CN117737060A - Non-coding RNA CsrC mutant, genetically engineered bacterium and application - Google Patents
Non-coding RNA CsrC mutant, genetically engineered bacterium and application Download PDFInfo
- Publication number
- CN117737060A CN117737060A CN202311640429.2A CN202311640429A CN117737060A CN 117737060 A CN117737060 A CN 117737060A CN 202311640429 A CN202311640429 A CN 202311640429A CN 117737060 A CN117737060 A CN 117737060A
- Authority
- CN
- China
- Prior art keywords
- csrc
- sialyllactose
- fucosyllactose
- coding rna
- genetically engineered
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000042567 non-coding RNA Human genes 0.000 title claims abstract description 42
- 108091027963 non-coding RNA Proteins 0.000 title claims abstract description 41
- 241000894006 Bacteria Species 0.000 title claims abstract description 27
- OIZGSVFYNBZVIK-FHHHURIISA-N 3'-sialyllactose Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@@H]1[C@@H](O)[C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@@H]1O OIZGSVFYNBZVIK-FHHHURIISA-N 0.000 claims abstract description 31
- 241000588724 Escherichia coli Species 0.000 claims abstract description 31
- RTVRUWIBAVHRQX-PMEZUWKYSA-N Fucosyllactose Chemical compound C([C@H]1O[C@@H]([C@H]([C@@H](O[C@@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@@H]1O)O)OC)O[C@H]1OC[C@@H](O)[C@H](O)[C@@H]1O RTVRUWIBAVHRQX-PMEZUWKYSA-N 0.000 claims abstract description 31
- 239000002773 nucleotide Substances 0.000 claims description 22
- 125000003729 nucleotide group Chemical group 0.000 claims description 20
- 238000000855 fermentation Methods 0.000 claims description 19
- 230000004151 fermentation Effects 0.000 claims description 19
- CILYIEBUXJIHCO-UHFFFAOYSA-N 102778-91-6 Natural products O1C(C(O)C(O)CO)C(NC(=O)C)C(O)CC1(C(O)=O)OC1C(O)C(OC2C(C(O)C(O)OC2CO)O)OC(CO)C1O CILYIEBUXJIHCO-UHFFFAOYSA-N 0.000 claims description 9
- DVGKRPYUFRZAQW-UHFFFAOYSA-N 3 prime Natural products CC(=O)NC1OC(CC(O)C1C(O)C(O)CO)(OC2C(O)C(CO)OC(OC3C(O)C(O)C(O)OC3CO)C2O)C(=O)O DVGKRPYUFRZAQW-UHFFFAOYSA-N 0.000 claims description 9
- CILYIEBUXJIHCO-UITFWXMXSA-N N-acetyl-alpha-neuraminyl-(2->3)-beta-D-galactosyl-(1->4)-beta-D-glucose Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)O[C@@H]2CO)O)O[C@H](CO)[C@@H]1O CILYIEBUXJIHCO-UITFWXMXSA-N 0.000 claims description 9
- OIZGSVFYNBZVIK-UHFFFAOYSA-N N-acetylneuraminosyl-D-lactose Natural products O1C(C(O)C(O)CO)C(NC(=O)C)C(O)CC1(C(O)=O)OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1O OIZGSVFYNBZVIK-UHFFFAOYSA-N 0.000 claims description 9
- TYALNJQZQRNQNQ-UHFFFAOYSA-N #alpha;2,6-sialyllactose Natural products O1C(C(O)C(O)CO)C(NC(=O)C)C(O)CC1(C(O)=O)OCC1C(O)C(O)C(O)C(OC2C(C(O)C(O)OC2CO)O)O1 TYALNJQZQRNQNQ-UHFFFAOYSA-N 0.000 claims description 8
- 229940062827 2'-fucosyllactose Drugs 0.000 claims description 8
- HWHQUWQCBPAQQH-UHFFFAOYSA-N 2-O-alpha-L-Fucosyl-lactose Natural products OC1C(O)C(O)C(C)OC1OC1C(O)C(O)C(CO)OC1OC(C(O)CO)C(O)C(O)C=O HWHQUWQCBPAQQH-UHFFFAOYSA-N 0.000 claims description 8
- HWHQUWQCBPAQQH-BWRPKUOHSA-N 2-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O HWHQUWQCBPAQQH-BWRPKUOHSA-N 0.000 claims description 8
- SNFSYLYCDAVZGP-UHFFFAOYSA-N UNPD26986 Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(OC(O)C(O)C2O)CO)OC(CO)C(O)C1O SNFSYLYCDAVZGP-UHFFFAOYSA-N 0.000 claims description 8
- TYALNJQZQRNQNQ-JLYOMPFMSA-N alpha-Neup5Ac-(2->6)-beta-D-Galp-(1->4)-beta-D-Glcp Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)OC[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)O[C@@H]2CO)O)O1 TYALNJQZQRNQNQ-JLYOMPFMSA-N 0.000 claims description 8
- WJPIUUDKRHCAEL-UHFFFAOYSA-N 3FL Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(CO)OC(O)C1O WJPIUUDKRHCAEL-UHFFFAOYSA-N 0.000 claims description 6
- AUNPEJDACLEKSC-ZAYDSPBTSA-N 3-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)O[C@H](CO)[C@@H]1O AUNPEJDACLEKSC-ZAYDSPBTSA-N 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 32
- 238000010353 genetic engineering Methods 0.000 abstract description 10
- 239000013612 plasmid Substances 0.000 description 21
- 238000010276 construction Methods 0.000 description 17
- 239000012634 fragment Substances 0.000 description 16
- 238000004519 manufacturing process Methods 0.000 description 12
- 230000006801 homologous recombination Effects 0.000 description 10
- 238000002744 homologous recombination Methods 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 238000012795 verification Methods 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 108091033409 CRISPR Proteins 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 229960000723 ampicillin Drugs 0.000 description 5
- 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 5
- 238000005516 engineering process Methods 0.000 description 5
- 235000020256 human milk Nutrition 0.000 description 5
- 210000004251 human milk Anatomy 0.000 description 5
- 238000010362 genome editing Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 150000002482 oligosaccharides Chemical class 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 238000010354 CRISPR gene editing Methods 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 3
- 102000003838 Sialyltransferases Human genes 0.000 description 3
- 108090000141 Sialyltransferases Proteins 0.000 description 3
- 102000004357 Transferases Human genes 0.000 description 3
- 108090000992 Transferases Proteins 0.000 description 3
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 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 3
- 239000000463 material Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 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 2
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- LFTYTUAZOPRMMI-CFRASDGPSA-N UDP-N-acetyl-alpha-D-glucosamine Chemical compound O1[C@H](CO)[C@@H](O)[C@H](O)[C@@H](NC(=O)C)[C@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 LFTYTUAZOPRMMI-CFRASDGPSA-N 0.000 description 2
- LFTYTUAZOPRMMI-UHFFFAOYSA-N UNPD164450 Natural products O1C(CO)C(O)C(O)C(NC(=O)C)C1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 LFTYTUAZOPRMMI-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical compound C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- 108020003264 Cotransporters Proteins 0.000 description 1
- 102000034534 Cotransporters Human genes 0.000 description 1
- 108091023173 CsrC RNA family Proteins 0.000 description 1
- 108010036364 Deoxyribonuclease IV (Phage T4-Induced) Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 1
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 1
- 229920000855 Fucoidan Polymers 0.000 description 1
- 108010045674 Fucose-1-phosphate guanylyltransferase Proteins 0.000 description 1
- 102100037047 Fucose-1-phosphate guanylyltransferase Human genes 0.000 description 1
- 108010062427 GDP-mannose 4,6-dehydratase Proteins 0.000 description 1
- 101710097673 GDP-mannose pyrophosphatase Proteins 0.000 description 1
- 102000002312 GDPmannose 4,6-dehydratase Human genes 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Natural products O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- SHZGCJCMOBCMKK-PQMKYFCFSA-N L-Fucose Natural products C[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O SHZGCJCMOBCMKK-PQMKYFCFSA-N 0.000 description 1
- 108010018080 L-arabinose isomerase Proteins 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- 102100040648 L-fucose kinase Human genes 0.000 description 1
- 101710091950 L-fucose kinase Proteins 0.000 description 1
- 108091022912 Mannose-6-Phosphate Isomerase Proteins 0.000 description 1
- 102000048193 Mannose-6-phosphate isomerases Human genes 0.000 description 1
- BRGMHAYQAZFZDJ-ZTVVOAFPSA-N N-acetyl-D-mannosamine 6-phosphate Chemical compound CC(=O)N[C@@H]1C(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BRGMHAYQAZFZDJ-ZTVVOAFPSA-N 0.000 description 1
- 102100033341 N-acetylmannosamine kinase Human genes 0.000 description 1
- 108700023220 N-acetylneuraminate lyases Proteins 0.000 description 1
- 102000048245 N-acetylneuraminate lyases Human genes 0.000 description 1
- 108010035265 N-acetylneuraminate synthase Proteins 0.000 description 1
- 108010029147 N-acylmannosamine kinase Proteins 0.000 description 1
- 241000080590 Niso Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 102100029954 Sialic acid synthase Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 101710152366 Sugar efflux transporter A Proteins 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- -1 UDP-glucose lipid Chemical class 0.000 description 1
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 1
- GBXZONVFWYCRPT-KVTDHHQDSA-N [(2s,3s,4r,5r)-3,4,5,6-tetrahydroxy-1-oxohexan-2-yl] dihydrogen phosphate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](C=O)OP(O)(O)=O GBXZONVFWYCRPT-KVTDHHQDSA-N 0.000 description 1
- 101150014383 adhE gene Proteins 0.000 description 1
- HXXFSFRBOHSIMQ-RWOPYEJCSA-L alpha-D-mannose 1-phosphate(2-) Chemical compound OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@@H](O)[C@@H](O)[C@@H]1O HXXFSFRBOHSIMQ-RWOPYEJCSA-L 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000006860 carbon metabolism Effects 0.000 description 1
- 230000008131 children development Effects 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229940101691 thiamine 10 mg Drugs 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a non-coding RNA CsrC mutant, a genetic engineering bacterium and application. The non-coding RNA CsrC mutant is obtained on the basis of wild-type non-coding RNA CsrC of Escherichia coli with Gene ID 2847776, and at least the 144 th a is mutated to t. The wild non-coding RNA CsrC in the escherichia coli genetic engineering bacteria for producing fucosyllactose and/or sialyllactose is mutated, so that after the 144 th a of the non-coding RNA CsrC is mutated to t, the yield of the fucosyllactose and/or sialyllactose of the obtained escherichia coli genetic engineering bacteria can be further improved, thereby better meeting the market demand.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a non-coding RNA CsrC mutant, a genetic engineering bacterium and application.
Background
Breast milk oligosaccharides are beneficial components present in breast milk that contribute to infant development, and include fucosyllactoses such as 2'-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL), and sialyllactoses such as 3'-sialyllactose (3' -sialyllactose,3 '-SL), 6' -sialyllactose (6 '-sialyllactose,6' -SL). At present, a microbial fermentation method is mainly adopted to produce fucosyllactose and sialyllactose, wherein escherichia coli is a common engineering strain for producing fucosyllactose and sialyllactose.
In the aspect of microbial fermentation production of fucosyllactose, related regulatory genes have been intensively studied in the industry, and patent document CN112501106A, CN115786220A and the like have disclosed various escherichia coli genetically engineered bacteria for improving fucosyllactose yield and related genes, such as beta-galactosidase coding geneslacZUDP-glucose lipid carrier transferase coding genewcaJRegulatory genes in lactose lac operon sequencelacICoding gene of L-fucose isomerasefucIL-fucoidan encoding genefucK、L-fucoidan-1-phosphate aldolase encoding genefucAGDP-fucose synthase encoding genewcaG、GDP-mannose-4, 6-dehydratase coding genegmd、β-Coding gene of galactosidaselacY、Phosphomannose isomerase coding genemanA、Coding gene of phosphomannose mutasemanB、Sugar efflux transporter A coding genesetA、Mannose-1-phosphate guanine transferase coding genemanC、L-arabinose isomerase coding genearaA、Rhamnose isomerase coding generhaA、2' -fucosyllactose encoding genefutC、L-fucose kinase/GDP-L-fucose pyrophosphorylase encoding genefkpEtc.
In the microbial fermentative production of sialyllactose, N-acetylneuraminic acid lyase NanA, N-acetylneuraminic acid H have been found + Cotransporter NanT, N-acetylmannosamine-6-phosphate 2-pimelic acidThe enzymes NanE, N-acetylmannosamine kinase NanK, UDP-GlcNAc epimerase NeuC, N-acetylneuraminic acid synthase NeuB, N-acetylneuraminic acid cytidylyltransferase NeuA, sialyltransferase SiaT and the like are important genes affecting the fermentation yield of sialyllactose of E.coli (Zhu YY, et al Recent progress on health effects and biosynthesis of two key sialylated human milk oligosaccharides, 3'-sialyllactose and' -sialylactose [ J ]]. Biotechnology Advances, 2023, 62:108058.)。
The research provides a good basis for the fermentation production and industrialization of fucosyllactose and sialyllactose. However, changes in metabolic flux during fermentation production of microorganisms affect physiological properties of microorganisms, thereby affecting fucosyllactose and sialyllactose yields. CsrC is a novel non-coding RNA component of a carbon storage regulation system of Escherichia coli, is a non-coding RNA molecule containing 246 bases, and 1 CsrC binds to 9 CsrA proteins in Escherichia coli (Chan E et al Endonuclease IV ofEscherichia coli is Induced by Paraquat[J]PNAS, 1987, 84 (10): 3189-3193.). CsrA is an important regulatory protein for carbon metabolism of microorganisms. After CsrC binds to CsrA, the regulatory function of CsrA on the regulated gene is reduced (Thomas Weilbacher et al.A novel sRNA component of the carbon storage regulatory system of)Escherichia coli[J]Mol Microbiol, 2003, 48 (3): 657-670.). Based on the function of non-coding RNA CsrC, it has been used to increase the fermentation yield of amino acids, such as the technology disclosed in patent literature such as CN111770993A, US8759042B2, wherein the technology of introducing exogenous non-coding RNA CsrC into host cells or shortening non-coding RNA CsrC nucleotide sequence is involved.
However, no report is made about the relation between the expression of non-coding RNA CsrC and the fermentation production of breast milk oligosaccharide, and no report is made about the improvement of the production level of related industrial strains by the non-coding RNA CsrC point mutant, and how to improve the fermentation yield of the breast milk oligosaccharide of escherichia coli by the point mutation of the gene of the non-coding RNA CsrC is still lack of research.
Disclosure of Invention
In order to solve the technical problems, the invention obtains the non-coding RNA CsrC mutant capable of improving the yield of fucosyllactose and/or sialyllactose through gene editing, and can be applied to the production of fucosyllactose and/or sialyllactose.
To achieve the above object, one of the technical solutions of the present invention provides a non-coding RNA CsrC mutant, the nucleotide sequence of which corresponds to the wild-type non-coding RNA CsrC of E.coli K12 MG1655 (Gene ID: 2847776), the 144 th a of which is mutated to t, i.e. has the nucleotide sequence shown in SEQ ID NO. 1; or the nucleotide sequence of the non-coding RNA CsrC mutant corresponds to the nucleotide sequence shown in SEQ ID NO.1, at least 144 th base is t, and has at least 90% homology with the nucleotide sequence shown in SEQ ID NO. 1. Compared with RNA transcribed from the original non-coding CsrC gene, the binding capacity of the mutant RNA and the transcription regulating factor CsrA is changed, so that the metabolic regulation of the CsrA on microorganisms is changed, and the fermentation yield of fucosyllactose and sialyllactose is improved.
The second technical scheme provided by the invention is to provide a genetic engineering bacterium for producing fucosyllactose and/or sialyllactose, wherein the genetic engineering bacterium is escherichia coli genetic engineering bacterium, and the genome of the escherichia coli genetic engineering bacterium carries the non-coding RNA CsrC mutant nucleotide sequence. Wherein the fucosyllactose can be any one of 2' -fucosyllactose and 3-fucosyllactose; sialyllactose is any one of 3'-sialyllactose and 6' -sialyllactose. The "genetically engineered bacterium producing fucosyllactose and/or sialyllactose" refers to a genetically engineered bacterium which has the ability to produce fucosyllactose and/or sialyllactose by itself even if it does not carry the non-coding RNA CsrC mutant nucleotide sequence.
The third technical scheme provided by the invention is to provide the application of the non-coding RNA CsrC mutant, in particular to the application in the production of fucosyllactose and sialyllactose, namely the application in the preparation of fucosyllactose and/or sialyllactose by fermentation of escherichia coli. Wherein the fucosyllactose can be any one of 2' -fucosyllactose and 3-fucosyllactose; sialyllactose is any one of 3'-sialyllactose and 6' -sialyllactose.
Advantageous effects
The invention obtains a non-coding RNA CsrC mutant by a gene editing technology, and can further improve the yield of fucosyllactose and sialyllactose by applying the non-coding RNA CsrC mutant to escherichia coli for producing fucosyllactose and/or sialyllactose.
The non-coding RNA CsrC mutant for improving the yield of fucosyllactose and/or sialyllactose is simpler to prepare compared with CsrC RNA gene editing fragments obtained by techniques such as insertion induction genes and the like; in particular to a non-coding RNA CsrB mutant prepared by single point mutation, which is simpler to prepare.
Drawings
FIG. 1 is a diagram showing the PCR verification of the first step homologous recombination colony of the strain W5; m is Marker,1, 2 are PCR verification strip;
FIG. 2 is a diagram showing PCR verification of a second step homologous recombination colony of the strain W5; m is Marker,1, 2 are PCR verification bands.
Detailed Description
The invention is further described below by means of specific embodiments. Unless otherwise indicated, the technical means, materials, etc. to which the following embodiments relate may be known to those skilled in the art, and appropriate ones may be selected among known means and materials capable of solving the respective technical problems. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
The non-coding RNA CsrC mutant is obtained by mutating the 144 th a into t on the basis of wild non-coding RNA CsrC (Gene ID is 2847776) of escherichia coli K12 MG1655, and the nucleotide sequence of the obtained non-coding RNA CsrC mutant is shown as SEQ ID NO. 1.
The invention will be further explained by means of specific embodiments.
EXAMPLE 1 construction of Strain W5
Based on the E.coli TKYW1 described in patent document CN115786220A, a non-coding RNA CsrC gene on the genome was mutated, and the 144 th a was mutated to t, to construct a strain W5.
The construction of E.coli TKYW1 was briefly described based on the content of patent document CN115786220A, and the E.coli TKYW1 construction method was introduced into this example. Wherein the Escherichia coli TKYW1 in CN115786220A is Escherichia coli W2% described in patent document CN112501106AE.coli K12 MG1655△lacIZ::Ptrc-wcaG-gmd-lacy,△adhE::Ptrc-manB-manA) To construct the original strain, the UDP-glucose lipid carrier transferase coding gene on the genome of the original strain is knocked outwcaJAnd GDP-mannose hydrolase encoding genenudDObtained. Construction of E.coli TKYW1wcaJIs 946583;nudDis 946559.
The wild-type non-coding RNA CsrC nucleotide sequence Gene ID of E.coli K12 MG1655 was 2847776. Based on the strain TKYW1, the CRISPR/Cas9 technology is utilized to mutate a non-coding RNA CsrC gene on a genome, the 144 th site a is mutated to t, the nucleotide sequence of the obtained non-coding RNA CsrC mutant is shown as SEQ ID NO.1, and the constructed strain is named as W5. The CRISPR/Cas9 technology used in the experiments was referred to earlier studies report (Zhao D, et al, CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency, sci Rep, 2017, 7 (1): 16624). The specific construction method of the strain W5 is as follows:
1. construction of homologous recombination fragments
The MG1655 wild strain stored in the laboratory is used as a template, and primer pairs CsrC-up-F/R and CsrC-down-F/R in the table 1 are respectively used as primers, and the upstream homology arm and the downstream homology arm of homologous recombination are obtained through PCR amplification. PCR obtained by constructing the strain TKYW1cat-Fragments of the N20 sequence are used as templates, and primer pairs Cs are usedPCR amplification is carried out by taking rC-cat-F/R as a primer to obtain a novel primer withcat-Fragments of the N20 sequence. The homologous arm above and downstream, new withcat-Fragments of N20 sequence, 3 fragments as templates, were subjected to overlap PCR using the primers CsrC-up-F and CsrC-down-R to obtain homologous recombination fragments containingCsrCMutation of genes, i.e. wild typeCsrCThe 144 th base a of the gene is mutated into t.
2. First step homologous recombination
The pCAGO plasmid was transformed into the strain TKYW1 using a conventional plasmid transformation method to obtain strain TKYW1 (pCAGO). TKYW1 (pCAGO) was competent in preparation of the strain by using LB medium containing 1% glucose and 0.1. 0.1 mM% IPTG (isopropyl-. Beta. -D-thiogalactoside), the homologous recombinant fragment was introduced by electrotransformation, and the transformed strain was spread on LB plates containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, and 1% glucose, and cultured at 30 ℃. And selecting the transformant to perform colony PCR verification, and if the recombination is correct, obtaining the homologous recombination strain in the first step, wherein the size of the stripe is about 2700 bp, and the verification result is shown in figure 1, and the stripe is correct.
3. Second step homologous recombination
The grown monoclonal was subjected to colony PCR validation. If the recombination is correct, the band size is about 1700 bp, the verification result is shown in figure 2, the band is correct, the PCR product of the band is sequenced, and the sequencing result is correct, so that the homologous recombination strain of the second step is obtained. The second homologous recombination strain is further cultivated at 37 ℃ to lose pCAGO plasmid therein, thereby obtaining the recombinant strain with the functions ofCsrCThe strain with the gene mutation (SEQ ID NO. 1) was designated as W5.
TABLE 1 constructionCsrCPrimers for gene mutant strains
Example 2 construction of plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ist
Construction of plasmid pTrc99a-P Using plasmid pTrc99a as template J23119 -neuB-neuC-P trc -neuA-ist. P involved in construction of the plasmid trc The nucleotide sequence of the promoter is shown as SEQ ID NO. 2; n-acetylneuraminic acid synthaseneuBNucleotide sequence of gene and UDP-N-acetylglucosamine 2-differential enzymeneuCNucleotide sequence of gene and N-acyl neuraminic acid cytidylyltransferase geneneuAShares a GenBank accession number with the nucleotide sequence of (A): AF400048.1; sialyltransferase geneistIs 61281137.
PCR amplification was performed using the plasmid pTrc99a as a template and the primers 99a-p119-F/R in Table 2 as primers to obtain linear vectors pTrc99a-p119; PCR amplification is carried out by taking neuB-F/R as a primer to obtain a linear gene fragmentneuBThe method comprises the steps of carrying out a first treatment on the surface of the PCR amplification is carried out by taking neuC-F/R as a primer to obtain a linear gene fragmentneuCThe method comprises the steps of carrying out a first treatment on the surface of the PCR amplification is carried out by taking ptrc-neuA-F/R as a primer to obtain a linear gene fragment P trc The method comprises the steps of carrying out a first treatment on the surface of the PCR amplification is carried out by taking neuA-F/R as a primer to obtain a linear gene fragmentneuAThe method comprises the steps of carrying out a first treatment on the surface of the To be used foristPCR amplification is carried out by taking F/R as a primer to obtain linear gene fragmentsist。Purifying and recovering the linear vector and the linear gene fragment obtained by the PCR, and using ClonExpress cube ® II recombinant ligation kit (Novain Biotechnology Co., ltd.) was ligated, transformed into E.coli DH 5. Alpha. Competent cells, cultured on LB plate containing 100 mg/L ampicillin, and transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid designated plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ist。
TABLE 2 construction of plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-istPrimers used
Example 3 construction of plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ST6
With plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-istConstruction of plasmid pTrc99a-P for template J23119 -neuB-neuC-P trc -neuA-ST6. Sialyltransferase involved in construction of the plasmidST6Is AB293985.1.
With plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-istPCR amplification was performed using the bst-AB-supported-F/R in Table 3 as a primer to obtain the linear vector pTrc99a-ST6; PCR amplification is carried out by taking bst-AB-F/R as a primer to obtain a linear gene fragmentST6The method comprises the steps of carrying out a first treatment on the surface of the Purifying and recovering the linear vector and the linear gene fragment obtained by the PCR, and using ClonExpress cube ® II recombinant ligation kit (Novain Biotechnology Co., ltd.) was ligated, transformed into E.coli DH 5. Alpha. Competent cells, cultured on LB plate containing 100 mg/L ampicillin, and transformants were picked for colony PCR and sequencing verification to obtain the correct recombinant plasmid designated plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA- ST6。
TABLE 3 construction of plasmid pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ST6Primers used
Example 4 construction of fucosyllactose and sialyllactose producing Strain and fermentation test
Plasmid pTrc99a-P was transformed by electrotransformation trc -futC-manC、pTrc99a-P trc -futA-manC、pTrc99a-P J23119 -neuB-neuC-P trc -neuA-istAnd pTrc99a-P J23119 -neuB-neuC-P trc -neuA-ST6TKYW1 and W5 were introduced, respectively, to construct 2' -fucosyllactose-producing strain F1 (TKYW 1 (pTrc 99 a-P) trc -futC-manC) F2 (W5 (pTrc 99 a-P) trc -futC-manC) 3-fucoseGlycosyllactose producing strain F3 (TKYW 1 (pTrc 99 a-P) trc -futA-manC) F4 (W5 (pTrc 99 a-P) trc -futA-manC) 3' -sialyllactose-producing Strain F5 (TKYW 1 (pTrc 99 a-P) J23119 -neuB-neuC-P trc -neuA-ist) F6 (W5 (pTrc 99 a-P) J23119 -neuB-neuC-P trc -neuA-ist) 6' -sialyllactose-producing strain F7 (TKYW 1 (pTrc 99 a-P) J23119 -neuB-neuC-P trc -neuA-ST6) And F8 (W5 (pTrc 99 a-P) J23119 -neuB-neuC-P trc -neuA-ST6) And the fermentation production level of the above strain was tested. Wherein plasmid pTrc99a-P trc -futC-manC、pTrc99a-P trc -futA-manCThe specific construction procedure of (a) is described in examples 3 and 4 of patent document CN116334025 a.
The culture medium used was:
LB medium: naCl 10 g/L, yeast powder 5 g/L, peptone 10 g/L and pH 7.0.
Fermentation medium: KH (KH) 2 PO 4 3 g/L, yeast powder 8 g/L, (NH) 4 ) 2 SO 4 4. 4 g/L, citric acid 1.7 g/L, mgSO 4 ·7H 2 O2 g/L, thiamine 10 mg/L, glycerol 10 g/L, lactose 5 g/L,1 ml/L trace elements (FeCl) 3 ·6H 2 O 25 g/L,MnCl 2 ·4H 2 O 9.8 g/L,CoCl 2 ·6H 2 O 1.6 g/L,CuCl 2 ·H 2 O 1 g/L,H 3 BO 3 1.9 g/L, ZnCl 2 2.6 g/L,Na 2 M O O 4 ·2H 2 O 1.1 g/L,Na 2 SeO 3 1.5 g/L,NiSO 4 ·6H 2 O1.5. 1.5 g/l), pH was adjusted to 7.2 with ammonia.
The fermentation test process comprises the following steps:
single colonies of fucosyllactose or sialyllactose producing strains were picked up, respectively, and cultured overnight at 37℃at 220 rpm/min in LB liquid medium containing 50 mg/L of ampicillin.The bacterial liquid cultured overnight is taken as seed liquid, the bacterial liquid is transferred into a 24-well plate containing 2 mL fermentation culture medium with the inoculum size of 1 percent, 50 mg/L of ampicillin and 0.1 mmol/L of IPTG are contained in the fermentation culture medium, and fermentation is carried out at 37 ℃ and 800 rpm/min. 3 samples were grown in parallel for each strain. During fermentation, the growth (OD) of the cells was measured 600 ) The concentration of fucosyllactose or sialyllactose in the sample was measured by HPLC using a chromatographic column of Carbohydrate ES 5u 250mm x 4.6mm, a detector of evaporative light detector and a mobile phase of 70% acetonitrile (acetonitrile: water), the flow rate is 0.8 mL/min, the column temperature is 30 ℃, and the sample injection amount is 5 mu L. The sample concentration was quantified using fucosyllactose or sialyllactose standards. The results are shown in tables 4 to 7:
as can be seen from tables 4 to 7, mutation of nucleotide a at position 144 of the non-coding RNA CsrC gene to t greatly increases the yield of fucosyllactose or sialyllactose.
TABLE 4 results of 2'-fucosyllactose (2' -FL) production test by different strains
TABLE 5 results of 3-fucosyllactose (3-FL) production test by different strains
TABLE 6 results of 3'-sialyllactose (3' -SL) production test by different strains
TABLE 7 results of 6'-sialyllactose (6' -SL) production test by different strains
Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims (10)
1. The non-coding RNA CsrC mutant is characterized in that the nucleotide sequence of the non-coding RNA CsrC mutant is shown as SEQ ID NO. 1; or the nucleotide sequence of the non-coding RNA CsrC mutant corresponds to the nucleotide sequence shown in SEQ ID NO.1, at least 144 th base is t, and has at least 90% homology with the nucleotide sequence shown in SEQ ID NO. 1.
2. A genetically engineered bacterium for producing fucosyllactose and/or sialyllactose, wherein the genetically engineered bacterium is an escherichia coli genetically engineered bacterium, and the genome of the escherichia coli genetically engineered bacterium carries the non-coding RNA CsrB mutant nucleotide sequence of claim 1.
3. The genetically engineered bacterium of claim 2, wherein the genome of the genetically engineered bacterium of escherichia coli carries a non-coding RNA CsrC mutant nucleotide sequence as set forth in SEQ ID No. 1.
4. The genetically engineered bacterium of claim 2, wherein the genetically engineered bacterium of escherichia coli is a genetically engineered bacterium of escherichia coli that produces fucosyllactose.
5. The genetically engineered bacterium of claim 4, wherein the fucosyllactose is any one of 2' -fucosyllactose and 3-fucosyllactose.
6. The genetically engineered bacterium of claim 2, wherein the genetically engineered bacterium of escherichia coli is a genetically engineered bacterium of escherichia coli that produces sialyllactose.
7. The genetically engineered bacterium of claim 6, wherein the sialyllactose is any one of 3'-sialyllactose and 6' -sialyllactose.
8. Use of a non-coding RNA CsrC mutant according to claim 1, for increasing the yield of fucosyllactose and/or sialyllactose produced by fermentation of e.
9. The use according to claim 8, wherein the fucosyllactose is any one of 2' -fucosyllactose and 3-fucosyllactose.
10. The use according to claim 8, wherein the sialyllactose is any one of 3'-sialyllactose and 6' -sialyllactose.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311640429.2A CN117737060A (en) | 2023-12-04 | 2023-12-04 | Non-coding RNA CsrC mutant, genetically engineered bacterium and application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311640429.2A CN117737060A (en) | 2023-12-04 | 2023-12-04 | Non-coding RNA CsrC mutant, genetically engineered bacterium and application |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117737060A true CN117737060A (en) | 2024-03-22 |
Family
ID=90280406
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311640429.2A Pending CN117737060A (en) | 2023-12-04 | 2023-12-04 | Non-coding RNA CsrC mutant, genetically engineered bacterium and application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117737060A (en) |
-
2023
- 2023-12-04 CN CN202311640429.2A patent/CN117737060A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
TW201819636A (en) | Improved process for the production of fucosylated oligosaccharides | |
CN114480240B (en) | Genetic engineering bacterium for producing fucosyllactose and production method thereof | |
US11761021B2 (en) | Recombinant Bacillus subtilis for synthesizing lacto-N-neotetraose and application thereof | |
CN113186142B (en) | Escherichia coli engineering strain for efficiently producing 2' -fucosyllactose | |
CN117363553B (en) | Genetically engineered bacterium for producing 2' -deoxyadenosine, construction method and application thereof | |
CN115786220B (en) | Recombinant strain for producing 2' -fucosyllactose, construction method and application | |
WO2012025780A1 (en) | Improved glycolic acid fermentative production with a modified microorganism | |
CN113151133B (en) | Recombinant host bacterium for producing sialyllactose and construction method and application thereof | |
DK181242B1 (en) | GENETICALLY ENGINEERED CELLS COMPRISING A RECOMBINANT NUCLEIC ACID SEQUNCE ENCODING AN α-1,2-FUCOSYLTRANSFERASE CAPABLE OF PRODUCING LNFP-I, NUCLEIC ACID SEQUENCES ENCODING SAME AND METHODS FOR USE OF SAME | |
CN116355819A (en) | Construction method and application of engineering escherichia coli for efficiently synthesizing lactoyl-N-tetraose | |
CN114410561B (en) | Genetically engineered strain for producing thymidine and construction method and application thereof | |
CN117737060A (en) | Non-coding RNA CsrC mutant, genetically engineered bacterium and application | |
CN114806991A (en) | Engineering escherichia coli for improving yield of fucosyllactose and production method | |
CN115927148A (en) | Genetic engineering bacterium for efficiently producing lactoyl-N-neotetraose without lactose addition and application thereof | |
CN117737061A (en) | Non-coding RNA CsrB mutant, genetically engineered bacterium and application | |
CN117736280A (en) | SecY protein mutant, genetically engineered bacterium and application | |
CN116334025A (en) | RNA polymerase beta subunit mutant and application thereof | |
CN118207145A (en) | Engineering strain for producing 2' -fucosyllactose, construction method and application | |
CN116948928B (en) | Seed culture medium and fermentation production method of 2' -fucosyllactose without antibiotics and IPTG inducer | |
CN117586938B (en) | Construction method and application of recombinant escherichia coli for synthesizing lactoyl-N-neotetraose | |
CN118207191A (en) | Methionine aminopeptidase mutant, escherichia coli engineering bacteria and application | |
CN118325929A (en) | Escherichia coli for producing fucosyllactose by using mannose and application thereof | |
CN117535258A (en) | Alpha 1, 2-fucosyltransferase mutant, recombinant escherichia coli expressing same and application thereof | |
CN117305211A (en) | Construction and application of genetic engineering bacteria for efficiently synthesizing 2' -fucosyllactose | |
CN118028202A (en) | Construction method and application of recombinant Escherichia coli for efficiently synthesizing lactoyl-N-disaccharide |
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 |