CN115927325B - Citrate synthase promoter mutant and application thereof - Google Patents
Citrate synthase promoter mutant and application thereof Download PDFInfo
- Publication number
- CN115927325B CN115927325B CN202211145762.1A CN202211145762A CN115927325B CN 115927325 B CN115927325 B CN 115927325B CN 202211145762 A CN202211145762 A CN 202211145762A CN 115927325 B CN115927325 B CN 115927325B
- Authority
- CN
- China
- Prior art keywords
- gene
- host cell
- promoter
- amino acid
- corynebacterium glutamicum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010030844 2-methylcitrate synthase Proteins 0.000 title claims abstract description 25
- 108010071536 Citrate (Si)-synthase Proteins 0.000 title claims abstract description 25
- 102000006732 Citrate synthase Human genes 0.000 title claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 52
- 238000000034 method Methods 0.000 claims abstract description 29
- 150000001413 amino acids Chemical class 0.000 claims abstract description 21
- 101150106096 gltA gene Proteins 0.000 claims abstract description 21
- 235000001014 amino acid Nutrition 0.000 claims abstract description 19
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 19
- 230000014509 gene expression Effects 0.000 claims abstract description 18
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 17
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 17
- 230000000694 effects Effects 0.000 claims abstract description 16
- 239000004220 glutamic acid Substances 0.000 claims abstract description 16
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 claims abstract description 14
- 230000002708 enhancing effect Effects 0.000 claims abstract description 10
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 51
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 31
- 239000013598 vector Substances 0.000 claims description 20
- 239000002773 nucleotide Substances 0.000 claims description 15
- 125000003729 nucleotide group Chemical group 0.000 claims description 15
- 230000000813 microbial effect Effects 0.000 claims description 12
- 230000035772 mutation Effects 0.000 claims description 7
- 101100024384 Escherichia coli (strain K12) mscS gene Proteins 0.000 claims description 5
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims description 4
- 101150019455 gdh gene Proteins 0.000 claims description 4
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 3
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 claims description 3
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 claims description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 claims description 3
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 claims description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 3
- 229960002749 aminolevulinic acid Drugs 0.000 claims description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 3
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 claims description 3
- 229960002591 hydroxyproline Drugs 0.000 claims description 3
- 229960003104 ornithine Drugs 0.000 claims description 3
- 108010004621 phosphoketolase Proteins 0.000 claims description 3
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 claims description 3
- 102000006589 Alpha-ketoglutarate dehydrogenase Human genes 0.000 claims description 2
- 108020004306 Alpha-ketoglutarate dehydrogenase Proteins 0.000 claims description 2
- 108091006146 Channels Proteins 0.000 claims description 2
- 102000034573 Channels Human genes 0.000 claims description 2
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 claims description 2
- 241000807905 Corynebacterium glutamicum ATCC 14067 Species 0.000 claims description 2
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims description 2
- 150000008575 L-amino acids Chemical class 0.000 claims description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 2
- 108010092528 Phosphate Transport Proteins Proteins 0.000 claims description 2
- 102000016462 Phosphate Transport Proteins Human genes 0.000 claims description 2
- 108091000041 Phosphoenolpyruvate Carboxylase Proteins 0.000 claims description 2
- 108010053763 Pyruvate Carboxylase Proteins 0.000 claims description 2
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 claims description 2
- 102000019259 Succinate Dehydrogenase Human genes 0.000 claims description 2
- 108010012901 Succinate Dehydrogenase Proteins 0.000 claims description 2
- 101150107204 asd gene Proteins 0.000 claims description 2
- 101150070145 aspB gene Proteins 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 101150011371 dapA gene Proteins 0.000 claims description 2
- 101150073654 dapB gene Proteins 0.000 claims description 2
- 101150057904 ddh gene Proteins 0.000 claims description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 2
- 101150112623 hemA gene Proteins 0.000 claims description 2
- 101150063051 hom gene Proteins 0.000 claims description 2
- 101150035025 lysC gene Proteins 0.000 claims description 2
- 101150115974 metX gene Proteins 0.000 claims description 2
- 101150021317 odhA gene Proteins 0.000 claims description 2
- 101150109148 pitA gene Proteins 0.000 claims description 2
- 101150023641 ppc gene Proteins 0.000 claims description 2
- 101150118057 proA gene Proteins 0.000 claims description 2
- 101150046501 proB gene Proteins 0.000 claims description 2
- 101150108812 proC gene Proteins 0.000 claims description 2
- 101150068263 putA gene Proteins 0.000 claims description 2
- 101150096049 pyc gene Proteins 0.000 claims description 2
- 101150111745 sucA gene Proteins 0.000 claims description 2
- 235000018102 proteins Nutrition 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 238000013518 transcription Methods 0.000 abstract description 5
- 230000035897 transcription Effects 0.000 abstract description 5
- 239000013604 expression vector Substances 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 238000003259 recombinant expression Methods 0.000 abstract description 2
- 229960002989 glutamic acid Drugs 0.000 description 30
- 239000002609 medium Substances 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 24
- 239000013612 plasmid Substances 0.000 description 19
- 239000012634 fragment Substances 0.000 description 17
- 229940024606 amino acid Drugs 0.000 description 11
- 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 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 10
- 238000012512 characterization method Methods 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 description 8
- 229930006000 Sucrose Natural products 0.000 description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 8
- 101150042350 gltA2 gene Proteins 0.000 description 8
- 108091033319 polynucleotide Proteins 0.000 description 8
- 102000040430 polynucleotide Human genes 0.000 description 8
- 239000002157 polynucleotide Substances 0.000 description 8
- 239000005720 sucrose Substances 0.000 description 8
- 238000000855 fermentation Methods 0.000 description 7
- 230000004151 fermentation Effects 0.000 description 7
- 238000010276 construction Methods 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 239000004202 carbamide Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 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 5
- 229960000318 kanamycin Drugs 0.000 description 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 238000005215 recombination Methods 0.000 description 5
- 230000006798 recombination Effects 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 239000007993 MOPS buffer Substances 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000013256 coordination polymer Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 229940049906 glutamate Drugs 0.000 description 3
- 229930195712 glutamate Natural products 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- 229960003121 arginine Drugs 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229960003646 lysine Drugs 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 229960002429 proline Drugs 0.000 description 2
- 101150094969 rfp1 gene Proteins 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000006941 Amino Acid Transport System X-AG Human genes 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101100315256 Brassica campestris PEC-2 gene Proteins 0.000 description 1
- 108010075254 C-Peptide Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 238000012366 Fed-batch cultivation Methods 0.000 description 1
- 108091006151 Glutamate transporters Proteins 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 238000012365 batch cultivation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 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
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 229960002743 glutamine Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of molecular biology, and in particular relates to a mutant of a citrate synthase gene promoter, a transcription expression cassette containing the promoter mutant, a recombinant expression vector, a recombinant microorganism host cell, and a method for enhancing the expression of a target gene, preparing protein and producing a target compound by using the promoter. The nucleic acid molecule with enhanced promoter activity provided by the invention has higher promoter activity than wild type, can be used for enhancing the expression of a target gene, can be operably connected with a gltA gene for example, and can enhance the expression intensity of citrate synthase, thereby improving the production efficiency of amino acid such as glutamic acid of a recombinant strain and having higher application value.
Description
Technical Field
The invention belongs to the field of molecular biology, and in particular relates to a mutant of a citrate synthase gene promoter, a transcription expression cassette containing the promoter mutant, a recombinant expression vector, a recombinant microorganism host cell, and a method for enhancing the expression of a target gene, preparing protein and producing a target compound by using the promoter.
Background
Glutamic acid is an amino acid variety with the greatest global yield, is one of 20 common amino acids constituting proteins, is a basic substance constituting proteins required for animal nutrition, and is widely applied to industries such as medicine, health, food, animal feed, cosmetics and the like, and is mainly produced by fermentation of corynebacterium glutamicum. With the continuous development of biotechnology, reports of metabolic engineering on corynebacteria to improve the glutamic acid yield of the corynebacteria are increasing, such as enhancing the activity of pyruvate dehydrogenase, enhancing the activity of glutamate transporter, introducing phosphoketolase activity and the like.
Citrate synthase, encoded by the gltA gene, catalyzes the production of both oxaloacetate and acetyl-CoA to citrate and CoA. It is reported in CN1261627A that the introduction of citrate synthase from Brevibacterium lactofermentum into E.coli can increase the glutamic acid yield of E.coli. CN105695383B reports that overexpression of citrate synthase in corynebacterium glutamicum using endogenous strong constitutive promoter P tuf can effectively increase glutamate yield. Those skilled in the art will appreciate that there are many ways to enhance the expression of enzymes, such as increasing copy number, replacing strong promoters, and the like. Since increasing the copy number increases the burden of the strain to some extent, it may cause the genome of the strain to be unstable, and the promoter does not have the above drawbacks for the expression control of genes, there is a need in the art for improving the productivity of coryneform glutamic acid by developing a promoter adapted to a target gene such as gltA so as to express citrate synthase more efficiently, thereby improving the productivity of glutamic acid of the strain and reducing the production cost.
Disclosure of Invention
In a first aspect, the invention provides a nucleic acid molecule having promoter activity, the nucleotide sequence of which is shown in SEQ ID NO. 2.
Wherein the terms "polynucleotide", "nucleic acid molecule" and "nucleic acid" are used interchangeably and refer to a polymer of nucleotides. Polynucleotides may be in the form of individual fragments or may be an integral part of a larger nucleotide sequence structure, derived from nucleotide sequences that are separated at least once in number or concentration, and capable of identifying, manipulating and recovering sequences and their constituent nucleotide sequences by standard molecular biological 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), where "U" replaces "T". In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (individual fragments or whole fragments), or may be a component or constituent of a larger nucleotide structure, such as an expression vector or polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.
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 synonymous. In some embodiments, a wild-type promoter in the present disclosure refers to a promoter of a wild-type gltA gene, i.e., a promoter as set forth in SEQ ID NO:1, and a polynucleotide having a sequence shown in seq id no.
By "mutant" is meant a polynucleotide comprising an alteration (i.e., substitution, insertion, and/or deletion) at one or more (e.g., several) positions relative to a "wild-type", or "comparable" polynucleotide or polypeptide, wherein substitution refers to the replacement of a nucleotide occupying a position with a different nucleotide, deletion refers to the removal of a nucleotide occupying a position, insertion refers to the addition of a nucleotide immediately following the nucleotide occupying a position.
The term "promoter" refers to a nucleic acid molecule, typically located upstream of the coding sequence of the gene of interest, which provides a recognition site for RNA polymerase and is located 5' upstream of the transcription initiation site of mRNA. It is a nucleic acid sequence that is not translated, and RNA polymerase, when bound to this nucleic acid sequence, initiates transcription of the gene of interest. The term "RBS" refers to a ribosome binding site (ribosomebinding site) which is an untranslated region upstream of the initial AUG of mRNA, recognized by 16SrRNA, and initiates translation.
The mutant of the promoter of the present invention is a modified promoter of the gltA gene of Corynebacterium glutamicum, which has a higher promoter activity than the wild-type promoter, such as an increase of at least 1.6 times.
In a second aspect, the invention provides an expression cassette and a recombinant vector comprising a mutant of the citrate synthase gene promoter.
The expression cassette has the meaning generally understood by those skilled in the art, i.e., an element which contains a promoter, a gene of interest and is capable of expressing the gene of interest. In a specific embodiment, the gene of interest is a gene encoding a citrate synthase, more preferably the gene encoding a citrate synthase having an amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4.
The term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable control sequences to express a gene of interest in a suitable host. The vector used in the present invention is not particularly limited, and may be any vector known in the art as long as it can replicate in a host. That is, the vectors include, but are not limited to, plasmids, phages, such as pEC-XK99E plasmids used in the specific examples of the invention. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases integrate into the genome itself.
In a third aspect, the invention provides a recombinant host cell comprising a mutant of the citrate synthase gene promoter.
Recombinant host cells are achieved in particular by transformation. "transformation" here has the meaning generally understood by those skilled in the art, i.e.the process of introducing exogenous DNA into a host. The transformation method includes any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate (CaPO 4) precipitation, calcium chloride (CaCl 2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.
The term "microbial host cell" as used herein is intended to have a meaning which is generally understood by a person skilled in the art, i.e.a cell into which a nucleic acid molecule having promoter activity according to the invention can be introduced, which is referred to as a recombinant microbial host cell after introduction. In other words, the present invention can utilize any host cell as long as the nucleic acid having promoter activity of the present invention is contained in the cell and operably linked to a gene to mediate transcription of the gene. The host cell of the present invention may be a prokaryotic cell or eukaryotic cell, preferably enterobacter or corynebacterium, more preferably corynebacterium glutamicum, including but not limited to corynebacterium glutamicum ATCC 13869, corynebacterium glutamicum ATCC 13032, corynebacterium glutamicum B253, corynebacterium glutamicum ATCC 14067, and mutants or strains producing L-amino acids prepared from the above strains.
Illustratively, the host cell producing glutamic acid may be one in which, based on Corynebacterium glutamicum ATCC 13869, one or more genes selected from the group consisting of:
a. an odhA gene encoding an alpha-ketoglutarate dehydrogenase.
B. the sucA gene encoding succinate dehydrogenase.
In some embodiments, illustratively, one or more genes selected from the group consisting of:
a. The pyc gene encoding pyruvate carboxylase;
b. a gdh gene encoding glutamate dehydrogenase;
c. The gltA gene encoding citrate synthase;
d. Fxpk gene encoding phosphoketolase;
e. a ppc gene encoding a phosphoenolpyruvate carboxylase;
f. pitA gene encoding phosphate transporter;
g. yggB gene encoding mechanociceptive channel protein.
In the present invention, the cultivation of the host cells may be performed according to a conventional method in the art, including but not limited to well plate cultivation, shake flask cultivation, batch cultivation, continuous cultivation, fed-batch cultivation, etc., and various cultivation conditions such as temperature, time, pH value of the medium, etc., may be appropriately adjusted according to the actual situation.
In a fourth aspect, the invention provides a method of enhancing expression of a gene of interest, the method comprising operably linking a mutant of the glutamate dehydrogenase gene promoter to the gene of interest.
The term "operably linked" in the present invention means that the mutant of the citrate synthase gene promoter of the present invention is functionally linked to the coding gene to initiate and mediate transcription of the gene, indicating that the mutant of the citrate synthase gene promoter of the present invention is operably linked to the coding gene to control the transcriptional activity of the operator gene. The means of operative connection may be any means described by those skilled in the art. The method for enhancing the expression of a target gene according to the present invention is carried out by using the mutant of the glutamate dehydrogenase gene promoter according to the present invention, and the method is generally used by those skilled in the art.
In one embodiment, the coding gene is a citrate synthase gene, which is shown in SEQ ID NO. 3 or SEQ ID NO. 4.
Furthermore, in a fifth aspect, the present invention provides a method for producing an amino acid, the method comprising culturing a host cell comprising a mutant of the citrate synthase gene promoter operably linked to a gene encoding citrate synthase, and collecting the amino acid produced. Wherein the amino acid comprises proline, lysine, glutamic acid, hydroxyproline, arginine, ornithine, glutamine, 5-aminolevulinic acid and the like. More preferably, the amino acid production-related genes include, but are not limited to, gdh gene, lysC gene, thrABC gene, asd gene, aspB gene, hom gene, metX gene, dapA gene, dapB gene, ddh gene, proB gene, proA gene, proC gene, putA gene, hemA gene, etc. By the method of the invention, the yield of amino acids of the strain, in particular proline, lysine, glutamic acid, hydroxyproline, arginine, ornithine, glutamate, 5-aminolevulinic acid, can be increased.
In the method for producing an amino acid according to the present invention, a method generally used by those skilled in the art is employed on the basis of the mutant of the citrate synthase gene promoter according to the present invention, and the step of recovering an amino acid from cells or a culture broth is also included. Methods for recovering amino acids from cells or culture media are well known in the art and include, but are not limited to: filtration, anion exchange chromatography, crystallization and HPLC.
In a specific embodiment of the present invention, the host cell is Corynebacterium glutamicum, which is further modified, specifically by introducing an A111V mutation into the NCgl1221 homologous gene (BBD29_ 06760 or yggB) in said bacterium, to obtain glutamic acid-producing strain SCgGC.
The invention has the beneficial effects that: the nucleic acid molecule with enhanced promoter activity provided by the invention has higher promoter activity than wild type, can be used for enhancing the expression of a target gene, can be operably connected with a gltA gene for example, and can enhance the expression intensity of citrate synthase, thereby improving the production efficiency of amino acid such as glutamic acid of a recombinant strain and having higher application value.
Detailed Description
Unless defined otherwise, 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental techniques and methods used in the examples, unless otherwise specified, are conventional techniques, such as those not specified in the examples below, and are generally performed under conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.
The media used in the examples are as follows:
The TSB plate medium composition is (g/L): glucose, 5 g/L; yeast powder, 5 g/L; soybean peptone, 9 g/L; urea, 3 g/L; succinic acid, 0.5 g/L; k 2HPO4·3H2O,1 g/L;MgSO4·7H2 O,0.1 g/L; biotin, 0.01 mg/L; vitamin B1,0.1 mg/L; MOPS,20 g/L; agar powder, 15 g/L.
The TSB liquid culture medium comprises the following components (g/L): glucose, 5 g/L; yeast powder, 5 g/L; soybean peptone, 9 g/L; urea, 3 g/L; succinic acid, 0.5 g/L; k 2HPO4·3H2O,1 g/L;MgSO4·7H2 O,0.1 g/L; biotin, 0.01 mg/L; vitamin B1,0.1 mg/L; MOPS,20 g/L.
The seed culture medium used in the glutamic acid fermentation experiment comprises the following components: glucose, 25 g/L; KH 2PO4·3H2 O,2.2 g/L; urea, 3 g/L; corn steep liquor, 33 mL; mgSO 4·7H2 O,0.9 g/L; bean cake hydrolysate, 22 mL; MOPS,20 g/L; initial pH7.2.
The fermentation medium used in the glutamic acid fermentation experiment comprises the following components: glucose, 80 g/L; KH 2PO4, 1 g/L; urea, 10 g/L; the corn steep liquor dry powder ,5 g/L;MgSO4·7H2O,0.4 g/L;FeSO4·7H2O,10 mg/L;MnSO4·4H2O,10 mg/L;VB1,200 μg/L;MOPS,40 g/L; is initially pH7.5.
EXAMPLE 1 characterization of the intensity of the Corynebacterium glutamicum gltA promoter
(1) Construction of the gltA promoter characterization vector of Corynebacterium glutamicum
According to the disclosed Corynebacterium glutamicum ATCC 13869 genome (GenBank: CP 016335.1) sequence, an amplification primer gltA-1/2 was designed, and a fragment comprising the wild type promoter P gltA-WT (sequence shown as SEQ ID NO: 1) of the gltA gene and the N-terminal 162 bp sequence was amplified using the ATCC 13869 genome as a template. The pEC-XK99E-RFP plasmid reported in the literature (Wang Yingchun et al. Screening of endogenous high efficiency constitutive promoter of Corynebacterium glutamicum based on time series transcriptome [ J ]. Bioengineering report, 2018,34 (11): 1760-1771) was used as template to amplify pEC-XK99E plasmid backbone with PEC-1/2 as primer and RFP-1/2 as primer to amplify red fluorescent protein gene DNA fragment containing connecting peptide (DNA sequence: GGCGGTGGCTCTGGAGGTGGTGGGTCCGGCGGTGGCTCT). The primers used in this example are shown in Table 1. And (3) cloning and connecting the fragments by using a one-step recombination kit of the nuprazan after recovering and purifying the fragments to obtain the pEC-XK99E-P gltA-WT -rfp characterization vector.
(2) Construction of the gla mutant promoter characterization vector of Corynebacterium glutamicum
Random mutation of the wild type gltA promoter was performed, and the mutation at 475-476 of the wild type promoter was found to have higher promoter activity. Subsequently, the promoter mutants were characterized as follows. Designing a primer PgltA-Ts24-F/R, reversely amplifying by taking the pEC-XK99E-P gltA-WT -rfp characterization vector as a template, carrying out terminal phosphorylation treatment on fragments by using T4 PNK, and connecting by using T4 ligase to obtain the characterization vector pEC-XK99E-P gltA-Ts24 -rfp of a mutant promoter P gltA-Ts24 (the sequence of which is shown as SEQ ID NO: 2).
TABLE 1 construction of characterization vector primers
| Primer(s) | Nucleotide sequence | SEQ ID NO： |
| gltA-1 | CCTGATGCGGTATTTTCTCCCCCAAACGATGAAAAACGCCC | SEQ ID NO：5 |
| gltA-2 | CCACCTCCAGAGCCACCGCCGGAGCCAGTGCTCACATAACCTG | SEQ ID NO：6 |
| PEC-1 | CTGCAGGCATGCAAGCTTGG | SEQ ID NO：7 |
| PEC-2 | GGAGAAAATACCGCATCAGGC | SEQ ID NO：8 |
| RFP-1 | GGCGGTGGCTCTGGAGGTGGTGGGTCCGGCGGTGGCTCTGCTTCCTCCGAAGACGTTATCAAAG | SEQ ID NO：9 |
| RFP-2 | CCAAGCTTGCATGCCTGCAGTTAAGCACCGGTGGAGTGACGAC | SEQ ID NO：10 |
| PgltA-Ts24-F | TATCGGTATAATGTGTTAACCGGACCA | SEQ ID NO：11 |
| PgltA-Ts24-R | AATGCGGTTTAGCCATATCCGAA | SEQ ID NO：12 |
(3) Characterization of the gltA mutant promoter Strength of Corynebacterium glutamicum
The above characterization vectors were transformed into Corynebacterium glutamicum ATCC 13869, respectively, and plated on TSB solid plates containing 25. Mu.g/mL kanamycin to obtain recombinant strains. Transformants were inoculated into 24 well plates containing 800. Mu.L of TSB liquid medium per well, 3 strains per well were parallel, the rotation speed of the well plates was 800 rpm, and after incubation at 30℃for 16 h, the fluorescence intensity of the strains was measured by means of an enzyme-labeled instrument. The fluorescence measurement excitation wavelength is 560 nm, and the emission wavelength is 607 nm; meanwhile, the bacterial liquid OD 600 is measured, the fluorescence intensity of the bacterial strain is calculated, and the result is shown in Table 2. It can be seen that the activity of the mutant promoter P gltA-Ts24 is significantly increased by 1.6 times compared with the wild-type promoter.
Table 2 gltA fluorescence characterization of promoter mutants
| Promoter numbering | Fluorescence intensity (RFU/OD 600) | Fold increase over wild type | Sequence number of complete sequence of promoter |
| PgltA-WT | 2452±90 | —— | SEQ ID NO：1 |
| PgltA-Ts24 | 6393±160 | 1.6 | SEQ ID NO：2 |
EXAMPLE 2 application of Corynebacterium glutamicum gltA Gene promoter mutant GltA C361Y to L-glutamic acid production
(1) Construction of the gltA Gene overexpression plasmid of Corynebacterium glutamicum
Primers PgltA-UH-F/R and PgltA-DH-F/R were designed based on the published Corynebacterium glutamicum ATCC 13869 genome (GenBank: CP 016335.1) and the ATCC 13869 genome was used as a template to amplify the upstream and downstream recombinant fragments comprising the mutant promoter P gltA-Ts24. The primer pK-F/R is designed according to the sequence information of the plasmid pK18mobsacB, and the linearized vector fragment is obtained by PCR reverse amplification by taking the plasmid pK18mobsacB as a template. The three fragments are recovered and recombined to obtain an editing plasmid pK18-P gltA-Ts24 with a P gltA-Ts24 mutant promoter.
(2) Construction of Corynebacterium glutamicum GltA C361Y Gene mutant plasmid
By simulating the structure of the citrate synthase GltA protein in the early stage, predicting that 361 site is a key target of the protein activity, and by mutating the site, a GltA C361Y mutant is obtained, so the embodiment further verifies the condition that the P gltA-Ts24 mutant promoter expresses GltA C361Y mutant genes.
First, according to the published Corynebacterium glutamicum ATCC 13869 genome (GenBank: CP 016335.1) sequences, primers gltA-F1/R1 and gltA-F2/R2 were designed, and the ATCC 13869 genome was used as a template to amplify upstream and downstream recombinant fragments comprising GltA C361Y mutants, and the wild gltA gene sequence was as shown in SEQ ID NO:3, the sequence of the mutated gltA gene is shown as SEQ ID NO: 4. The primer pK-F/R is designed according to the sequence information of the plasmid pK18mobsacB, and the linearized vector fragment is obtained by PCR reverse amplification by taking the plasmid pK18mobsacB as a template. The three fragments are recovered and recombined to obtain the editing plasmid pK18-GltA C361Y with GltA C361Y mutant.
(3) Construction of L-glutamic acid-producing Strain
The introduction of the A111V mutation in the Corynebacterium glutamicum ATCC 13869 genome NCgl1221 homologous gene (BBD29_ 06760 or yggB) has been reported in the literature to confer the ability to constitutively synthesize L-glutamic acid. To verify the use of the above promoter in L-glutamic acid production, the above mutation was first introduced into the Corynebacterium glutamicum ATCC 13869 genome.
Primers A111V-UH-F/R and A111V-DH-F/R were designed according to the published Corynebacterium glutamicum ATCC13869 genome (GenBank: CP 016335.1) sequence. Using ATCC13869 genome as a template, and respectively obtaining DNA fragments with YggB A111V mutation by PCR amplification by using the primers; designing a primer pK-F/R according to the sequence information of the plasmid pK18mobsacB, and obtaining a linearization vector fragment by PCR reverse amplification by taking the plasmid pK18mobsacB as a template; the three fragments are recovered and recombined, clones obtained after transformation are collected and plasmids are extracted, and the YggB A111V mutated editing vector pK18-YggB A111V is obtained.
C.glutamicum ATCC 13869 competent cells (Biotechnology Letters, 2015, 37: 2445-52.) were prepared by the method reported in the literature, 1. Mu.g of pK18-YggB A111V plasmid was electrotransformed into the obtained 13869 competent cells, 1 mL of TSB medium preheated at 46℃was added, incubated at 46℃for 6 min, incubated at 30℃for 3h, and TSB solid medium containing 25. Mu.g/mL of kanamycin was applied and incubated at 30℃for 1 day to obtain transformants of the first recombination. The correct transformant was inoculated with TSB medium containing 5 g/L glucose overnight, then with TSB medium containing 100 g/L sucrose, cultured at 30℃for 6 h, and then plated on TSB medium supplemented with 100 g/L sucrose for selection to obtain L-glutamic acid producing strain SCgGC5. The primers used in this example are shown in Table 3.
Preparation of L-glutamic acid-producing Strain SCgGC competent cells （Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity, Biotechnology Letters, 2015, 37: 2445-52.）, by the method reported in the literature 1. Mu.g of pK18-GltA C361Y plasmid was electrotransformed into SCgGC competent cells obtained by the above preparation, 1 mL of TSB medium preheated at 46℃was added, 6 min incubated at 46℃and 3h incubated at 30℃were applied to TSB solid medium containing 25. Mu.g/mL kanamycin, and 24h cultured at 30℃to obtain transformants of the first recombination. The correct transformant was inoculated with 5 g/L of glucose in TSB medium overnight, then with 100 g/L of sucrose in TSB medium, cultured at 30℃for 4h and then plated on 100 g/L of sucrose in TSB medium for selection, whereby strain SCgGC7 with GltA C361Y mutant was obtained.
L-glutamic acid-producing strain SCgGC competent cells were prepared by the method reported in the literature, 1. Mu.g of pK18-P gltA-Ts24 plasmid was electrotransformed into SCgGC competent cells obtained by the above preparation, 1mL of TSB medium preheated at 46℃was added, 6 min of incubation at 46℃and 3 h of incubation at 30℃were performed, TSB solid medium containing 25. Mu.g/mL kanamycin was applied, and 24 h of culture at 30℃was used to obtain transformants of the first recombination. The correct transformant was inoculated with TSB medium containing 5g/L glucose overnight, then with TSB medium containing 100 g/L sucrose, cultured at 30℃for 4 h and then plated on TSB medium supplemented with 100 g/L sucrose for selection to obtain L-glutamic acid producing strain SCgGC8 harboring mutant promoter P gltA-Ts24.
Competent cells of L-glutamic acid-producing strain SCgGC8 were prepared by the same manner as described above, 1. Mu.g of pK18-GltA C361Y plasmid was electrotransformed into the competent cells, 1mL of TSB medium preheated at 46℃was added, incubation was performed at 46℃for 6 min, incubation was performed at 30℃for 3h, TSB solid medium containing 25. Mu.g/mL kanamycin was applied, and culture was performed at 30℃for 24 h to obtain transformants of the first recombination. The correct transformant was inoculated with TSB medium containing 5 g/L glucose overnight, then with TSB medium containing 100 g/L sucrose, cultured at 30℃for 6 h and then plated on TSB medium supplemented with 100 g/L sucrose for selection to obtain L-glutamic acid producing strain SCgGC with mutant promoters P gltA-Ts24 and GltA C361Y.
TABLE 3 primers used in this example
| Primer(s) | Nucleotide sequence | SEQ ID NO： |
| A111V-UH-F | tgacatgattacgaattcATCCACTGGAGTTTTGCCAATTCTC | SEQ ID NO：13 |
| A111V-UH-R | gtcttggtGTGcagtcgattgttgcg | SEQ ID NO：14 |
| A111V-DH-F | atcgactgCACaccaagaccaatggc | SEQ ID NO：15 |
| A111V-DH-R | cgacggccagtgccaagcttTGGAGGAATAGAGCGGGTCATACAC | SEQ ID NO：16 |
| pK-F | AAGCTTGGCACTGGCCGTCG | SEQ ID NO：17 |
| pK-R | GAATTCGTAATCATGTCATAGCTGT | SEQ ID NO：18 |
| PgltA-UH-F | tgacatgattacgaattcATTGAACCCACGACGTTTTTTACTG | SEQ ID NO：19 |
| PgltA-UH-R | tataccgataaatgcggtttagcca | SEQ ID NO：20 |
| PgltA-DH-F | gcatttatcggtataATgtgttaaccggacca | SEQ ID NO：21 |
| PgltA-DH-R | cgacggccagtgccaagcttATGTCTTCGAGCATCTCCAGAACAG | SEQ ID NO：22 |
| gltA-F1 | tgacatgattacgaattcTTTGACCCAGGTTATGTGAG | SEQ ID NO：23 |
| gltA-R1 | aatcatcagccagtgcaatttct | SEQ ID NO：24 |
| gltA-F2 | cactggctgatgattActtcatctcccgcaa | SEQ ID NO：25 |
| gltA-R2 | cgacggccagtgccaagcttCGGAAATCATAGAGCGACAA | SEQ ID NO：26 |
| pK-F | AAGCTTGGCACTGGCCGTCG | SEQ ID NO：27 |
| pK-R | GAATTCGTAATCATGTCATAGCTGT | SEQ ID NO：28 |
(4) Effect of gltA Gene promoter mutant and combination GltA C361Y mutant on L-glutamic acid Synthesis
To verify the effect of the gltA promoter mutant and the combination GltA C361Y mutant on glutamate production, fermentation tests were performed on the SCgGC and SCgGC strains constructed as described above, with strain SCgGC as a control.
The strains were first inoculated into a seed medium for cultivation of 8 h, the cultures were inoculated as seeds into 24-well plates containing 800. Mu.L of fermentation medium per well, the initial OD 600 was controlled to be about 0.5, the rotation speed of the well plates was 800 rpm, 3 strains were grown in parallel, at 30℃for cultivation, 5 g/L urea was fed in 19 h and 22 hours, the fermentation of 25 h was completed, the yield of L-glutamic acid and the glucose consumption were examined, and the sugar acid conversion rate from glucose to L-glutamic acid was calculated. The results are shown in Table 4.
TABLE 4L-glutamic acid production
| Strain | L-glutamic acid (g/L) | Conversion of sugar acid (g/g,%) |
| SCgGC5 | 4.50±0.22 | 5.86±0.33 |
| SCgGC7 | 8.53±0.31 | 12.03±0.0.56 |
| SCgGC8 | 5.15±0.25 | 6.70±0.31 |
| SCgGC9 | 10.53±0.81 | 15.48±1.28 |
As shown in the table, P gltA-Ts24 can obviously improve the yield of L-glutamic acid and the sugar acid conversion rate, and on the basis, the GltA C361Y mutant is integrated, so that the yield of L-glutamic acid and the sugar acid conversion rate can be further improved. Therefore, the gltA promoter mutant has a good application prospect in the production of L-glutamic acid and derivatives thereof.
Claims (15)
1. A nucleic acid molecule with promoter activity is characterized in that the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2.
2. An expression cassette comprising the nucleic acid molecule of claim 1.
3. A recombinant vector comprising the nucleic acid molecule of claim 1.
4. A recombinant microbial host cell comprising the nucleic acid molecule of claim 1.
5. The recombinant microbial host cell of claim 4 wherein the recombinant microbial host cell is corynebacterium glutamicum.
6. The recombinant microbial host cell of claim 5 wherein the recombinant microbial host cell is corynebacterium glutamicum ATCC 13869, corynebacterium glutamicum ATCC 13032, corynebacterium glutamicum B253, corynebacterium glutamicum ATCC 14067, or an L-amino acid producing strain prepared from the above strains.
7. The recombinant microbial host cell of claim 6 wherein the recombinant microbial host cell is based on corynebacterium glutamicum ATCC 13869 wherein one or more genes selected from the group consisting of:
An odhA gene encoding alpha-ketoglutarate dehydrogenase;
A sucA gene encoding succinate dehydrogenase;
Or one or more genes selected from the group consisting of:
a. The pyc gene encoding pyruvate carboxylase;
b. a gdh gene encoding glutamate dehydrogenase;
c. The gltA gene encoding citrate synthase;
d. Fxpk gene encoding phosphoketolase;
e. a ppc gene encoding a phosphoenolpyruvate carboxylase;
f. pitA gene encoding phosphate transporter;
g. yggB gene encoding mechanociceptive channel protein.
8. The recombinant microbial host cell according to claim 6 or 7, wherein the recombinant microbial host cell is a glutamic acid-producing strain obtained by introducing an a111V mutation into the NCgl1221 gene or a homologous gene bbd29_06760 or yggB thereof in corynebacterium glutamicum ATCC 13869.
9. A method of enhancing expression of a gene of interest, the method comprising operably linking the nucleic acid molecule of claim 1 to a gene of interest.
10. The method of claim 9, wherein the gene of interest is a gene encoding citrate synthase.
11. The method of claim 10, wherein the citrate synthase has an amino acid sequence as set forth in SEQ ID No. 3 or SEQ ID No. 4.
12. A method of producing an amino acid, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 1 operably linked to a gene encoding an amino acid production, and collecting the amino acid produced.
13. The method of claim 12, wherein the amino acid is selected from the group consisting of proline, lysine, glutamic acid, hydroxyproline, arginine, ornithine, glutamine, 5-aminolevulinic acid; the amino acid production related gene is selected from gdh gene, lysC gene, thrABC gene, asd gene, aspB gene, hom gene, metX gene, dapA gene, dapB gene, ddh gene, proB gene, proA gene, proC gene, putA gene, hemA gene.
14. The method of claim 13, further comprising the step of recovering the amino acid from the cells or the culture broth.
15. Use of the nucleic acid molecule of claim 1, the expression cassette of claim 2, the recombinant vector of claim 3, the recombinant microbial host cell of any one of claims 4-8 for enhancing expression of a gene or producing an amino acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211145762.1A CN115927325B (en) | 2022-09-20 | 2022-09-20 | Citrate synthase promoter mutant and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211145762.1A CN115927325B (en) | 2022-09-20 | 2022-09-20 | Citrate synthase promoter mutant and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115927325A CN115927325A (en) | 2023-04-07 |
| CN115927325B true CN115927325B (en) | 2024-07-09 |
Family
ID=86654613
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211145762.1A Active CN115927325B (en) | 2022-09-20 | 2022-09-20 | Citrate synthase promoter mutant and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115927325B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695383A (en) * | 2016-03-02 | 2016-06-22 | 廊坊梅花生物技术开发有限公司 | Recombinant strain and application thereof |
| CN113201535A (en) * | 2020-07-20 | 2021-08-03 | 中国科学院天津工业生物技术研究所 | Mutant of glutamate dehydrogenase gene promoter and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1655374B1 (en) * | 2003-06-10 | 2014-10-15 | Ajinomoto Co., Inc. | Process for producing l-glutamic acid |
| CN113755492B (en) * | 2020-07-20 | 2023-05-30 | 中国科学院天津工业生物技术研究所 | Mutant of pyruvate carboxylase gene promoter and application thereof |
-
2022
- 2022-09-20 CN CN202211145762.1A patent/CN115927325B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695383A (en) * | 2016-03-02 | 2016-06-22 | 廊坊梅花生物技术开发有限公司 | Recombinant strain and application thereof |
| CN113201535A (en) * | 2020-07-20 | 2021-08-03 | 中国科学院天津工业生物技术研究所 | Mutant of glutamate dehydrogenase gene promoter and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115927325A (en) | 2023-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113201535B (en) | Mutant of glutamate dehydrogenase gene promoter and application thereof | |
| JP5266252B2 (en) | Promoter nucleic acid molecule derived from Corynebacterium glutamicum, recombinant vector containing the promoter, host cell containing the recombinant vector, and method for expressing a gene using the host cell | |
| WO2022037338A1 (en) | Polynucleotide having promoter activity and application of polynucleotide in producing amino acid | |
| TW201712117A (en) | Microorganisms for producing putrescine or ornithine and process for producing putrescine or ornithine using them | |
| HUE030771T2 (en) | Improved promoter, and a production method for l-lysine using the same | |
| KR102277407B1 (en) | Novel glutamate synthase subunit alpha variant and a method for producing L-glutamic acid using the same | |
| WO2022017223A1 (en) | Mutant of pyruvate carboxylase gene promoter and use thereof | |
| CN115927325B (en) | Citrate synthase promoter mutant and application thereof | |
| CN115948396B (en) | Glutamate dehydrogenase promoter mutant and application thereof | |
| RU2812048C1 (en) | Pyruvate carboxylase gene promoter mutant and its application | |
| RU2812048C9 (en) | Pyruvate carboxylase gene promoter mutant and its application | |
| CN115449518B (en) | Polynucleotide with promoter activity based on mdh gene and application thereof | |
| CN116042591A (en) | Methyl pyrimidine phosphate synthase mutant and application thereof in construction of glutamic acid production strain | |
| CN116144564A (en) | Construction method of glutamic acid production strain and application of glutamic acid production strain in production of glutamic acid | |
| KR102616694B1 (en) | Shewanella atlantica-Drived Protein Expressing Microorganism and Method of L-Amino Acid Production Using the Same | |
| WO2013154182A1 (en) | Method for producing amino acid | |
| KR102339271B1 (en) | Novel promoter and use thereof | |
| KR102339264B1 (en) | Novel promoter and use thereof | |
| CN116004501A (en) | NADP-ferredoxin reductase mutant and application thereof in production of glutamic acid | |
| EP4332230A1 (en) | Corynebacterium glutamicum variant with improved l-lysine production ability, and method for producing l-lysine using same | |
| KR102031886B1 (en) | Novel promoter and uses thereof | |
| CN117737021A (en) | Mutant citrate synthase and use thereof | |
| CN115851802A (en) | Construction method of glutamic acid high-producing strain and application of glutamic acid high-producing strain in glutamic acid production | |
| CN116445379A (en) | Inactivation of N-acetylglucosamine 6-phosphate deacetylase and application thereof in glutamic acid production | |
| CN116334112A (en) | Construction method and application of amino acid production strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |