (ru) | Новый полипептид и способ получения l-лейцина с его использованием | |
| JP2024515391A (ja) | Ｌ－リシン生産能が向上したコリネバクテリウム・グルタミカム変異株及びそれを用いたｌ－リシンの生産方法 | |
| CN111676205A (zh) | L-赖氨酸生产力提高的微生物和利用其生产l-赖氨酸的方法 | |
| JP2024515389A (ja) | Ｌ－リシン生産能が向上したコリネバクテリウム・グルタミカム変異株及びそれを用いたｌ－リシンの生産方法 | |
| JP2023506053A (ja) | クエン酸シンターゼの活性が弱化した新規な変異型ポリペプチド及びそれを用いたｌ－アミノ酸生産方法 |
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 |