CN114874958B - 生产l-脯氨酸的菌株及其构建方法和应用 - Google Patents
生产l-脯氨酸的菌株及其构建方法和应用 Download PDFInfo
- Publication number
- CN114874958B CN114874958B CN202110161475.9A CN202110161475A CN114874958B CN 114874958 B CN114874958 B CN 114874958B CN 202110161475 A CN202110161475 A CN 202110161475A CN 114874958 B CN114874958 B CN 114874958B
- Authority
- CN
- China
- Prior art keywords
- proline
- membrane transporter
- strain
- seq
- corynebacterium glutamicum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 title claims abstract description 80
- 229960002429 proline Drugs 0.000 title claims abstract description 80
- 229930182821 L-proline Natural products 0.000 title claims abstract description 78
- 238000010276 construction Methods 0.000 title claims abstract description 20
- 108090000301 Membrane transport proteins Proteins 0.000 claims abstract description 69
- 102000003939 Membrane transport proteins Human genes 0.000 claims abstract description 65
- 238000004519 manufacturing process Methods 0.000 claims abstract description 39
- 108090000623 proteins and genes Proteins 0.000 claims description 74
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 45
- 230000000694 effects Effects 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 34
- 102000004169 proteins and genes Human genes 0.000 claims description 32
- 150000001413 amino acids Chemical group 0.000 claims description 31
- 239000002773 nucleotide Substances 0.000 claims description 25
- 125000003729 nucleotide group Chemical group 0.000 claims description 25
- 230000035772 mutation Effects 0.000 claims description 21
- 230000014509 gene expression Effects 0.000 claims description 18
- 230000002829 reductive effect Effects 0.000 claims description 16
- 238000000855 fermentation Methods 0.000 claims description 15
- 230000004151 fermentation Effects 0.000 claims description 15
- 102000005133 Glutamate 5-kinase Human genes 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 9
- 230000005764 inhibitory process Effects 0.000 claims description 8
- 108700023479 Glutamate 5-kinases Proteins 0.000 claims description 7
- 108010016106 Glutamate-5-semialdehyde dehydrogenase Proteins 0.000 claims description 7
- 230000002238 attenuated effect Effects 0.000 claims description 5
- 210000000349 chromosome Anatomy 0.000 claims description 4
- 238000012239 gene modification Methods 0.000 claims description 4
- 230000005017 genetic modification Effects 0.000 claims description 4
- 235000013617 genetically modified food Nutrition 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 230000001771 impaired effect Effects 0.000 claims 2
- 230000009466 transformation Effects 0.000 abstract description 7
- 238000012216 screening Methods 0.000 abstract description 6
- 230000002779 inactivation Effects 0.000 abstract description 5
- 241000894006 Bacteria Species 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 230000037361 pathway Effects 0.000 abstract description 2
- 238000006467 substitution reaction Methods 0.000 description 30
- 235000018102 proteins Nutrition 0.000 description 27
- 108020004414 DNA Proteins 0.000 description 25
- 108090000765 processed proteins & peptides Proteins 0.000 description 18
- 239000013612 plasmid Substances 0.000 description 17
- 229920001184 polypeptide Polymers 0.000 description 17
- 102000004196 processed proteins & peptides Human genes 0.000 description 17
- 229940024606 amino acid Drugs 0.000 description 14
- 235000001014 amino acid Nutrition 0.000 description 14
- 125000000539 amino acid group Chemical group 0.000 description 12
- 108091033319 polynucleotide Proteins 0.000 description 11
- 102000040430 polynucleotide Human genes 0.000 description 11
- 239000002157 polynucleotide Substances 0.000 description 11
- 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 10
- 210000004027 cell Anatomy 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- 230000003313 weakening effect Effects 0.000 description 9
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 101000619151 Dickeya chrysanthemi Serralysin B Proteins 0.000 description 7
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 7
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 7
- 238000003209 gene knockout Methods 0.000 description 7
- 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 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 101710198928 Gamma-glutamyl phosphate reductase Proteins 0.000 description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 5
- 229960002685 biotin Drugs 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- 230000014616 translation Effects 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 241000186216 Corynebacterium Species 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 244000068988 Glycine max Species 0.000 description 4
- 235000010469 Glycine max Nutrition 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 239000007993 MOPS buffer Substances 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 238000012300 Sequence Analysis Methods 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 108010050848 glycylleucine Proteins 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 108091033409 CRISPR Proteins 0.000 description 3
- 238000010446 CRISPR interference Methods 0.000 description 3
- 241000186031 Corynebacteriaceae Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000588722 Escherichia Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- 125000000393 L-methionino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C(SC([H])([H])[H])([H])[H] 0.000 description 3
- 125000002068 L-phenylalanino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- 241000186146 Brevibacterium Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 102220605874 Cytosolic arginine sensor for mTORC1 subunit 2_D10A_mutation Human genes 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- KFKWRHQBZQICHA-STQMWFEESA-N L-leucyl-L-phenylalanine Natural products CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-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
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 241000319304 [Brevibacterium] flavum Species 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 231100000221 frame shift mutation induction Toxicity 0.000 description 2
- 230000037433 frameshift Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 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
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 101150046501 proB gene Proteins 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 108010048818 seryl-histidine Proteins 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- NTUPOKHATNSWCY-PMPSAXMXSA-N (2s)-2-[[(2s)-1-[(2r)-2-amino-3-phenylpropanoyl]pyrrolidine-2-carbonyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound C([C@@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C1=CC=CC=C1 NTUPOKHATNSWCY-PMPSAXMXSA-N 0.000 description 1
- NXOIMAMHRHDCFR-UHFFFAOYSA-N 2,3-dihydro-1h-pyrrole-2-carboxylic acid Chemical compound OC(=O)C1CC=CN1 NXOIMAMHRHDCFR-UHFFFAOYSA-N 0.000 description 1
- WXHLLJAMBQLULT-UHFFFAOYSA-N 2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-n-(2-methyl-6-sulfanylphenyl)-1,3-thiazole-5-carboxamide;hydrate Chemical compound O.C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1S WXHLLJAMBQLULT-UHFFFAOYSA-N 0.000 description 1
- WDIYWDJLXOCGRW-ACZMJKKPSA-N Ala-Asp-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WDIYWDJLXOCGRW-ACZMJKKPSA-N 0.000 description 1
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 description 1
- NHLAEBFGWPXFGI-WHFBIAKZSA-N Ala-Gly-Asn Chemical compound C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N NHLAEBFGWPXFGI-WHFBIAKZSA-N 0.000 description 1
- QJABSQFUHKHTNP-SYWGBEHUSA-N Ala-Ile-Trp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QJABSQFUHKHTNP-SYWGBEHUSA-N 0.000 description 1
- AWZKCUCQJNTBAD-SRVKXCTJSA-N Ala-Leu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN AWZKCUCQJNTBAD-SRVKXCTJSA-N 0.000 description 1
- UWIQWPWWZUHBAO-ZLIFDBKOSA-N Ala-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)N)CC(C)C)C(O)=O)=CNC2=C1 UWIQWPWWZUHBAO-ZLIFDBKOSA-N 0.000 description 1
- NINQYGGNRIBFSC-CIUDSAMLSA-N Ala-Lys-Ser Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CO)C(O)=O NINQYGGNRIBFSC-CIUDSAMLSA-N 0.000 description 1
- RUXQNKVQSKOOBS-JURCDPSOSA-N Ala-Phe-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RUXQNKVQSKOOBS-JURCDPSOSA-N 0.000 description 1
- 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 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 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 1
- BAVDUESNGSMLPI-CIUDSAMLSA-N Arg-Asn-Gly-Ser Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O BAVDUESNGSMLPI-CIUDSAMLSA-N 0.000 description 1
- RFXXUWGNVRJTNQ-QXEWZRGKSA-N Arg-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N RFXXUWGNVRJTNQ-QXEWZRGKSA-N 0.000 description 1
- 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 1
- IRRMIGDCPOPZJW-ULQDDVLXSA-N Arg-His-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IRRMIGDCPOPZJW-ULQDDVLXSA-N 0.000 description 1
- HJDNZFIYILEIKR-OSUNSFLBSA-N Arg-Ile-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HJDNZFIYILEIKR-OSUNSFLBSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- KSGAFDTYQPKUAP-GMOBBJLQSA-N Asn-Met-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KSGAFDTYQPKUAP-GMOBBJLQSA-N 0.000 description 1
- UGXYFDQFLVCDFC-CIUDSAMLSA-N Asn-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O UGXYFDQFLVCDFC-CIUDSAMLSA-N 0.000 description 1
- XBQSLMACWDXWLJ-GHCJXIJMSA-N Asp-Ala-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XBQSLMACWDXWLJ-GHCJXIJMSA-N 0.000 description 1
- DBWYWXNMZZYIRY-LPEHRKFASA-N Asp-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N)C(=O)O DBWYWXNMZZYIRY-LPEHRKFASA-N 0.000 description 1
- HOQGTAIGQSDCHR-SRVKXCTJSA-N Asp-Asn-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HOQGTAIGQSDCHR-SRVKXCTJSA-N 0.000 description 1
- PXLNPFOJZQMXAT-BYULHYEWSA-N Asp-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O PXLNPFOJZQMXAT-BYULHYEWSA-N 0.000 description 1
- SEMWSADZTMJELF-BYULHYEWSA-N Asp-Ile-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O SEMWSADZTMJELF-BYULHYEWSA-N 0.000 description 1
- WNGZKSVJFDZICU-XIRDDKMYSA-N Asp-Leu-Trp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(=O)O)N WNGZKSVJFDZICU-XIRDDKMYSA-N 0.000 description 1
- LBOVBQONZJRWPV-YUMQZZPRSA-N Asp-Lys-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O LBOVBQONZJRWPV-YUMQZZPRSA-N 0.000 description 1
- OTKUAVXGMREHRX-CFMVVWHZSA-N Asp-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 OTKUAVXGMREHRX-CFMVVWHZSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- OYTPNWYZORARHL-XHNCKOQMSA-N Gln-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N OYTPNWYZORARHL-XHNCKOQMSA-N 0.000 description 1
- BTSPOOHJBYJRKO-CIUDSAMLSA-N Gln-Asp-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O BTSPOOHJBYJRKO-CIUDSAMLSA-N 0.000 description 1
- GPISLLFQNHELLK-DCAQKATOSA-N Gln-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](CCC(=O)N)N GPISLLFQNHELLK-DCAQKATOSA-N 0.000 description 1
- XQDGOJPVMSWZSO-SRVKXCTJSA-N Gln-Pro-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)N)N XQDGOJPVMSWZSO-SRVKXCTJSA-N 0.000 description 1
- NHMRJKKAVMENKJ-WDCWCFNPSA-N Gln-Thr-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NHMRJKKAVMENKJ-WDCWCFNPSA-N 0.000 description 1
- HPBKQFJXDUVNQV-FHWLQOOXSA-N Gln-Tyr-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O HPBKQFJXDUVNQV-FHWLQOOXSA-N 0.000 description 1
- VGUYMZGLJUJRBV-YVNDNENWSA-N Glu-Ile-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O VGUYMZGLJUJRBV-YVNDNENWSA-N 0.000 description 1
- OQXDUSZKISQQSS-GUBZILKMSA-N Glu-Lys-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OQXDUSZKISQQSS-GUBZILKMSA-N 0.000 description 1
- FMBWLLMUPXTXFC-SDDRHHMPSA-N Glu-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(=O)O)N)C(=O)O FMBWLLMUPXTXFC-SDDRHHMPSA-N 0.000 description 1
- FVGOGEGGQLNZGH-DZKIICNBSA-N Glu-Val-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 FVGOGEGGQLNZGH-DZKIICNBSA-N 0.000 description 1
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 1
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 1
- NTBOEZICHOSJEE-YUMQZZPRSA-N Gly-Lys-Ser Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O NTBOEZICHOSJEE-YUMQZZPRSA-N 0.000 description 1
- JPVGHHQGKPQYIL-KBPBESRZSA-N Gly-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 JPVGHHQGKPQYIL-KBPBESRZSA-N 0.000 description 1
- GLACUWHUYFBSPJ-FJXKBIBVSA-N Gly-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN GLACUWHUYFBSPJ-FJXKBIBVSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- JQFILXICXLDTRR-FBCQKBJTSA-N Gly-Thr-Gly Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)NCC(O)=O JQFILXICXLDTRR-FBCQKBJTSA-N 0.000 description 1
- HUFUVTYGPOUCBN-MBLNEYKQSA-N Gly-Thr-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HUFUVTYGPOUCBN-MBLNEYKQSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- ORZGPQXISSXQGW-IHRRRGAJSA-N His-His-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C(C)C)C(O)=O ORZGPQXISSXQGW-IHRRRGAJSA-N 0.000 description 1
- ORERHHPZDDEMSC-VGDYDELISA-N His-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N ORERHHPZDDEMSC-VGDYDELISA-N 0.000 description 1
- KAXZXLSXFWSNNZ-XVYDVKMFSA-N His-Ser-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KAXZXLSXFWSNNZ-XVYDVKMFSA-N 0.000 description 1
- VSZALHITQINTGC-GHCJXIJMSA-N Ile-Ala-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)O)C(=O)O)N VSZALHITQINTGC-GHCJXIJMSA-N 0.000 description 1
- 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 1
- ZDNORQNHCJUVOV-KBIXCLLPSA-N Ile-Gln-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O ZDNORQNHCJUVOV-KBIXCLLPSA-N 0.000 description 1
- HOLOYAZCIHDQNS-YVNDNENWSA-N Ile-Gln-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HOLOYAZCIHDQNS-YVNDNENWSA-N 0.000 description 1
- YKLOMBNBQUTJDT-HVTMNAMFSA-N Ile-His-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YKLOMBNBQUTJDT-HVTMNAMFSA-N 0.000 description 1
- YNMQUIVKEFRCPH-QSFUFRPTSA-N Ile-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)O)N YNMQUIVKEFRCPH-QSFUFRPTSA-N 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- WSSGUVAKYCQSCT-XUXIUFHCSA-N Ile-Met-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(=O)O)N WSSGUVAKYCQSCT-XUXIUFHCSA-N 0.000 description 1
- UAELWXJFLZBKQS-WHOFXGATSA-N Ile-Phe-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(O)=O UAELWXJFLZBKQS-WHOFXGATSA-N 0.000 description 1
- YJRSIJZUIUANHO-NAKRPEOUSA-N Ile-Val-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)O)N YJRSIJZUIUANHO-NAKRPEOUSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- 125000000570 L-alpha-aspartyl group Chemical group [H]OC(=O)C([H])([H])[C@]([H])(N([H])[H])C(*)=O 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- 125000003290 L-leucino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- KVRKAGGMEWNURO-CIUDSAMLSA-N Leu-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(C)C)N KVRKAGGMEWNURO-CIUDSAMLSA-N 0.000 description 1
- 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 1
- CQGSYZCULZMEDE-SRVKXCTJSA-N Leu-Gln-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(O)=O CQGSYZCULZMEDE-SRVKXCTJSA-N 0.000 description 1
- CQGSYZCULZMEDE-UHFFFAOYSA-N Leu-Gln-Pro Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)N1CCCC1C(O)=O CQGSYZCULZMEDE-UHFFFAOYSA-N 0.000 description 1
- SGIIOQQGLUUMDQ-IHRRRGAJSA-N Leu-His-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](C(C)C)C(=O)O)N SGIIOQQGLUUMDQ-IHRRRGAJSA-N 0.000 description 1
- AUBMZAMQCOYSIC-MNXVOIDGSA-N Leu-Ile-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O AUBMZAMQCOYSIC-MNXVOIDGSA-N 0.000 description 1
- KUIDCYNIEJBZBU-AJNGGQMLSA-N Leu-Ile-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O KUIDCYNIEJBZBU-AJNGGQMLSA-N 0.000 description 1
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 1
- OVZLLFONXILPDZ-VOAKCMCISA-N Leu-Lys-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OVZLLFONXILPDZ-VOAKCMCISA-N 0.000 description 1
- ZAVCJRJOQKIOJW-KKUMJFAQSA-N Leu-Phe-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=CC=C1 ZAVCJRJOQKIOJW-KKUMJFAQSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- ILDSIMPXNFWKLH-KATARQTJSA-N Leu-Thr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ILDSIMPXNFWKLH-KATARQTJSA-N 0.000 description 1
- AIMGJYMCTAABEN-GVXVVHGQSA-N Leu-Val-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O AIMGJYMCTAABEN-GVXVVHGQSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- AAORVPFVUIHEAB-YUMQZZPRSA-N Lys-Asp-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O AAORVPFVUIHEAB-YUMQZZPRSA-N 0.000 description 1
- LLSUNJYOSCOOEB-GUBZILKMSA-N Lys-Glu-Asp Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O LLSUNJYOSCOOEB-GUBZILKMSA-N 0.000 description 1
- HYSVGEAWTGPMOA-IHRRRGAJSA-N Lys-Pro-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O HYSVGEAWTGPMOA-IHRRRGAJSA-N 0.000 description 1
- IOQWIOPSKJOEKI-SRVKXCTJSA-N Lys-Ser-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IOQWIOPSKJOEKI-SRVKXCTJSA-N 0.000 description 1
- QLFAPXUXEBAWEK-NHCYSSNCSA-N Lys-Val-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O QLFAPXUXEBAWEK-NHCYSSNCSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- CAODKDAPYGUMLK-FXQIFTODSA-N Met-Asn-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O CAODKDAPYGUMLK-FXQIFTODSA-N 0.000 description 1
- TZLYIHDABYBOCJ-FXQIFTODSA-N Met-Asp-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O TZLYIHDABYBOCJ-FXQIFTODSA-N 0.000 description 1
- GMMLGMFBYCFCCX-KZVJFYERSA-N Met-Thr-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O GMMLGMFBYCFCCX-KZVJFYERSA-N 0.000 description 1
- FSTWDRPCQQUJIT-NHCYSSNCSA-N Met-Val-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCSC)N FSTWDRPCQQUJIT-NHCYSSNCSA-N 0.000 description 1
- 241001467578 Microbacterium Species 0.000 description 1
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- HCTXJGRYAACKOB-SRVKXCTJSA-N Phe-Asn-Asp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N HCTXJGRYAACKOB-SRVKXCTJSA-N 0.000 description 1
- NHCKESBLOMHIIE-IRXDYDNUSA-N Phe-Gly-Phe Chemical compound C([C@H](N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 NHCKESBLOMHIIE-IRXDYDNUSA-N 0.000 description 1
- KDYPMIZMXDECSU-JYJNAYRXSA-N Phe-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 KDYPMIZMXDECSU-JYJNAYRXSA-N 0.000 description 1
- SMFGCTXUBWEPKM-KBPBESRZSA-N Phe-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 SMFGCTXUBWEPKM-KBPBESRZSA-N 0.000 description 1
- INHMISZWLJZQGH-ULQDDVLXSA-N Phe-Leu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 INHMISZWLJZQGH-ULQDDVLXSA-N 0.000 description 1
- GPLWGAYGROGDEN-BZSNNMDCSA-N Phe-Phe-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O GPLWGAYGROGDEN-BZSNNMDCSA-N 0.000 description 1
- GRVMHFCZUIYNKQ-UFYCRDLUSA-N Phe-Phe-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O GRVMHFCZUIYNKQ-UFYCRDLUSA-N 0.000 description 1
- 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 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- UMIHVJQSXFWWMW-JBACZVJFSA-N Phe-Trp-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N UMIHVJQSXFWWMW-JBACZVJFSA-N 0.000 description 1
- UTAUEDINXUMHLG-FXQIFTODSA-N Pro-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 UTAUEDINXUMHLG-FXQIFTODSA-N 0.000 description 1
- 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 1
- CGSOWZUPLOKYOR-AVGNSLFASA-N Pro-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 CGSOWZUPLOKYOR-AVGNSLFASA-N 0.000 description 1
- FDMCIBSQRKFSTJ-RHYQMDGZSA-N Pro-Thr-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O FDMCIBSQRKFSTJ-RHYQMDGZSA-N 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- NRCJWSGXMAPYQX-LPEHRKFASA-N Ser-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)N)C(=O)O NRCJWSGXMAPYQX-LPEHRKFASA-N 0.000 description 1
- OJPHFSOMBZKQKQ-GUBZILKMSA-N Ser-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CO OJPHFSOMBZKQKQ-GUBZILKMSA-N 0.000 description 1
- SMIDBHKWSYUBRZ-ACZMJKKPSA-N Ser-Glu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O SMIDBHKWSYUBRZ-ACZMJKKPSA-N 0.000 description 1
- IOVHBRCQOGWAQH-ZKWXMUAHSA-N Ser-Gly-Ile Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IOVHBRCQOGWAQH-ZKWXMUAHSA-N 0.000 description 1
- XXXAXOWMBOKTRN-XPUUQOCRSA-N Ser-Gly-Val Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXXAXOWMBOKTRN-XPUUQOCRSA-N 0.000 description 1
- IFPBAGJBHSNYPR-ZKWXMUAHSA-N Ser-Ile-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O IFPBAGJBHSNYPR-ZKWXMUAHSA-N 0.000 description 1
- HEUVHBXOVZONPU-BJDJZHNGSA-N Ser-Leu-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HEUVHBXOVZONPU-BJDJZHNGSA-N 0.000 description 1
- RRVFEDGUXSYWOW-BZSNNMDCSA-N Ser-Phe-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RRVFEDGUXSYWOW-BZSNNMDCSA-N 0.000 description 1
- MQUZANJDFOQOBX-SRVKXCTJSA-N Ser-Phe-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O MQUZANJDFOQOBX-SRVKXCTJSA-N 0.000 description 1
- NVNPWELENFJOHH-CIUDSAMLSA-N Ser-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)N NVNPWELENFJOHH-CIUDSAMLSA-N 0.000 description 1
- NERYDXBVARJIQS-JYBASQMISA-N Ser-Trp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CO)N)O NERYDXBVARJIQS-JYBASQMISA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 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
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 1
- GFDUZZACIWNMPE-KZVJFYERSA-N Thr-Ala-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O GFDUZZACIWNMPE-KZVJFYERSA-N 0.000 description 1
- AYCQVUUPIJHJTA-IXOXFDKPSA-N Thr-His-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O AYCQVUUPIJHJTA-IXOXFDKPSA-N 0.000 description 1
- MEJHFIOYJHTWMK-VOAKCMCISA-N Thr-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)O MEJHFIOYJHTWMK-VOAKCMCISA-N 0.000 description 1
- UUSQVWOVUYMLJA-PPCPHDFISA-N Thr-Lys-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UUSQVWOVUYMLJA-PPCPHDFISA-N 0.000 description 1
- JAWUQFCGNVEDRN-MEYUZBJRSA-N Thr-Tyr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N)O JAWUQFCGNVEDRN-MEYUZBJRSA-N 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- QNMIVTOQXUSGLN-SZMVWBNQSA-N Trp-Arg-Arg Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 QNMIVTOQXUSGLN-SZMVWBNQSA-N 0.000 description 1
- ADMHZNPMMVKGJW-BPUTZDHNSA-N Trp-Ser-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N ADMHZNPMMVKGJW-BPUTZDHNSA-N 0.000 description 1
- ZPFLBLFITJCBTP-QWRGUYRKSA-N Tyr-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O ZPFLBLFITJCBTP-QWRGUYRKSA-N 0.000 description 1
- UMSZZGTXGKHTFJ-SRVKXCTJSA-N Tyr-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 UMSZZGTXGKHTFJ-SRVKXCTJSA-N 0.000 description 1
- KHPLUFDSWGDRHD-SLFFLAALSA-N Tyr-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O KHPLUFDSWGDRHD-SLFFLAALSA-N 0.000 description 1
- HZYOWMGWKKRMBZ-BYULHYEWSA-N Val-Asp-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N HZYOWMGWKKRMBZ-BYULHYEWSA-N 0.000 description 1
- XKVXSCHXGJOQND-ZOBUZTSGSA-N Val-Asp-Trp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N XKVXSCHXGJOQND-ZOBUZTSGSA-N 0.000 description 1
- BRPKEERLGYNCNC-NHCYSSNCSA-N Val-Glu-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N BRPKEERLGYNCNC-NHCYSSNCSA-N 0.000 description 1
- UKEVLVBHRKWECS-LSJOCFKGSA-N Val-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](C(C)C)N UKEVLVBHRKWECS-LSJOCFKGSA-N 0.000 description 1
- MYLNLEIZWHVENT-VKOGCVSHSA-N Val-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](C(C)C)N MYLNLEIZWHVENT-VKOGCVSHSA-N 0.000 description 1
- BZWUSZGQOILYEU-STECZYCISA-N Val-Ile-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BZWUSZGQOILYEU-STECZYCISA-N 0.000 description 1
- RQOMPQGUGBILAG-AVGNSLFASA-N Val-Met-Leu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O RQOMPQGUGBILAG-AVGNSLFASA-N 0.000 description 1
- NHXZRXLFOBFMDM-AVGNSLFASA-N Val-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C NHXZRXLFOBFMDM-AVGNSLFASA-N 0.000 description 1
- LCHZBEUVGAVMKS-RHYQMDGZSA-N Val-Thr-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(O)=O LCHZBEUVGAVMKS-RHYQMDGZSA-N 0.000 description 1
- AEFJNECXZCODJM-UWVGGRQHSA-N Val-Val-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)NCC([O-])=O AEFJNECXZCODJM-UWVGGRQHSA-N 0.000 description 1
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000006227 byproduct Substances 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
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 108010054813 diprotin B Proteins 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 108010012204 glutamate aminotransferase Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 108010073628 glutamyl-valyl-phenylalanine Proteins 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010084389 glycyltryptophan Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- -1 his Chemical group 0.000 description 1
- 101150099953 ilvE gene Proteins 0.000 description 1
- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 239000002054 inoculum Substances 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
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 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 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 1
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000029226 lipidation Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 1
- 108010089198 phenylalanyl-prolyl-arginine Proteins 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 108010015385 valyl-prolyl-proline Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- 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
- C12P13/24—Proline; Hydroxyproline; Histidine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了生产L‑脯氨酸的菌株及其构建方法和应用。本发明经过筛选发现两种膜转运蛋白的失活可以提高在L‑脯氨酸生产菌的L‑脯氨酸的产量。本发明的发现可以为本领域技术人员构建L‑脯氨酸生产菌株提供新的思路和靶点,即不仅仅关注于L‑脯氨酸合成途径的改造,而是通过膜转运蛋白的失活来提高菌株的产量,为构建更高产L‑脯氨酸的生产菌株提供借鉴。
Description
技术领域
本发明属于分子生物学和生物工程领域,具体涉及膜转运蛋白在生产L-脯氨酸中的应用、生产L-脯氨酸的菌株、这种菌株的构建方法和应用。
背景技术
L-脯氨酸,是天然存在的一种人体的非必需氨基酸,在临床、生物材料和工业等方面有广泛的应用。L-脯氨酸的生产方法主要有化学法和发酵法,化学法由于化学提取法污染严重成本高,已逐渐失去市场,微生物发酵法由于具有生产成本低、生产强度高、高特异性和对环境污染小等优点而成为当今工业应用最广泛的方法。目前,常用的工业发酵菌株有棒杆菌和埃希氏菌,常用的埃希氏菌如大肠杆菌(Escherichia coli),常用的棒杆菌如谷氨酸棒杆菌(Corynebacterium glutamicum),短杆菌如黄色短杆菌(Brevibacteriumflavum)、乳酸发酵短杆菌(Brevibacterium lactofermentus),以及节杆菌属的某些种和微杆菌属的某些种。由于棒杆菌的生理优越性,已成为工业中最重要的生产菌株用来生产氨基酸等产品。
在棒杆菌中,主要以谷氨酸为底物经过γ-谷氨酰激酶(ProB)、谷氨酸半醛脱氢酶(ProA)、吡咯啉-5-羧酸还原酶(ProC)催化后生产L-脯氨酸。现有技术主要以L-脯氨酸合成途径的关键基因ProA、ProB作为改造靶点进行基因工程改造来生产L-脯氨酸,如CN101084312A报道了谷氨酸棒杆菌来源的ProB的149位突变可以解除L-脯氨酸的反馈抑制,提高工程菌株L-脯氨酸的产量。CN109402038A报道了在生产L-脯氨酸的棒杆菌中谷氨酸转氨酶编码基因ilvE启动子后插入终止子并过表达proB基因,获得了高产L-脯氨酸且降低副产物含量的重组菌株。可见,现有改造靶点较少,菌株产酸水平提升有限,本领域急需挖掘新的改造靶点,以便进一步提升菌株的L-脯氨酸产量,增强菌株的应用价值。
发明内容
为克服现有技术中存在的不足,本发明通过全基因组规模的膜转运蛋白抑制库进行筛选,发现谷氨酸棒杆菌的两种膜转运蛋白基因抑制后,L-脯氨酸的产量明显提升。因此,本发明的首要目的是提供一种膜转运蛋白在生产L-脯氨酸中的用途。
本发明的又一目的是提供一种L-脯氨酸的生产菌株。
本发明的另一目的是提供一种生产L-脯氨酸的方法。
本发明的再一目的是提供一种L-脯氨酸生产菌株的构建方法。
为了实现上述目的,本发明采用如下技术方案:
在本发明的第一方面,提供一种膜转运蛋白在微生物发酵生产L-脯氨酸中的用途,所述蛋白是:
(A)氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示;或
(B)与(A)所述蛋白氨基酸序列具有98%以上同源性,且来源于谷氨酸棒杆菌与(A)所述蛋白具有相同活性。
优选地,所述膜转运蛋白的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
优选地,上述用途中,(A)或(B)中的一种膜转运蛋白的活性减弱或完全去除。
优选地,其中编码所述膜转运蛋白的基因选自以下DNA:
(A)核苷酸序列如SEQ ID NO:3或SEQ ID NO:4所示;或
(B)与(A)所示核苷酸序列具有98%以上同源性,且来源于谷氨酸棒杆菌与(A)所述DNA编码的蛋白具有相同活性。
优选地,编码所述膜转运蛋白的基因的核苷酸序列如SEQ ID NO:3或SEQ ID NO:4所示。
本发明术语“同源性”指两个或更多个多核苷酸或多肽之间相同(即同一)的核苷酸或氨基酸的百分比。两个或更多个多核苷酸或多肽之间的序列同一性可通过以下方法测定:将多核苷酸或多肽的核苷酸或氨基酸序列对准且对经对准的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置数目进行评分,且将其与经对准的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸(即取代或突变)或缺失核苷酸(即一个或两个多核苷酸中的核苷酸插入或核苷酸缺失)而在一个位置处不同。多肽可例如通过含有不同氨基酸(即取代或突变)或缺失氨基酸(即一个或两个多肽中的氨基酸插入或氨基酸缺失)而在一个位置处不同。序列同一性可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中氨基酸残基的总数来计算。举例而言,可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中核苷酸或氨基酸残基的总数且乘以100来计算同一性百分比。
本公开中的术语“编码所述膜转运蛋白的基因”是指能够通过一定的规则指导膜转运蛋白的合成DNA分子,指导蛋白合成的过程一般包括以双链DNA为模板的转录过程和以mRNA为模板的翻译过程。编码基因含有CDS序列(Coding Sequence),能够指导编码蛋白质的mRNA的产生。
在本发明的第二方面,提供一种L-脯氨酸的生产菌株,所述菌株被修饰,使得膜转运蛋白的基因被去除或使膜转运蛋白相对于未改性的蛋白活性减弱,其中所述膜转运蛋白选自:
(A)氨基酸序列如SEQ ID NO:1和/或SEQ ID NO:2所示;或
(B)与(A)所述蛋白氨基酸序列具有98%以上同源性,且与(A)所述蛋白具有相同活性。
优选地,上述菌株中,位于菌株染色体上编码所述膜转运蛋白的基因经突变和/或其表达控制序列经突变使所述膜转运蛋白被去除或使膜转运蛋白相对于未改性的蛋白活性减弱。
进一步优选地,所述菌株中的膜转运蛋白相对于未改性的蛋白活性减弱,是指所述膜转运蛋白的编码基因的转录、表达降低至少30%,优选至少40%,更优选至少50%,例如至少60%、至少70%、至少80%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%;或者所述膜转运蛋白的活性降低至少30%、优选至少40%,更优选至少50%,例如至少60%、至少70%、至少80%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%。
优选地,所述生产菌株中的谷氨酸激酶(ProB,Glutamate-5-kinase)不受L-脯氨酸反馈抑制或L-脯氨酸反馈抑制减弱。
优选地,所述生产菌株选自谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌ATCC21799、谷氨酸棒杆菌WM001。
进一步优选地,所述的生产细菌中谷氨酸激酶(ProB,Glutamate-5-kinase)和/或谷氨酸半醛脱氢酶(ProA,Glutamate-semialdehyde dehydrogenase)的活性增强。
进一步优选地,所述膜转运蛋白为氨基酸序列如SEQ ID NO:1和/或SEQ ID NO:2所示。
优选地,所述生产菌株中,其中编码所述膜转运蛋白的基因选自以下DNA:
(A)核苷酸序列如SEQ ID NO:3和/或SEQ ID NO:4所示;或
(B)与(A)所示核苷酸序列具有98%以上同源性,且与(A)所述DNA编码的蛋白具有相同活性。
进一步优选地,编码所述膜转运蛋白的基因的核苷酸序列如SEQ ID NO:3和/或SEQ ID NO:4所示。
本文所用的术语“未改性的”、“野生型的”、“内源性的”、“天然存在的”指在自然界中可以找到的对象。例如,一种存在于生物体中,可以从自然界的一个来源中分离出来并且在实验室中没有被人类有意修改的多肽或多核苷酸序列是天然存在的。
本发明所述“修饰”是指任何对野生型菌株或亲本菌株进行的遗传操作,包括但不限于各种分子生物学手段。
在本发明的第三方面,提供一种L-脯氨酸的制备方法,所述方法包括:培养前面所述的生产菌株,使之生产L-脯氨酸。
在另一优选例中,所述方法还包括从发酵液中分离L-脯氨酸的步骤。
在本发明的第四方面,提供一种L-脯氨酸生产菌株的构建方法,所述方法包括通过遗传修饰,使得菌株中膜转运蛋白相对于未改性的蛋白活性减弱或完全去除,其中所述膜转运蛋白选自:
(A)氨基酸序列如SEQ ID NO:1和/或SEQ ID NO:2所示;或
(B)与(A)所述蛋白氨基酸序列具有98%以上同源性,且与(A)所述蛋白具有相同活性。
优选地,上述构建方法中,位于菌株染色体上编码所述膜转运蛋白的基因经突变和/或其表达控制序列经突变使所述膜转运蛋白被去除或使膜转运蛋白相对于未改性的蛋白活性减弱。
进一步优选地,上述构建方法中,所述菌株中的膜转运蛋白相对于未改性的蛋白活性减弱,是指所述膜转运蛋白的编码基因的转录、表达降低至少30%,优选至少40%,更优选至少50%,例如至少60%、至少70%、至少80%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%;或者所述膜转运蛋白的活性降低至少30%、优选至少40%,更优选至少50%,例如至少60%、至少70%、至少80%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%。
优选地,上述构建方法中,所述生产菌株中的谷氨酸激酶(ProB,Glutamate-5-kinase)不受L-脯氨酸反馈抑制或L-脯氨酸反馈抑制减弱。
优选地,上述构建方法中,所述生产菌株中的谷氨酸激酶(ProB,Glutamate-5-kinase)和/或谷氨酸半醛脱氢酶(ProA,Glutamate-semialdehyde dehydrogenase)的活性增强。
进一步优选地,上述构建方法中,所述生产菌株选自谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌ATCC21799、谷氨酸棒杆菌WM001。
进一步优选地,上述构建方法中,所述膜转运蛋白为氨基酸序列如SEQ ID NO:1和/或SEQ ID NO:2所示。
优选地,上述构建方法中,其中编码所述膜转运蛋白的基因选自以下DNA:
(A)核苷酸序列如SEQ ID NO:3和/或SEQ ID NO:4所示;或
(B)与(A)所示核苷酸序列具有98%以上同源性,且与(A)所述DNA编码的蛋白具有相同活性。
进一步优选地,编码所述膜转运蛋白的基因的核苷酸序列如SEQ ID NO:3和/或SEQ ID NO:4所示。
本发明中术语“活性减弱或完全去除”可以通过修饰来实现,包括但不限于通过删除部分或全部编码基因、基因阅读框移码突变、弱化转录或翻译强度、或对基因的编码序列进行突变、或使用编码具有较低活性的相应蛋白质的基因或等位基因,或使对应酶或蛋白失去活性,及任选地组合使用这些方法。采用合适的培养方法或基因表达的信号结构的遗传修饰(突变)可以实现基因表达的降低,例如基因表达的信号结构是阻遏基因,活性基因,操纵基因,启动子,弱化子,核糖体结合位点,起始密码子和终止子。
本发明中术语“遗传修饰”可以通过以下方法之一或组合实现:部分敲除或完全敲除多肽的编码基因;编码基因突变,如移码突变、错义突变、缺失、起始密码子改变等;改变编码基因的启动子、表达调控序列或编码区密码子令其转录或翻译弱化;改变编码基因序列使其mRNA稳定性减弱或编码的蛋白的结构不稳定;或其他任何通过修饰基因编码区及其临近的上下游区域使其失活的方式等。
本发明的术语“表达控制序列”,即调控基因表达的核苷酸序列,是指能提高或降低个体中特定基因的表达的片段,包括但不限于启动子、转录因子结合位点、核糖体结合位点、调控中转录和翻译终止的序列等。
在本发明的第五方面,提供上述L-脯氨酸生产菌株或上述方法构建的L-脯氨酸生产菌株在生产L-脯氨酸中的应用。
本发明的有益效果
1、本发明经过筛选发现两种膜转运蛋白的失活可以提高谷氨酸棒杆菌ATCC13869菌株基础上构建的L-脯氨酸生产菌SZCgP1的L-脯氨酸的产量,提高幅度可达2.1倍以上。因此,在实践上可用于细菌发酵生产L-脯氨酸,便于推广应用,具备重要的工业应用价值。
2、本发明经过筛选发现两种膜转运蛋白的失活有助于菌株L-脯氨酸的积累,本发明的发现可以为本领域技术人员构建L-脯氨酸生产菌株提供新的思路和靶点,即不仅仅关注于L-脯氨酸合成途径的改造,而是通过膜转运蛋白的失活来提高菌株的产量,为构建更高产的L-脯氨酸生产菌株提供借鉴。
附图说明
图1.pdCas9gRNA-ccdB质粒图谱。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解,下列实施例仅为示例性地说明和解释本发明,而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明,以下实施例中使用的原料和试剂均为市售商品,或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
定义与说明:
本文所用的术语“蛋白”、“多肽”和“肽”可互换使用,并具有本领域普通技术人员通常理解的含义。在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的,它可以包含修饰的氨基酸,并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如,二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作,如以标记组分缀合)的氨基酸聚合物。
本文中的术语“宿主细胞”或“生产菌株”是具有本领域普通技术人员通常理解的含义,即,本发明所述的膜转运蛋白中的一种或两种经修饰后基因被去除或使膜转运蛋白相对于未改性的蛋白活性减弱,且能够生产L-脯氨酸的细胞。换言之,本发明可以利用任何宿主细胞,只要所述细胞中的本发明所述的膜转运蛋白中的一种或两种经修饰后基因被去除或使膜转运蛋白相对于未改性的蛋白活性减弱,且能够生产L-脯氨酸的细胞。例如,适用于本发明的宿主细胞包括但不限于埃希氏菌属、棒杆菌属,优选大肠杆菌或谷氨酸棒杆菌,更优选谷氨酸棒杆菌,更具体的是谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌ATCC21799、谷氨酸棒杆菌WM001。
本文中的术语“转化”具有本领域技术人员普遍理解的意思,即将外源性的DNA导入宿主的过程。所述转化的方法包括任何将核酸导入细胞的方法,这些方法包括但不限于电穿孔法、磷酸钙沉淀法、氯化钙(CaCl2)沉淀法、微注射法、聚乙二醇(PEG)法、DEAE-葡聚糖法、阳离子脂质体法以及乙酸锂-DMSO法。
本文的宿主细胞的培养可以根据本领域的常规方法进行,包括但不限于孔板培养、摇瓶培养、批次培养、连续培养和分批补料培养等,并可以根据实际情况适当地调整各种培养条件如温度、时间和培养基的pH值等。
本文所用的术语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的,并不排除额外的、未引述的元件或方法步骤。
本文所用的“约”表示:一个值包括测定该值所使用的装置或方法的误差的标准偏差。
本文所用的“或”的定义仅为替代物以及“和/或”,但除非明确表示仅为替代物或替代物之间相互排斥外,权利要求中的术语“或”是指“和/或”。
本文所用的选择/可选/优选的“数值范围”既包括范围两端的数值端点,也包括相对于前述数值端点而言,所述数值端点中间所覆盖的所有自然数。
本文所用的术语“未改性的”、“野生型的”、“内源性的”、“天然存在的”指在自然界中可以找到的对象。例如,一种存在于生物体中,可以从自然界的一个来源中分离出来并且在实验室中没有被人类有意修改的多肽或多核苷酸序列是天然存在的。
本文所用的术语“氨基酸突变”或“核苷酸突变”,包括“取代、重复、缺失或添加一个或多个氨基酸或核苷酸”。在本发明中,术语“突变”是指核苷酸序列或者氨基酸序列的改变。
本文所用的术语“自然状态”是指微生物中多肽处于未修饰状态的活性,即自然状态下的活性。
本领域技术人员知晓,为提升活性而对野生型多肽进行突变,找到能实现所需目的的位点更为重要。因此,基于本发明的教导,本领域技术人员会对序列1或2所示氨基酸序列进行突变,并检测突变体的相关活性。此外,本领域普通技术人员也不难知晓,在多肽的某些区域,例如非重要区域改变少数氨基酸残基基本上不会改变生物活性,例如,适当替换某些氨基酸得到的序列并不会影响其活性(可参见Watson等,Molecular Biology of TheGene,第四版,1987,The Benjamin/Cummings Pub.Co.P224)。因此,本领域普通技术人员能够实施这种替换并且确保所得分子仍具有所需生物活性。
因此,对本发明的膜转运蛋白作进一步突变而得到仍具备相应功能和活性的进一步突变体是显而易见的。例如,本领域技术人员公知在多肽的任一端增加或减少数个氨基酸残基,例如优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基不会影响得到的突变体的功能。因此,本发明应包括在本发明基础上得到的保守性突变。
术语“保守突变”是指可正常维持蛋白质的功能的突变。保守突变的代表性例子为保守置换。
如本公开所使用的,术语“保守置换”涉及用具有类似侧链的氨基酸残基替换氨基酸残基。本领域已经定义了具有类似侧链的氨基酸残基家族,并且包括具有碱性侧链(例如赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸和谷氨酸)、不带电极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、和半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸和色氨酸)、β-支链(例如苏氨酸、缬氨酸和异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸和组氨酸)。
如本公开所使用的,“保守置换”通常在蛋白质的一个或多个位点上交换一种氨基酸。这种取代可以是保守的。作为被视作保守置换的置换,示例性的,可以举出Ala向Ser或Thr的置换、Arg向Gln、His或Lys的置换、Asn向Glu、Gln、Lys、His或Asp的置换、Asp向Asn、Glu或Gln的置换、Cys向Ser或Ala的置换、Gln向Asn、Glu、Lys、His、Asp或Arg的置换、Glu向Gly、Asn、Gln、Lys或Asp的置换、Gly向Pro的置换、His向Asn、Lys、Gln、Arg或Tyr的置换、Ile向Leu、Met、Val或Phe的置换、Leu向Ile、Met、Val或Phe的置换、Lys向Asn、Glu、Gln、His或Arg的置换、Met向Ile、Leu、Val或Phe的置换、Phe向Trp、Tyr、Met、Ile或Leu的置换、Ser向Thr或Ala的置换、Thr向Ser或Ala的置换、Trp向Phe或Tyr的置换、Tyr向His、Phe或Trp的置换、及Val向Met、Ile或Leu的置换。此外,保守突变还包括起因于基因所来源的个体差异、株、种的差异等天然产生的突变。
在具体的实施方式中,所述同源性或序列相同性可以是98%以上。本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于:计算机分子生物学(Computational Molecular Biology),Lesk,A.M.编,牛津大学出版社,纽约,1988;生物计算:信息学和基因组项目(Biocomputing:Informatics and Genome Projects),Smith,D.W.编,学术出版社,纽约,1993;序列数据的计算机分析(Computer Analysis ofSequence Data),第一部分,Griffin,A.M.和Griffin,H.G.编,Humana Press,新泽西,1994;分子生物学中的序列分析(Sequence Analysis in Molecular Biology),vonHeinje,G.,学术出版社,1987和序列分析引物(Sequence Analysis Primer),Gribskov,M.与Devereux,J.编M Stockton Press,纽约,1991和Carillo,H.与Lipman,D.,SIAMJ.Applied Math.,48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于:GCG程序包(Devereux,J.等,1984)、BLASTP、BLASTN和FASTA(Altschul,S,F.等,1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册,Altschul,S.等,NCBI NLM NIH Bethesda,Md.20894;Altschul,S.等,1990)。熟知的Smith Waterman算法也可用于测定相同性。
除非另外定义或由背景清楚指示,否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
实施例1.在谷氨酸棒杆菌中筛选表达弱化可以提高L-脯氨酸产量的膜转运蛋白
本发明通过全基因组规模膜转运蛋白基因的弱化库,筛选基因表达弱化后可以提高谷氨酸棒杆菌L-脯氨酸产量的膜转运蛋白。将pCas9(LIU,Jiao,et al.Development ofa CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum.Microbialcell factories,2017,16.1:205.)质粒的Cas9基因进行D10A和H840A突变,同时将质粒骨架中的Bsa I酶切位点去除,获得pdCas9质粒;再从pnCas9(D10A)-AID-gRNA-ccdBTS(WANG,Yu,et al.Expanding targeting scope,editing window,and base transitioncapability of base editing in Corynebacterium glutamicum.Biotechnology andbioengineering,2019,116:3016-3029)质粒上扩增gRNA-ccdB表达盒克隆至pdCas9的相同位置,获得可以高效构建的CRISPRi质粒pdCas9gRNA-ccdB,质粒序列如SEQ ID NO:5所示,质粒图谱如图1所示。该系统的弱化效率可以达到96%。基于以上巧妙设计,弱化目标基因时只需要设计20bp的sgRNA靶DNA结合区,再合成2条24bp互补引物,退火成双链并通过Golden Gate克隆构建目标基因的弱化质粒。本发明同时构建了没有20bp定位序列的对照质粒pdCas9gRNA-control。将谷氨酸棒杆菌ATCC13869全基因组规模的蛋白质序列在TransportDB数据库进行膜转运蛋白预测,获得全基因组规模的397个膜转运蛋白;再基于CRISPRi系统构建397个膜转运蛋白基因的弱化质粒,将弱化库质粒和对照质粒分别导入一个脯氨酸生产菌SZCgP1(谷氨酸棒杆菌ATCC13869引入proB基因的G149D突变,密码子从GGT突变为GAT,具体参见CN101084312A),获得弱化库菌株;最后通过96孔板发酵评价所有菌株的L-脯氨酸产量。基于以上筛选,本发明发现2个功能没有明确实验验证和功能注释的蛋白WP_060563805.1(其氨基酸序列如SEQ ID NO:1所示,基因序列如SEQ ID NO:3所示)、WP_060563806.1(其氨基酸序列如SEQ ID NO:2所示,基因序列如SEQ ID NO:4所示)的编码基因表达弱化后,L-脯氨酸产量明显提高。构建编码WP_060563805.1、WP_060563806.1基因的CRISPRi质粒的引物分别为060563805.1-F/R、060563806.1-F/R(表1),构建的质粒分别命名为pdCas9gRNA1、pdCas9gRNA2。以上质粒及对照质粒分别转化SZCgP1菌株,获得弱化菌株SZCgP1(pdCas9gRNA1)、SZCgP1(pdCas9gRNA2)和对照菌株SZCgP1(pdCas9gRNA-control)。
表1
引物 | 核苷酸序列 | SEQ ID NO. |
060563805.1-F | TTCAGAGATGTAGTCGAAGAACGA | 6 |
060563805.1-R | AAACTCGTTCTTCGACTACATCTC | 7 |
060563806.1-F | TTCACTTCTCGTTACCCGCAATGA | 8 |
060563806.1-R | AAACTCATTGCGGGTAACGAGAAG | 9 |
为了进一步确认WP_060563805.1、WP_060563806.1的编码基因的弱化对L-脯氨酸生产的影响,采用24孔深孔板进行发酵评价。种子培养基成份为(g/L):葡萄糖,5g/L;酵母粉,1g/L;大豆蛋白胨,3g/L;尿素,3g/L;丁二酸,0.5g/L;K2HPO4·3H2O,1g/L;MgSO4·7H2O,0.1g/L;生物素,0.01mg/L;维生素B1,0.1mg/L;MOPS,20g/L;初始pH7.2。发酵培养基成份为:葡萄糖,80g/L;酵母粉,1g/L;大豆蛋白胨,1g/L;NaCl,1g/L;硫酸铵,1g/L;尿素,6g/L;K2HPO4·3H2O,1g/L;MgSO4·7H2O,0.45g/L;FeSO4·7H2O,0.05g/L;生物素,0.4mg/L;维生素B1,0.1mg/L;MOPS,40g/L;初始pH7.2。首先将菌株接种到含有15μg/ml氯霉素的种子培养基中培养8h,培养物作为种子接种到含有15μg/ml氯霉素和0.03mM IPTG发酵培养基的24孔板中,培养基装液量为800μl,接种量控制在初始OD600为0.03(酶标仪检测),30℃培养21h,孔板摇床转速为800rpm,每个菌株3个平行,发酵结束后检测OD600和L-脯氨酸产量。L-脯氨酸的检测方法:用3%(W/V)磺基水杨酸稀释到合适浓度;取1mL稀释液,加入1mL酸合茚三酮(1.25g茚三酮溶于30mL冰醋酸和20mL 6M H3PO4中,70℃加热溶解)和1mL冰醋酸,100℃沸水浴反应45min;冷却后测定OD520。采用0-100mg/L浓度的L-脯氨酸绘制标准曲线,根据标准曲线计算待测样品的浓度。结果如表2所示,WP_060563805.1、WP_060563806.1的编码基因的弱化分别提高L-脯氨酸产量74%、77%。
表2膜转运蛋白基因表达弱化对L-脯氨酸产量的影响
实施例2.膜转运蛋白基因敲除用于提高L-脯氨酸产量
(1)谷氨酸棒杆菌膜转运蛋白基因敲除的重组载体构建
根据已报道的谷氨酸棒杆菌ATCC13869基因组序列,分别以ATCC13869基因组为模板,以060563805.1-UF/060563805.1-UR和060563805.1-DF/060563805.1-DR,060563806.1-UF/060563806.1-UR和060563806.1-DF10/060563806.1-DR为引物,PCR扩增WP_060563805.1、WP_060563806.1编码基因敲除的上下游同源臂;同时以pK18-1/2引物扩增pK18mobsacB(GenBank:FJ437239.1)的质粒骨架。上述对应的3个PCR片段回收后,通过诺唯赞的一步重组试剂盒克隆连接,分别获得WP_060563805.1和WP_060563806.1编码基因敲除的重组载体pK18-1和pK18-2。以上所用引物序列如表3所示。
表3
(2)谷氨酸棒杆菌膜转运蛋白基因敲除突变体的构建
将上述构建的重组载体pK181和pK18-2分别转化谷氨酸棒杆菌L-脯氨酸生产菌SZCgP1,涂布含有5g/L葡萄糖和25μg/mL卡那霉素的LBHIS固体培养基上,30℃培养获得第一次重组的转化子。正确的一次重组转化子分别接种含有5g/L葡萄糖的LB培养基,过夜培养,分别稀释涂布添加100g/L蔗糖的LB固体培养基平板进行筛选,分别获得WP_060563805.1和WP_060563806.1编码基因敲除的菌株SZCgP4和SZCgP5。
(3)谷氨酸棒杆菌膜转运蛋白基因敲除突变体的L-脯氨酸生产能力评价
为了测试谷氨酸棒杆菌中膜转运蛋白基因敲除对菌株产L-脯氨酸的影响,分别对SZCgP1、SZCgP4和SZCgP5菌株进行发酵测试。种子培养基成份为(g/L):葡萄糖,5g/L;酵母粉,1g/L;大豆蛋白胨,3g/L;尿素,3g/L;丁二酸,0.5g/L;K2HPO4·3H2O,1g/L;MgSO4·7H2O,0.1g/L;生物素,0.01mg/L;维生素B1,0.1mg/L;MOPS,20g/L;初始pH7.2。发酵培养基成份为:葡萄糖,80g/L;酵母粉,1g/L;大豆蛋白胨,1g/L;NaCl,1g/L;硫酸铵,1g/L;尿素,6g/L;K2HPO4·3H2O,1g/L;MgSO4·7H2O,0.45g/L;FeSO4·7H2O,0.05g/L;生物素,0.4mg/L;维生素B1,0.1mg/L;MOPS,40g/L;初始pH7.2。首先将菌株接种到种子培养基中培养8h,培养物作为种子接种到每孔含有800μl发酵培养基的24孔板中,初始OD600控制约为0.03(酶标仪检测),30℃培养21h,孔板摇床转速为800rpm,每个菌株3个平行,发酵结束后检测L-脯氨酸产量,方法与实施例1相同。结果如表4所示,WP_060563805.1和WP_060563806.1编码基因敲除均可以提高L-脯氨酸产量,WP_060563805.1编码基因敲除L-脯氨酸产量提高106%,WP_060563806.1编码基因敲除L-脯氨酸产量提高109%。以上结果表明敲除WP_060563805.1和WP_060563806.1的编码基因可应用于L-脯氨酸生产。
表4膜转运蛋白基因敲除对L-脯氨酸产量的影响
菌株 | OD600 | L-脯氨酸产量(g/L) |
SZCgP1 | 14.17±2.14 | 4.95±0.59 |
SZCgP4 | 12.96±0.32 | 10.71±1.90 |
SZCgP5 | 12.75±0.47 | 10.79±2.35 |
以上,对本发明的实施方式进行了说明。但是,本发明不限定于上述实施方式。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 生产L-脯氨酸的菌株及其构建方法和应用
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 294
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 1
Met Thr Ala Ala Asp Glu Ser Gly Val Lys Glu Asp Gln Thr Leu Leu
1 5 10 15
Val Asp Asp Thr His Leu Arg Ala Leu Trp Ser Arg Pro Ser Phe Phe
20 25 30
Asp Tyr Ile Ser Gln Leu Trp Arg Arg Arg His Phe Ile Gln Ala Glu
35 40 45
Ala Lys Ser Lys Ser Leu Gly Thr Gly Arg Gly Thr Phe Leu Gly Lys
50 55 60
Leu Trp Ile Ile Leu Asp Pro Leu Leu Gln Val Ala Val Tyr Ala Val
65 70 75 80
Ile Phe Gly Leu Ile Leu Lys Val Asp Arg Gly Ile Asp Asn Phe Ile
85 90 95
Gly Phe Leu Val Ile Gly Val Ile Tyr Phe Gly Phe Leu Thr Ser Gly
100 105 110
Leu Ser Ala Gly Ser Gly Leu Ile Gln Asn Ser Lys Asn Met Ile Ser
115 120 125
Ser Phe Ser Phe Pro Arg Ala Ser Leu Ala Phe Ser Val Thr Leu Arg
130 135 140
Asn Met Ile Asp Asn Leu Ala Pro Ala Leu Val Ala Val Ile Leu Ala
145 150 155 160
Leu Ala Thr Gln Tyr Tyr Gln Ala Pro Ser Trp Thr Val Met Leu Val
165 170 175
Val Pro Leu Phe Leu Val Leu His Val Phe Thr Leu Gly Leu Thr Leu
180 185 190
Ile Val Ala Arg Ile Thr Ala Phe Ile Pro Asp Ala Lys Pro Leu Val
195 200 205
Ala Leu Leu Gln Arg Ala Leu Phe Phe Val Ser Gly Ile Phe Phe Ser
210 215 220
Val Glu Arg Phe Asn Asp Gln Pro Leu Ile Gln Glu Ile Met Leu Ala
225 230 235 240
Asn Pro Phe Tyr Lys Phe Leu Ser Ala Phe Arg Ile Cys Val Leu His
245 250 255
Gly Glu Val Pro Pro Leu Asp Leu Trp Ile Ala Leu Val Ile Trp Ser
260 265 270
Gly Gly Thr Val Val Val Gly Ile Leu Phe Phe Trp Gln Ala Glu Ala
275 280 285
Arg Tyr Ser Gly Val Arg
290
<210> 2
<211> 247
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 2
Met Val Glu Ala Leu Lys Pro Met Ser Leu Ala Cys Phe Thr Gly Glu
1 5 10 15
Ser Ile Gly Ile Ile Gly Arg Asn Gly Ser Gly Lys Ser Thr Leu Leu
20 25 30
Ser Leu Ile Ala Gly Asn Glu Lys Pro Thr Ala Gly Glu Val Phe Val
35 40 45
Ser Ser His Pro Thr Leu Leu Ser Val Ser Ala Ala Leu Gln Pro His
50 55 60
Leu Asn Ala Leu Asp Asn Val Arg Leu Gly Leu Leu Ala Lys Gly Ala
65 70 75 80
Ala Pro Ser Leu Val Glu Glu Ile Glu His His Val Val Asp Trp Ala
85 90 95
Asp Ile Gly Asp Ala Ile Asp Arg Pro Leu Lys Thr Tyr Ser Ser Gly
100 105 110
Met Ala Ala Arg Leu Lys Phe Ala Ile Ala Thr Ala Ile Arg Pro Gln
115 120 125
Ile Leu Leu Val Asp Glu Ala Leu Ala Thr Gly Asp Ala Ala Phe Asn
130 135 140
Glu Lys Ala Gln Asp Arg Met Asn Ser Phe Leu Glu Lys Asp Gly Thr
145 150 155 160
Val Leu Val Val Ser His Ser Ala Gly Thr Ile Gln Gln His Cys Ser
165 170 175
Arg Ala Ile Trp Ile His Glu Gly Glu Val Ile Ala Asp Gly Pro Thr
180 185 190
Asp Asp Val Thr Tyr Leu Tyr Ser Gly Trp Ser Arg His Ile Ser Asn
195 200 205
Arg Asp Arg Ile Ser Ala Ala Lys Met Ile Arg Arg Ala Gln Lys Ser
210 215 220
Tyr Tyr Pro Thr Lys Ile Leu Phe Asp Ser Glu Ala Thr Ala Met Leu
225 230 235 240
Asp Lys Gly Met Asp Ser His
245
<210> 3
<211> 885
<212> DNA
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 3
atgacagctg ctgacgagag tggagtaaaa gaagatcaaa ctcttctggt agacgacacc 60
cacctgcgag cgttgtggag tcgtccgtcg ttcttcgact acatctcaca gttgtggcga 120
cgtcgccact ttattcaagc tgaggccaaa agcaaatctc ttggtaccgg tcgaggtaca 180
tttcttggaa agctatggat tattctcgat cctctccttc aggttgccgt gtatgcggtt 240
atttttggat taattttgaa agtggaccga gggatagata atttcatcgg tttcttggtc 300
attggtgtga tttatttcgg atttctaacc agtggactct cagccggtag cgggttaatc 360
caaaactcga agaatatgat ttcatccttt tcgttcccac gagcctcgct ggccttctcc 420
gtaactttac gcaatatgat tgataatctt gcgccagcat tggtggctgt aattttggcg 480
ttggctacac agtattacca agctccaagt tggacagtga tgctggtagt acctcttttc 540
ttagttctac atgttttcac tcttggattg accttgatcg tggcgagaat aactgctttc 600
attcccgatg cgaaaccttt ggttgccctg ttgcagagag ccttgttttt tgtatcggga 660
atattcttct cggtggaaag attcaacgat caacctttaa tccaagagat aatgctagct 720
aatcctttct acaaatttct cagtgctttt agaatctgtg tcctgcacgg tgaggtgccc 780
ccactggatt tatggatcgc tttagtaatc tggtctggtg gaactgttgt tgtaggtatt 840
ctgttcttct ggcaggcgga agcaaggtat tcaggtgtta ggtaa 913
<210> 4
<211> 744
<212> DNA
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 4
atggttgaag ccttaaagcc gatgtctcta gcatgtttta ccggagagtc tattggcatt 60
attgggcgta atggttctgg taaatccaca ttgctatccc tcattgcggg taacgagaag 120
ccaacagcag gcgaggtttt tgtatcctca catcccacat tgcttagtgt ttctgcagca 180
ctccagcccc atctgaatgc ccttgataat gtgcgtctgg ggctcttggc taagggggca 240
gcacctagct tggtcgaaga gattgaacat catgttgtgg attgggcaga cataggggac 300
gccatagacc gcccattgaa aacctattcg tccggaatgg cggctcgttt aaagtttgcc 360
atcgctactg caatcagacc tcaaatacta ttggtagatg aagcgctggc tacaggggat 420
gcggcattca acgaaaaggc tcaagatcga atgaactctt ttcttgaaaa ggacggtacc 480
gtattagtag tttcgcacag tgcggggact atccagcaac attgctctcg tgcgatttgg 540
atccatgagg gtgaagtaat cgcagatggt cctacagacg acgtcacata tttatatagt 600
ggctggagtc gacatatttc taacagagat cgaattagcg cagcaaagat gatccgaaga 660
gcacaaaaat cttactatcc gacaaaaatt ctttttgatt ctgaggctac agcgatgctg 720
gataaaggga tggatagcca ttag 768
<210> 5
<211> 11773
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aattaagctt aaaggagttg agaatggata agaaatactc aataggctta gctatcggca 60
caaatagcgt cggatgggcg gtgatcactg atgaatataa ggttccgtct aaaaagttca 120
aggttctggg aaatacagac cgccacagta tcaaaaaaaa tcttataggg gctcttttat 180
ttgacagtgg agagacagcg gaagcgactc gtctcaaacg gacagctcgt agaaggtata 240
cacgtcggaa gaatcgtatt tgttatctac aggagatttt ttcaaatgag atggcgaaag 300
tagatgatag tttctttcat cgacttgaag agtctttttt ggtggaagaa gacaagaagc 360
atgaacgtca tcctattttt ggaaatatag tagatgaagt tgcttatcat gagaaatatc 420
caactatcta tcatctgcga aaaaaattgg tagattctac tgataaagcg gatttgcgct 480
taatctattt ggccttagcg catatgatta agtttcgtgg tcattttttg attgagggag 540
atttaaatcc tgataatagt gatgtggaca aactatttat ccagttggta caaacctaca 600
atcaattatt tgaagaaaac cctattaacg caagtggagt agatgctaaa gcgattcttt 660
ctgcacgatt gagtaaatca agacgattag aaaatctcat tgctcagctc cccggtgaga 720
agaaaaatgg cttatttggg aatctcattg ctttgtcatt gggtttgacc cctaatttta 780
aatcaaattt tgatttggca gaagatgcta aattacagct ttcaaaagat acttacgatg 840
atgatttaga taatttattg gcgcaaattg gagatcaata tgctgatttg tttttggcag 900
ctaagaattt atcagatgct attttacttt cagatatcct aagagtaaat actgaaataa 960
ctaaggctcc cctatcagct tcaatgatta aacgctacga tgaacatcat caagacttga 1020
ctcttttaaa agctttagtt cgacaacaac ttccagaaaa gtataaagaa atcttttttg 1080
atcaatcaaa aaacggatat gcaggttata ttgatggggg agctagccaa gaagaatttt 1140
ataaatttat caaaccaatt ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac 1200
taaatcgtga agatttgctg cgcaagcaac ggacctttga caacggctct attccccatc 1260
aaattcactt gggtgagctg catgctattt tgagaagaca agaagacttt tatccatttt 1320
taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct tattatgttg 1380
gtccattggc gcgtggcaat agtcgttttg catggatgac tcggaagtct gaagaaacaa 1440
ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg 1500
aacgcatgac aaactttgat aaaaatcttc caaatgaaaa agtactacca aaacatagtt 1560
tgctttatga gtattttacg gtttataacg aattgacaaa ggtcaaatat gttactgaag 1620
gaatgcgaaa accagcattt ctttcaggtg aacagaagaa agccattgtt gatttactct 1680
tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga agattatttc aaaaaaatag 1740
aatgttttga tagtgttgaa atttcaggag ttgaagatag atttaatgct tcattaggta 1800
cctaccatga tttgctaaaa attattaaag ataaagattt tttggataat gaagaaaatg 1860
aagatatctt agaggatatt gttttaacat tgaccttatt tgaagatagg gagatgattg 1920
aggaaagact taaaacatat gctcacctct ttgatgataa ggtgatgaaa cagcttaaac 1980
gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt gattaatggt attagggata 2040
agcaatctgg caaaacaata ttagattttt tgaaatcaga tggttttgcc aatcgcaatt 2100
ttatgcagct gatccatgat gatagtttga catttaaaga agacattcaa aaagcacaag 2160
tgtctggaca aggcgatagt ttacatgaac atattgcaaa tttagctggt agccctgcta 2220
ttaaaaaagg tattttacag actgtaaaag ttgttgatga attggtcaaa gtaatggggc 2280
ggcataagcc agaaaatatc gttattgaaa tggcacgtga aaatcagaca actcaaaagg 2340
gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa 2400
gtcagattct taaagagcat cctgttgaaa atactcaatt gcaaaatgaa aagctctatc 2460
tctattatct ccaaaatgga agagacatgt atgtggacca agaattagat attaatcgtt 2520
taagtgatta tgatgtcgat gccattgttc cacaaagttt ccttaaagac gattcaatag 2580
acaataaggt cttaacgcgt tctgataaaa atcgtggtaa atcggataac gttccaagtg 2640
aagaagtagt caaaaagatg aaaaactatt ggagacaact tctaaacgcc aagttaatca 2700
ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata 2760
aagctggttt tatcaaacgc caattggttg aaactcgcca aatcactaag catgtggcac 2820
aaattttgga tagtcgcatg aatactaaat acgatgaaaa tgataaactt attcgagagg 2880
ttaaagtgat taccttaaaa tctaaattag tttctgactt ccgaaaagat ttccaattct 2940
ataaagtacg tgagattaac aattaccatc atgcccatga tgcgtatcta aatgccgtcg 3000
ttggaactgc tttgattaag aaatatccaa aacttgaatc ggagtttgtc tatggtgatt 3060
ataaagttta tgatgttcgt aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa 3120
ccgcaaaata tttcttttac tctaatatca tgaacttctt caaaacagaa attacacttg 3180
caaatggaga gattcgcaaa cgccctctaa tcgaaactaa tggggaaact ggagaaattg 3240
tctgggataa agggcgagat tttgccacag tgcgcaaagt attgtccatg ccccaagtca 3300
atattgtcaa gaaaacagaa gtacagacag gcggattctc caaggagtca attttaccaa 3360
aaagaaattc ggacaagctt attgctcgta aaaaagactg ggatccaaaa aaatatggtg 3420
gttttgatag tccaacggta gcttattcag tcctagtggt tgctaaggtg gaaaaaggga 3480
aatcgaagaa gttaaaatcc gttaaagagt tactagggat cacaattatg gaaagaagtt 3540
cctttgaaaa aaatccgatt gactttttag aagctaaagg atataaggaa gttaaaaaag 3600
acttaatcat taaactacct aaatatagtc tttttgagtt agaaaacggt cgtaaacgga 3660
tgctggctag tgccggagaa ttacaaaaag gaaatgagct ggctctgcca agcaaatatg 3720
tgaatttttt atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg 3780
aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa 3840
tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg 3900
catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat 3960
ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc 4020
gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca 4080
ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgactga gcttggctgt 4140
tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt 4200
ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg 4260
aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctgccca tgcgagagta 4320
gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 4380
tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt 4440
gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag 4500
gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct 4560
tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 4620
aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 4680
ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 4740
aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 4800
acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 4860
ttgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4920
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4980
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5040
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5100
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5160
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5220
tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5280
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5340
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5400
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5460
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 5520
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5580
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5640
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5700
tgagattatc aaaaaggatc ttcacctaga tccttttggg gtgggcgaag aactccagca 5760
tgagatcccc gcgctggagg atcatccagc cattcggggt cgttcactgg ttcccctttc 5820
tgatttctgg catagaagaa cccccgtgaa ctgtgtggtt ccgggggttg ctgatttttg 5880
cgagacttct cgcgcaattc cctagcttag gtgaaaacac catgaaacac tagggaaaca 5940
cccatgaaac acccattagg gcagtagggc ggcttcttcg tctagggctt gcatttgggc 6000
ggtgatctgg tctttagcgt gtgaaagtgt gtcgtaggtg gcgtgctcaa tgcactcgaa 6060
cgtcacgtca tttaccgggt cacggtgggc aaagagaact agtgggttag acattgtttt 6120
cctcgttgtc ggtggtggtg agcttttcta gccgctcggt aaacgcggcg atcatgaact 6180
cttggaggtt ttcaccgttc tgcatgcctg cgcgcttcat gtcctcacgt agtgccaaag 6240
gaacgcgtgc ggtgaccacg acgggcttag cctttgcctg cgcttctagt gcttcgatgg 6300
tggcttgtgc ctgcgcttgc tgcgcctgta gtgcctgttg agcttcttgt agttgctgtt 6360
ctagctgtgc cttggttgcc atgctttaag actctagtag ctttcctgcg atatgtcatg 6420
cgcatgcgta gcaaacattg tcctgcaact cattcattat gtgcagtgct cctgttacta 6480
gtcgtacata ctcatattta cctagtctgc atgcagtgca tgcacatgca gtcatgtcgt 6540
gctaatgtgt aaaacatgta catgcagatt gctgggggtg cagggggcgg agccaccctg 6600
tccatgcggg gtgtggggct tgccccgccg gtacagacag tgagcaccgg ggcacctagt 6660
cgcggatacc ccccctaggt atcggacacg taaccctccc atgtcgatgc aaatctttaa 6720
cattgagtac gggtaagctg gcacgcatag ccaagctagg cggccaccaa acaccactaa 6780
aaattaatag tccctagaca agacaaaccc ccgtgcgagc taccaactca tatgcacggg 6840
ggccacataa cccgaagggg tttcaattga caaccatagc actagctaag acaacgggca 6900
caacacccgc acaaactcgc actgcgcaac cccgcacaac atcgggtcta ggtaacactg 6960
agtaacactg aaatagaagt gaacacctct aaggaaccgc aggtcaatga gggttctaag 7020
gtcactcgcg ctagggcgtg gcgtaggcaa aacgtcatgt acaagatcac caatagtaag 7080
gctctggcgg ggtgccatag gtggcgcagg gacgaagctg ttgcggtgtc ctggtcgtct 7140
aacggtgctt cgcagtttga gggtctgcaa aactctcact ctcgctgggg gtcacctctg 7200
gctgaattgg aagtcatggg cgaacgccgc attgagctgg ctattgctac taagaatcac 7260
ttggcggcgg gtggcgcgct catgatgttt gtgggcactg ttcgacacaa ccgctcacag 7320
tcatttgcgc aggttgaagc gggtattaag actgcgtact cttcgatggt gaaaacatct 7380
cagtggaaga aagaacgtgc acggtacggg gtggagcaca cctatagtga ctatgaggtc 7440
acagactctt gggcgaacgg ttggcacttg caccgcaaca tgctgttgtt cttggatcgt 7500
ccactgtctg acgatgaact caaggcgttt gaggattcca tgttttcccg ctggtctgct 7560
ggtgtggtta aggccggtat ggacgcgcca ctgcgtgagc acggggtcaa acttgatcag 7620
gtgtctacct ggggtggaga cgctgcgaaa atggcaacct acctcgctaa gggcatgtct 7680
caggaactga ctggctccgc tactaaaacc gcgtctaagg ggtcgtacac gccgtttcag 7740
atgttggata tgttggccga tcaaagcgac gccggcgagg atatggacgc tgttttggtg 7800
gctcggtggc gtgagtatga ggttggttct aaaaacctgc gttcgtcctg gtcacgtggg 7860
gctaagcgtg ctttgggcat tgattacata gacgctgatg tacgtcgtga aatggaagaa 7920
gaactgtaca agctcgccgg tctggaagca ccggaacggg tcgaatcaac ccgcgttgct 7980
gttgctttgg tgaagcccga tgattggaaa ctgattcagt ctgatttcgc ggttaggcag 8040
tacgttctcg attgcgtgga taaggctaag gacgtggccg ctgcgcaacg tgtcgctaat 8100
gaggtgctgg caagtctggg tgtggattcc accccgtgca tgatcgttat ggatgatgtg 8160
gacttggacg cggttctgcc tactcatggg gacgctacta agcgtgatct gaatgcggcg 8220
gtgttcgcgg gtaatgagca gactattctt cgcacccact aaaagcggca taaaccccgt 8280
tcgatatttt gtgcgatgaa tttatggtca atgtcgcggg ggcaaactat gatgggtctt 8340
gttgttggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg cggcggtgga 8400
atttttctcc acataagctg gcaatgttgc gacgcaacag gtacagtgta attcatgaga 8460
ccacgcgtgg atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt 8520
tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc 8580
tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata 8640
tatgatgtca atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg 8700
cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt 8760
gaaatgaacg gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta 8820
cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga 8880
cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt 8940
ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac 9000
cgatatggcc agtgtgccgg tatccgttat cggggaagaa gtggctgatc tcagccaccg 9060
cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataag gtctcagttt 9120
tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca 9180
ccgagtcggt gctttttttc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg 9240
gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg 9300
ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca 9360
taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt 9420
ctacaaactc tttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt 9480
aattccgcta gatgacgtgc ggcttcgaaa atctcgtgat ggcaggttgg gcgtcgcttg 9540
gtcggtcatt tcgaagggca ccaataactg ccttaaaaaa attacgcccc gccctgccac 9600
tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacagacg 9660
gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg 9720
cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg 9780
gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg 9840
aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc 9900
cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa 9960
acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata 10020
cggaactccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac 10080
ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg 10140
ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg 10200
gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct 10260
gaaaatctcg tcgaagctcg gcggatttgt cctactcaag ctgatccgac aaaatccaca 10320
cattatccca ggtgtccgga tcggtcaaat acgctgccag ctcatagacc gtatccaaag 10380
catccggggc tgatccccgg cgccagggtg gtttttcttt tcaccagtga gacgggcaac 10440
agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgtggttt 10500
gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt 10560
cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg 10620
taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa 10680
cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc 10740
cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca 10800
gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac 10860
ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac 10920
tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag 10980
cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc 11040
gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca 11100
tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt 11160
gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc 11220
ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca 11280
ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct 11340
gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca 11400
ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt 11460
cgatggtgtc aacgtaaatg ccgcttcgcc ttcgcgcgcg aattgcaagc tgatccgggc 11520
ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta 11580
tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct 11640
ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt 11700
tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac 11760
aggaaacaga att 12165
<210> 6
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttcagagatg tagtcgaaga acga 24
<210> 7
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaactcgttc ttcgactaca tctc 24
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ttcacttctc gttacccgca atga 24
<210> 9
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aaactcattg cgggtaacga gaag 24
<210> 10
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caggaaacag ctatgacatg gccagatggg tgcttattc 39
<210> 11
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
caacagttcc accagaccag atgtagtcga agaacgacgg 40
<210> 12
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tggtctggtg gaactgttg 19
<210> 13
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tgtaaaacga cggccagtgc gcagtagcga tggcaaactt 40
<210> 14
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caggaaacag ctatgacatg tggattgacc ttgatcgtgg 40
<210> 15
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gctcttcgga tcatctttgc ccagaaccat tacgcccaa 39
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gcaaagatga tccgaagagc 20
<210> 17
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tgtaaaacga cggccagtgc ggacttgacc ttcgtcagt 39
<210> 18
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gcactggccg tcgttttac 19
<210> 19
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
catgtcatag ctgtttcctg tgtg 24
Claims (11)
1.一种膜转运蛋白在微生物发酵生产L-脯氨酸中的用途,其特征在于,所述蛋白
的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示;所述微生物为谷氨酸棒杆菌,所述谷氨酸棒杆菌以ATCC13869为基础,其谷氨酸激酶ProB不受L-脯氨酸反馈抑制或L-脯氨酸反馈抑制减弱,并且膜转运蛋白的活性减弱或完全去除。
2. 根据权利要求1所述的用途,其特征在于,编码权利要求1所述膜转运蛋白的基因的核苷酸序列如SEQ ID NO:3或SEQ ID NO:4所示。
3. 一种L-脯氨酸的生产菌株,其特征在于,修饰谷氨酸棒杆菌,使得膜转运蛋白相对于未改性的蛋白活性减弱或完全去除,其中所述膜转运蛋白的氨基酸序列如SEQ ID NO:1和/或SEQ ID NO:2所示,所述谷氨酸棒杆菌以ATCC13869为基础,且谷氨酸激酶ProB不受L-脯氨酸反馈抑制或L-脯氨酸反馈抑制减弱。
4.根据权利要求3所述的生产菌株,其特征在于,位于谷氨酸棒杆菌染色体上编码所述膜转运蛋白的基因经突变和/或其表达控制序列经突变使所述膜转运蛋白被去除或使膜转运蛋白相对于未改性的蛋白活性减弱。
5.根据权利要求3或4所述的生产菌株,其特征在于,所述生产菌株中的谷氨酸激酶ProB和/或谷氨酸半醛脱氢酶ProA的活性增强。
6.一种L-脯氨酸的制备方法,其特征在于,所述方法包括:培养权利要求3-5任一项所述的生产菌株,使之生产L-脯氨酸。
7.根据权利要求6所述的制备方法,其特征在于,所述方法还包括从发酵液中分离L-脯氨酸的步骤。
8. 一种L-脯氨酸生产菌株的构建方法,其特征在于,所述方法包括通过遗传修饰,使得谷氨酸棒杆菌中膜转运蛋白相对于未改性的蛋白活性减弱或完全去除,其中所述膜转运蛋白的氨基酸序列如SEQ ID NO:1和/或SEQ ID NO:2所示,所述谷氨酸棒杆菌以ATCC13869为基础,且谷氨酸激酶ProB不受L-脯氨酸反馈抑制或L-脯氨酸反馈抑制减弱。
9.根据权利要求8所述的构建方法,其特征在于,位于谷氨酸棒杆菌染色体上编码所述膜转运蛋白的基因经突变和/或其表达控制序列经突变使所述膜转运蛋白被去除或使膜转运蛋白相对于未改性的蛋白活性减弱。
10.根据权利要求9所述的构建方法,其特征在于,所述生产菌株中的谷氨酸激酶ProB和/或谷氨酸半醛脱氢酶ProA的活性增强。
11.权利要求3-5任一项所述的生产菌株、权利要求8-10任一项所述的构建方法获得的菌株在生产L-脯氨酸中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110161475.9A CN114874958B (zh) | 2021-02-05 | 2021-02-05 | 生产l-脯氨酸的菌株及其构建方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110161475.9A CN114874958B (zh) | 2021-02-05 | 2021-02-05 | 生产l-脯氨酸的菌株及其构建方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114874958A CN114874958A (zh) | 2022-08-09 |
CN114874958B true CN114874958B (zh) | 2024-01-16 |
Family
ID=82667881
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110161475.9A Active CN114874958B (zh) | 2021-02-05 | 2021-02-05 | 生产l-脯氨酸的菌株及其构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114874958B (zh) |
Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1479789A (zh) * | 2000-12-22 | 2004-03-03 | �����ɷ� | 棒状杆菌基因 |
CN1578834A (zh) * | 2001-11-05 | 2005-02-09 | 巴斯福股份公司 | 编码代谢途径蛋白的基因 |
CN102471790A (zh) * | 2009-07-29 | 2012-05-23 | 味之素株式会社 | 产生l-氨基酸的方法 |
CN107603932A (zh) * | 2016-07-12 | 2018-01-19 | 中国科学院微生物研究所 | 一种提高谷氨酸棒杆菌氨基酸产量的方法及其应用 |
CN107674863A (zh) * | 2017-08-04 | 2018-02-09 | 中国科学院天津工业生物技术研究所 | 一种生产反式‑4‑羟基‑l‑脯氨酸的方法 |
CN109402038A (zh) * | 2018-12-13 | 2019-03-01 | 江南大学 | 一种高产l-脯氨酸的重组菌及其构建方法与应用 |
CN111349640A (zh) * | 2018-12-21 | 2020-06-30 | 中国科学院天津工业生物技术研究所 | 反式-4-羟基-l-脯氨酸的生产菌株及其构建方法和应用 |
CN111484997A (zh) * | 2019-01-25 | 2020-08-04 | 中国科学院天津工业生物技术研究所 | 反式-l-羟脯氨酸的生产菌株及其构建方法和应用 |
CN112111469A (zh) * | 2020-11-23 | 2020-12-22 | 中国科学院天津工业生物技术研究所 | γ-谷氨酰激酶突变体及其应用 |
CN112251391A (zh) * | 2019-05-13 | 2021-01-22 | 中国科学院天津工业生物技术研究所 | 一种赖氨酸生产菌株的构建方法和应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050244935A1 (en) * | 1999-06-25 | 2005-11-03 | Basf Ag | Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport |
-
2021
- 2021-02-05 CN CN202110161475.9A patent/CN114874958B/zh active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1479789A (zh) * | 2000-12-22 | 2004-03-03 | �����ɷ� | 棒状杆菌基因 |
CN1578834A (zh) * | 2001-11-05 | 2005-02-09 | 巴斯福股份公司 | 编码代谢途径蛋白的基因 |
CN102471790A (zh) * | 2009-07-29 | 2012-05-23 | 味之素株式会社 | 产生l-氨基酸的方法 |
CN107603932A (zh) * | 2016-07-12 | 2018-01-19 | 中国科学院微生物研究所 | 一种提高谷氨酸棒杆菌氨基酸产量的方法及其应用 |
CN107674863A (zh) * | 2017-08-04 | 2018-02-09 | 中国科学院天津工业生物技术研究所 | 一种生产反式‑4‑羟基‑l‑脯氨酸的方法 |
CN109402038A (zh) * | 2018-12-13 | 2019-03-01 | 江南大学 | 一种高产l-脯氨酸的重组菌及其构建方法与应用 |
CN111349640A (zh) * | 2018-12-21 | 2020-06-30 | 中国科学院天津工业生物技术研究所 | 反式-4-羟基-l-脯氨酸的生产菌株及其构建方法和应用 |
CN111484997A (zh) * | 2019-01-25 | 2020-08-04 | 中国科学院天津工业生物技术研究所 | 反式-l-羟脯氨酸的生产菌株及其构建方法和应用 |
CN112251391A (zh) * | 2019-05-13 | 2021-01-22 | 中国科学院天津工业生物技术研究所 | 一种赖氨酸生产菌株的构建方法和应用 |
CN112111469A (zh) * | 2020-11-23 | 2020-12-22 | 中国科学院天津工业生物技术研究所 | γ-谷氨酰激酶突变体及其应用 |
Non-Patent Citations (5)
Title |
---|
Increasing L-lysine production in Corynebacterium glutamicum by engineering amino acid transporters;Jing Xiao et al.;《Amino Acids》;第52卷;1363-1374 * |
WP_060563805.1;Reizer , j. et al.;《Gene Bank》;1-2 * |
WP_060563806.1;Gene Bank;《Gene Bank》;1-2 * |
大肠杆菌氨基酸转运蛋白的研究进展;马蓉 等;《科技通报》;第28卷(第3期);49-56、99 * |
植物ABCB转运蛋白研究进展;王暄 等;《生物技术通报》;第36卷(第6期);223-229 * |
Also Published As
Publication number | Publication date |
---|---|
CN114874958A (zh) | 2022-08-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108271384B (zh) | 用于特异性转变靶向dna序列的核酸碱基的革兰氏阳性菌的基因组序列的转变方法、及其使用的分子复合体 | |
KR20220076467A (ko) | 신규 핵염기 편집기 및 이의 사용 방법 | |
CN101855233B (zh) | 合成5’utr、表达载体以及增强转基因表达的方法 | |
CN113913356B (zh) | 高产l-谷氨酰胺的谷氨酸棒杆菌菌株及其构建方法与应用 | |
CN109486803B (zh) | 工程化苯丙氨酸解氨酶多肽 | |
CN113403294B (zh) | 一种融合蛋白、碱基编辑工具及其应用 | |
CN110951767B (zh) | 一种具有高拷贝能力的棒状杆菌和大肠杆菌双表达载体及其构建方法 | |
CN108085287B (zh) | 一种重组谷氨酸棒状杆菌、其制备方法及其应用 | |
KR20220010540A (ko) | 프로그래밍가능한 염기 편집기 시스템을 이용하여 단일염기다형성을 편집하는 방법 | |
KR20210126680A (ko) | 알파-1 항트립신 결핍증을 치료하기 위한 조성물 및 방법 | |
CN109206496B (zh) | 蛋白质GhFLS1在调控植物耐热性中的应用 | |
CN113265413B (zh) | 一种假病毒的制备方法 | |
KR20210129108A (ko) | 글리코겐 저장 질환 1a형을 치료하기 위한 조성물 및 방법 | |
CN114874958B (zh) | 生产l-脯氨酸的菌株及其构建方法和应用 | |
US20120094338A1 (en) | Continuous production and excretion of waxy products from photosynthetic organisms | |
KR102081699B1 (ko) | 스쿠알렌 생성능을 가지는 형질전환된 코리네박테리움 글루타미쿰 | |
CN110408646B (zh) | 一种植物遗传转化筛选载体及其应用 | |
CN113604412A (zh) | L-谷氨酸亚适量高产菌株及其构建方法与nh4+分阶段控制发酵方法 | |
CN110835631B (zh) | 一种改造的sgRNA及其在提高碱基编辑效率中的应用 | |
CN110835630B (zh) | 一种高效的sgRNA及其在基因编辑中的应用 | |
CN101985631B (zh) | 一种棒状杆菌启动子探测载体及其构建方法和应用 | |
KR101834286B1 (ko) | 다중 유전자 발현 조절능이 있는 크리스퍼 간섭을 이용한 코리네박테리움 글루타미쿰의 형질전환용 키트 | |
CN110878321B (zh) | 一种用于肺炎克雷伯菌基因编辑的表达载体 | |
CN110923263B (zh) | 水稻β-淀粉酶BA1及其编码基因与应用 | |
CN112592930B (zh) | 一种提高透明质酸产量的方法及菌株 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |