CN116515723A - PheP的活性增强及生产L-谷氨酸的方法 - Google Patents
PheP的活性增强及生产L-谷氨酸的方法 Download PDFInfo
- Publication number
- CN116515723A CN116515723A CN202310125356.7A CN202310125356A CN116515723A CN 116515723 A CN116515723 A CN 116515723A CN 202310125356 A CN202310125356 A CN 202310125356A CN 116515723 A CN116515723 A CN 116515723A
- Authority
- CN
- China
- Prior art keywords
- phep
- corynebacterium glutamicum
- glutamic acid
- gene
- strain
- 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
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 title claims abstract description 132
- 229960002989 glutamic acid Drugs 0.000 title claims abstract description 70
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 29
- 230000000694 effects Effects 0.000 title claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 65
- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 46
- 235000018102 proteins Nutrition 0.000 claims abstract description 23
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 23
- 108091033319 polynucleotide Proteins 0.000 claims description 27
- 102000040430 polynucleotide Human genes 0.000 claims description 27
- 239000002157 polynucleotide Substances 0.000 claims description 27
- 150000001413 amino acids Chemical group 0.000 claims description 22
- 230000035772 mutation Effects 0.000 claims description 16
- 230000002018 overexpression Effects 0.000 claims description 16
- 238000006467 substitution reaction Methods 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 7
- 230000014509 gene expression Effects 0.000 claims description 7
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 6
- 239000004474 valine Substances 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 101100024384 Escherichia coli (strain K12) mscS gene Proteins 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 4
- 150000007523 nucleic acids Chemical group 0.000 claims description 4
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 claims description 3
- 229940049906 glutamate Drugs 0.000 claims description 3
- 229930195712 glutamate Natural products 0.000 claims description 3
- 241000807905 Corynebacterium glutamicum ATCC 14067 Species 0.000 claims description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 150000003680 valines Chemical class 0.000 claims description 2
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 claims 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims 1
- 108010092528 Phosphate Transport Proteins Proteins 0.000 claims 1
- 102000016462 Phosphate Transport Proteins Human genes 0.000 claims 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims 1
- 235000004279 alanine Nutrition 0.000 claims 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims 1
- 230000002238 attenuated effect Effects 0.000 claims 1
- 229930029653 phosphoenolpyruvate Natural products 0.000 claims 1
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 claims 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 14
- 238000006243 chemical reaction Methods 0.000 abstract description 13
- 239000004220 glutamic acid Substances 0.000 abstract description 13
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 abstract description 12
- 235000013922 glutamic acid Nutrition 0.000 abstract description 12
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 23
- 239000002773 nucleotide Substances 0.000 description 22
- 125000003729 nucleotide group Chemical group 0.000 description 22
- 239000012634 fragment Substances 0.000 description 17
- 239000002609 medium Substances 0.000 description 17
- 239000013612 plasmid Substances 0.000 description 13
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 12
- 238000000855 fermentation Methods 0.000 description 12
- 230000004151 fermentation Effects 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- 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 11
- 239000008103 glucose Substances 0.000 description 11
- 238000010276 construction Methods 0.000 description 9
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 229940024606 amino acid Drugs 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 101100352026 Escherichia coli (strain K12) pheP gene Proteins 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- 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 6
- 239000004202 carbamide Substances 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 229920000642 polymer Polymers 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
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000007993 MOPS buffer Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229960005091 chloramphenicol Drugs 0.000 description 4
- 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 4
- 238000002474 experimental method Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 241000186216 Corynebacterium Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 229930195714 L-glutamate Natural products 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 239000013256 coordination polymer Substances 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 108050005273 Amino acid transporters Proteins 0.000 description 2
- 102000034263 Amino acid transporters Human genes 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 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
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 108091006146 Channels Proteins 0.000 description 2
- 102000034573 Channels Human genes 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 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
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000009655 industrial fermentation Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 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
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 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 2
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000006589 Alpha-ketoglutarate dehydrogenase Human genes 0.000 description 1
- 108020004306 Alpha-ketoglutarate dehydrogenase Proteins 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- 102000003846 Carbonic anhydrases Human genes 0.000 description 1
- 108090000209 Carbonic anhydrases Proteins 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 238000012366 Fed-batch cultivation Methods 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 102100025169 Max-binding protein MNT Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 108010053763 Pyruvate Carboxylase Proteins 0.000 description 1
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 101150067371 acnR gene Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 238000012365 batch cultivation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 101150019455 gdh gene Proteins 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 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
- 238000002372 labelling Methods 0.000 description 1
- 230000029226 lipidation Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 101150021317 odhA gene Proteins 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 108010004621 phosphoketolase Proteins 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 101150096049 pyc gene Proteins 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
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 108091006107 transcriptional repressors Proteins 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000013519 translation Methods 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
- 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
- C12N15/11—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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
-
- 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
- C12N9/88—Lyases (4.)
-
- 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
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/04—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a disulfide as acceptor (1.2.4)
- C12Y102/04002—Oxoglutarate dehydrogenase (succinyl-transferring) (1.2.4.2), i.e. alpha-ketoglutarat dehydrogenase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/99—Oxidoreductases acting on the CH-CH group of donors (1.3) with other acceptors (1.3.99)
- C12Y103/99001—Succinate dehydrogenase (1.3.99.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01031—Phosphoenolpyruvate carboxylase (4.1.1.31)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/02—Aldehyde-lyases (4.1.2)
- C12Y401/02009—Phosphoketolase (4.1.2.9)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
- C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
- C12Y604/01001—Pyruvate carboxylase (6.4.1.1)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于分子生物学领域,具体涉及一种产L‑谷氨酸的谷氨酸棒杆菌,所述菌株中的PheP蛋白的活性增强。此外,还包括利用上述菌株生产L‑谷氨酸、提高L‑谷氨酸产量和转化率的方法。本发明的产L‑谷氨酸的谷氨酸棒杆菌相对于野生型菌株而言,L‑谷氨酸的产量和转化率均有所提高,在生产中可降低谷氨酸的生产成本,为大规模生产提供了新的策略。
Description
技术领域
本发明涉及微生物与生物技术领域,具体涉及PheP的活性增强,以及PheP活性增强的谷氨酸棒杆菌,以及利用上述谷氨酸棒杆菌生产L-谷氨酸的方法。
背景技术
L-谷氨酸是世界第一大氨基酸产品,是构成动物营养所需蛋白质的基本物质,在生物体内的蛋白质代谢过程中占重要地位,参与动物、植物和微生物中的许多重要化学反应,主要用于生产味精、鸡精等调味料以及各种食品,并在医药、化工、畜牧等领域应用广泛。由于需求量大,产能高,L-谷氨酸的工业发酵一直是我国重要的发酵产业之一。
当前,L-谷氨酸的生产主要采用微生物发酵法来生产,通常使用的工业发酵微生物包括棒杆菌属(Corynebacterium)、短杆菌属(Brevibacterium)的菌株。由于谷氨酸棒杆菌(Corynebacterium glutamicum)的生理优越性,其已成为工业中最重要的谷氨酸生产菌株。通过代谢工程手段,使得谷氨酸棒杆菌L-谷氨酸的生产性能也不断提升。
现有技术发现谷氨酸棒杆菌胞内L-谷氨酸浓度过高是制约高产菌株的最终产量的关键因素之一。因此,自改造机械敏感通道蛋白YggB可提高菌株L-谷氨酸的产量被报道后,利用L-谷氨酸运输蛋白降低胞内谷氨酸的浓度,从而提高重组菌的产量的改造方法受到广泛关注(CN101090911A,CN105695383A,CN108250278A)。此后,第二个具有L-谷氨酸外排功能的蛋白MscCG2也被报道,研究表明加强MscCG2的表达可以提高L-谷氨酸的产量(Wang Y,Cao G,Xu D,et al.A novel Corynebacterium glutamicum L-glutamateexporter.Applied and Environment Microbiology,2018,84(6):e02691–02617)。然后,对于工业高产菌株而言,以上外排蛋白并不能完全解决胞内L-谷氨酸浓度过高的问题。
同时,由于L-谷氨酸合成途径较短,可改造靶点和策略相对有限,因此工业菌种水平进一步改造提升的难度很大,因此,本领域仍需进一步鉴定能够影响L-谷氨酸合成或转运的新靶点,以提高L-谷氨酸的产量。
发明内容
本发明的目的是提供一种新的构建L-谷氨酸生产菌株的方法。基于其目的,本发明通过对谷氨酸棒杆菌13869中的pheP(Cgl1155/NCgl1108)的同源基因BBD29_06185进行突变和过表达,发现可以提高菌株的L-谷氨酸产量,进而提升L-谷氨酸的生产效率,降低生产成本。在此基础上,完成本发明。
第一方面,本发明提供了一种产L-谷氨酸的谷氨酸棒杆菌,所述菌株中PheP活性增强。更具体的,所述菌株中SEQ ID NO:4所示序列的多核苷酸的表达增强(如替换更强的启动子),或导入PheP蛋白编码基因以过表达;或所述菌株中PheP发生突变,或PheP发生突变且该突变体的表达增强。更进一步地,所述PheP发生突变是SEQ ID NO:3所示氨基酸序列的194位甘氨酸被缬氨酸取代。
其中,本文所述的“PheP”,是指来源于谷氨酸棒杆菌的苯丙氨酸摄入蛋白(编码基因为Cgl1155/NCgl1108),其在谷氨酸棒杆菌ATCC13869中的编码基因(Gene ID)为BBD29_06185,被注释为氨基酸渗透酶。如本文所使用,PheP不被具体限制,只要其具有相应的活性,并且其可以是衍生自谷氨酸棒状杆菌,但是不限于此。例如,PheP可以是如SEQ ID NO:3的氨基酸序列的野生型序列或者与SEQ ID NO:3的氨基酸序列具有至少75%,具体地至少80%,更具体地85%,并且甚至更具体地90%、91%、92%、93%、94%、95%、96%、97%、98%、或者99%或更高的同源性的氨基酸序列。另外,如果氨基酸序列与上述序列具有同源性并且与SEQ ID NO:3的蛋白质具有基本上相同或相应的生物学活性,则具有缺失、修饰、置换或添加的氨基酸序列也应当属于本公开内容的范围是显而易见的。在本发明中,编码PheP的任何多核苷酸序列可以属于本公开内容的范围。例如,多核苷酸序列可以是与SEQID NO:4的多核苷酸序列具有至少75%,具体地至少80%,更具体地85%,并且甚至更具体地90%、91%、92%、93%、94%、95%、96%、97%、98%、或者99%或更高的同源性的多核苷酸序列。另外,基于密码子简并性或考虑生物体表达蛋白质优选的密码子,编码蛋白质的多核苷酸序列可以在不改变从编码区表达的蛋白质的氨基酸序列的范围内具有编码区上的各种变体。
所述“多肽”、“肽”和“蛋白质”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。
所述“活性增强”是指与野生型菌株相比,PheP蛋白活性增强,或PheP编码基因的转录表达水平增强,包括以下述基因工程的方法:如向微生物的细胞中引入强启动子、强核糖体结合位点;引入非整合型蛋白的重组表达载体;引入染色体整合型蛋白的重组表达载体;改变编码基因的启动子、翻译调控区或编码区密码子令其转录或翻译加强;改变编码基因序列使其mRNA稳定性增强或编码蛋白的结构稳定;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活性增强的方式等。
所述“野生型的”指在自然界中可以找到的对象,包括天然的菌株、天然的基因和蛋白等等。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。如本公开所用的,“天然存在的”和“野生型的”是同义词。在一些实施方式中,本公开中野生型的PheP是指谷氨酸棒杆菌中天然存在的蛋白,也即如SEQ ID NO:3所示氨基酸序列的多肽。
所述“突变体”是指相对于“野生型”,或者“相比较的”多核苷酸或多肽,在一个或多个(例如,若干个)位置处包含改变(即,取代、***和/或缺的多核苷酸,其中,取代是指用不同的核苷酸置换占用一个位置的核苷酸。缺失是指去除占据某一位置的核苷酸。***是指在邻接并且紧随占据位置的核苷酸之后添加核苷酸。在具体的实施方式中,所述“突变”为“取代”,是由一个或多个核苷酸中的碱基被另一个不同的碱基取代所引起的突变,也称为碱基置换突变(substitution)或点突变(point mutation)。
所述“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。在一个具体的实施方式中,术语“突变”是指“取代”。
在一些实施方式中,所述的“突变”包含在SEQ ID NO:3所示序列的第194位甘氨酸被缬氨酸取代。与出发菌株相比,含有该突变体的菌株其L-谷氨酸产量提高8.7%左右,糖酸转化率提高10%。
本发明所述的L-谷氨酸生产菌株的构建,是通过将PheP或其编码基因转化进入宿主细胞来实现的。此处“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(CaPO4)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。
本发明所述“宿主细胞”是具有本领域普通技术人员通常理解的含义,即,能够导入本发明的具有启动子活性的核酸的细胞,导入之后称为重组宿主细胞。换言之,本发明可以利用任何宿主细胞,只要其细胞中含有本发明的具有启动子活性的核酸,并且与某一基因可操作性地连接介导该基因的转录。本发明的宿主细胞可以是原核细胞,优选肠杆菌或棒杆菌,更优选谷氨酸棒杆菌,包括但不限于谷氨酸棒杆菌ATCC 13869、谷氨酸棒杆菌ATCC13032、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067,以及由上述菌株制备的产生L-谷氨酸的突变体或菌株。
在一些实施方式中,对于谷氨酸生产菌株,可以是在谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC13869基础上改造获得的菌株,这些改造包括但不限于选自以下的一个或多个基因被增强或过表达:
a.编码机械敏感通道蛋白的yggB基因(CN108250278A);
b.编码磷酸转酮酶的fxpk基因(WO2006016705A1);
c.编码丙酮酸羧化酶的pyc基因(WO2004069996A2);
d.编码谷氨酸脱氢酶的gdh基因(CN103205390A);
e.编码碳酸酐酶的基因(WO2011024583A1)。
在一些实施方式中,所述谷氨酸生产菌株中还可以包括但不限于选自以下的一个或多个基因被弱化或表达降低:
a.编码α-酮戊二酸脱氢酶的odhA基因(WO2006028298A2);
b.编码转录调控基因的amtR基因(EP2276845A1);
c.编码转录抑制子的acnR基因(CN111334535A)。
在本发明的一个具体实施方式中,所述宿主细胞是谷氨酸棒杆菌,进一步地经过改良,具体地是在所述菌中的BBD29_06760基因或其同源基因(NCgl1221或yggB)中引入A111V突变,获得谷氨酸生产菌株SCgGC5。
第二方面,本发明提供了一种PheP突变体,所述突变体在对应于SEQ ID NO:3的194位的甘氨酸被缬氨酸取代。
进一步地,本发明还提供了编码所述PheP突变体的多核苷酸。
所述“多核苷酸”指由核苷酸组成的聚合物。多核苷酸可以是单独片段的形式,也可以是更大的核苷酸序列结构的一个组成部分,其是从至少在数量或浓度上分离一次的核苷酸序列衍生而来的,能够通过标准分子生物学方法(例如,使用克隆载体)识别、操纵以及恢复序列及其组分核苷酸序列。当一个核苷酸序列通过一个DNA序列(即A、T、G、C)表示时,这也包括一个RNA序列(即A、U、G、C),其中“U”取代“T”。换句话说,“多核苷酸”指从其他核苷酸(单独的片段或整个片段)中去除的核苷酸聚合物,或者可以是一个较大核苷酸结构的组成部分或成分,如表达载体或多顺反子序列。多核苷酸包括DNA、RNA和cDNA序列。
具体地,本发明的PheP的编码多核苷酸包括SEQ ID NO:2所示的多核苷酸。此外,本发明的多核苷酸还包括与SEQ ID NO:2所示多核苷酸具有75%或更高,具体地80%或更高,更具体地85%或更高,并且甚至更具体地90%、91%、92%、93%、94%、95%、96%、97%、98%、和99%或更高的同源性的任何多核苷酸。
本发明的术语“同源性”指的是两个多核苷酸或多肽部分之间的同一性的百分比。一个部分与另一个部分的序列之间的同源性可以通过本领域中已知的技术测定。例如,同源性可以通过使用容易可获得的计算机程序直接排列两个多核苷酸分子或两个多肽分子的序列信息来测定。计算机程序的实例可以包括BLAST(NCBI)、CLC Main Workbench(CLCbio)、MegAlignTM(DNASTAR Inc.)等。另外,多核苷酸之间的同源性可以通过如下步骤来测定:在同源区之间形成稳定双链的条件下杂交多核苷酸,利用单链特异性核酸酶分解,然后对分解的片段进行大小测定。
第三方面,本发明提供所述PheP突变体或其编码核苷酸在提高L-谷氨酸产量和转化率中的应用。
第四方面,本发明提供了一种生产L-谷氨酸的方法,所述方法包括培养第一方面的宿主细胞使之生产L-谷氨酸,进一步包括从培养基中分离提取或回收L-谷氨酸的步骤。
本发明的有益效果:本发明提供的PheP活性增强的菌株,或含有PheP突变体的菌株,或含有PheP突变体且该突变体过表达的菌株,其相对于野生型菌株而言,L-谷氨酸的产量和转化率均有所提高,在生产中可降低谷氨酸的生产成本,为大规模生产提供了新的策略。
在本发明中,所述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。
具体实施方式
除非另有定义,本文所用的所有技术和科学术语与本发明所属领域普通技术人员通常理解的意义相同。虽然可利用与本文所述相似或等价的任何方法和材料来实施或检验本发明,但优选此处提供的方法和材料。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。实施例中所用到的实验技术与实验方法,如无特殊说明均为常规技术方法,例如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 LaboratoryPress,1989)中所述的条件,或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等,如无特殊说明,均可通过正规商业渠道获得。
实施例中所用的培养基如下:
TSB平板培养基成份为(g/L):葡萄糖,5g/L;酵母粉,5g/L;大豆蛋白胨,9g/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;琼脂粉,15g/L。
TSB液体培养基成份为(g/L):葡萄糖,5g/L;酵母粉,5g/L;大豆蛋白胨,9g/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。
谷氨酸发酵实验使用的种子培养基成份为:葡萄糖,25g/L;KH2PO4·3H2O,2.2g/L;尿素,3g/L;玉米浆,33mL;MgSO4·7H2O,0.9g/L;豆饼水解液,22mL;MOPS,20g/L;初始pH7.2。
谷氨酸发酵实验使用的发酵培养基成份为:葡萄糖,80g/L;KH2PO4,1g/L;尿素,10g/L;玉米浆干粉,5g/L;MgSO4·7H2O,0.4g/L;FeSO4·7H2O,10mg/L;MnSO4·4H2O,10mg/L;VB1,200μg/L;MOPS,40g/L;初始pH7.5。
实施例1谷氨酸棒杆菌PheP缺失和过表达菌株的构建
谷氨酸棒杆菌来源的PheP蛋白(编码基因Cgl1155或NCgl1108),其在谷氨酸棒杆菌ATCC13869中由Gene ID为BBD29_06185的基因编码,被注释为氨基酸渗透酶,是潜在的氨基酸转运蛋白。目前,对该酶的研究较少,现有技术报道该酶具有摄入苯丙氨酸的功能(Zhao Z,etal.,The ncgl1108(PheP(Cg))gene encodes a new L-Phe transporter inCorynebacterium glutamicum.Applied Microbiology and Biotechnology volume 90,pages2005–2013(2011))。我们推测对蛋白的改造可能影响L-谷氨酸的合成或转运,因此,对谷氨酸棒杆菌13869中的该酶进行敲除和过表达改造,确认其对L-谷氨酸产量的影响。
(1)生产L-谷氨酸的基础菌株构建
已有文献报道了在谷氨酸棒杆菌ATCC 13869基因组NCgl1221同源基因(BBD29_06760或yggB)中引入A111V突变,可以使菌株具备组成型合成分泌L-谷氨酸的能力。根据已公开的谷氨酸棒杆菌ATCC 13869基因组(GenBank:CP016335.1)序列,设计引物A111V-UH-F/R和A111V-DH-F/R。以ATCC13869基因组为模板,分别利用上述引物通过PCR扩增获得带有YggBA111V突变的DNA片段;根据质粒pK18mobsacB的序列信息设计引物pK-F/R,以质粒pK18mobsacB为模板,通过PCR反向扩增获得线性化载体片段;上述三片段回收后重组连接,对化转后所获得的克隆进行收集并提取质粒,获得YggBA111V突变的编辑载体pK18-YggBA111V。
采用文献报道的方法制备C.glutamicum ATCC 13869感受态细胞(BiotechnologyLetters,2015,37:2445-52.),向上述制备获得的13869感受态细胞电转化1μg的pK18-YggBA111V质粒,加入1mL 46℃预热的TSB培养基,46℃孵育6min,30℃孵育3h,涂布含有25μg/mL卡那霉素的TSB固体培养基,30℃培养1天,获得第一次重组的转化子。正确转化子转接含有5g/L葡萄糖的TSB培养基过夜培养,然后转接含有100g/L蔗糖的TSB培养基,30℃培养6h后涂布于添加100g/L蔗糖的TSB培养基进行筛选,获得L-谷氨酸生产菌株SCgGC5。本实施例所用引物见表1。
表1本实施例所用引物
引物 | 核苷酸序列 |
A111V-UH-F | tgacatgattacgaattcATCCACTGGAGTTTTGCCAATTCTC |
A111V-UH-R | gtcttggtGTGcagtcgattgttgcg |
A111V-DH-F | atcgactgCACaccaagaccaatggc |
A111V-DH-R | cgacggccagtgccaagcttTGGAGGAATAGAGCGGGTCATACAC |
(2)PheP缺失菌株的构建
根据已报道的谷氨酸棒杆菌ATCC 13869中的野生型PheP序列(氨基酸序列如SEQID NO:3所示,核苷酸如SEQ ID NO:4所示),分别设计引物PheP-F3/R3和PheP-F4/R4,以ATCC 13869基因组为模板通过PCR扩增获得用于PheP缺失的上下游同源臂。上述PCR片段回收后,与EcoRI和BamHI酶切处理的pK18mobsacB载体进行重组连接,获得PheP基因的缺失载体pK18-ΔPheP。
其中,SEQ ID NO:3:
MNASPAPTRSFRGLRARHIHFIALGSAIGTGLFYGSAGAIQAAGPSVLLVYLLGGAVVYFMLRALGEMAVHHPVRGSFAVYTRAHLGGWAGYITGWMFAFEMLIVCLADLTAIGIYMNFWFPGTPQWTWVVATLLIVGGANLASVRWFGELEFIFTIIKVTAVVAMIVGGAAILAFGLGANAEVAGVSNLWEHGGFFPNGVEGMIAAFILVLFAFGGTEIIGVAGSEAEDPEKSIPKAVNTVPVRILLFYVGAILVILALNPWPSITGEESPFVQIFDTLGVNWAAGLLNAVVITAALSAINADLFGAGRVLTGLAKENLAPKAMGKIAKNGVPVMTTTIMIIVLIVGVILNAVLPERVFEIVASLATFATVYVWLMILLAQVGSRRNMPADEVKSLKFPVPFYPFGQYFAILFIAFTFGIMVWYDNYHLPLAVGVGFLVLMTILYYATGRPKAIAPINYEELDPRRD。
SEQ ID NO:4:
atgaatgcctcccctgccccaacccgatcttttagaggattgcgggctcgacacattcacttcatcgcgctgggttccgcgatcggcaccggcttgttctacggttccgctggcgcaatccaagcagctggtccatcagtactcttggtctaccttctcggtggcgccgtcgtgtacttcatgctgcgcgcactcggcgagatggctgtccaccacccagtccgtggctcctttgccgtttacacccgcgcacaccttggcggatgggcaggctacatcaccggctggatgttcgcctttgagatgctcatcgtctgtctggctgacctcacagccatcggcatctatatgaacttctggttcccaggcaccccgcaatggacttgggtggtagccacccttcttattgtcggtggcgcaaacctcgcatcagtgcgttggttcggtgagctcgagttcatcttcaccatcattaaggtcaccgcagttgtcgccatgatcgtcggcggcgcagccatcctcgcattcggtctcggcgccaacgctgaagttgccggcgtatccaacctctgggagcacggcggattcttccccaacggtgttgaaggcatgatcgcagccttcatccttgttctcttcgcattcggtggcaccgaaatcatcggtgttgcaggctctgaagctgaagatcctgagaagtccatccccaaggctgttaatactgtcccagtacgcatcctcctcttctatgtgggtgccatcctggtgatccttgcccttaatccttggccttccatcaccggcgaagaatccccattcgtccagatcttcgacaccctcggcgtcaactgggctgctggtctcctcaacgccgtggtcatcaccgctgcactgtctgccatcaacgctgacctcttcggcgctggccgcgttctcactggtcttgcgaaggaaaacctcgcaccaaaggccatgggcaagatcgccaagaacggcgttccagtcatgaccaccaccatcatgatcatcgtcttgatcgtgggagtaatcctcaacgcagtgcttcccgagcgcgtcttcgagatcgtcgcttccctagcaactttcgccacagtttacgtctggctgatgatcctgctcgcacaggtgggatcccgccgaaacatgcctgccgacgaggtcaagtccctgaagttccctgtccccttctaccccttcggacaatacttcgcgatcctatttatcgccttcaccttcggcatcatggtctggtacgacaactaccacctgccactcgccgtcggcgttggattccttgtcctgatgacaatcctttactacgccacaggccgaccaaaggcgatcgcaccgatcaattatgaagagttagatccgcgacgcgactaa。
采用文献报道的方法制备C.glutamicumSCgGC5感受态细胞(BiotechnologyLetters,2015,37:2445-52.),向上述制备获得的感受态细胞电转化1μg的pK18-ΔPheP质粒,加入1mL 46℃预热的TSB培养基,46℃孵育6min,30℃孵育3h,涂布含有25μg/mL卡那霉素的TSB固体培养基,30℃培养1天,获得第一次重组的转化子。正确转化子转接含有5g/L葡萄糖的TSB培养基过夜培养,然后转接含有100g/L蔗糖的TSB培养基,30℃培养6h后涂布于添加100g/L蔗糖的TSB培养基进行筛选,获得L-谷氨酸生产菌株SCgGC5-ΔPheP。本实施例所用引物见表2。
(3)PheP过表达菌株构建
根据已公开的谷氨酸棒杆菌ATCC 13869基因组(GenBank:CP016335.1)序列及野生型PheP的序列,设计扩增引物PheP-F和PheP-R,以ATCC 13869基因组为模板,扩增获得野生型pheP片段。根据质粒pXMJ19(Biotechnology Techniques,1999,13(6),437-441)序列,设计引物pXMJ19-rev-F和pXMJ19-rev-R,以质粒pXMJ19为模板,通过PCR扩增得到pXMJ19线性化载体片段。将纯化回收的pheP片段与pXMJ19线性化载体片段回收后重组连接,连接产物转化Trans T1感受态细胞,涂布于含34μg/mL氯霉素的LB抗性平板,过夜培养,挑阳性克隆进行菌落PCR和测序验证,验证正确的重组载体命名为pXMJ19-PhePWT。将pXMJ19和pXMJ19-PhePWT质粒分别转化SCgGC5感受态细胞,得到重组菌株SCgGC5/pXMJ19和SCgGC5/pXMJ19-PhePWT。本实施例所用引物见表2。
表2本实施例所用引物
引物 | 核苷酸序列 |
PheP-F3 | tgacatgattacgaattcTTGCCAAAAGTATAAATGGGACC |
PheP-R3 | gtcctgatgacaatcctttactacg |
PheP-F4 | aaggattgtcatcaggacggtgccgatcgcggaacc |
PheP-R4 | cgacggccagtgccaagcttGATGACTCGTTTGAAGGCTGGTTAG |
PheP-F | gaaggagatatacatATGAATGCCTCCCCTGCCCC |
PheP-R | gggatcctctagagtTTAGTCGCGTCGCGGATCTA |
pXMJ19-rev-F | ACTCTAGAGGATCCCCGGGTAC |
pXMJ19-rev-R | ATGTATATCTCCTTCCTGCAGGCATGCAAGCTT |
实施例2 PheP缺失或过表达对L-谷氨酸生产的影响
为了验证敲除或过表达PheP对谷氨酸产量的效果,对上述构建的SCgGC5-ΔPheP、SCgGC5/pXMJ19-PhePW菌株进行发酵测试,分别以菌株SCgGC5和SCgGC5/pXMJ19作为对照。
将菌株SCgGC5和SCgGC5-ΔPheP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0.5,孔板摇床转速为800rpm,每个菌株3个平行,30℃培养,17h、20h和23h补加5g/L尿素,25h发酵结束,检测L-谷氨酸产量和葡萄糖消耗量,并计算从葡萄糖到L-谷氨酸的糖酸转化率。结果如表4所示。
将菌株SCgGC5/pXMJ19和SCgGC5/pXMJ19-PhePWT接种到含有5μg/mL氯霉素的种子培养基中培养8h,培养物作为种子接种到每孔含有5μg/mL氯霉素和10μM IPTG的800μL发酵培养基的24孔板中,初始OD600控制约为0.5,孔板摇床转速为800rpm,每个菌株3个平行,30℃培养,20h和23h补加5g/L尿素,25h发酵结束,检测L-谷氨酸产量和葡萄糖消耗量,并计算从葡萄糖到L-谷氨酸的糖酸转化率。结果如表3所示。从表中可知,PheP缺失菌株中L-谷氨酸产量略有下降,而过表达PheP能够明显提高L-谷氨酸产量及糖酸转化率。
表3 L-谷氨酸生产量和糖酸转化率
实施例3 PheP突变菌株和突变型PheP过表达菌株构建
进一步,我们发现在发明人前期诱变筛选的一株高产谷氨酸的菌株SL4(LiuJiao,et al.,Mutations in Peptidoglycan Synthesis Gene ponAImproveElectrotransformation Efficiency of Corynebacterium glutamicum ATCC13869.Appl.Environ.Microbiol.,2018,84,e02225-02218.)中,研究发现其中PheP的第194位甘氨酸被缬氨酸取代。鉴于前述实施例2已经证实PheP的过表达可以增强菌株的L-谷氨酸产量,因此,推测该突变体可能也对L-谷氨酸的生产有影响。
(1)PheP突变的谷氨酸生产菌株构建
首先,根据已公开的谷氨酸棒杆菌ATCC 13869基因组(GenBank:CP016335.1)序列及野生型PheP的序列(其氨基酸序列如SEQ ID NO:3所示,其核苷酸序列如SEQ ID NO:4所示),设计扩增引物PheP-F1/R1和PheP-F2/R2,以ATCC 13869基因组为模板,扩增包含PhePG194V突变体的上游和下游重组片段。根据质粒pK18mobsacB的序列信息设计引物pK-F/R,以质粒pK18mobsacB为模板,通过PCR反向扩增获得线性化载体片段。本实施例所用引物见表4。上述三片段回收后重组连接,获得带有PhePG194V突变体的编辑质粒pK18-PhePG194V。
表4构建突变体编辑质粒引物
引物 | 核苷酸序列 |
pK-F | AAGCTTGGCACTGGCCGTCG |
pK-R | GAATTCGTAATCATGTCATAGCTGT |
PheP-F1 | tgacatgattacgaattcAAACTACAGTGACTGCTTCC |
PheP-R1 | ctgggagcacgtcggattcttccccaacggtgt |
PheP-F2 | ggaagaatccgacgtgctcccagaggttggata |
PheP-R2 | cgacggccagtgccaagcttCCGACGAAATGACGTTATTC |
向SCgGC5感受态细胞电转化1μg的pK18-PhePG194V质粒,加入1mL 46℃预热的TSB培养基,46℃孵育6min,30℃孵育3h,涂布含有25μg/mL卡那霉素的TSB固体培养基,30℃培养24h,获得第一次重组的转化子。正确转化子转接含有5g/L葡萄糖的TSB培养基过夜培养,然后转接含有100g/L蔗糖的TSB培养基,30℃培养4h后涂布于添加100g/L蔗糖的TSB培养基进行筛选,获得带有突变型PheP的L-谷氨酸生产菌株SCgGC5-PhePG194V。即该菌株中含有了突变后的PheP(其氨基酸序列如SEQ ID NO:1所示,其核苷酸序列如SEQ ID NO:2所示)。
SEQ ID NO:1:
MNASPAPTRSFRGLRARHIHFIALGSAIGTGLFYGSAGAIQAAGPSVLLVYLLGGAVVYFMLRALGEMAVHHPVRGSFAVYTRAHLGGWAGYITGWMFAFEMLIVCLADLTAIGIYMNFWFPGTPQWTWVVATLLIVGGANLASVRWFGELEFIFTIIKVTAVVAMIVGGAAILAFGLGANAEVAGVSNLWEHVGFFPNGVEGMIAAFILVLFAFGGTEIIGVAGSEAEDPEKSIPKAVNTVPVRILLFYVGAILVILALNPWPSITGEESPFVQIFDTLGVNWAAGLLNAVVITAALSAINADLFGAGRVLTGLAKENLAPKAMGKIAKNGVPVMTTTIMIIVLIVGVILNAVLPERVFEIVASLATFATVYVWLMILLAQVGSRRNMPADEVKSLKFPVPFYPFGQYFAILFIAFTFGIMVWYDNYHLPLAVGVGFLVLMTILYYATGRPKAIAPINYEELDPRRD。
SEQ ID NO:2:
atgaatgcctcccctgccccaacccgatcttttagaggattgcgggctcgacacattcacttcatcgcgctgggttccgcgatcggcaccggcttgttctacggttccgctggcgcaatccaagcagctggtccatcagtactcttggtctaccttctcggtggcgccgtcgtgtacttcatgctgcgcgcactcggcgagatggctgtccaccacccagtccgtggctcctttgccgtttacacccgcgcacaccttggcggatgggcaggctacatcaccggctggatgttcgcctttgagatgctcatcgtctgtctggctgacctcacagccatcggcatctatatgaacttctggttcccaggcaccccgcaatggacttgggtggtagccacccttcttattgtcggtggcgcaaacctcgcatcagtgcgttggttcggtgagctcgagttcatcttcaccatcattaaggtcaccgcagttgtcgccatgatcgtcggcggcgcagccatcctcgcattcggtctcggcgccaacgctgaagttgccggcgtatccaacctctgggagcacgtcggattcttccccaacggtgttgaaggcatgatcgcagccttcatccttgttctcttcgcattcggtggcaccgaaatcatcggtgttgcaggctctgaagctgaagatcctgagaagtccatccccaaggctgttaatactgtcccagtacgcatcctcctcttctatgtgggtgccatcctggtgatccttgcccttaatccttggccttccatcaccggcgaagaatccccattcgtccagatcttcgacaccctcggcgtcaactgggctgctggtctcctcaacgccgtggtcatcaccgctgcactgtctgccatcaacgctgacctcttcggcgctggccgcgttctcactggtcttgcgaaggaaaacctcgcaccaaaggccatgggcaagatcgccaagaacggcgttccagtcatgaccaccaccatcatgatcatcgtcttgatcgtgggagtaatcctcaacgcagtgcttcccgagcgcgtcttcgagatcgtcgcttccctagcaactttcgccacagtttacgtctggctgatgatcctgctcgcacaggtgggatcccgccgaaacatgcctgccgacgaggtcaagtccctgaagttccctgtccccttctaccccttcggacaatacttcgcgatcctatttatcgccttcaccttcggcatcatggtctggtacgacaactaccacctgccactcgccgtcggcgttggattccttgtcctgatgacaatcctttactacgccacaggccgaccaaaggcgatcgcaccgatcaattatgaagagttagatccgcgacgcgactaa
(2)突变型PheP过表达菌株构建
以pXMJ19-PhePWT重组载体为模板,利用引物PheP-F5和PheP-R5扩增包含PhePG194V突变体的片段。本实施例所用引物见表5。将上述获得的突变体的片段分别纯化回收后利用T4多聚合核苷酸激酶(T4 PNK)进行末端磷酸化处理,然后使用T4连接酶自身环化连接,将连接产物转化Trans T1感受态细胞,涂布于含34μg/mL氯霉素的LB抗性平板,过夜培养,挑阳性克隆进行菌落PCR和测序验证,正确的重组载体分别命名为pXMJ19-PhePG194V。将上述重组载体转化SCgGC5,得到重组菌株SCgGC5/pXMJ19-PhePG194V。
表5本实施例所用引物
引物 | 核苷酸序列 |
PheP-F5 | CTCTGGGAGCACGTCGGATTCTTCCCCAACGGTGT |
PheP-R5 | GTTGGATACGCCGGCAACTTCAG |
实施例4 PheP突变及突变型PheP过表达对L-谷氨酸生产的影响
为了验证PhePG194V突变以及过表达该突变体对谷氨酸产量的效果,对上述构建的SCgGC5-PhePG194V和SCgGC5/pXMJ19-PhePG194V菌株进行发酵测试,分别以菌株SCgGC5和SCgGC5/pXMJ19作为对照。
发酵过程同实施例2,发酵结果如表6所示。从表中可知,PhePG194V突变能够明显提高L-谷氨酸产量及糖酸转化率,而该突变体的过表达相对于对照菌株(SCgGC5/pXMJ19)的L-谷氨酸产量和糖酸转化率也有小幅提升,说明PhePG194V突变体以及该突变体的过表达在L-谷氨酸及其衍生物生产中具有较好的应用前景。
表6不各菌株的L-谷氨酸生产量和糖酸转化率
/>
Claims (10)
1.一种产L-谷氨酸的谷氨酸棒杆菌,其特征在于,所述菌株中的PheP蛋白的活性增强,以提高所述菌株产L-谷氨酸的能力。
2.如权利要求1所述的谷氨酸棒杆菌,其特征在于,所述PheP蛋白的活性增强是SEQ IDNO:4所示序列的多核苷酸的表达增强,或导入PheP蛋白编码基因以过表达。
3.如权利要求1所述的谷氨酸棒杆菌,其特征在于,所述PheP蛋白的活性增强是PheP发生突变而活性增加,或PheP发生突变且该突变体的表达增强。
4.如权利要求3所述的谷氨酸棒杆菌,其特征在于,所述PheP发生突变是SEQ ID NO:3所示氨基酸序列的194位甘氨酸被缬氨酸取代。
5.如权利要求1至4任一项所述的谷氨酸棒杆菌,其特征在于,所述菌株包括但不限于谷氨酸棒杆菌ATCC 13869、谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067,以及由上述菌株制备的产生L-谷氨酸的菌株。
6.如权利要求5所述的谷氨酸棒杆菌,其特征在于,所述菌株进一步(优选为谷氨酸棒杆菌ATCC 13869)存在选自以下的一个或多个基因被弱化或表达降低:
a. 编码α-酮戊二酸脱氢酶的odhA基因;
b. 编码琥珀酸脱氢酶的sucA基因。
7.如权利要求6所述的谷氨酸棒杆菌,其特征在于,所述菌株中还选自以下的一个或多个基因被增强或过表达:
a. 编码丙酮酸羧化酶的pyc基因;
b. 编码谷氨酸脱氢酶的gdh基因;
c. 编码柠檬酸合酶的gltA基因;
d. 编码天磷酸转酮酶的fxpk基因;
e.编码磷酸烯醇丙酮酸羧化酶的ppc基因;
f. 编码磷酸盐转运蛋白的pitA基因;
g.编码机械敏感通道蛋白的yggB基因。
8.如权利要求5至7任一项所述的谷氨酸棒杆菌,其特征在于,所述谷氨酸棒杆菌中的BBD29_06760基因或其同源基因(如NCgl1221或yggB)编码的氨基酸序列的111位丙氨酸被缬氨酸取代。
9.一种多核苷酸在提高谷氨酸棒杆菌L-谷氨酸产量中的应用,其特征在于,在谷氨酸棒杆菌中将所述多核苷酸过表达,所述多核苷酸的核酸序列如SEQ ID NO:2或SEQ ID NO:4所示。
10.一种生产L-谷氨酸的方法,其特征在于,包括培养如权利要求1至8任一项所述的谷氨酸棒杆菌使之生产L-谷氨酸,进一步包括从培养基中分离提取或回收L-谷氨酸的步骤。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310125356.7A CN116515723A (zh) | 2023-02-16 | 2023-02-16 | PheP的活性增强及生产L-谷氨酸的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310125356.7A CN116515723A (zh) | 2023-02-16 | 2023-02-16 | PheP的活性增强及生产L-谷氨酸的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116515723A true CN116515723A (zh) | 2023-08-01 |
Family
ID=87394683
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310125356.7A Pending CN116515723A (zh) | 2023-02-16 | 2023-02-16 | PheP的活性增强及生产L-谷氨酸的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116515723A (zh) |
-
2023
- 2023-02-16 CN CN202310125356.7A patent/CN116515723A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1154020A2 (en) | Arginine repressor deficient strain of coryneform bacterium and method for producing L-arginine | |
TWI632237B (zh) | 用於製造腐胺或鳥胺酸之微生物及使用該微生物製造腐胺或鳥胺酸之方法 | |
JP7203475B2 (ja) | 新規なポリペプチド及びそれを用いたオルニチン系産物の生産方法 | |
KR20160043890A (ko) | L-아르기닌을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아르기닌의 제조 방법 | |
JP7308891B2 (ja) | 新規なポリペプチド及びこれを用いたオルニチン系産物の生産方法 | |
KR101640711B1 (ko) | 글루코네이트 리프레서 변이체, 이를 포함하는 l-라이신을 생산하는 미생물 및 이를 이용한 l-라이신 생산방법 | |
WO2022017223A1 (zh) | 丙酮酸羧化酶基因启动子的突变体及其应用 | |
KR20190003401A (ko) | 신규한 o-숙시닐 호모세린 트랜스퍼라제 변이체 및 이를 이용한 o-숙시닐 호모세린의 제조방법 | |
AU2022206623B2 (en) | Glxr protein variant or threonine production method using same | |
KR20190003402A (ko) | 신규한 o-숙시닐 호모세린 트랜스퍼라제 변이체 및 이를 이용한 o-숙시닐 호모세린의 제조방법 | |
WO2008088149A1 (en) | Corynebacterium glutamicum variety producing l-arginine and method for fabricating the same | |
CN116515723A (zh) | PheP的活性增强及生产L-谷氨酸的方法 | |
CN117321193A (zh) | 新型支链氨基酸转氨酶变体及使用其生产异亮氨酸的方法 | |
KR101687474B1 (ko) | L-아르기닌 생산능이 향상된 코리네박테리움속 미생물 및 이를 이용한 l-아르기닌의 생산 방법 | |
CN115725532A (zh) | 一种生物素合酶突变体及其在构建谷氨酸生产菌株中的应用 | |
KR102339271B1 (ko) | 신규 프로모터 및 이의 용도 | |
KR102339264B1 (ko) | 신규 프로모터 및 이의 용도 | |
CN115927325B (zh) | 柠檬酸合酶启动子突变体及其应用 | |
CN116004501A (zh) | Nadp-铁氧还蛋白还原酶突变体及其在生产谷氨酸中的应用 | |
RU2812048C1 (ru) | Мутант промотора гена пируваткарбоксилазы и его применение | |
RU2812048C9 (ru) | Мутант промотора гена пируваткарбоксилазы и его применение | |
CN115851802A (zh) | 谷氨酸高产菌株的构建方法及其在谷氨酸生产中的应用 | |
JP2003102490A (ja) | L−アルギニンの製造法 | |
CN116144564A (zh) | 一种谷氨酸生产菌株的构建方法及其在生产谷氨酸中的用途 | |
CN116042591A (zh) | 磷酸甲基嘧啶合酶突变体及其在构建谷氨酸生产菌株中的应用 |
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 |