CN116769748A - 5-aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA - Google Patents
5-aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA Download PDFInfo
- Publication number
- CN116769748A CN116769748A CN202310831702.3A CN202310831702A CN116769748A CN 116769748 A CN116769748 A CN 116769748A CN 202310831702 A CN202310831702 A CN 202310831702A CN 116769748 A CN116769748 A CN 116769748A
- Authority
- CN
- China
- Prior art keywords
- aminolevulinic acid
- ala
- mutant
- escherichia coli
- 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.)
- Granted
Links
- 229960002749 aminolevulinic acid Drugs 0.000 title claims abstract description 50
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 30
- 108090000364 Ligases Proteins 0.000 title claims abstract description 13
- 102000003960 Ligases Human genes 0.000 title claims abstract description 11
- 239000002243 precursor Substances 0.000 title abstract description 8
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 claims abstract description 41
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 229930003779 Vitamin B12 Natural products 0.000 claims abstract description 14
- 239000011715 vitamin B12 Substances 0.000 claims abstract description 14
- 235000019163 vitamin B12 Nutrition 0.000 claims abstract description 14
- 108020000161 polyphosphate kinase Proteins 0.000 claims abstract description 6
- 150000001413 amino acids Chemical group 0.000 claims description 15
- 101710084741 5-aminolevulinate synthase Proteins 0.000 claims description 13
- 101710188223 5-aminolevulinate synthase, mitochondrial Proteins 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 108010075728 Succinate-CoA Ligases Proteins 0.000 claims description 5
- 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 claims description 4
- 102000011929 Succinate-CoA Ligases Human genes 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 239000004475 Arginine Substances 0.000 claims description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 2
- 229960000310 isoleucine Drugs 0.000 claims description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 20
- 102000004190 Enzymes Human genes 0.000 abstract description 18
- 230000015572 biosynthetic process Effects 0.000 abstract description 13
- 238000003786 synthesis reaction Methods 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 11
- 230000006835 compression Effects 0.000 abstract description 5
- 238000007906 compression Methods 0.000 abstract description 5
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 abstract description 3
- 241000191023 Rhodobacter capsulatus Species 0.000 abstract description 3
- 238000000329 molecular dynamics simulation Methods 0.000 abstract description 3
- 210000003370 receptor cell Anatomy 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 2
- 101150044508 key gene Proteins 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 10
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 7
- 229940024606 amino acid Drugs 0.000 description 7
- 101150112623 hemA gene Proteins 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000002609 medium Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 4
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 4
- 241000024188 Andala Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 101150001140 ppk gene Proteins 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000168053 Pseudomonas denitrificans (nomen rejiciendum) Species 0.000 description 2
- 241000190950 Rhodopseudomonas palustris Species 0.000 description 2
- 241000589196 Sinorhizobium meliloti Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910001453 nickel ion Inorganic materials 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 2
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 2
- 229960001327 pyridoxal phosphate Drugs 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- VNOYUJKHFWYWIR-ITIYDSSPSA-N succinyl-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)CCC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 VNOYUJKHFWYWIR-ITIYDSSPSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 101100284769 Drosophila melanogaster hemo gene Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- VEQPNABPJHWNSG-UHFFFAOYSA-N Nickel(2+) Chemical compound [Ni+2] VEQPNABPJHWNSG-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000186429 Propionibacterium Species 0.000 description 1
- 241000186428 Propionibacterium freudenreichii Species 0.000 description 1
- 241000589180 Rhizobium Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 235000010633 broth Nutrition 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 150000001722 carbon compounds Chemical class 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- WUPRCGRRQUZFAB-DEGKJRJSSA-N corrin Chemical group N1C2CC\C1=C\C(CC/1)=N\C\1=C/C(CC\1)=N/C/1=C\C1=NC2CC1 WUPRCGRRQUZFAB-DEGKJRJSSA-N 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000003041 virtual screening Methods 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a 5-aminolevulinic acid synthetase mutant and escherichia coli producing a B12 precursor ALA, and belongs to the technical field of biology. The invention modifies key enzyme 5-aminolevulinic acid synthetase for ALA synthesis based on isothermal compression coefficient disturbance engineering of high-pressure molecular dynamics simulation to obtain a mutant with improved specific activity, and then synthesizes ALA by multi-gene combined expression fermentation in escherichia coli, in particular, two key genes succinyl-CoA synthetase gene SUC and polyphosphate kinase gene PPK from escherichia coli MG1655 and key gene 5-aminolevulinic acid synthetase gene mutant from rhodobacter capsulatus are introduced into receptor cells, thereby establishing a synthesis path of ALA in the receptor cells and laying a foundation for developing health-care food rich in vitamin B12.
Description
Technical Field
The invention relates to a 5-aminolevulinic acid synthetase mutant and escherichia coli producing a B12 precursor ALA, belonging to the technical field of biology.
Background
Vitamin B12, also known as cobalamin, is one of the smallest but structurally complex molecules recognized in the field of bioscience, and is the only water-soluble vitamin containing metal ions, which has wide application in medicine and cosmetics, plays an important cofactor role in fat biochemical reactions, protein synthesis and regulation, and it has now been found that only microorganisms have the ability to synthesize cobalamin through aerobic and anaerobic pathways.
5-aminolevulinic acid (ALA) is an important intermediate for the in vivo synthesis of tetrapyrroles (precursors of vitamin B12, chlorophyll and heme). ALA is synthesized by two routes, namely C4 and C5, wherein the C5 route is mainly found in higher plants, algae and various bacteria, and is obtained by three enzymatic reactions of glutamic acid. The C4 pathway is the condensation of two precursor substances glycine and succinyl-CoA, four carbon compounds, in a 1:1 ratio, under the catalysis of ALA synthase (ALAS, encoded by the hemA gene) to form ALA. The aerobic and anaerobic vitamin B12 synthesis pathway can be divided into four parts: synthesis of ALA, synthesis of corrin ring component, construction of lower axial ligand and synthesis of cobalamin. Industrial microbial fermentation is used as an alternative to the initially established chemical synthesis of vitamin B12, which requires at least 60 steps.
Industrial production of vitamin B12 is mainly carried out by fermentation of microorganisms such as Pseudomonas denitrificans (Pseudomonas denitrificans), propionibacterium freudenreichii (Propionibacterium freichi) and Sinorhizobium meliloti (Sinorhizobium meliloti). In recent years, escherichia coli constructed by synthetic biology and metabolic engineering techniques can be used for producing vitamin B12, and the escherichia coli has the advantages of fast growth, mature genetic operation tools and the like and is widely applied. For example, in 2003, lee et al used hemA from a slow-growing rhizobium sojae to transfer into E.coli BL21 (DE 3), optimizing the C, N ratio in the medium and increasing the yield to 3.8g/L. In 2004, kang et al introduced hemA derived from rhodobacter capsulatus into E.coli BL21 (DE 3), and measured ALA yield of 2.8g/L. In 2008, choi et al transferred hemA gene derived from Rhodopseudomonas palustris KUGB306 into E.coli BL21 (DE 3) via pGEX-KG vector for expression, succinic acid, glycine and glucose were added into the medium, and the ALA yield was 5.2g/L. In 2009, lin et al transferred the hemA gene from Agrobacterium radiobacter zju-0121 into E.coli Rosetta (DE 3) via pET28a vector, and ALA yields as high as 7.3g/L were measured under optimal fermentation conditions. In 2013, zhang et al cloned hemA and hemO genes from Rhodopseudomonas palustris and successfully expressed in E.coli, and measured ALA yields in the fermentation broths were 5.7g/L and 6.3g/L, respectively. In 2014, lou et al were introduced into E.coli Rosetta (DE 3) using hemA from rhodobacter capsulatus, and ALA yield was 8.8g/l measured under optimal culture conditions. However, as shown in patent CN102206606a, the modified escherichia coli is fermented in a 3L fermentation tank for 56 hours, and the final ALA yield only reaches 4.13g/L, so that the effect of the microorganism on synthesizing the vitamin B12 precursor ALA needs to be further improved.
Disclosure of Invention
In order to solve the problems, the invention provides a 5-aminolevulinic acid synthetase mutant with obviously improved specific enzyme activity, and a method for efficiently preparing a precursor substance ALA of vitamin B12 is developed based on the mutant, so that a foundation is laid for developing health-care food rich in vitamin B12.
The first object of the present invention is to provide a 5-aminolevulinic acid synthase mutant obtained by mutating arginine at position 151 of a 5-aminolevulinic acid synthase having an amino acid sequence as shown in SEQ ID NO.1 into isoleucine (R151I).
It is a second object of the present invention to provide a gene encoding the above-mentioned 5-aminolevulinic acid synthase mutant.
A third object of the present invention is to provide a recombinant plasmid carrying the above gene.
It is a fourth object of the present invention to provide host cells expressing the above-described mutants of 5-aminolevulinic acid synthase.
Further, the host cell is a bacterial, fungal, plant cell or animal cell.
It is a fifth object of the present invention to provide the use of the above-described 5-aminolevulinic acid synthase mutants, genes, recombinant plasmids or host cells for the preparation of 5-aminolevulinic acid or vitamin B12.
A sixth object of the present invention is to provide a recombinant E.coli producing 5-aminolevulinic acid, which heterologously expresses the above-mentioned 5-aminolevulinic acid synthase mutant.
Further, the recombinant E.coli also overexpresses succinyl-CoA synthetase and polyphosphate kinase.
Further, the amino acid sequence of the succinyl-CoA synthetase is shown as SEQ ID NO. 2; the amino acid sequence of the polyphosphate kinase is shown as SEQ ID NO. 3.
Furthermore, E.coli BL21 (DE 3) was used as starting strain.
A seventh object of the present invention is to provide the use of the recombinant E.coli described above for the preparation of 5-aminolevulinic acid or vitamin B12.
An eighth object of the present invention is to provide a method for producing 5-aminolevulinic acid, comprising the step of fermentation production using the recombinant E.coli as described above.
Further, glucose is used as a substrate for fermentation production.
The invention has the beneficial effects that:
the invention reforms the key enzyme ALAS (5-aminolevulinic acid synthetase) for ALA synthesis through isothermal compression coefficient disturbance engineering based on high-pressure molecular dynamics simulation, and obtains mutants with improved specific enzyme activity. And then, a polygene combined expression engineering technology is used for introducing a 5-aminolevulinic acid synthesis key enzyme gene into an escherichia coli expression system, and an ALA biosynthesis path is established in a receptor cell body, so that high-level production of ALA is realized, and escherichia coli is easy to realize high-density large-scale culture, thereby greatly reducing the production cost. Finally, the OD600 reaches 23 after fermentation in a 5L fermentation tank for 32 hours, and the ALA yield reaches 28 g.L -1 。
Drawings
FIG. 1 is a schematic representation of the synthesis of 5-aminolevulinic acid via the C4 pathway.
FIG. 2 shows the specific enzyme activities of the wild type and mutant.
FIG. 3 is a graph showing the yield of ALA produced by shake flask fermentation of a wild strain and a mutant strain.
FIG. 4 is a graph showing the yield of ALA produced by fermentation in a 5L fermenter of a wild strain and a mutant strain.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The materials and methods involved in the following examples are as follows:
(1) Sequence:
the wild type amino acid sequence of the 5-aminolevulinic acid synthetase is shown as SEQ ID NO. 1.
The amino acid sequence of succinyl-CoA synthetase is shown as SEQ ID NO. 2.
The amino acid sequence of the polyphosphate kinase is shown as SEQ ID NO. 3.
(2) Culture medium:
seed medium (g.L-1): yeast extract 5.0, peptone 10.0, sodium chloride 5.0, ph7.0.
Fermentation medium (g.L-1): yeast extract 2.0, ammonium sulfate 16.0, potassium dihydrogen phosphate 3.0, disodium hydrogen phosphate dodecahydrate 16.0, magnesium sulfate heptahydrate 1.0, manganese sulfate monohydrate 0.01, mgSO 4 ·7H 2 O and MnSO 4 ·H 2 O was sterilized separately and 0.09M glycine and 0.1M succinic acid were added as reaction substrates. Antibiotics were added according to different resistance requirements at concentrations of 100. Mu.g.mL-1 for ampicillin, 50. Mu.g.mL-1 for kanamycin and 50. Mu.g.mL-1 for spectinomycin.
(3) The detection method of the ALA product comprises the following steps:
1mL of the bacterial liquid is taken and centrifuged for 1min under the condition of 12000 r.min < -1 >, and the extracellular supernatant is taken for ALA concentration measurement. Preparing a reaction system: 400. Mu.L of sodium acetate buffer, 400. Mu.L of supernatant or supernatant diluent and 100. Mu.L of acetylacetone were added in this order, and after thorough mixing, the mixture was reacted in a boiling water bath (100 ℃ C.) for 15 minutes. After taking out, cooling to room temperature, adding 900 mu LModiiedEhrlich's reagent (adding 16mL of 70% perchloric acid into 2g of p-dimethylaminobenzaldehyde, fixing the volume to 100mL with glacial acetic acid), fully mixing for 15min, measuring the absorbance of the reaction solution at 540nm by using an ultraviolet spectrophotometer, and calculating the ALA concentration according to a standard curve.
Example 1
According to the sequence of escherichia coli MG1655 succinyl-CoA synthetase gene and polyphosphate kinase gene ppk published in Genebank, designing proper primers, taking escherichia coli MG1655 genome DNA as a template, and cloning algae haematochrome synthesis key enzyme genes, the gene and the ppk by adopting a genetic engineering method;
and (3) applying a polygene combination expression engineering method, respectively adding BamHI and HindIII enzyme cutting sites at two ends of the ppk gene, and inserting the BamHI and HindIII enzyme cutting sites into a first multicloning site of the pACYC-dure plasmid to obtain a recombinant vector pACYC-dure-ppk. NdeL and XhoI restriction sites are added at two ends of the gene suc respectively, and the gene is cloned into another polyclonal site of pACYC-dur-ppk, so that a recombinant vector pACYC-dur-ppk-suc is obtained.
Example 2
1. Virtual screening of high stability and active mutants
The method comprises the steps of screening out a high-plasticity area which is positioned on the surface of a protein and far away from an active center through isothermal compression coefficient disturbance of high-pressure molecular dynamics simulation (namely, applying gradient pressure to a 5-aminolevulinic acid synthase system, calculating isothermal compression coefficients under different pressures, selecting an area with large isothermal compression coefficient fluctuation as the high-plasticity area under a pressurizing disturbance mode), calculating Dynamic Flexibility Index (DFI) of amino acid on the high-plasticity area of the 5-aminolevulinic acid synthase, screening out the amino acid with the% DFI of more than 0.2, then calculating Dynamic Coupling Index (DCI) of the amino acid with the active center (H138-H143-E184), screening out the amino acid with the DCI of more than 0.8, comparing the amino acid with an amino acid sequence of the 5-aminolevulinic acid synthase reported by Genbank, and mutating the amino acid as a conservative amino acid. 7 mutants K150E, R151I, G192L, R374V, V390F, V390W, L395F were obtained.
2. Construction of Single-Point mutant recombinant plasmid
Primers shown in Table 1 were designed. And carrying out full plasmid PCR amplification by taking plasmid pET-28a-RphemA as an original template, and constructing to obtain a mutant recombinant plasmid. The PCR reaction system was 50. Mu.L: ddH 2 O18. Mu.L; 2xMaxBuffer 25. Mu.L; dNTPMix (10 mM) 1. Mu.L; 1 μl of pET-28 a-RphhemA template; 2. Mu.L of each of the upstream and downstream primers (10 mM); phantaMaxSuper-FidelityDNAPloymerase 1. Mu.L. PCR reaction conditions: 95 ℃ for 30s; 15s at 95 ℃, 15s at 68 ℃, 5min at 72 ℃,30 cycles; stored at 72℃for 5min and at 4 ℃. After the reaction is finished, the PCR product is digested by DpnI enzyme, the digested product is transferred into E.coliJM109, and finally recombinants with correct sequence are amplified and cultured, plasmids are extracted, and transferred into E.coliBL21 (DE 3) and stored at the temperature of minus 20 ℃.
TABLE 1PCR primers
3. Characterization of the enzymatic Properties of the mutants
The 5-aminolevulinic acid synthetase gene is subjected to protein expression by adopting an escherichia coli expression system, wherein a selected plasmid is pET-28a, and escherichia coli E.coli BL21 (DE 3) is taken as a host.
E.coliBL21 (DE 3) transformant carrying mutant recombinant plasmid is selected for small-scale expansion culture, and bacterial liquid PCR and sequencing verification are carried out. After verification, E.coli BL21 (DE 3) of the 9 mutant recombinant plasmids were subjected to expansion culture and induced expression, and after the induction culture was completed, the cells were collected, washed with 20mM phosphate buffer (pH 7.4) for one time to remove the remaining medium as much as possible, resuspended in this buffer, and subjected to cell disruption using an ultrasonic disruption instrument (450 w,5s/5s,25 min). After the crushing, centrifuging the crushed solution at 4 ℃ (10000 r.min < -1 >) for 1h, collecting the supernatant, and filtering the supernatant by a 0.22 mu m microporous filter to obtain crude enzyme solution.
The crude enzyme solution of the 9 mutant 5-aminolevulinic acid synthetases was purified using a nickel ion affinity column (1 mLHistTrapFF) packed with Ni-NTA and an AKTA protein purifier as follows: (1) equilibration column: the column (20 volumes) was equilibrated with ultrapure water and then equilibrated with a final concentration of imidazole of 20mM in the binding solution (20 volumes). (2) sample loading: and (3) adopting a sample injection pump to automatically sample, and loading the crude enzyme liquid at a flow rate of 1 mL/min. (3) elution: washing 10 column volumes with buffer solution with imidazole final concentration of 20mM to remove part of the impurity protein, washing 30 column volumes with eluent with imidazole final concentration of 500mM, collecting the eluted product under the target peak and labeling. (4) column regeneration: due to the loss of nickel ions in the purification process, after the purification is finished, the nickel column is regenerated by the pre-prepared regeneration solution, so that the next use is convenient. (5) And performing SDS-PAGE verification and enzyme activity detection on the purified and collected enzyme solution.
The enzyme activity of the product 5-ALA obtained by the reaction was measured by a chromogenic method. After induction, the thalli are subjected to ultrasonic wall breaking, the wall is placed at 12000rpm for 5min, 1000 mu L of supernatant is taken and 1000 mu L of reaction buffer solution is added: 50mM Tris-HC1, pH=7.5, 0.2mM succinyl CoA,0.1mM pyridoxal phosphate (PLP), were mixed and reacted at 37℃for 20min, respectively. The reaction was stopped by adding 500. Mu.L of 10% trichloroacetic acid at the end of the reaction time. After the reaction was completed, the mixture was centrifuged at 12000rpm for 5 minutes, and the supernatant was transferred to another tube. To the supernatant, 2mL of sodium acetate (pH 4.6) at a concentration of 2mo1/L, 500. Mu.L of acetylacetone was added, followed by mixing, boiling for 15min, and cooling to room temperature. The enzyme activity was calculated by adding 2mL of modified Ehrich's reagent, and measuring the absorbance at 554nm after 15min of reaction at room temperature. Definition of enzyme activity: the amount of enzyme required for converting the substrate to 1nmol of 5-ALA was converted at 37℃and pH7.5 for 1 min. The results of the enzyme activities are shown in FIG. 2.
Example 3
The wild-type recombinant strain constructed in example 2 and 7 mutant recombinant strains were subjected to shake flask fermentation. When ALA is produced by shake flask fermentation, 0.1 mmol.L final concentration is required to be added -1 IPTG induces the expression of exogenous genes, and the shake flask fermentation yields of wild type and mutant are shown in FIG. 3.
Example 5
In order to further evaluate and verify the ALA production capability of the recombinant engineering bacteria (recombinant bacteria containing mutant R151I) with the optimal shake flask fermentation effect, the strain is subjected to amplification culture in a 5L fermentation tank, and cell growth, carbon source consumption and ALA synthesis conditions are analyzed.
In a 5L tank, the concentration of the recombinant strain is greatly improved compared with that of shake flask culture, the OD600 reaches 23 at most, and more and denser thalli are provided for ALA production; with the consumption of glucose as a carbon source and the increase of the bacterial body quantity, the ALA yield steadily increases along with time, reaches the peak of 28 g.L-1 at about 32h, and is improved by about 255.5 percent compared with the yield of a wild strain. The fermentation yield of the wild type and mutant at 32h of fermentation is shown in FIG. 4.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. A mutant 5-aminolevulinic acid synthase, characterized by: is obtained by mutating arginine at position 151 of 5-aminolevulinic acid synthetase with an amino acid sequence shown as SEQ ID NO.1 into isoleucine.
2. A gene encoding the 5-aminolevulinic acid synthase mutant according to claim 1.
3. A recombinant plasmid carrying the gene of claim 2.
4. A host cell expressing the 5-aminolevulinic acid synthase mutant of claim 1.
5. Use of a 5-aminolevulinic acid synthase mutant according to claim 1, a gene according to claim 2, a recombinant plasmid according to claim 3 or a host cell according to claim 4 for the preparation of 5-aminolevulinic acid or vitamin B12.
6. A recombinant escherichia coli producing 5-aminolevulinic acid, characterized by: the recombinant escherichia coli heterologously expresses the 5-aminolevulinic acid synthetase mutant as defined in claim 1.
7. The recombinant E.coli according to claim 6, wherein: the recombinant E.coli also overexpresses succinyl-CoA synthetase and polyphosphate kinase.
8. Use of the recombinant escherichia coli of claim 6 or 7 for the preparation of 5-aminolevulinic acid or vitamin B12.
9. A process for producing 5-aminolevulinic acid, characterized by: comprising the step of fermentation production using the recombinant E.coli of claim 6 or 7.
10. The method according to claim 9, wherein: and fermenting and producing by taking glucose as a substrate.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310831702.3A CN116769748B (en) | 2023-07-07 | 5-Aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310831702.3A CN116769748B (en) | 2023-07-07 | 5-Aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116769748A true CN116769748A (en) | 2023-09-19 |
CN116769748B CN116769748B (en) | 2024-06-28 |
Family
ID=
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012129748A1 (en) * | 2011-03-31 | 2012-10-04 | 山东大学 | Recombinant e.coli and use thereof in producing 5-aminolevulinic acid |
CN103146694A (en) * | 2013-02-28 | 2013-06-12 | 河北科技大学 | Gene for constructing 5-aminolevulinic acid C4 biosynthesis pathway in Escherichia coli and construction method thereof |
CN104004701A (en) * | 2014-06-18 | 2014-08-27 | 江南大学 | Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains |
CN104830748A (en) * | 2015-06-02 | 2015-08-12 | 江南大学 | Method for weakening hemB gene expression to increase yield of 5-aminolevulinic acid synthesized by escherichia coli |
US20150376661A1 (en) * | 2013-02-07 | 2015-12-31 | Tianjin Institute Of Industrial Biotechnology, Chinese Academy Of Sciences | 5-aminolevulinic acid high-yield bacterial strain, preparation method and use thereof |
CN106497858A (en) * | 2016-12-02 | 2017-03-15 | 江南大学 | A kind of colibacillus engineering of 5 amino-laevulic acids of production |
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012129748A1 (en) * | 2011-03-31 | 2012-10-04 | 山东大学 | Recombinant e.coli and use thereof in producing 5-aminolevulinic acid |
US20150376661A1 (en) * | 2013-02-07 | 2015-12-31 | Tianjin Institute Of Industrial Biotechnology, Chinese Academy Of Sciences | 5-aminolevulinic acid high-yield bacterial strain, preparation method and use thereof |
CN103146694A (en) * | 2013-02-28 | 2013-06-12 | 河北科技大学 | Gene for constructing 5-aminolevulinic acid C4 biosynthesis pathway in Escherichia coli and construction method thereof |
CN104004701A (en) * | 2014-06-18 | 2014-08-27 | 江南大学 | Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains |
CN104830748A (en) * | 2015-06-02 | 2015-08-12 | 江南大学 | Method for weakening hemB gene expression to increase yield of 5-aminolevulinic acid synthesized by escherichia coli |
CN106497858A (en) * | 2016-12-02 | 2017-03-15 | 江南大学 | A kind of colibacillus engineering of 5 amino-laevulic acids of production |
Non-Patent Citations (2)
Title |
---|
JIA-WEI LOU等: "High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties", J ZHEJIANG UNIV SCI B ., vol. 15, no. 5, 31 May 2014 (2014-05-31), pages 491 - 499 * |
刘艳;江海洋;马伟;范军;程备久;朱苏文;: "表达浑球红细菌hemA基因生产5-氨基乙酰丙酸的研究", 激光生物学报, no. 04, 15 August 2010 (2010-08-15), pages 453 - 457 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111433357B (en) | Alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohol | |
CN111019878B (en) | Recombinant escherichia coli with improved L-threonine yield as well as construction method and application thereof | |
CN109370998B (en) | Omega-transaminase mutant I215F with improved catalytic efficiency | |
CN112746067B (en) | Lysine decarboxylase mutants for preparing D-ornithine | |
CN111454918B (en) | Enol reductase mutant and application thereof in preparation of (R) -citronellal | |
CN113201516A (en) | P-nitrobenzyl esterase mutant and application thereof | |
CN116769748B (en) | 5-Aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA | |
CN109486780B (en) | Omega-transaminase mutant with improved catalytic efficiency | |
US11492607B2 (en) | Highly active S-cyanohydrin lyase and application thereof | |
CN108034646B (en) | PvEH3 mutant with improved catalytic activity and improved enantiotropic normalization | |
CN101668850B (en) | Novel n-acetylglucosamine-2-epimerase and method for producing CMP-neuraminic acid using the same | |
CN116769748A (en) | 5-aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA | |
CN113604453B (en) | Marine streptomycete phospholipase D mutant and application | |
CN113444702B (en) | Enone reductase mutant and application thereof | |
KR20020029767A (en) | Cyclic depsipeptide synthases, genes thereof and mass production system of cyclic depsipeptide | |
CN112921012B (en) | Corynebacterium glutamicum meso-2, 6-diaminopimelate dehydrogenase mutant and application thereof | |
CN112375725B (en) | Metabolic engineering strain for producing vitamin B6 and construction method and application thereof | |
CN109402188B (en) | Omega-transaminase from bacillus pumilus and application of omega-transaminase in biological amination | |
CN112481226A (en) | Alcohol dehydrogenase mutant and application thereof | |
CN109609473A (en) | A kind of carbonyl reductase DmCR and its encoding gene, recombinant expression carrier, recombinant expression cell and its application | |
US9637761B2 (en) | Recombinant microorganism metabolizing 3,6-anhydride-L-galactose and a use thereof | |
CN113999827B (en) | Leucine dehydrogenase mutant and preparation method and application thereof | |
CN116790536B (en) | Bud ketone methyltransferases EnEMT and EnEMT from lithocarpus erythropolis and genes and applications thereof | |
CN109486789B (en) | Soybean epoxide hydrolase mutant with improved stereoselectivity | |
CN110904066B (en) | Recombinant R-type transaminase, mutant and application thereof |
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 |