CN115746110A - Mutant of transcriptional regulatory factor LysG and application thereof - Google Patents
Mutant of transcriptional regulatory factor LysG and application thereof Download PDFInfo
- Publication number
- CN115746110A CN115746110A CN202111026806.4A CN202111026806A CN115746110A CN 115746110 A CN115746110 A CN 115746110A CN 202111026806 A CN202111026806 A CN 202111026806A CN 115746110 A CN115746110 A CN 115746110A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- mutant
- lysg
- basic amino
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 101
- 230000002103 transcriptional effect Effects 0.000 title claims abstract description 52
- 150000001413 amino acids Chemical class 0.000 claims abstract description 185
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 145
- 238000013518 transcription Methods 0.000 claims abstract description 63
- 230000035897 transcription Effects 0.000 claims abstract description 63
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 60
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 52
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 52
- 239000002157 polynucleotide Substances 0.000 claims abstract description 52
- 230000035772 mutation Effects 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 45
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 38
- 239000013604 expression vector Substances 0.000 claims abstract description 36
- 230000014509 gene expression Effects 0.000 claims abstract description 36
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 35
- 230000004044 response Effects 0.000 claims abstract description 34
- 238000003259 recombinant expression Methods 0.000 claims abstract description 32
- 238000012216 screening Methods 0.000 claims abstract description 28
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 27
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 27
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 27
- 230000001976 improved effect Effects 0.000 claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 61
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 60
- 239000004472 Lysine Substances 0.000 claims description 55
- 239000002773 nucleotide Substances 0.000 claims description 40
- 125000003729 nucleotide group Chemical group 0.000 claims description 40
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 35
- 229920001184 polypeptide Polymers 0.000 claims description 34
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 34
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 25
- 230000000694 effects Effects 0.000 claims description 21
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 18
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 16
- 239000004475 Arginine Substances 0.000 claims description 15
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 15
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 14
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 241000186216 Corynebacterium Species 0.000 claims description 7
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 7
- 241000186146 Brevibacterium Species 0.000 claims description 6
- 241000588698 Erwinia Species 0.000 claims description 6
- 241000588722 Escherichia Species 0.000 claims description 6
- 241000589516 Pseudomonas Species 0.000 claims description 6
- 241000607142 Salmonella Species 0.000 claims description 6
- 241000607720 Serratia Species 0.000 claims description 6
- 241000187747 Streptomyces Species 0.000 claims description 6
- 241000588768 Providencia Species 0.000 claims description 5
- 108091006104 gene-regulatory proteins Proteins 0.000 claims description 4
- 102000034356 gene-regulatory proteins Human genes 0.000 claims description 4
- 230000005026 transcription initiation Effects 0.000 claims description 4
- 241000807905 Corynebacterium glutamicum ATCC 14067 Species 0.000 claims description 3
- 241000588914 Enterobacter Species 0.000 claims description 3
- 102000003939 Membrane transport proteins Human genes 0.000 claims description 2
- 108090000301 Membrane transport proteins Proteins 0.000 claims description 2
- 230000009061 membrane transport Effects 0.000 claims description 2
- 230000033228 biological regulation Effects 0.000 abstract description 17
- 230000006698 induction Effects 0.000 abstract description 11
- 230000037361 pathway Effects 0.000 abstract description 10
- 108700026220 vif Genes Proteins 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 9
- 108090000790 Enzymes Proteins 0.000 abstract description 9
- 238000010276 construction Methods 0.000 abstract description 9
- 238000013537 high throughput screening Methods 0.000 abstract description 9
- 238000001514 detection method Methods 0.000 abstract description 5
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 230000001276 controlling effect Effects 0.000 abstract 1
- 235000001014 amino acid Nutrition 0.000 description 161
- 229940024606 amino acid Drugs 0.000 description 154
- 210000004027 cell Anatomy 0.000 description 57
- 235000018977 lysine Nutrition 0.000 description 53
- 235000018102 proteins Nutrition 0.000 description 45
- 238000006467 substitution reaction Methods 0.000 description 38
- 108020004414 DNA Proteins 0.000 description 25
- 239000012634 fragment Substances 0.000 description 18
- 229960002885 histidine Drugs 0.000 description 16
- 239000013598 vector Substances 0.000 description 16
- 125000000539 amino acid group Chemical group 0.000 description 15
- 239000002609 medium Substances 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 10
- 101150044424 lysE gene Proteins 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
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 description 8
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 8
- 230000001965 increasing effect Effects 0.000 description 8
- 229930027917 kanamycin Natural products 0.000 description 8
- 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 8
- 229960000318 kanamycin Drugs 0.000 description 8
- 229930182823 kanamycin A Natural products 0.000 description 8
- 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 7
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 7
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 108010064711 Homoserine dehydrogenase Proteins 0.000 description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 6
- 108700008625 Reporter Genes Proteins 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- -1 his Chemical compound 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 5
- 108010055400 Aspartate kinase Proteins 0.000 description 5
- 108010078791 Carrier Proteins Proteins 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 5
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 5
- 239000007993 MOPS buffer Substances 0.000 description 5
- 108090000854 Oxidoreductases Proteins 0.000 description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 5
- 239000004473 Threonine Substances 0.000 description 5
- 239000004202 carbamide Substances 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 5
- 229960000310 isoleucine Drugs 0.000 description 5
- 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
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 4
- 108050005273 Amino acid transporters Proteins 0.000 description 4
- 102000034263 Amino acid transporters Human genes 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 4
- 101710088194 Dehydrogenase Proteins 0.000 description 4
- 102000004316 Oxidoreductases Human genes 0.000 description 4
- 239000001110 calcium chloride Substances 0.000 description 4
- 229910001628 calcium chloride Inorganic materials 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- OJJHFKVRJCQKLN-YFKPBYRVSA-N (4s)-4-acetamido-5-oxo-5-phosphonooxypentanoic acid Chemical compound OC(=O)CC[C@H](NC(=O)C)C(=O)OP(O)(O)=O OJJHFKVRJCQKLN-YFKPBYRVSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- VBNGJYNPUFWTKX-UHFFFAOYSA-N 10,10-diamino-1,6-dioxacyclotridecane-2,5,7,13-tetrone Chemical compound NC1(CCC(=O)OC(CCC(=O)OC(CC1)=O)=O)N VBNGJYNPUFWTKX-UHFFFAOYSA-N 0.000 description 3
- 108700021045 Acetylglutamate kinase Proteins 0.000 description 3
- 101710191958 Amino-acid acetyltransferase Proteins 0.000 description 3
- 108700024106 Argininosuccinate synthases Proteins 0.000 description 3
- 108020004652 Aspartate-Semialdehyde Dehydrogenase Proteins 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 241000305071 Enterobacterales Species 0.000 description 3
- 150000008575 L-amino acids Chemical class 0.000 description 3
- 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 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- 101710113020 Ornithine transcarbamylase, mitochondrial Proteins 0.000 description 3
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- 229930003451 Vitamin B1 Natural products 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 108010056578 diaminopimelate dehydrogenase Proteins 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 108010050322 glutamate acetyltransferase Proteins 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 101150076679 lysG gene Proteins 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 238000012269 metabolic engineering Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 229960003495 thiamine Drugs 0.000 description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- 239000011691 vitamin B1 Substances 0.000 description 3
- 235000010374 vitamin B1 Nutrition 0.000 description 3
- RVLOMLVNNBWRSR-KNIFDHDWSA-N (2s)-2-aminopropanoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound C[C@H](N)C(O)=O.NCCCC[C@H](N)C(O)=O RVLOMLVNNBWRSR-KNIFDHDWSA-N 0.000 description 2
- GMKMEZVLHJARHF-UHFFFAOYSA-N 2,6-diaminopimelic acid Chemical compound OC(=O)C(N)CCCC(N)C(O)=O GMKMEZVLHJARHF-UHFFFAOYSA-N 0.000 description 2
- 108010017192 4-hydroxy-4-methyl-2-oxoglutarate aldolase Proteins 0.000 description 2
- 108091000044 4-hydroxy-tetrahydrodipicolinate synthase Proteins 0.000 description 2
- 108020001657 6-phosphogluconate dehydrogenase Proteins 0.000 description 2
- 108010049445 Acetylornithine transaminase Proteins 0.000 description 2
- 102100029589 Acylpyruvase FAHD1, mitochondrial Human genes 0.000 description 2
- WVRUNFYJIHNFKD-WDSKDSINSA-N Arg-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N WVRUNFYJIHNFKD-WDSKDSINSA-N 0.000 description 2
- 102000009042 Argininosuccinate Lyase Human genes 0.000 description 2
- 102000053640 Argininosuccinate synthases Human genes 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 102100032948 Aspartoacylase Human genes 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- FRJIAZKQGSCKPQ-FSPLSTOPSA-N His-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CN=CN1 FRJIAZKQGSCKPQ-FSPLSTOPSA-N 0.000 description 2
- 101000797251 Homo sapiens Aspartoacylase Proteins 0.000 description 2
- 108010075869 Isocitrate Dehydrogenase Proteins 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 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 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 108010048581 Lysine decarboxylase Proteins 0.000 description 2
- 108010069823 Oxaloacetate decarboxylase Proteins 0.000 description 2
- 108091000041 Phosphoenolpyruvate Carboxylase Proteins 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
- 108010053763 Pyruvate Carboxylase Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108010043652 Transketolase Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 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 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 101150035025 lysC gene Proteins 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 108010073086 succinyl-CoA-tetrahydrodipicolinate N-succinyltransferase Proteins 0.000 description 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- XVZCXCTYGHPNEM-IHRRRGAJSA-N (2s)-1-[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O XVZCXCTYGHPNEM-IHRRRGAJSA-N 0.000 description 1
- UFUQINYQINJWIL-UHFFFAOYSA-N 1,2,3,4-tetrahydropyridine-2,3-dicarboxylic acid Chemical compound OC(=O)C1CC=CNC1C(O)=O UFUQINYQINJWIL-UHFFFAOYSA-N 0.000 description 1
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 description 1
- 108010092060 Acetate kinase Proteins 0.000 description 1
- 101710165738 Acetylornithine aminotransferase Proteins 0.000 description 1
- XCVRVWZTXPCYJT-BIIVOSGPSA-N Ala-Asn-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N XCVRVWZTXPCYJT-BIIVOSGPSA-N 0.000 description 1
- XYTNPQNAZREREP-XQXXSGGOSA-N Ala-Glu-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XYTNPQNAZREREP-XQXXSGGOSA-N 0.000 description 1
- NYDBKUNVSALYPX-NAKRPEOUSA-N Ala-Ile-Arg Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CCCN=C(N)N NYDBKUNVSALYPX-NAKRPEOUSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- DEWWPUNXRNGMQN-LPEHRKFASA-N Ala-Met-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N1CCC[C@@H]1C(=O)O)N DEWWPUNXRNGMQN-LPEHRKFASA-N 0.000 description 1
- BHTBAVZSZCQZPT-GUBZILKMSA-N Ala-Pro-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N BHTBAVZSZCQZPT-GUBZILKMSA-N 0.000 description 1
- IOFVWPYSRSCWHI-JXUBOQSCSA-N Ala-Thr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)N IOFVWPYSRSCWHI-JXUBOQSCSA-N 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- OQCWXQJLCDPRHV-UWVGGRQHSA-N Arg-Gly-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O OQCWXQJLCDPRHV-UWVGGRQHSA-N 0.000 description 1
- MTYLORHAQXVQOW-AVGNSLFASA-N Arg-Lys-Met Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(O)=O MTYLORHAQXVQOW-AVGNSLFASA-N 0.000 description 1
- LQJAALCCPOTJGB-YUMQZZPRSA-N Arg-Pro Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O LQJAALCCPOTJGB-YUMQZZPRSA-N 0.000 description 1
- CPTXATAOUQJQRO-GUBZILKMSA-N Arg-Val-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O CPTXATAOUQJQRO-GUBZILKMSA-N 0.000 description 1
- 101710149879 Arginine repressor Proteins 0.000 description 1
- VILLWIDTHYPSLC-PEFMBERDSA-N Asp-Glu-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VILLWIDTHYPSLC-PEFMBERDSA-N 0.000 description 1
- QSFHZPQUAAQHAQ-CIUDSAMLSA-N Asp-Ser-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O QSFHZPQUAAQHAQ-CIUDSAMLSA-N 0.000 description 1
- JSNWZMFSLIWAHS-HJGDQZAQSA-N Asp-Thr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O JSNWZMFSLIWAHS-HJGDQZAQSA-N 0.000 description 1
- XOASPVGNFAMYBD-WFBYXXMGSA-N Asp-Trp-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O XOASPVGNFAMYBD-WFBYXXMGSA-N 0.000 description 1
- GIKOVDMXBAFXDF-NHCYSSNCSA-N Asp-Val-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GIKOVDMXBAFXDF-NHCYSSNCSA-N 0.000 description 1
- 101710113083 Carbamoyl-phosphate synthase Proteins 0.000 description 1
- VIRYODQIWJNWNU-NRPADANISA-N Cys-Glu-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)N VIRYODQIWJNWNU-NRPADANISA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010001625 Diaminopimelate epimerase Proteins 0.000 description 1
- 108010014468 Dihydrodipicolinate Reductase Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 208000000666 Fowlpox Diseases 0.000 description 1
- 101710198928 Gamma-glutamyl phosphate reductase Proteins 0.000 description 1
- YJIUYQKQBBQYHZ-ACZMJKKPSA-N Gln-Ala-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YJIUYQKQBBQYHZ-ACZMJKKPSA-N 0.000 description 1
- FNAJNWPDTIXYJN-CIUDSAMLSA-N Gln-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCC(N)=O FNAJNWPDTIXYJN-CIUDSAMLSA-N 0.000 description 1
- UTKUTMJSWKKHEM-WDSKDSINSA-N Glu-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O UTKUTMJSWKKHEM-WDSKDSINSA-N 0.000 description 1
- BPDVTFBJZNBHEU-HGNGGELXSA-N Glu-Ala-His Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 BPDVTFBJZNBHEU-HGNGGELXSA-N 0.000 description 1
- WIKMTDVSCUJIPJ-CIUDSAMLSA-N Glu-Ser-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WIKMTDVSCUJIPJ-CIUDSAMLSA-N 0.000 description 1
- MWTGQXBHVRTCOR-GLLZPBPUSA-N Glu-Thr-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MWTGQXBHVRTCOR-GLLZPBPUSA-N 0.000 description 1
- KIEICAOUSNYOLM-NRPADANISA-N Glu-Val-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O KIEICAOUSNYOLM-NRPADANISA-N 0.000 description 1
- VIPDPMHGICREIS-GVXVVHGQSA-N Glu-Val-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O VIPDPMHGICREIS-GVXVVHGQSA-N 0.000 description 1
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 description 1
- 102000005731 Glucose-6-phosphate isomerase Human genes 0.000 description 1
- 102000005133 Glutamate 5-kinase Human genes 0.000 description 1
- 108010016106 Glutamate-5-semialdehyde dehydrogenase Proteins 0.000 description 1
- LJPIRKICOISLKN-WHFBIAKZSA-N Gly-Ala-Ser Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O LJPIRKICOISLKN-WHFBIAKZSA-N 0.000 description 1
- JRDYDYXZKFNNRQ-XPUUQOCRSA-N Gly-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN JRDYDYXZKFNNRQ-XPUUQOCRSA-N 0.000 description 1
- UPOJUWHGMDJUQZ-IUCAKERBSA-N Gly-Arg-Arg Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UPOJUWHGMDJUQZ-IUCAKERBSA-N 0.000 description 1
- 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 1
- MHXKHKWHPNETGG-QWRGUYRKSA-N Gly-Lys-Leu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O MHXKHKWHPNETGG-QWRGUYRKSA-N 0.000 description 1
- IGOYNRWLWHWAQO-JTQLQIEISA-N Gly-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IGOYNRWLWHWAQO-JTQLQIEISA-N 0.000 description 1
- BXDLTKLPPKBVEL-FJXKBIBVSA-N Gly-Thr-Met Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(O)=O BXDLTKLPPKBVEL-FJXKBIBVSA-N 0.000 description 1
- PYFIQROSWQERAS-LBPRGKRZSA-N Gly-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)CN)C(=O)NCC(O)=O)=CNC2=C1 PYFIQROSWQERAS-LBPRGKRZSA-N 0.000 description 1
- CGAMSLMBYJHMDY-ONGXEEELSA-N His-Val-Gly Chemical compound CC(C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N CGAMSLMBYJHMDY-ONGXEEELSA-N 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- QADCTXFNLZBZAB-GHCJXIJMSA-N Ile-Asn-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C)C(=O)O)N QADCTXFNLZBZAB-GHCJXIJMSA-N 0.000 description 1
- IDAHFEPYTJJZFD-PEFMBERDSA-N Ile-Asp-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N IDAHFEPYTJJZFD-PEFMBERDSA-N 0.000 description 1
- GYAFMRQGWHXMII-IUKAMOBKSA-N Ile-Asp-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N GYAFMRQGWHXMII-IUKAMOBKSA-N 0.000 description 1
- KUHFPGIVBOCRMV-MNXVOIDGSA-N Ile-Gln-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N KUHFPGIVBOCRMV-MNXVOIDGSA-N 0.000 description 1
- HPCFRQWLTRDGHT-AJNGGQMLSA-N Ile-Leu-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O HPCFRQWLTRDGHT-AJNGGQMLSA-N 0.000 description 1
- IVXJIMGDOYRLQU-XUXIUFHCSA-N Ile-Pro-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O IVXJIMGDOYRLQU-XUXIUFHCSA-N 0.000 description 1
- JNLSTRPWUXOORL-MMWGEVLESA-N Ile-Ser-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N JNLSTRPWUXOORL-MMWGEVLESA-N 0.000 description 1
- RQZFWBLDTBDEOF-RNJOBUHISA-N Ile-Val-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N RQZFWBLDTBDEOF-RNJOBUHISA-N 0.000 description 1
- 235000019766 L-Lysine Nutrition 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
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 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
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 1
- NTRAGDHVSGKUSF-AVGNSLFASA-N Leu-Arg-Arg Chemical compound CC(C)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 NTRAGDHVSGKUSF-AVGNSLFASA-N 0.000 description 1
- HFBCHNRFRYLZNV-GUBZILKMSA-N Leu-Glu-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HFBCHNRFRYLZNV-GUBZILKMSA-N 0.000 description 1
- IWTBYNQNAPECCS-AVGNSLFASA-N Leu-Glu-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IWTBYNQNAPECCS-AVGNSLFASA-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
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 1
- ZRHDPZAAWLXXIR-SRVKXCTJSA-N Leu-Lys-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O ZRHDPZAAWLXXIR-SRVKXCTJSA-N 0.000 description 1
- RGUXWMDNCPMQFB-YUMQZZPRSA-N Leu-Ser-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RGUXWMDNCPMQFB-YUMQZZPRSA-N 0.000 description 1
- MVHXGBZUJLWZOH-BJDJZHNGSA-N Leu-Ser-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MVHXGBZUJLWZOH-BJDJZHNGSA-N 0.000 description 1
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- JCFYLFOCALSNLQ-GUBZILKMSA-N Lys-Ala-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JCFYLFOCALSNLQ-GUBZILKMSA-N 0.000 description 1
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- IHITVQKJXQQGLJ-LPEHRKFASA-N Met-Asn-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N IHITVQKJXQQGLJ-LPEHRKFASA-N 0.000 description 1
- 101000859568 Methanobrevibacter smithii (strain ATCC 35061 / DSM 861 / OCM 144 / PS) Carbamoyl-phosphate synthase Proteins 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 1
- 108010001609 NADP Transhydrogenases Proteins 0.000 description 1
- 102000004132 Ornithine aminotransferases Human genes 0.000 description 1
- 102000007981 Ornithine carbamoyltransferase Human genes 0.000 description 1
- NPLGQVKZFGJWAI-QWHCGFSZSA-N Phe-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O NPLGQVKZFGJWAI-QWHCGFSZSA-N 0.000 description 1
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 1
- 108700023175 Phosphate acetyltransferases Proteins 0.000 description 1
- JFBJPBZSTMXGKL-JYJNAYRXSA-N Pro-Met-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JFBJPBZSTMXGKL-JYJNAYRXSA-N 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 101100217185 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) aruC gene Proteins 0.000 description 1
- 102000004518 Pyrroline-5-carboxylate reductase Human genes 0.000 description 1
- 108010042687 Pyruvate Oxidase Proteins 0.000 description 1
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 108020000772 Ribose-Phosphate Pyrophosphokinase Proteins 0.000 description 1
- 102000000439 Ribose-phosphate pyrophosphokinase Human genes 0.000 description 1
- SRTCFKGBYBZRHA-ACZMJKKPSA-N Ser-Ala-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SRTCFKGBYBZRHA-ACZMJKKPSA-N 0.000 description 1
- HBZBPFLJNDXRAY-FXQIFTODSA-N Ser-Ala-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O HBZBPFLJNDXRAY-FXQIFTODSA-N 0.000 description 1
- OYEDZGNMSBZCIM-XGEHTFHBSA-N Ser-Arg-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OYEDZGNMSBZCIM-XGEHTFHBSA-N 0.000 description 1
- RNMRYWZYFHHOEV-CIUDSAMLSA-N Ser-Gln-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O RNMRYWZYFHHOEV-CIUDSAMLSA-N 0.000 description 1
- BUYHXYIUQUBEQP-AVGNSLFASA-N Ser-Phe-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CO)N BUYHXYIUQUBEQP-AVGNSLFASA-N 0.000 description 1
- OJFFAQFRCVPHNN-JYBASQMISA-N Ser-Thr-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O OJFFAQFRCVPHNN-JYBASQMISA-N 0.000 description 1
- 108010009197 Succinyldiaminopimelate transaminase Proteins 0.000 description 1
- 101100022072 Sulfolobus acidocaldarius (strain ATCC 33909 / DSM 639 / JCM 8929 / NBRC 15157 / NCIMB 11770) lysJ gene Proteins 0.000 description 1
- UKBSDLHIKIXJKH-HJGDQZAQSA-N Thr-Arg-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O UKBSDLHIKIXJKH-HJGDQZAQSA-N 0.000 description 1
- XOWKUMFHEZLKLT-CIQUZCHMSA-N Thr-Ile-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O XOWKUMFHEZLKLT-CIQUZCHMSA-N 0.000 description 1
- AMXMBCAXAZUCFA-RHYQMDGZSA-N Thr-Leu-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AMXMBCAXAZUCFA-RHYQMDGZSA-N 0.000 description 1
- 102100033451 Thyroid hormone receptor beta Human genes 0.000 description 1
- 102000014701 Transketolase Human genes 0.000 description 1
- PXYJUECTGMGIDT-WDSOQIARSA-N Trp-Arg-Leu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(C)C)C(O)=O)=CNC2=C1 PXYJUECTGMGIDT-WDSOQIARSA-N 0.000 description 1
- XZLHHHYSWIYXHD-XIRDDKMYSA-N Trp-Gln-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O XZLHHHYSWIYXHD-XIRDDKMYSA-N 0.000 description 1
- JVTHMUDOKPQBOT-NSHDSACASA-N Trp-Gly-Gly Chemical compound C1=CC=C2C(C[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O)=CNC2=C1 JVTHMUDOKPQBOT-NSHDSACASA-N 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 1
- ROLGIBMFNMZANA-GVXVVHGQSA-N Val-Glu-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N ROLGIBMFNMZANA-GVXVVHGQSA-N 0.000 description 1
- OTJMMKPMLUNTQT-AVGNSLFASA-N Val-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N OTJMMKPMLUNTQT-AVGNSLFASA-N 0.000 description 1
- RWOGENDAOGMHLX-DCAQKATOSA-N Val-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N RWOGENDAOGMHLX-DCAQKATOSA-N 0.000 description 1
- NZGOVKLVQNOEKP-YDHLFZDLSA-N Val-Phe-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N NZGOVKLVQNOEKP-YDHLFZDLSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 101150006213 ackA gene Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 101150014383 adhE gene Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- VFRROHXSMXFLSN-SLPGGIOYSA-N aldehydo-D-glucose 6-phosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O VFRROHXSMXFLSN-SLPGGIOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229960002749 aminolevulinic acid Drugs 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 101150008194 argB gene Proteins 0.000 description 1
- 101150070427 argC gene Proteins 0.000 description 1
- 101150089042 argC2 gene Proteins 0.000 description 1
- 101150050866 argD gene Proteins 0.000 description 1
- 101150056313 argF gene Proteins 0.000 description 1
- 101150118463 argG gene Proteins 0.000 description 1
- 101150054318 argH gene Proteins 0.000 description 1
- 101150029940 argJ gene Proteins 0.000 description 1
- 101150089004 argR gene Proteins 0.000 description 1
- 108010091818 arginyl-glycyl-aspartyl-valine Proteins 0.000 description 1
- 108010094001 arginyl-tryptophyl-arginine Proteins 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 101150107204 asd gene Proteins 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 101150008667 cadA gene Proteins 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
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 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
- 239000000470 constituent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 101150011371 dapA gene Proteins 0.000 description 1
- 101150073654 dapB gene Proteins 0.000 description 1
- 101150064923 dapD gene Proteins 0.000 description 1
- 101150000582 dapE gene Proteins 0.000 description 1
- 101150057904 ddh gene Proteins 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
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 101150077334 focA gene Proteins 0.000 description 1
- 108010008221 formate C-acetyltransferase Proteins 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010077435 glycyl-phenylalanyl-glycine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 108010028295 histidylhistidine Proteins 0.000 description 1
- 108010071598 homoserine kinase Proteins 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 101150041134 ldcC gene Proteins 0.000 description 1
- 101150041530 ldha gene Proteins 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
- 101150094164 lysY gene Proteins 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 101150111581 pflB gene Proteins 0.000 description 1
- 101150053253 pgi gene Proteins 0.000 description 1
- 108010071189 phosphoenolpyruvate-glucose phosphotransferase Proteins 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 101150000475 pntAB gene Proteins 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 101150060030 poxB gene Proteins 0.000 description 1
- 101150023641 ppc gene Proteins 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 101150070305 prsA gene Proteins 0.000 description 1
- 208000009305 pseudorabies Diseases 0.000 description 1
- 101150108780 pta gene Proteins 0.000 description 1
- 108020001898 pyrroline-5-carboxylate reductase Proteins 0.000 description 1
- 230000018612 quorum sensing Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 101150014006 thrA gene Proteins 0.000 description 1
- 101150072448 thrB gene Proteins 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 108091008023 transcriptional regulators Proteins 0.000 description 1
- 230000014621 translational initiation 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
- 108700004896 tripeptide FEG Proteins 0.000 description 1
- 108010058119 tryptophyl-glycyl-glycine Proteins 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- UZVNCLCLJHPHIF-NOJKMYKQSA-J zinc;(1e)-2-(ethylcarbamoylamino)-n-methoxy-2-oxoethanimidoyl cyanide;manganese(2+);n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[Zn+2].[S-]C(=S)NCCNC([S-])=S.[S-]C(=S)NCCNC([S-])=S.CCNC(=O)NC(=O)C(\C#N)=N\OC UZVNCLCLJHPHIF-NOJKMYKQSA-J 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The disclosure belongs to the field of genetic engineering and molecular biology, and relates to a mutant of a transcriptional regulatory factor LysG, a polynucleotide encoding the mutant, a nucleic acid construct, a recombinant expression vector, a recombinant host cell, a biosensor, application, a method for regulating and controlling expression of a target gene, a method for producing basic amino acid, a basic amino acid high-yield strain and a screening method for synthesizing a protein or a protein encoding gene with the basic amino acid. The mutant of the transcription regulation and control factor LysG in the disclosure is a mutant of the transcription regulation and control factor LysG after mutation of an EBD structural domain, has improved strength of response to induction of basic amino acid, can realize high-sensitivity detection of basic amino acid high-yield strains and proteins related to synthesis of the basic amino acid or encoding genes thereof, and has important significance for high-throughput screening of the basic amino acid high-yield strains and proteins related to synthesis pathways and expression regulation and control of key enzymes in construction of the basic amino acid high-yield strains.
Description
Technical Field
The disclosure belongs to the field of genetic engineering and molecular biology, and particularly relates to a mutant of a transcription regulatory factor LysG, a polynucleotide for encoding the mutant, a nucleic acid construct, a recombinant expression vector, a recombinant host cell, a biosensor, application, a method for regulating expression of a target gene, a method for producing basic amino acid, a basic amino acid high-yield strain and a screening method for synthesizing a related protein or a protein encoding gene by the basic amino acid.
Background
The LysR family of transcription regulators (LysR-type transcription regulators, LLTRs) are widely distributed in various species of microorganisms, and the regulated genes have various functions, and are involved in anabolism, quorum sensing, virulence, and the like [1] . Along with biological informationWith the development of science and protein structure, more and more LysR type transcription regulators are discovered. To date, the LysR family of transcriptional regulators has become the largest family of transcriptional factors in prokaryotes.
LysG identified in Corynebacterium glutamicum belongs to a typical LysR family transcription regulatory factor, a Helix-turn-Helix motif (HTH) combined with DNA exists at the N end, a region (EBD) combined with an Effector exists at the C end, and the middle part is connected with the HTH through flexible Linker Helix [2] . The transcription regulation factor LysG can respond to three intracellular basic amino acids (lysine, arginine and histidine), and can regulate and control the lysE promoter to further activate the expression of the downstream transport protein LysE so as to maintain the balance of the intracellular basic amino acids [3] 。
Due to the sensing characteristics of the transcription regulatory factor, the current biosensors based on the transcription regulatory factor are widely used for detecting the concentration of intracellular small molecule metabolites and high-throughput screening of high-yield strains of the small molecule metabolites, such as high-throughput screening of high-yield strains of amino acids and key enzymes of the synthesis pathway thereof [3 -5]. However, biosensors based on natural transcription regulators often have low response intensity to small molecules, and are difficult to apply to high-throughput screening of high-producing strains and key enzymes of synthetic pathways. Therefore, how to modify the natural transcription regulatory factor and optimize the response strength of the biosensor is an important problem to be solved urgently.
The transcription regulation factor LysG can regulate the expression of a downstream target gene-amino acid transporter through a lysE promoter [6] The transcription regulation and control effect generated by the response of the basic amino acid has important significance for improving the regulation and control performance of the response element. However, the wild type LysG has low response strength to three basic amino acids, has limited transcriptional activation effect on downstream amino acid transporters and other regulatory proteins, cannot be used for gene expression regulation in a wider range, and cannot be used for constructing a biosensor with high sensitivity.
Cited documents:
[1]Maddocks,S.E.and Oyston,P.C.F.(2008)Structure and function of the LysR-type transcriptional regulator(LTTR)family proteins.Microbiology,154,3609-3623.
[2]Della Corte,D.,van Beek,H.L.,Syberg,F.,Schallmey,M.,Tobola,F.,Cormann,K.U.,Schlicker,C.,Baumann,P.T.,Krumbach,K.,Sokolowsky,S.et al.(2020)Engineering and application of a biosensor with focused ligand specificity.Nat.Commun.,11,4851.
[3]Binder,S.,et al.(2012)A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.Genome biology,13,R40.
[4]Kortmann,M.,Mack,C.,Baumgart,M.and Bott,M.(2019)Pyruvate carboxylase variants enabling improved lysine production from glucose identified by biosensor-based high-throughput fluorescence-activated cell sorting screening.ACS Synth.Biol.,8,274-281.
[5]Schendzielorz,G.,et al.(2014)Taking control over control:use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways.ACS Synth.Biol.,3,21-29.
[6]Zhou LB,Zeng AP.(2015)Engineering a lysine-ON riboswitch for metabolic control of lysine production in Corynebacterium glutamicum.ACS Synth.Biol.,4,1335-1340.
disclosure of Invention
Problems to be solved by the invention
In view of the problems in the prior art, for example, the wild type LysG has low response strength to basic amino acids, cannot efficiently regulate the expression of target genes, and cannot construct a biosensor with high sensitivity. Therefore, the polypeptide with the activity of the transcription regulatory factor is provided, and is a mutant of the transcription regulatory factor LysG, compared with the wild type transcription regulatory factor LysG, the mutant has improved strength of response to induction of basic amino acids, can realize high-sensitivity detection of a biosensor on basic amino acid high-yield strains and protein coding genes related to synthesis of the basic amino acids, and has important significance for high-throughput screening of the basic amino acid high-yield strains and proteins related to synthesis pathways and expression regulation of key enzymes in construction of the basic amino acid high-yield strains.
Means for solving the problems
(1) A mutant of the transcriptional regulator LysG having one or more mutated amino acids located in the EBD domain; and, the mutant has improved basic amino acid response strength compared to the wild-type transcription regulatory factor LysG.
(2) The mutant of the transcription regulatory factor LysG according to (1), wherein the mutant is selected from any one of the following groups (i) to (iii):
(i) Comprises a nucleotide sequence as set forth in SEQ ID NO:1, which mutant has a sequence which does not substantially hybridize to a polypeptide corresponding to SEQ ID NO:1 with a mutated amino acid at one or more positions from position 111 to position 270;
(ii) (ii) a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the amino acid sequence set forth in (i);
(iii) (iii) a polypeptide having an amino acid sequence as shown in (i) or (ii) in which one or more amino acids are added or deleted at least one of the N-terminus and the C-terminus.
(3) The mutant of the transcription regulatory factor LysG according to (1) or (2), wherein the mutant corresponds to the amino acid sequence of SEQ ID NO:1, having mutated amino acids at one or more of the following positions:
143, 146, 125, 133, 136, 141, 152, 165, 169, 177, 194, 198, 209, 212, 241, 248, 255, 259, 260, 266, 269, 270, 111, 115.
Preferably, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, or a variant thereof, and at least 1, at least 2, or at least 3 positions of the sequence set forth in 1.
(4) The mutant of the transcriptional regulatory factor LysG according to any one of (1) to (3), wherein the mutant corresponds to the nucleotide sequence of SEQ ID NO:1, mutated amino acids having one or more of the following:
R143C、N146D、E125V、R133Q、D136N、V141I、E152G、A165T、L169M、G177E、V194A、R198H、G209E、R212H、A241V、E248G、A255S、I259M、P260S、W266C、E269G、S270P、A111P、G115R。
(5) The mutant of the transcriptional regulatory factor LysG according to any one of (1) to (4), wherein the mutant corresponds to the nucleotide sequence of SEQ ID NO:1, having the following sequence (m) 1 )-(m 20 ) Any one of the mutated amino acids:
(m 1 )R143C；
(m 2 )N146D；
(m 3 )E248G；
(m 4 ) E125V and a165T;
(m 5 )G115R；
(m 6 )S270P；
(m 7 ) E125V and G209E;
(m 8 )V194A；
(m 9 )R212H；
(m 10 ) E152G and A241V;
(m 11 )V141I；
(m 12 )R198H；
(m 13 ) A255S and W266C;
(m 14 ) E248G, P260S and E269G;
(m 15 )I259M；
(m 16 )A111P；
(m 17 ) D136N and L169M;
(m 18 ) R143C and G177E;
(m 19 )R133Q；
(m 20 ) D136N and L169M.
(6) The mutant of the transcriptional regulatory factor LysG according to any one of (1) to (5), wherein the mutant has a mutation in comparison with the mutant of SEQ ID NO:1, has 1.2-3.8 times of improved basic amino acid response intensity compared with the polypeptide of the sequence shown in 1.
(7) An isolated polynucleotide, wherein said polynucleotide encodes a mutant of the transcriptional regulator LysG of any one of (1) to (6).
(8) A nucleic acid construct, wherein the nucleic acid construct comprises the polynucleotide of (7) operably linked to a LysG-regulated promoter that directs expression of a polypeptide.
(9) A recombinant expression vector, wherein said recombinant expression vector comprises the polynucleotide of (7), or the nucleic acid construct of (7).
(10) A recombinant host cell comprising a mutant of the transcriptional regulator LysG of any one of (1) to (6), the polynucleotide of (7), the nucleic acid construct of (8), or the recombinant expression vector of (9).
(11) The recombinant host cell according to (10), wherein the host cell is derived from a microorganism of the genus Escherichia (Escherichia), erwinia (Erwinia), serratia (Serratia), providencia (Providencia), enterobacteria (Enterobacteria), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium) or Corynebacterium (Corynebacterium);
preferably, the host cell is the corynebacterium glutamicum ATCC13032, the corynebacterium glutamicum ATCC13869, the corynebacterium glutamicum ATCC 14067 strain, or a derivative of any of the foregoing.
(12) A biosensor comprising a mutant of the transcriptional regulator LysG according to any one of (1) to (6), or a polynucleotide according to (7), a nucleic acid construct according to (8), a recombinant expression vector according to (9), or a polypeptide expressed by a recombinant host cell according to any one of (10) to (11).
(13) Use of a mutant of the transcriptional regulatory factor LysG according to any one of (1) to (6), a polynucleotide according to (7), a nucleic acid construct according to (8), a recombinant expression vector according to (9), or a recombinant host cell according to any one of (10) to (11) in at least one of the following (a) to (d):
(a) Regulating the transcription level of a target gene, or preparing a reagent or a kit for regulating the transcription level of a target gene;
(b) Producing basic amino acids, or preparing reagents or kits for producing basic amino acids;
(c) Screening basic amino acid high-producing strains, or preparing a reagent, a kit or a biosensor for screening the basic amino acid high-producing strains;
(d) Screening proteins or protein coding genes related to the synthesis of basic amino acids, or preparing a reagent, a kit or a biosensor for screening proteins or protein coding genes related to the synthesis of basic amino acids.
(14) A method for regulating the expression of a target gene, wherein the method comprises the step of operably linking the polynucleotide according to (7) to a transcription initiation sequence, the target gene; preferably, the transcription initiation sequence is a polynucleotide having a LysG-regulated promoter activity. Optionally, the target gene includes at least one of a gene encoding a protein associated with basic amino acid synthesis, a gene encoding a gene expression regulatory protein, and a gene encoding a protein associated with membrane transport.
(15) A method for producing a basic amino acid, which comprises the step of producing a basic amino acid in the presence of the mutant of the transcription regulatory factor LysG according to any one of (1) to (6), the polynucleotide according to (7), the nucleic acid construct according to (8), and the recombinant expression vector according to (9); alternatively, the method comprises the step of producing a basic amino acid using the recombinant host cell according to any one of (10) to (11);
optionally, the method further comprises the step of isolating or purifying the basic amino acid.
(16) A method for screening a basic amino acid highly productive strain, wherein said method comprises the step of screening a basic amino acid highly productive strain using the mutant of the transcription regulatory factor LysG according to any one of (1) to (6), the polynucleotide according to (7), the nucleic acid construct according to (8), the recombinant expression vector according to (9), the recombinant host cell according to any one of (10) to (11), or the biosensor according to (12).
(17) A method for screening a protein or a gene encoding a protein involved in basic amino acid synthesis, which comprises screening a protein or a gene encoding a protein involved in basic amino acid synthesis using the mutant of the transcriptional regulator LysG according to any one of (1) to (6), the polynucleotide according to (7), the nucleic acid construct according to (8), the recombinant expression vector according to (9), the recombinant host cell according to any one of (10) to (11), or the biosensor according to (12).
ADVANTAGEOUS EFFECTS OF INVENTION
In some embodiments, the mutant of the transcriptional regulatory factor LysG provided by the present disclosure, which is obtained by mutating the EBD domain by the transcriptional regulatory factor LysG, has a significantly increased strength of response to induction of basic amino acids compared to the wild-type transcriptional regulatory factor LysG; the mutant is used for screening the basic amino acid high-yield strain and dominant mutation of protein and protein coding genes related to basic amino acid synthesis, and can effectively improve the sensitivity of screening the basic amino acid high-yield strain or protein and protein coding genes related to basic amino acid synthesis; on the other hand, the LysG mutant and the promoter element regulated by the LysG mutant can also be used for inducing expression of a target gene of a basic amino acid anabolism pathway, and have important significance for constructing a basic amino acid high-yield strain, a basic amino acid high-yield strain and high-throughput screening of synthetic pathway related proteins.
In some embodiments, the mutant of the transcriptional regulatory factor LysG provided by the present disclosure has an enhanced transcriptional activation effect on a LysG-regulated promoter due to its increased strength in response to basic amino acids, and the mutant of LysG and the promoter regulated thereby can activate expression of a key gene in a synthesis pathway to construct an amino acid high-producing strain, thereby effectively increasing the yield of a target amino acid.
In some embodiments, the polynucleotides, nucleic acid constructs, recombinant expression vectors provided by the present disclosure may express mutants of the transcriptional regulator LysG with increased basic amino acid response strength, be used for high throughput screening of basic amino acid production strains and proteins related to basic amino acid synthesis, or be used to increase the yield of amino acids.
In some embodiments, the recombinant host cell provided by the present disclosure, which comprises a mutant of the transcriptional regulatory factor LysG, or a polynucleotide, a nucleic acid construct, or a recombinant expression vector encoding the mutant, can effectively increase the yield and the conversion rate of the target amino acid, and realize large-scale industrial production of the target amino acid.
In some embodiments, the biosensor provided by the present disclosure has a mutant of the transcriptional regulatory factor LysG with increased basic amino acid response strength, and the biosensor has increased strength in screening of basic amino acid high-producing strains or basic amino acid synthesis-related protein coding genes, and can realize high-throughput industrial screening, and on the other hand, can also be used for expression regulation of basic amino acid anabolism pathway key genes to construct efficient basic amino acid high-producing strains.
Drawings
FIG. 1 shows the results of the induced responses of LysG and its mutants to three basic amino acids.
FIG. 2 shows the effect of regulating expression of lysE by LysG and its mutants on lysine accumulation.
Detailed Description
Definition of
The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.
As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Throughout this application, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.
Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.
As used in the claims or specification, the term "range of values" is selected/preferred to include both the end points of the range and all natural numbers subsumed in the middle of the end points with respect to the preceding end points of the range.
As used in this disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and are polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
As used in this disclosure, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, can be isolated from a source in nature, and is not intentionally modified by man in the laboratory, is naturally occurring. As used in this disclosure, "naturally occurring" and "wild-type" are synonyms.
As used in this disclosure, the term "transcriptional regulator" is a class of proteins that control the transcription of DNA into RNA, the first stage of gene expression. Which regulates the transcription process of a target gene by specifically binding to a DNA sequence of a regulatory region. In some embodiments, the transcription regulatory factor is a transcription regulatory factor LysG belonging to a typical LysR family, which has a Helix-turn-Helix motif (HTH) bound to DNA at the N-terminus and an Effector Binding Domain (EBD) at the C-terminus, and is linked to the middle by a flexible Linker Helix. Illustratively, the DNA sequence of the regulatory region which binds to LysG is a promoter sequence of the gene lysE encoding the basic amino acid transporter LysE, or a promoter sequence whose transcription is regulated by LysG in any other type.
As used in this disclosure, the term "response strength" refers to the level of transcription at which a transcriptional regulator initiates transcription of a target gene upon induction of an amino acid. In some embodiments, the response intensity characterization of the transcription regulatory factor induced by a specific amino acid is realized by connecting a fluorescent reporter gene to the downstream of the transcription regulatory factor and detecting the intensity of a fluorescent signal under the induction action of the specific amino acid.
As used in the present disclosure, the term "LysG-regulated promoter" refers to a promoter that binds to LysG and whose transcriptional activity is regulated by LysG. In some embodiments, the LysG-regulated promoter is not particularly limited as long as its activity is regulated by LysG. Illustratively, the lysG-regulated promoter may be a wild-type lysE promoter, and includes promoters that are engineered to remain under the regulation of LysG, and also includes unknown or unidentified promoters under the regulation of LysG.
The transcription regulatory factor LysG in the present disclosure is not particularly limited as long as it has a corresponding transcription regulatory activity. In some embodiments, the LysG is derived from a microorganism of the genus corynebacterium, and further, the LysG is derived from corynebacterium glutamicum. Illustratively, lysG derived from corynebacterium glutamicum has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence substantially identical to SEQ ID NO:1, having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity (including all ranges and percentages between these values).
As used in this disclosure, the term "mutant" refers to a polynucleotide or polypeptide that comprises an alteration (i.e., a substitution, insertion, and/or deletion) at one or more (e.g., several) positions relative to a "wild-type," or "comparable" polynucleotide or polypeptide, wherein a substitution refers to the replacement of a nucleotide or amino acid that occupies a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid that occupies a position. Insertion refers to the addition of nucleotides or amino acids next to and immediately following the nucleotide or amino acid occupying a position.
As used in this disclosure, the term "mutated amino acid" includes "amino acid that is substituted, repeated, deleted, or added with one or more". In the present disclosure, the term "mutation" refers to a change in the amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".
The mutant of the transcription regulatory factor LysG in the present disclosure is a mutant obtained by mutating the EBD domain of the transcription regulatory factor LysG, and has improved basic amino acid response strength as compared with the wild-type transcription regulatory factor LysG. Further, a mutant of the transcriptional regulator LysG has a sequence corresponding to SEQ ID NO:1 at one or more positions of 111-270 in the EBD region of the transcriptional regulatory factor LysG, wherein the mutation of one or more amino acids thereof results in the mutant having the transcriptional regulatory activity of the transcriptional regulatory factor LysG and having a significantly increased strength of induced response to basic amino acids (lysine, histidine or arginine). In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, and substituted amino acids at one or more positions 111-270 of the sequence shown in 1.
In the present disclosure, "mutations" may also be comprised in the sequences corresponding to SEQ ID NOs: 1, or an amino acid which does not affect the activity of the transcription regulatory factor at one or more positions of the sequence shown in 1. It is well known that the alteration of a few amino acid residues in certain regions, e.g., non-essential regions, of a polypeptide does not substantially alter the biological activity, e.g., the sequence resulting from appropriate substitutions, additions or deletions of certain amino acids does not affect its activity. Illustratively, a "mutation" of the present disclosure is contained in the corresponding SEQ ID NO:1, and at least one amino acid residue at least one of the C-terminal and N-terminal ends of the polypeptide, and the polypeptide has a transcriptional regulator activity. In some embodiments, a "mutation" of the present disclosure corresponds to a sequence as set forth in SEQ ID NO:1, from the N-terminus or C-terminus, from which 1 to 15 amino acids, preferably 1 to 10, more preferably 1 to 6, amino acids are deleted or added, and has a transcription regulatory factor activity.
In some embodiments, a "mutation" of the present disclosure may be selected from a "conservative mutation". In the present disclosure, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. A representative example of a conservative mutation is a conservative substitution.
As used in this disclosure, the term "conservative substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branches (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).
As used in this disclosure, "conservative substitutions" typically exchange one amino acid at one or more positions in a protein. Such substitutions may be conservative. Examples of substitutions regarded as conservative substitutions include substitutions of Ala with Ser or Thr, arg with Gln, his or Lys, substitution of Asn with Glu, gln, lys, his or Asp, substitution of Asp with Asn, glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, glu, lys, his, asp or Arg, substitution of Glu with Gly, asn, gln, lys or Asp, substitution of Gly with Pro, substitution of His with Asn, lys, gln, arg or Tyr, substitution of Ile with Leu, met, val or Phe, substitution of Leu with Ile, met, val or Phe, substitution of Lys with Asn, glu, his or Arg, substitution of Met with Ile, leu, val or Phe, substitution of Phe with Trp, tyr, val, ile or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe, tyr, val with Phe, or Phe, and substitution of Met with Phe, met, and Met. Furthermore, conservative mutations include naturally occurring mutations due to individual differences in the origin of the gene, differences in strain, species, and the like.
As used in this disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments, or may be a component of a larger nucleotide sequence structure, derived from nucleotide sequences that have been isolated at least once in quantity or concentration, and which are capable of being recognized, manipulated, and recovered in sequence, and their component nucleotide sequences, by standard molecular biology methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (either individual fragments or whole fragments), or may be a component or constituent of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.
As used in this disclosure, the terms "sequence identity" and "percent identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides may be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides containing different nucleotide or amino acid residues. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or deleted nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or both polynucleotides). Polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or deleting amino acids (i.e., amino acid insertions or amino acid deletions in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
In some embodiments, two or more sequences or subsequences have "sequence identity" or "percent identity" of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotides when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the biopolymers (e.g., polynucleotides) being compared.
The term "corresponding to" as used herein has the meaning commonly understood by a person of ordinary skill in the art. Specifically, "corresponding to" means the position of one sequence corresponding to a specified position in the other sequence after alignment of the two sequences by homology or sequence identity. Thus, for example, in the case of "amino acid residue corresponding to position 150 of the amino acid sequence shown in SEQ ID No. 1", if a 6 XHis tag is added to the terminus of the amino acid sequence shown in SEQ ID No. 1, position 150 of the resulting mutant corresponding to the amino acid sequence shown in SEQ ID No. 1 may be position 156.
As used in this disclosure, the term "expression" includes any step involving RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
As used in this disclosure, the term "nucleic acid construct" is a recombinant expression element comprising a polynucleotide having a sequence encoding a transcription factor mutant polypeptide. In some embodiments, a LysG-regulated promoter is also included in the nucleic acid construct. By "operably linked" to a LysG-regulated promoter, it is meant that the polynucleotide encoding the transcription factor mutant polypeptide is functionally linked to the LysG-regulated promoter to initiate and mediate transcription of a target gene, and that the operably linking can be by any means described by one of skill in the art.
As used in this disclosure, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable control sequences for the expression of a gene of interest in a suitable host. "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors can include, for example, vectors comprising i) a collection of genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) transcriptional subunits of appropriate transcriptional and translational initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not critical and any vector may be used, including plasmids, viruses, phages and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies. In the present disclosure, "recombinant expression vector" and "recombinant vector" may be used interchangeably.
The term "protein-encoding gene" in the present disclosure refers to a synthetic DNA molecule capable of directing a protein through a certain rule, and the process of directing protein synthesis by a protein-encoding gene generally includes a transcription process using double-stranded DNA as a template and a translation process using mRNA as a template. The protein-encoding gene contains a CDS Sequence (Coding Sequence) capable of directing the production of mRNA encoding the protein.
Illustratively, the protein coding gene is a gene coding for a protein involved in the synthesis of a basic amino acid.
In an exemplary manner, the first and second electrodes are, the protein-encoding gene may be a pyruvate carboxylase gene, a phosphoenolpyruvate carboxylase gene, a gamma-glutamyl kinase gene, a glutamate semialdehyde dehydrogenase gene, a pyrroline-5-carboxylate reductase gene, an amino acid transporter gene, a PtsG system-related gene, a pyruvate dehydrogenase gene, a homoserine dehydrogenase gene, an oxaloacetate decarboxylase gene, a gluconate repressor gene, a glucose dehydrogenase gene, an aspartokinase gene, an aspartate semialdehyde dehydrogenase gene, an aspartate ammonia lyase gene, a dihydrodipicolinate synthase gene, a dihydropicolinate reductase gene, a succinyldiaminopimelate aminotransferase gene, a tetrahydropyridinedicarboxylate succinylase gene, a succinyldiaminopimelate deacylase gene, a diaminopimelate epimerase gene, diaminopimelate deacylase gene, glyceraldehyde-3-phosphate dehydrogenase gene, transketolase gene, diaminopimelate dehydrogenase gene, N-acetylglutamylphosphate reductase gene, ornithine acetyltransferase gene, N-acetylglutamate kinase gene, acetylornithine transaminase gene, ornithine carbamoyl transferase gene, argininosuccinate synthetase gene, argininosuccinate lyase gene and carbamoyl phosphate synthase gene, glyceraldehyde-3-phosphate dehydrogenase gene, isocitrate dehydrogenase gene, malate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, glucose-6-phosphate isomerase gene and pyridine nucleotide transhydrogenase gene.
The term "host cell" in the present disclosure means any cell type susceptible to use comprising a mutant of the transcriptional regulatory factor LysG of the present disclosure, or comprising a polynucleotide, nucleic acid construct or recombinant expression vector encoding the mutant. The term "recombinant host cell" encompasses host cells which differ from the parent cell upon introduction of a polynucleotide, nucleic acid construct or recombinant expression vector, the recombinant host cell being specifically achieved by transformation.
The term "transformation" in the present disclosure has the meaning commonly understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate precipitation, calcium chloride (CaCl) 2 ) Precipitation method, microInjection method, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.
The host cell of the present disclosure may be a prokaryotic cell, as long as it is a cell capable of containing a mutant of the transcriptional regulatory factor LysG of the present disclosure, or containing a polynucleotide, a nucleic acid construct or a recombinant expression vector encoding the mutant. In some embodiments, the host cell is derived from a microorganism suitable for the fermentative production of an amino acid, including, but not limited to, strains that may include Escherichia (Escherichia), erwinia (Erwinia), serratia (Serratia), providence (Providencia), enterobacter (Enterobacteria), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium), corynebacterium (Corynebacterium), and the like. In some embodiments, the host cell is a corynebacterium glutamicum ATCC13032, a corynebacterium glutamicum ATCC13869, a corynebacterium glutamicum ATCC 14067 strain, or a derivative of any of the foregoing. In some embodiments, the host cell in the present disclosure may also be other types of strains having amino acid-producing ability, including wild-type strains and recombinant strains.
Illustratively, the host cell is a lysine-producing host cell. In some embodiments, for lysine-producing host cells, can be in the Corynebacterium glutamicum ATCC13032 based on the expression of feedback inhibition of aspartate kinase strain. In addition, lysine-producing host cells may also be other kinds of strains having lysine-producing ability.
In some embodiments, the lysine producing host cell may further include, but is not limited to, attenuated or reduced expression of one or more genes selected from the group consisting of:
a. the adhE gene encoding alcohol dehydrogenase;
b. the ackA gene encoding acetate kinase;
c. a pta gene encoding a phosphate acetyltransferase;
d. an ldhA gene encoding lactate dehydrogenase;
e. the focA gene encoding the formate transporter;
f. the pflB gene encoding pyruvate formate lyase;
g. a poxB gene encoding pyruvate oxidase;
h. the thrA gene of bifunctional enzyme for coding aspartokinase I/homoserine dehydrogenase I;
i. the thrB gene encoding homoserine kinase;
j. an ldcC gene encoding lysine decarboxylase; and
h. the cadA gene which codes for lysine decarboxylase.
In some embodiments, one or more genes selected from the group consisting of, but not limited to:
a. the dapA gene encoding dihydrodipicolinate synthase which relieves feedback inhibition by lysine;
b. a dapB gene encoding dihydrodipicolinate reductase;
c. a ddh gene encoding diaminopimelate dehydrogenase;
d. dapD encoding a tetrahydrodipicolinate succinylase and dapE encoding a succinyldiaminopimelate deacylase;
e. an asd gene encoding aspartate-semialdehyde dehydrogenase;
f. the ppc gene encoding phosphoenolpyruvate carboxylase;
g. the pntAB gene encoding nicotinamide adenine dinucleotide transhydrogenase;
i. the lysE gene of the transport protein which codes for lysine.
In some embodiments, the arginine producing host cell may include, but is not limited to, enhanced or overexpressed by one or more genes selected from the group consisting of:
a) The gene encoding N-acetylglutamyl phosphate reductase argC;
b) Encoding an ornithine acetyltransferase argJ gene;
c) Encoding the N-acetylglutamate kinase argB gene;
d) The gene encoding acetylornithine transaminase argD;
e) Encoding an ornithine carbamoyltransferase argF gene;
f) Encoding argininosuccinate synthetase argG gene;
g) Encoding the argininosuccinate lyase argH gene;
h) Encoding a carbamoyl phosphate synthetase gene.
In some embodiments, the arginine producing host cell may further comprise an inactivation of the argR gene encoding an arginine repressor.
In some embodiments, the histidine-producing host cell may include, but is not limited to, enhanced or overexpressed by one or more genes selected from:
a) Encoding the L-histidine synthetic operon hisEG gene and hisDCB gene;
b) The gene encoding the 6-phosphoglucose dehydrogenase zwf-opcA;
c) Encoding PRPP synthetase prsA gene;
in some embodiments, the histidine-producing host cell may further comprise a reduction in the expression of the glucose-6-phosphate isomerase pgi gene in the producing strain.
The cultivation of the host cell of the present disclosure may be performed according to a conventional method in the art, including, but not limited to, a well plate culture, a shake flask culture, a batch culture, a continuous culture, a fed-batch culture, and the like, and various culture conditions such as temperature, time, pH of a medium, and the like may be appropriately adjusted according to actual circumstances.
The term "amino acid" or "L-amino acid" in the present disclosure refers to the basic building block of a protein in which an amino group and a carboxyl group are bound to the same carbon atom. Illustratively, the amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, 5-aminolevulinic acid, or a derivative of any of the above amino acids. In addition, the amino acid may be other kinds of amino acids in the art. In a specific embodiment of the present disclosure, the amino acids are basic amino acids, i.e., lysine, histidine and arginine.
The term "culturing" of the present disclosure may be carried out according to a conventional method in the art, including, but not limited to, a well plate culture, a shake flask culture, a batch culture, a continuous culture, a fed-batch culture, and the like, and various culture conditions such as temperature, time, pH of a medium, and the like may be appropriately adjusted according to actual circumstances.
Unless defined otherwise herein or clearly indicated by the background, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Mutant of transcription regulatory factor LysG
Random mutation is carried out on an EBD region (amino acid from 90 to 290) of the LysG, a mutant library of the EBD region of the LysG is obtained, the mutant is found to be capable of greatly improving the induced response strength of the biosensor to lysine, histidine and arginine, and the mutant can be used as the biosensor to be applied to screening of basic amino acid high-yield strains and basic amino acid synthesis related proteins; in addition, the mutant and the lysE gene promoter regulated by the mutant can be further utilized to strengthen the expression of key genes in an amino acid synthesis pathway and improve the synthesis and yield of amino acids.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, and the mutant has a transcriptional regulatory activity of a transcriptional regulatory factor LysG. The present disclosure found that following mutation of the amino acid at the above positions, the amino acid sequence of SEQ ID NO: compared with the wild polypeptide with the sequence shown in 1, the mutant has obviously improved strength for responding lysine, histidine and arginine, and can improve the sensitivity, dynamic range, operation and the like of a biosensor for screening basic amino acid high-yield strains or key genes in a basic amino acid synthesis path.
In some more specific embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, and amino acids substituted at one or more positions of 111-270 of the sequence shown in 1, wherein the amino acid substitution can improve the strength of induced response to basic amino acids while maintaining the transcriptional regulatory activity of LysG.
In some embodiments, the mutant corresponds to SEQ ID NO:1, having a mutated amino acid at one or more of the following positions: 111 th, 115 th, 125 th, 133 th, 136 th, 141 th, 143 th, 146 th, 152 th, 165 th, 169 th, 177 th, 194 th, 198 th, 209 th, 212 th, 241 th, 248 th, 255 th, 259 th, 260 th, 266 th, 269 th, 270 th. The present disclosure has found that mutation of the amino acid residues at each of the above positions can improve the strength of the induced response of LysG to a basic amino acid to various degrees.
In some more specific embodiments, the mutant has a substituted amino acid at one or more of the positions described above. The present disclosure finds that substitution of amino acids at the above positions has a positive effect on improving the induction responsiveness to basic amino acids by screening mutant libraries.
In some preferred embodiments, the mutant corresponds to SEQ ID NO:1, or a variant thereof, and at least 1, at least 2, or at least 3 positions of the sequence set forth in 1. Exemplary, the mutant is represented in SEQ ID NO:1, 2, 3, 4, 5, etc. of positions 111, 115, 125, 133, 136, 141, 143, 146, 152, 165, 169, 177, 194, 198, 209, 212, 241, 248, 255, 259, 260, 266, 269, and 270 of the sequence shown in 1.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1 at any one of positions 248, 115, 270, 194, 212, 146, 141, 198, 259, 111, 133 and 143 of the sequence shown in 1.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, and the 125 th and 165 th positions of the sequence shown in 1 have amino acid substitutions at the same time.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, wherein the amino acid residues at positions 125 and 209 are substituted.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, having amino acid substitutions at positions 152 and 241.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, has amino acid substitutions at positions 255 and 266.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1 has an amino acid substitution at position 248, 260 and 269 of the sequence shown in 1.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, having amino acid substitutions at positions 136 and 169.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, having amino acid substitutions at positions 143 and 177.
In some embodiments, the mutant has a mutation in a nucleotide sequence corresponding to SEQ ID NO:1, having amino acid substitutions at positions 136 and 169.
In some preferred embodiments, the mutant corresponds to SEQ ID NO:1, mutated amino acids having one or more of the following:
A111P、G115R、E125V、R133Q、D136N、V141I、R143C、N146D、E152G、A165T、L169M、G177E、V194A、R198H、G209E、R212H、A241V、E248G、A255S、I259M、P260S、W266C、E269G、S270P。
in some preferred embodiments, the mutant corresponds to SEQ ID NO:1, and has any 1, any 2 or any 3 of a111P, G115R, E125V, R133Q, D136N, V141I, R143C, N146D, E152G, a165T, L169M, G177E, V194A, R198H, G209E, R212H, a241V, E248G, a255S, I259M, P260S, W266C, E269G and S270P. After the amino acid mutation, the mutant of the transcription regulatory factor LysG can be used for constructing a biosensor with improved detection sensitivity, and high-throughput screening of high-yield strains of basic amino acids is realized; or used for improving the expression of key genes in the synthetic pathway of the basic amino acid and improving the synthetic efficiency and the yield of the amino acid.
In some embodiments, the mutant corresponds to SEQ ID NO:1, having the following sequence (m) 1 )-(m 20 ) Any one of the mutated amino acids:
(m 1 )E248G；(m 2 ) E125V and a165T; (m) 3 )G115R；(m 4 )S270P；(m 5 ) E125V and G209E; (m) 6 )V194A；(m 7 )R212H；(m 8 )N146D；(m 9 ) E152G and a241V; (m) 10 )V141I；(m 11 )R198H；(m 12 ) A255S and W266C; (m) 13 ) E248G, P260S and E269G; (m) 14 )I259M；(m 15 )A111P；(m 16 ) D136N and L169M; (m) 17 ) R143C and G177E; (m) 18 )R133Q；(m 19 ) D136N and L169M; (m) 20 ) And R143C. For (m) 1 )-(m 20 ) Any of the mutants shown has improved induction responsiveness to basic amino acids.
In some embodiments, the mutant of the transcriptional regulator LysG has the transcriptional regulator activity of the transcriptional regulator LysG and an increased strength of basic amino acid response after deletion or addition of at least one amino acid, preferably 1 to 15 amino acids, preferably 1 to 10, more preferably 1 to 6 amino acids, at least one of the N-terminus and the C-terminus of the mutant.
In some embodiments, the mutant of the transcriptional regulatory factor LysG has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a mutant of any of the above, and the amino acid sequence is not SEQ ID NO:1, has the transcription regulatory factor activity of the transcription regulatory factor LysG and improved response intensity of basic amino acids.
In some embodiments, the mutant of the transcriptional regulator LysG of the present disclosure is identical to SEQ ID NO:1, 1.21-fold, 1.34-fold, 1.35-fold, 1.41-fold, 1.54-fold, 1.74-fold, 1.84-fold, 2.51-fold, 2.17-fold, 2.33-fold, 2.36-fold, 2.41-fold, 2.51-fold, 2.62-fold, 2.64-fold, 2.70-fold, 2.74-fold, 3.84-fold.
In some embodiments, the mutant of the transcription regulatory factor LysG is encoded by a polynucleotide, and the sequence of the polynucleotide is not particularly limited in the present disclosure as long as it can encode a polypeptide having the transcription regulatory factor activity of any of the above.
Construction of mutant libraries and mutant screening
In some embodiments, the present disclosure uses an artificially synthesized eyfp gene (GeneBank No. am774338.1) as a template and eyfp-F and eyfp-R as primers to amplify an eyfp gene fragment; carrying out PCR by taking a Corynebacterium glutamicum 13032 genome as a template and lysGE-F and lysGE-R as primers, and amplifying to obtain a fragment of a lysGE gene; carrying out PCR amplification by taking pTRCmob as a template and pTRCmob-rev-F/R as a primer to obtain a plasmid framework; and (3) carrying out homologous recombination and connection on the fragments to obtain a recombinant expression vector pLysWT.
In some embodiments, the present disclosure uses an expression vector pLysWT containing a lysine biosensor as a template, and utilizes random mutation primers EBD-mut-F and EBD-mut-R to obtain encoding gene segments of EBD domains with different mutation rates through error-prone PCR amplification; using pLysWT as a template, and removing an EBD region of LysG by utilizing primers EBD-rev-F and EBD-rev-R through PCR reverse amplification to obtain a linearized vector fragment; and (3) carrying out homologous recombination and connection on the fragments to obtain a recombinant expression vector containing the gene fragments with different mutation rates.
In some embodiments, the recombinant expression vector is transformed into a host cell to produce a library of mutated strains. In some more specific embodiments, the host cell is a competent cell of c. In other embodiments, the host cell may be of other cell types.
In some embodiments, the present disclosure transforms the vectors pTRCmob and pLysWT into ATCC13032 strain to obtain control recombinant host cells 13032 (pTRCmob) and 13032 (pLysWT), respectively. The recombinant host cells are cultured in the presence of a basic amino acid (e.g., lysine), and then the strain library is subjected to fluorescence sorting to obtain target recombinant host cells having higher fluorescence intensity compared with the control recombinant host cells, wherein the target recombinant host cells contain a mutant of the target transcription regulatory factor LysG.
Biosensor and method for measuring the same
The principle of the biosensor is as follows: at a low concentration of a basic amino acid (e.g., lysine) as an inducer, the transcription regulatory factor LysG cannot bind to the amino acid. When the concentration of basic amino acids is reached, the transcription regulatory factor LysG binds to basic amino acids (e.g., lysine) as inducers, and then the promoter of lysE gene can be regulated. Therefore, when a reporter gene such as a fluorescent protein is ligated downstream of the lysE gene promoter, expression of the downstream reporter gene can be initiated, and the concentration of the basic amino acid can be evaluated by the intensity of the fluorescent signal of the reporter gene, thereby screening an amino acid-producing strain.
In some embodiments, the present disclosure constructs a biosensor using a mutant of the transcriptional regulatory factor LysG, which has a greatly improved induced response intensity, and thus, the fluorescence intensity of the mutant that initiates the expression of a reporter gene is significantly improved, resulting in a greatly improved detection sensitivity of the biosensor.
In some embodiments, the biosensor is used to screen for a basic amino acid-producing strain, or to screen for a gene encoding a protein involved in the synthesis of a basic amino acid. Wherein the basic amino acid high-producing strain is lysine, arginine or histidine high-producing strain. The biosensor disclosed by the invention can realize high-throughput screening of basic amino acid high-producing strains or proteins related to basic amino acid synthesis.
In some preferred embodiments, the amino acid-producing strain is a lysine-producing strain.
In some embodiments, the biosensor is cultured under the following culture medium conditions:CGXII medium, comprising the following components: 50g/L glucose,16.5g/L NH 4 Cl，5g/L urea，1g/L KH 2 PO 4 ，1g/L K 2 HPO 4 ，42g/L MOPS，0.25g/L MgSO 4 ，0.01g/L FeSO 4 ·2H 2 O，0.01g/L MnSO 4 ·H 2 O，0.001g/L ZnSO 4 ·7H 2 O，0.2mg/L CuSO 4 ，0.02mg/L NiCl 2 ·6H 2 O，0.01g/L CaCl 2 0.03g/L protocatechuic acid,0.2 mg/Lthiontin and 0.1mg/L vitamin B1. The medium contained 25. Mu.g/mL kanamycin and various concentrations of lysine-alanine, arginine-alanine or histidine-alanine dipeptide (0 mM, 0.5mM, 1.0mM, 1.5mM, 2mM, 4mM, 6mM and 8 mM). Culturing at 30 deg.C and 800rpm for 10h, diluting by a certain fold, and detecting fluorescence value and OD of bacteria solution with enzyme-linked standard instrument (SpectraMax M5, molecular Devices, lambda excitation =488nm, lambda emission =520 nm) 600 。
Process for producing objective amino acid
(1) Operably connecting the gene of the mutant of the coding transcription regulatory factor LysG with a LysG regulatory promoter and a protein coding gene related to the synthesis of the target amino acid to obtain a recombinant expression vector capable of expressing the protein related to the synthesis of the target amino acid, and transforming a host cell by using the recombinant expression vector to obtain the recombinant host cell.
(2) And (3) carrying out fermentation culture on the recombinant host cells, and collecting the target compound from the recombinant host cells or a culture solution of the recombinant host cells to finish the production process of the target compound.
In the production process, because the mutant of the transcription regulation factor LysG has improved induction influence strength, the transcription regulation effect of the mutant on the LysG regulation promoter is enhanced, and the expression level of a protein coding gene related to target amino acid synthesis can be further improved, so that the yield of a target compound is obviously improved.
In some embodiments, the LysG-regulated promoter is a lysE gene promoter; in other embodiments, the gene encoding a mutant of the transcriptional regulator LysG may also be linked to other types of promoters that initiate the transcriptional process under the transcriptional regulation of LysG.
In some embodiments, the target amino acid is an L-amino acid, and the protein-encoding gene associated with a synthetic amino acid refers to a protein-encoding gene associated with a synthetic L-amino acid.
Illustratively, the target amino acid includes one or a combination of two or more of: lysine, arginine, histidine, or a derivative of any of the foregoing amino acids.
Exemplary genes encoding proteins associated with synthetic amino acids include genes encoding enzymes that include one or a combination of two or more of the following: pyruvate carboxylase, phosphoenolpyruvate carboxylase, gamma-glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, amino acid transporter, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, gluco-repressor, glucose dehydrogenase, aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, transketolase, diaminopimelate dehydrogenase, N-acetylglutamyl phosphate reductase, ornithine acetyltransferase, N-acetylglutamate kinase, acetylornithine aminotransferase, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase and carbamoylphosphomalate synthase, glyceraldehyde-3-phosphate dehydrogenase gene, isocitrate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, and the pyruvate dehydrogenase gene.
In some embodiments, the host cell is Corynebacterium glutamicum (Corynebacterium glutamicum), which is an important strain for the production of amino acids, organic acids, and other target compounds.
In some embodiments, the host cell is corynebacterium glutamicum modified as follows: the threonine at position 311 of the aspartokinase (encoded by the lysC gene) in the genome of Corynebacterium glutamicum ATCC13032 was mutated to isoleucine.
In some embodiments, the target amino acid is lysine, and the lysine fermentation medium comprises: 80g/L glucose,8g/L yeast powder, 9g/L urea and 1.5g/L K 2 HPO 4 ·3H 2 O，0.01g/L MnSO 4 ，0.6g/LMgSO 4 ·7H 2 O，0.01g/L FeSO 4 ·7H 2 O，42g/L MOPS。
In some embodiments, the compound of interest is recovered from the recombinant host cell or the culture broth of the recombinant cell by methods commonly used in the art, including, but not limited to: filtration, anion exchange chromatography, crystallization or HPLC.
Methods for manipulating microorganisms are known in the art, such as modern methods in molecular biology (Online ISBN:9780471142720, john Wiley and sons, inc.), [ metabolic engineering of microorganisms: methods and protocols (Qiong Cheng ed., springer) and "systemic metabolic engineering: methods and protocols (Hal s. Alper ed., springer) etc. publications.
Examples
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
The experimental techniques and experimental procedures used in this example are, unless otherwise specified, conventional techniques, e.g., those in the following examples, in which specific conditions are not specified, and generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The materials, reagents and the like used in the examples are commercially available from normal sources unless otherwise specified.
Example 1 construction of random mutant library of EBD Domain of LysG protein
(1) Construction of lysine biosensor
The method comprises the steps of performing whole Gene synthesis according to reported eyfp Gene sequence information (GeneBank No. AM774338.1), performing PCR amplification by utilizing an eyfp-F/eyfp-R primer to obtain an eyfp Gene fragment, performing PCR amplification by utilizing a Corynebacterium glutamicum 13032 genome (Gene ID: 2830649) as a template and utilizing lysGE-F/lysGE-R as a primer to obtain a lysGE Gene fragment, performing PCR amplification by utilizing pTRCmob as a template and pTRCmob-rev-F/R as a primer to obtain a plasmid framework, purifying and recovering the fragment, recombining and connecting to obtain a recombinant vector pLysWT containing a lysine biosensor.
(2) Construction of random mutation library of EBD domain of LysG protein
Based on the protein crystal structure (PDB: 6 XTU) of LysG, random mutation primer EBD-mut-F/R was designed for the EBD domain (amino acids 90-290) using expression vector pLysWT containing lysine biosensor as template, to which 0.05-0.5mM MnCl was added 2 Under the condition of (1), EBD structural domain coding gene segments with different mutation rates are obtained by error-prone PCR amplification. Meanwhile, pLysWT is used as a template, and the EBD region of LysG is removed by PCR reverse amplification by using a primer EBD-rev-F/R to obtain a linearized vector fragment. After the Dpn I treatment to remove the template plasmid and recovery, the two fragments are connected by a recombinase, chemically transformed into E.coli DH5 alpha, and cultured overnight at 37 ℃. Scraping colony from a plate, extracting plasmids with different mutation rates, mixing the plasmids with different mutation rates according to an equimolar ratio, electrically pulsing into competent cells of the strain C.glutamicum ATCC13032, recovering for 2h at 30 ℃, transferring into a TSB liquid medium containing 25 mug/mL kanamycin, and culturing for 16h at 30 ℃ to obtain a strain library for screening.
Example 2 LysG mutant library flow cytometry sorting
According to the response principle of lysine biosensor, the regulatory protein LysG cannot bind to lysine when the concentration of lysine is low, and thus cannot initiate fluorescent protein by regulating the lysE promoterTranscription of the white gene; when the lysine reaches a certain concentration, the LysG is combined with the lysine, so that the conformation of the LysG is changed, and then the LysG is combined with a lysE promoter to start the expression of a downstream fluorescent reporter gene. The different mutants have different induced response intensities to lysine, and the expression intensities of the fluorescent protein are different. Based on this principle, we screened a library of random mutations of the EBD domain of the LysG protein. The ATCC13032 strain was first transformed with the empty plasmid pTRCmob and the negative control plasmid pLysWT, respectively, to obtain control strains 13032 (pTRCmob) and 13032 (pLysWT). Then, the above strains and mutant strain library were inoculated into TSB liquid medium containing 25. Mu.g/mL kanamycin, cultured at 30 ℃ for 8 hours, and then transferred to CGXIIY medium (50 g/L glucose,2g/L yeast extract,16.5g/L NH) supplemented with 0.8M lysine 4 Cl，5g/L urea，1g/L KH 2 PO 4 ，1g/L K 2 HPO 4 ，42g/L MOPS，0.25g/L MgSO 4 ，0.01g/L FeSO 4 ·2H 2 O，0.01g/L MnSO 4 ·H 2 O，0.001g/L ZnSO 4 ·7H 2 O，0.2mg/L CuSO 4 ，0.02mg/L NiCl 2 ·6H 2 O，0.01g/LCaCl 2 0.03g/L of procatechuic acid,0.2mg/L of biotin,0.1mg/L of vitamin B1), cultured at 30 ℃ for 6 hours, and then the cell concentration was diluted to 0.1 with PBS buffer. The fluorescence intensity of the above strain was analyzed by a flow cytometer, a region having a higher fluorescence intensity than 13032 (pLysWT) was selected as a sorting region, and cells in the region were sorted to obtain a single clone.
Example 3 EBD Domain mutant library rescreening of LysG protein
The single-cell colonies obtained by flow cytometry sorting were randomly picked up and a portion of the single clones were inoculated into a 96-well plate containing 200. Mu.L of TSB broth (containing 25. Mu.g/mL kanamycin) together with control strains 13032 (pTRCmob) and 13032 (pLysWT) and cultured for 8 hours at 30 ℃ at 800rpm in a well plate shaker. The seed solution was transferred to a 96-well plate containing 200. Mu.L of CGXIII Y medium (25. Mu.g/mL kanamycin was added) at 5% (V/V) and 0M or 0.8M lysine was added, the seed solution was cultured at 30 ℃ and 800rpm in a well plate shaker for 12 hours, the bacterial solution was diluted 20-fold with PBS buffer, the fluorescence intensity and OD of eYFP were measured with an enzyme-linked calibrator (SpectraMax M5, molecular Max Devices,. Lambda.excitation =488nm,. Lambda.emision =520 nm), and the mutant having a fluorescence value stronger than that of control strain 13032 (pLysWT) was sequenced to analyze the mutation site. As shown in Table 1, the mutation of the amino acid residues at different positions in the EBD region could improve the induced response of LysG to lysine to various degrees.
TABLE 1 summary of LysG mutants
A: fluorescence value at the time of induction of LysG mutant sensor/fluorescence value at the time of induction of LysG wild-type biosensor;
b: fluorescence value when LysG mutant sensor was induced/fluorescence value when LysG mutant sensor was not induced
Example 4 response of LysG mutants to histidine and arginine
To test the induction response of the mutant LysG EBD region to three basic amino acids, the strains 13032 (pTRCmob), 13032 (pLysWT) were first tested N146D ) And 13032 (pLysWT) R143C ) After inoculating a 24-well plate containing 1mL of TSB (containing 25. Mu.g/mL kanamycin) and culturing at 30 ℃ and 800rpm for 8 hours, the seed solution was transferred to a 96-well plate containing 200. Mu.L of CGXII medium with the following composition at an initial OD 0.5: 50g/L glucose,16.5g/L NH 4 Cl，5g/L urea，1g/L KH 2 PO 4 ，1g/L K 2 HPO 4 ，42g/L MOPS，0.25g/L MgSO 4 ，0.01g/L FeSO 4 ·2H 2 O，0.01g/L MnSO 4 ·H 2 O，0.001g/L ZnSO 4 ·7H 2 O，0.2mg/L CuSO 4 ，0.02mg/L NiCl 2 ·6H 2 O，0.01g/L CaCl 2 0.03g/L protocatechuic acid,0.2mg/L biotin,0.1mg/L vitamin B1. The medium contained 25. Mu.g/mL kanamycin and various concentrations of lysine-alanine, arginine-alanine or histidine-alanine dipeptide (0 mM, 0.5mM, 1.0 m)M, 1.5mM, 2mM, 4mM, 6mM and 8 mM). After incubation at 30 ℃ for 10h at 800rpm and dilution by a certain fold, fluorescence and OD of the cells were measured using an enzyme calibrator (SpectraMax M5, molecular Devices,. Lamda. Excitation =488nm,. Lamda. Emission =520 nm). The detection results are shown in fig. 1, and the data show that the induced response strength of the two mutants to lysine is obviously enhanced, and the induced response strength to arginine and histidine is also greatly improved. It was shown that the LysG mutants of the present disclosure can be applied as biosensors for lysine, arginine and histidine.
Example 5 application of LysG protein EBD Domain mutant to construction of amino acid high-producing Strain
(1) Construction of lysE Gene overexpression vector
To test the effect of the LysG protein EBD mutant in the amino acid-producing strain, pLysWT and pLysWT were prepared separately N146D And pLysWT R143C The fluorescent protein-encoding gene eyfp above was replaced with lysine transporter-encoding gene lysE, and the effect of lysine-induced overexpression of lysE on lysine synthesis was tested. Primers lysE-F/R were designed, and lysE gene was obtained by PCR amplification using ATCC13032 genome as a template. In addition, primers W8-Rev-F/R were designed, and plasmids pLysWT and pLysWT were used respectively N146D And pLysWT R143C Using PCR reverse amplification to remove eyfp gene to obtain linearized vector fragment, treating vector fragment PCR product with Dpn I to remove template plasmid, recovering fragment, connecting the above two fragments with recombinase, and chemically transforming into E.coli DH5 alpha competent cell to obtain expression vector pTRCmob-lysG-lysE, pTRCmob-lysG of lysE gene N146D lysE and pTRCmob-lysG R143C -lysE。
(2) Effect of overexpression of LysE on lysine accumulation
According to the construction method of lysine strains disclosed in the literature (Becker, J., zelder, O.,
S.,
H.and Wittmann, C. (2011) From zero to halo-design-based systems of metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metal. Eng.,13, 159-168.), a strain SCgL30 with certain lysine synthesis capacity was constructed by mutating threonine at position 311 of aspartokinase (encoded by lysC gene) in Corynebacterium glutamicum ATCC13032 genome to isoleucine using pK18 mobsacB-based homologous recombination technology. To examine the effect of regulation of expression of lysE gene by LysG and its mutant on lysine synthesis, the above expression vector and empty vector pTRCmob were transformed into the strain C.glutamicum SCgL30, respectively, to obtain LysE overexpression strains SCgL30 (pTRCmob), SCgL30 (pTRCmob-lysG-lysE) and SCgL30 (pTRCmob-lysG) with different regulation of LysG mutant N146D lysE) and its control strain SCgL30 (pTRCmob-lysG) N146D -lysE)。
The above test strains were inoculated into 24-well plates containing 1mL of TSB (supplemented with 25. Mu.g/mL of kanamycin), cultured at 30 ℃ for 8 hours at 800rpm in a well plate shaker, transferred to 24-well plates containing 1mL of lysine fermentation medium at an initial OD 0.5, cultured for 24 hours at 30 ℃ in an 800rpm well plate shaker, and the OD of the bacterial liquid was measured and the lysine concentration and the residual glucose concentration were measured by a biosensor SBA-40D. The lysine fermentation medium comprises the following components: 80g/L glucose,8g/L yeast powder, 9g/L urea and 1.5g/L K 2 HPO 4 ·3H 2 O，0.01g/L MnSO 4 ，0.6g/L MgSO 4 ·7H 2 O，0.01g/L FeSO 4 ·7H 2 O,42g/L MOPS. The fermentation result is shown in figure 2, and the data show that the yield of lysine is 2.07g/L after the expression of the lysine transport protein LysE regulated and controlled by the wild type LysG, which is only 5.1 percent higher than that of the control strain; the lysine yield of the LysE expression strain regulated and controlled by the LysG mutant N146D and R143C respectively reaches 2.57g/L and 2.7g/L, which are respectively improved by 30.5 percent and 37.1 percent compared with the control strain; meanwhile, the glucose conversion rate is respectively improved by 37.8 percent and 44.0 percent compared with the control strain. The above results indicate that the use of lysG mutant and lysE promoter having improved response strength to lysine can be used for expression of target genes of metabolic pathways to promote synthesis and accumulation of lysine.
The primers used in the examples of the disclosure are shown in table 2 below:
all technical features disclosed in the present specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
Furthermore, from the foregoing description, one skilled in the art can readily appreciate the key features of the disclosure from the present disclosure, that numerous modifications can be made to adapt the invention to various usages and conditions without departing from the spirit and scope of the disclosure, and therefore, such modifications are intended to fall within the scope of the appended claims.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> mutant of transcription regulatory factor LysG and application thereof
<130> 6A17-2173363I
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 290
<212> PRT
<213> Corynebacterium glutamicum
<400> 1
Met Asn Pro Ile Gln Leu Asp Thr Leu Leu Ser Ile Ile Asp Glu Gly
1 5 10 15
Ser Phe Glu Gly Ala Ser Leu Ala Leu Ser Ile Ser Pro Ser Ala Val
20 25 30
Ser Gln Arg Val Lys Ala Leu Glu His His Val Gly Arg Val Leu Val
35 40 45
Ser Arg Thr Gln Pro Ala Lys Ala Thr Glu Ala Gly Glu Val Leu Val
50 55 60
Gln Ala Ala Arg Lys Met Val Leu Leu Gln Ala Glu Thr Lys Ala Gln
65 70 75 80
Leu Ser Gly Arg Leu Ala Glu Ile Pro Leu Thr Ile Ala Ile Asn Ala
85 90 95
Asp Ser Leu Ser Thr Trp Phe Pro Pro Val Phe Asn Glu Val Ala Ser
100 105 110
Trp Gly Gly Ala Thr Leu Thr Leu Arg Leu Glu Asp Glu Ala His Thr
115 120 125
Leu Ser Leu Leu Arg Arg Gly Asp Val Leu Gly Ala Val Thr Arg Glu
130 135 140
Ala Asn Pro Val Ala Gly Cys Glu Val Val Glu Leu Gly Thr Met Arg
145 150 155 160
His Leu Ala Ile Ala Thr Pro Ser Leu Arg Asp Ala Tyr Met Val Asp
165 170 175
Gly Lys Leu Asp Trp Ala Ala Met Pro Val Leu Arg Phe Gly Pro Lys
180 185 190
Asp Val Leu Gln Asp Arg Asp Leu Asp Gly Arg Val Asp Gly Pro Val
195 200 205
Gly Arg Arg Arg Val Ser Ile Val Pro Ser Ala Glu Gly Phe Gly Glu
210 215 220
Ala Ile Arg Arg Gly Leu Gly Trp Gly Leu Leu Pro Glu Thr Gln Ala
225 230 235 240
Ala Pro Met Leu Lys Ala Gly Glu Val Ile Leu Leu Asp Glu Ile Pro
245 250 255
Ile Asp Thr Pro Met Tyr Trp Gln Arg Trp Arg Leu Glu Ser Arg Ser
260 265 270
Leu Ala Arg Leu Thr Asp Ala Val Val Asp Ala Ala Ile Glu Gly Leu
275 280 285
Arg Pro
290
<210> 2
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 2
gtaatagatg aactttaaga aggagatata catatggtga gcaagggcga g 51
<210> 3
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 3
cagcggccgc tactagttta ttacttgtac agctcgtcca tg 42
<210> 4
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 4
gcccttagtg actcgaattc ctaaggccgc aatccctc 38
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 5
tcttaaagtt catctattac ggtccgatgg acagtaaaag a 41
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 6
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 7
gaattcgagt cactaagggc ta 22
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 8
cccttagtga ctcgaattcc ta 22
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 9
ctggacgcct tgctgaaatc 20
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 10
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 11
<210> 12
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 12
tttaagaagg agatatacat atggtgatca tggaaatctt cattac 46
<210> 13
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 13
cagcggccgc tactagttta ctaacccatc aacatcagtt tgat 44
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 14
<210> 15
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> primer sequences
<400> 15
atgtatatct ccttcttaaa gttcatctag g 31
Claims (17)
1. A mutant of the transcriptional regulator LysG having one or more mutated amino acids located in the EBD domain; and, the mutant has improved basic amino acid response strength compared to the wild-type transcription regulatory factor LysG.
2. The mutant of the transcriptional regulatory factor LysG according to claim 1, wherein the mutant is selected from any one of the group consisting of the following (i) to (iii):
(i) Comprises the amino acid sequence shown as SEQ ID NO:1, which mutant has a sequence which, in the polypeptide corresponding to SEQ ID NO:1, has a mutated amino acid at one or more positions from position 111 to 270;
(ii) (ii) a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the amino acid sequence set forth in (i);
(iii) (iii) a polypeptide having an amino acid sequence as shown in (i) or (ii) in which one or more amino acids are added or deleted at least one of the N-terminus and the C-terminus.
3. The mutant of the transcription regulatory factor LysG according to claim 1 or 2, wherein the mutant corresponds to SEQ ID NO:1, having a mutated amino acid at one or more of the following positions:
143, 146, 125, 133, 136, 141, 152, 165, 169, 177, 194, 198, 209, 212, 241, 248, 255, 259, 260, 266, 269, 270, 111, 115;
preferably, the mutant has a mutation in a region corresponding to SEQ ID NO:1, or a variant thereof, and at least 1, at least 2, or at least 3 positions of the sequence set forth in 1.
4. The mutant of the transcription regulatory factor LysG according to any one of claims 1 to 3, wherein the mutant corresponds to the amino acid sequence of SEQ ID NO:1, mutated amino acids having one or more of the following:
R143C、N146D、E125V、R133Q、D136N、V141I、E152G、A165T、L169M、G177E、V194A、R198H、G209E、R212H、A241V、E248G、A255S、I259M、P260S、W266C、E269G、S270P、A111P、G115R。
5. the mutant of the transcriptional regulatory factor LysG according to any one of claims 1 to 4, wherein the mutant corresponds to the amino acid sequence of SEQ ID NO:1, having the following sequence (m) 1 )-(m 20 ) Any one of the mutated amino acids:
(m 1 )R143C；
(m 2 )N146D；
(m 3 )E248G；
(m 4 ) E125V and a165T;
(m 5 )G115R；
(m 6 )S270P；
(m 7 ) E125V and G209E;
(m 8 )V194A；
(m 9 )R212H；
(m 10 ) E152G and a241V;
(m 11 )V141I；
(m 12 )R198H；
(m 13 ) A255S and W266C;
(m 14 ) E248G, P260S and E269G;
(m 15 )I259M；
(m 16 )A111P；
(m 17 ) D136N and L169M;
(m 18 ) R143C and G177E;
(m 19 )R133Q；
(m 20 ) D136N and L169M.
6. The mutant of the transcription regulatory factor LysG according to any one of claims 1 to 5, wherein the mutant has a mutation in comparison with the nucleotide sequence of SEQ ID NO:1, has 1.2-3.8 times of improved basic amino acid response intensity compared with the polypeptide with the sequence shown in 1.
7. An isolated polynucleotide encoding a mutant of the transcriptional regulator LysG of any of claims 1-6.
8. A nucleic acid construct comprising the polynucleotide of claim 7 operably linked to a LysG-regulated promoter that directs expression of a polypeptide.
9. A recombinant expression vector comprising the polynucleotide of claim 7, or the nucleic acid construct of claim 8.
10. A recombinant host cell comprising a mutant of the transcriptional regulator LysG of any of claims 1-6, the polynucleotide of claim 7, the nucleic acid construct of claim 8, or the recombinant expression vector of claim 9.
11. The recombinant host cell according to claim 10, wherein the host cell is derived from a microorganism of the genus Escherichia (Escherichia), erwinia (Erwinia), serratia (Serratia), providencia (Providencia), enterobacter (enterobacter), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium), or Corynebacterium (Corynebacterium);
preferably, the host cell is the corynebacterium glutamicum ATCC13032, the corynebacterium glutamicum ATCC13869, the corynebacterium glutamicum ATCC 14067 strain, or a derivative of any of the foregoing.
12. A biosensor comprising a mutant of the transcriptional regulator LysG of any of claims 1-6, or a polynucleotide of claim 7, a nucleic acid construct of claim 8, a recombinant expression vector of claim 9, or a polypeptide expressed by a recombinant host cell of any of claims 10-11.
13. The mutant of the transcriptional regulator LysG according to any one of claims 1 to 6, the polynucleotide according to claim 7, the nucleic acid construct according to claim 8, the recombinant expression vector according to claim 9, or the recombinant host cell according to any one of claims 10 to 11 for use in at least one of the following (a) to (d):
(a) Regulating the transcription level of the target gene, or preparing a reagent or a kit for regulating the transcription level of the target gene;
(b) Producing basic amino acids, or preparing reagents or kits for producing basic amino acids;
(c) Screening basic amino acid high-producing strains, or preparing a reagent, a kit or a biosensor for screening the basic amino acid high-producing strains;
(d) Screening proteins or protein coding genes related to basic amino acid synthesis, or preparing a reagent, a kit or a biosensor for screening proteins or protein coding genes related to basic amino acid synthesis;
optionally, the basic amino acid is histidine, lysine or arginine.
14. A method for regulating expression of a target gene, wherein the method comprises the step of operably linking the polynucleotide of claim 7 to a transcription initiation sequence, the target gene; preferably, the transcription initiation sequence is a polynucleotide having LysG-regulated promoter activity; optionally, the target gene includes at least one of a gene encoding a protein associated with basic amino acid synthesis, a gene encoding a gene expression regulatory protein, and a gene encoding a protein associated with membrane transport.
15. A method for producing a basic amino acid, wherein the method comprises the step of producing a basic amino acid in the presence of the mutant of the transcriptional regulator LysG according to any one of claims 1 to 6, the polynucleotide according to claim 7, the nucleic acid construct according to claim 8, the recombinant expression vector according to claim 9; alternatively, the method comprises the step of producing a basic amino acid using a recombinant host cell according to any one of claims 10-11;
optionally, the method further comprises the step of isolating or purifying the basic amino acid.
16. A method for screening a basic amino acid highly productive strain, wherein the method comprises the step of screening a basic amino acid highly productive strain using the mutant of the transcription regulatory factor LysG according to any one of claims 1 to 6, the polynucleotide according to claim 7, the nucleic acid construct according to claim 8, the recombinant expression vector according to claim 9, the recombinant host cell according to any one of claims 10 to 11, or the biosensor according to claim 12.
17. A method for screening a protein or a gene encoding a protein, which is involved in the synthesis of a basic amino acid, wherein the method comprises the step of screening the protein or the gene encoding a protein, which is involved in the synthesis of a basic amino acid, using the mutant of the transcriptional regulator LysG according to any one of claims 1 to 6, the polynucleotide according to claim 7, the nucleic acid construct according to claim 8, the recombinant expression vector according to claim 9, the recombinant host cell according to any one of claims 10 to 11, or the biosensor according to claim 12.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111026806.4A CN115746110A (en) | 2021-09-02 | 2021-09-02 | Mutant of transcriptional regulatory factor LysG and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111026806.4A CN115746110A (en) | 2021-09-02 | 2021-09-02 | Mutant of transcriptional regulatory factor LysG and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115746110A true CN115746110A (en) | 2023-03-07 |
Family
ID=85332679
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111026806.4A Pending CN115746110A (en) | 2021-09-02 | 2021-09-02 | Mutant of transcriptional regulatory factor LysG and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115746110A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1209169A (en) * | 1995-12-22 | 1999-02-24 | 于利奇研究中心有限公司 | Process for microbial prodn. of amino acid by boosted activity of export carriers |
| CN101054593A (en) * | 1999-07-01 | 2007-10-17 | Basf公司 | Corynebacterium glutamicum gene encoding hpr of phosphoenolpyruvate:sugar phosphotransferase system |
-
2021
- 2021-09-02 CN CN202111026806.4A patent/CN115746110A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1209169A (en) * | 1995-12-22 | 1999-02-24 | 于利奇研究中心有限公司 | Process for microbial prodn. of amino acid by boosted activity of export carriers |
| CN101054593A (en) * | 1999-07-01 | 2007-10-17 | Basf公司 | Corynebacterium glutamicum gene encoding hpr of phosphoenolpyruvate:sugar phosphotransferase system |
Non-Patent Citations (2)
| Title |
|---|
| MARBANIANG, CARMELITA N.等: "ranscriptional Cross-Regulation between Gram-Negative and Gram-Positive Bacteria, Demonstrated Using ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum", JOURNAL OF BACTERIOLOGY,, vol. 194, no. 20, 31 October 2012 (2012-10-31), pages 5657 - 5666 * |
| PU, WEI等: "Directed evolution of linker helix as an efficient strategy for engineering LysR-type transcriptional regulators as whole-cell biosensors", BIOSENSORS & BIOELECTRONICS, vol. 122, 15 February 2023 (2023-02-15), pages 115004 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU764189B2 (en) | Method for producing L-amino acid | |
| EP4183880A1 (en) | Mutant of glutamate dehydrogenase gene promoter and application thereof | |
| US20230399667A1 (en) | Polynucleotide Having Promoter Activity and Application of Polynucleotide in Producing Amino Acid | |
| CN114540261B (en) | Gene engineering bacteria for producing amino adipic acid | |
| CN116731131A (en) | Amino acid biosensor and application thereof | |
| CN113278620B (en) | Mutant hypertonic inducible promoter Pprox and application thereof | |
| WO2023231547A1 (en) | Ncgl2747 gene mutant and use thereof in preparation of l-lysine | |
| CN115746110A (en) | Mutant of transcriptional regulatory factor LysG and application thereof | |
| CN113201538B (en) | Polynucleotides having promoter activity and use thereof for producing target compounds | |
| EP4230723A1 (en) | Polypeptide with aspartate kinase activity and use thereof in production of amino acid | |
| CN113201539B (en) | Polynucleotides having promoter activity and use thereof for producing target compounds | |
| CN115746111A (en) | Mutant of transcription regulatory factor LysG and application thereof | |
| CN118063573A (en) | Arginine-specific transcription regulatory factor mutant and application thereof | |
| US20240084314A1 (en) | Mdh gene-based polynucleotide having promoter activity and use thereof | |
| CN115449519B (en) | Polynucleotide having promoter activity based on dapB gene and use thereof | |
| CN114478724B (en) | ramB mutant and method for constructing lysine production strain by using same | |
| CN115490761B (en) | Recombinant microorganism constructed based on lysine efflux protein and method for producing lysine | |
| EP4293115A1 (en) | Polynucleotide having promoter activity and use thereof in production of traget compounds | |
| CN113493500B (en) | Glutamic acid efflux protein mutant and application thereof | |
| CN115322989A (en) | Polynucleotide having promoter activity, use thereof, and method for producing target compound | |
| CN115806962A (en) | Phosphoenolpyruvate carboxylase mutant and application thereof | |
| CN115322990A (en) | Polynucleotides having promoter activity and use thereof for producing target compounds | |
| CN116891868A (en) | Osmotic pressure tolerance functional gene identification method and application of functional gene | |
| CN115506035A (en) | Promoter mutant library construction method and promoter mutant library | |
| TW202030327A (en) | Ornithine decarboxylase variants，methods for producing putrescine using the same, polynucleotide, microorganism, method for preparing polyamine, and composition for preparing polyamide |
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 |