CN116286919A - 基因工程菌及利用其制备岩藻糖基化寡糖的方法 - Google Patents
基因工程菌及利用其制备岩藻糖基化寡糖的方法 Download PDFInfo
- Publication number
- CN116286919A CN116286919A CN202111468092.2A CN202111468092A CN116286919A CN 116286919 A CN116286919 A CN 116286919A CN 202111468092 A CN202111468092 A CN 202111468092A CN 116286919 A CN116286919 A CN 116286919A
- Authority
- CN
- China
- Prior art keywords
- genetically engineered
- fucose
- engineered bacterium
- donor
- fucosyltransferase
- 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
- 241000894006 Bacteria Species 0.000 title claims abstract description 63
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 59
- 150000002482 oligosaccharides Chemical class 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 22
- 108010019236 Fucosyltransferases Proteins 0.000 claims abstract description 36
- 102000006471 Fucosyltransferases Human genes 0.000 claims abstract description 36
- 230000000694 effects Effects 0.000 claims abstract description 21
- 239000002773 nucleotide Substances 0.000 claims abstract description 17
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 125000002446 fucosyl group Chemical group C1([C@@H](O)[C@H](O)[C@H](O)[C@@H](O1)C)* 0.000 claims abstract description 12
- 101710098620 Alpha-1,2-fucosyltransferase Proteins 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims description 60
- 108090000790 Enzymes Proteins 0.000 claims description 60
- 108090000623 proteins and genes Proteins 0.000 claims description 40
- 230000037353 metabolic pathway Effects 0.000 claims description 38
- GUBGYTABKSRVRQ-QKKXKWKRSA-N lactose group Chemical group OC1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@H](O2)CO)[C@H](O1)CO GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 31
- 238000000855 fermentation Methods 0.000 claims description 23
- 230000004151 fermentation Effects 0.000 claims description 23
- 239000008101 lactose Substances 0.000 claims description 23
- 241000588724 Escherichia coli Species 0.000 claims description 20
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 19
- 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 claims description 19
- 108020003175 receptors Proteins 0.000 claims description 17
- 239000002243 precursor Substances 0.000 claims description 16
- LQEBEXMHBLQMDB-QIXZNPMTSA-N GDP-L-fucose Chemical group O[C@H]1[C@H](O)[C@H](O)[C@H](C)OC1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C3=C(C(NC(N)=N3)=O)N=C2)O1 LQEBEXMHBLQMDB-QIXZNPMTSA-N 0.000 claims description 15
- 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 claims description 15
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 15
- 230000001588 bifunctional effect Effects 0.000 claims description 14
- 239000000370 acceptor Substances 0.000 claims description 13
- 108090000992 Transferases Proteins 0.000 claims description 12
- -1 L-fucosyl Chemical group 0.000 claims description 11
- 102000004357 Transferases Human genes 0.000 claims description 11
- 230000015556 catabolic process Effects 0.000 claims description 11
- 238000006731 degradation reaction Methods 0.000 claims description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 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 9
- PTVXQARCLQPGIR-SXUWKVJYSA-N beta-L-fucose 1-phosphate Chemical compound C[C@@H]1O[C@H](OP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O PTVXQARCLQPGIR-SXUWKVJYSA-N 0.000 claims description 9
- 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
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- 101150066555 lacZ gene Proteins 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- HZZVJAQRINQKSD-UHFFFAOYSA-N Clavulanic acid Natural products OC(=O)C1C(=CCO)OC2CC(=O)N21 HZZVJAQRINQKSD-UHFFFAOYSA-N 0.000 claims description 5
- 102000004195 Isomerases Human genes 0.000 claims description 5
- 108090000769 Isomerases Proteins 0.000 claims description 5
- 102100040648 L-fucose kinase Human genes 0.000 claims description 5
- 101710091950 L-fucose kinase Proteins 0.000 claims description 5
- 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 claims description 5
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 5
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 5
- HZZVJAQRINQKSD-PBFISZAISA-N clavulanic acid Chemical compound OC(=O)[C@H]1C(=C/CO)/O[C@@H]2CC(=O)N21 HZZVJAQRINQKSD-PBFISZAISA-N 0.000 claims description 5
- 229960003324 clavulanic acid Drugs 0.000 claims description 5
- 230000002860 competitive effect Effects 0.000 claims description 5
- 230000037361 pathway Effects 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 101001027098 Arabidopsis thaliana Fucose-1-phosphate guanylyltransferase Proteins 0.000 claims description 4
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 229930182830 galactose Natural products 0.000 claims description 4
- 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 claims description 4
- IEQCXFNWPAHHQR-UHFFFAOYSA-N lacto-N-neotetraose Natural products OCC1OC(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)C(NC(=O)C)C(O)C1OC1OC(CO)C(O)C(O)C1O IEQCXFNWPAHHQR-UHFFFAOYSA-N 0.000 claims description 4
- 229940062780 lacto-n-neotetraose Drugs 0.000 claims description 4
- RBMYDHMFFAVMMM-PLQWBNBWSA-N neolactotetraose Chemical compound O([C@H]1[C@H](O)[C@H]([C@@H](O[C@@H]1CO)O[C@@H]1[C@H]([C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@@H]1O)O)NC(=O)C)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O RBMYDHMFFAVMMM-PLQWBNBWSA-N 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 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 3
- 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 3
- AXQLFFDZXPOFPO-UHFFFAOYSA-N UNPD216 Natural products O1C(CO)C(O)C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(=O)C)C1OC(C1O)C(O)C(CO)OC1OC1C(O)C(O)C(O)OC1CO AXQLFFDZXPOFPO-UHFFFAOYSA-N 0.000 claims description 3
- AXQLFFDZXPOFPO-UNTPKZLMSA-N beta-D-Galp-(1->3)-beta-D-GlcpNAc-(1->3)-beta-D-Galp-(1->4)-beta-D-Glcp Chemical compound O([C@@H]1O[C@H](CO)[C@H](O)[C@@H]([C@H]1O)O[C@H]1[C@@H]([C@H]([C@H](O)[C@@H](CO)O1)O[C@H]1[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O1)O)NC(=O)C)[C@H]1[C@H](O)[C@@H](O)[C@H](O)O[C@@H]1CO AXQLFFDZXPOFPO-UNTPKZLMSA-N 0.000 claims description 3
- 150000002402 hexoses Chemical class 0.000 claims description 3
- USIPEGYTBGEPJN-UHFFFAOYSA-N lacto-N-tetraose Natural products O1C(CO)C(O)C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(=O)C)C1OC1C(O)C(CO)OC(OC(C(O)CO)C(O)C(O)C=O)C1O USIPEGYTBGEPJN-UHFFFAOYSA-N 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 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 2
- PEOXGOPDODNWAZ-KZBIEJSGSA-N beta-D-Galp-(1->3)-[alpha-L-Fucp-(1->4)]-beta-D-GlcpNAc-(1->3)-beta-D-Galp-(1->4)-beta-D-Glcp 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]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O[C@@H]2[C@H]([C@H](O[C@@H]3[C@H](O[C@@H](O)[C@H](O)[C@H]3O)CO)O[C@H](CO)[C@@H]2O)NC(C)=O)O[C@@H]1CO PEOXGOPDODNWAZ-KZBIEJSGSA-N 0.000 claims 1
- 230000033581 fucosylation Effects 0.000 abstract description 6
- 239000000758 substrate Substances 0.000 abstract description 6
- 238000009776 industrial production Methods 0.000 abstract description 5
- 239000000386 donor Substances 0.000 description 21
- 108020004414 DNA Proteins 0.000 description 12
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- 235000020256 human milk Nutrition 0.000 description 10
- 210000004251 human milk Anatomy 0.000 description 10
- 239000002609 medium Substances 0.000 description 7
- PHTAQVMXYWFMHF-GJGMMKECSA-N alpha-L-Fucp-(1->2)-beta-D-Galp-(1->4)-D-GlcpNAc 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]2[C@H](OC(O)[C@H](NC(C)=O)[C@H]2O)CO)O[C@H](CO)[C@H](O)[C@@H]1O PHTAQVMXYWFMHF-GJGMMKECSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 238000004977 Hueckel calculation Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- LQEBEXMHBLQMDB-UHFFFAOYSA-N GDP-L-fucose Natural products OC1C(O)C(O)C(C)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C3=C(C(N=C(N)N3)=O)N=C2)O1 LQEBEXMHBLQMDB-UHFFFAOYSA-N 0.000 description 4
- LQEBEXMHBLQMDB-JGQUBWHWSA-N GDP-beta-L-fucose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C3=C(C(NC(N)=N3)=O)N=C2)O1 LQEBEXMHBLQMDB-JGQUBWHWSA-N 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 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 4
- 229960000268 spectinomycin Drugs 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 230000004186 co-expression Effects 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 108010001671 galactoside 3-fucosyltransferase Proteins 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 239000000348 glycosyl donor Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 101150028074 2 gene Proteins 0.000 description 1
- SNFSYLYCDAVZGP-OLAZETNGSA-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]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)O[C@H](CO)[C@H](O)[C@@H]1O SNFSYLYCDAVZGP-OLAZETNGSA-N 0.000 description 1
- 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 description 1
- 108010083651 3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase Proteins 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 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 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 101000928259 Homo sapiens NADPH:adrenodoxin oxidoreductase, mitochondrial Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 102100036777 NADPH:adrenodoxin oxidoreductase, mitochondrial Human genes 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 101001134239 Trigonella foenum-graecum Protein MANNAN SYNTHESIS-RELATED Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- NPPRJALWPIXIHO-PNCMPRLYSA-N beta-D-Gal-(1->4)-beta-D-GlcNAc-(1->3)-[beta-D-Gal-(1->4)-beta-D-GlcNAc-(1->6)]-beta-D-Gal-(1->4)-D-Glc Chemical compound O([C@H]1[C@H](O)[C@H]([C@@H](O[C@@H]1CO)OC[C@@H]1[C@@H]([C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O[C@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)[C@@H](CO)O2)NC(C)=O)[C@@H](O)[C@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)O1)O)NC(=O)C)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O NPPRJALWPIXIHO-PNCMPRLYSA-N 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 210000003022 colostrum Anatomy 0.000 description 1
- 235000021277 colostrum Nutrition 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 101150025078 fucK gene Proteins 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 235000021140 nondigestible carbohydrates Nutrition 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01069—Galactoside 2-alpha-L-fucosyltransferase (2.4.1.69)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/0703—Fucose-1-phosphate guanylyltransferase (2.7.7.30)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种基因工程菌及利用其制备岩藻糖基化寡糖的方法。所述方法包括:通过在基因工程菌中异源表达的岩藻糖基转移酶将供体的岩藻糖基转移至寡糖受体上;所述供体为核苷酸活化的供体,所述岩藻糖基转移酶具有α‑1,2‑岩藻糖基转移酶活性;其中,所述岩藻糖基转移酶选自NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的酶中的一种或多种。本发明的制备方法产率高、底物转化率和产物转化率得到了较大提高,具备应用于工业化生产的潜力。
Description
技术领域
本发明属于微生物发酵领域,具体涉及一种基因工程菌及利用其制备岩藻糖基化寡糖的方法。
背景技术
人乳由碳水化合物、蛋白质、脂类、激素和微量元素的混合物组成,不仅能为婴儿的生长发育提供所需要的营养成分,还可以提供保护剂,如免疫球蛋白等。除此之外,人乳还包含一系列具有保护特性的复合低聚糖——人乳寡糖。
人乳寡糖(Human milk oligosaccharides,HMOs)是人乳中一类结构复杂的非消化性碳水化合物,在人初乳中的含量为22~24g/L,在正常人乳中的含量为5~12g/L,是人乳中含量仅次于脂肪和乳糖的第三大固体成分。HMOs通过刺激新生儿中有益肠道细菌如双歧杆菌和乳酸杆菌的生长,平衡肠道菌群的发育。HMOs可能对调节新生儿出生后免疫系统起重要作用,作为先进婴儿配方食品的功能性成分非常重要。此外,HMOs可以抑制病原体与上皮细胞表面聚糖的粘附,从而限制一些病原体的毒力。
人乳中大概存在200多种不同的寡糖,目前已确定结构的人乳寡糖有115种。根据组成HMOs的单糖结构单元,HMOs可分为中性岩藻糖基乳糖,酸性唾液酸乳糖和中性非岩藻糖基化乳糖三种。
岩藻糖基转移酶(Fucosyltransferase,FucT)能够催化核苷二磷酸岩藻糖(通常是GDP-岩藻糖)将岩藻糖基转移至受体分子(如寡糖、糖蛋白、糖脂)。基于岩藻糖基添加部位不同,可将岩藻糖基转移酶分为α-1,2-岩藻糖基转移酶、α-1,3-岩藻糖基转移酶、α-1,4-岩藻糖基转移酶、α-1,6-岩藻糖基转移酶以及O-岩藻糖基转移酶。α-1,2-岩藻糖基转移酶广泛存在于脊椎动物、无脊椎动物、植物以及细菌中,但这些岩藻糖基转移酶在大多数细菌中的可溶性表达量很低,极大的限制了岩藻糖基化寡糖的生物合成。
目前岩藻糖基转移酶在制备岩藻糖基化寡糖中的活性低,严重限制了岩藻糖基化寡糖的生产水平,无法满足工业化生产的需求。因此本发明通过试验研究,以期筛选高活性的α-1,2-岩藻糖基转移酶,并能够在商业化生产中得以提高岩藻糖基化寡糖的产率。
发明内容
本发明所要解决的技术问题是为了克服现有技术缺少高活性、产率高的岩藻糖基转移酶用于岩藻糖基化寡糖的工业化生产,提供一种基因工程菌及利用其制备岩藻糖基化寡糖的方法。本发明基因工程菌及利用其的制备方法产率高、底物转化率和产物转化率得到了较大提高,具备应用于工业化生产的潜力。
本发明通过以下技术方案解决上述技术问题:
本发明第一方面提供一种制备岩藻糖基化寡糖的方法,所述方法包括:通过在基因工程菌中异源表达的岩藻糖基转移酶将供体的岩藻糖基转移至寡糖受体上;所述供体为核苷酸活化的供体,所述岩藻糖基转移酶具有α-1,2-岩藻糖基转移酶活性;
其中,所述岩藻糖基转移酶选自NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的酶中的一种或多种。
在本发明一些实施方案中,所述岩藻糖基转移酶为NCBI登录号为RTL12957.1和WP_120175093.1对应的酶。
在本发明一些实施方案中,所述寡糖受体选自乳糖、3-岩藻糖基乳糖、乳-N-四糖、乳-N-新四糖、乳-N-岩藻戊糖Ⅱ、乳-N-己糖和唾液酸乳-N-四糖b。
在本发明一些实施方案中,所述岩藻糖基化寡糖选自2'-岩藻糖基乳糖、2',3-二岩藻糖基乳糖、乳-N-岩藻戊糖I、乳-N-新岩藻戊糖I、乳-N-二岩藻糖己糖I、乳-N-岩藻糖基庚糖I和岩藻糖基乳-N-唾液酸戊糖b。
在本发明一些具体实施方案中,所述供体为鸟苷二磷酸岩藻糖。
在本发明一些实施方案中,所述基因工程菌为工程化改造的大肠杆菌或酵母。
在本发明一些较佳实施方案中,所述基因工程菌为工程化改造的大肠杆菌(E.coli)BL21(DE3)菌株。
在本发明一些实施方案中,所述基因工程菌还表达兼具L-岩藻糖激酶/岩藻糖-1-磷酸酯鸟嘌呤基转移酶的双功能酶;较佳地,所述双功能酶为NCBI登录号WP_010993080.1对应的酶。
和/或,所述基因工程菌中,所述寡糖受体的旁路代谢途径受到抑制;较佳地,所述寡糖受体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述寡糖受体为乳糖时,所述基因工程菌中编码β-半乳糖苷酶的基因例如lacZ基因敲除而失活,乳糖降解为半乳糖的代谢途径受到抑制。
本发明中,所述寡糖受体的旁路代谢途径是指作为岩藻糖基受体以外的代谢途径。
和/或,所述基因工程菌中,所述供体的前体的旁路代谢途径受到抑制;较佳地,所述前体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述供体为鸟苷二磷酸岩藻糖时,所述前体为L-岩藻糖,所述基因工程菌中编码L-岩藻糖基异构酶和/或L-墨角藻糖激酶的基因例如FucI和/或FucK敲除而失活,L-岩藻糖的旁路代谢途径受到抑制。
本发明中,所述供体的前体的旁路代谢途径是指转化为供体以外的代谢途径。
和/或,所述基因工程菌中,所述供体的旁路代谢途径受到抑制;较佳地,所述供体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述供体为鸟苷二磷酸岩藻糖时,所述基因工程菌中编码UDP-葡萄糖脂质载体转移酶的基因例如wacJ敲除而失活,鸟苷二磷酸岩藻糖降解为克拉酸的竞争性利用途径被阻断。
本发明中,所述供体的旁路代谢途径是指提供岩藻糖基以外的代谢途径。
在本发明一些实施方案中,所述方法还包括将所述基因工程菌在发酵培养基中进行发酵培养。
较佳地,所述发酵培养基包含:20~25g/L的甘油、10~12g/L的蛋白胨、5~6g/L的酵母粉、10~12g/L的NaCl,在所述发酵培养基的OD600=0.6-0.8时加入0.1~0.2mM的IPTG、5~6g/L的用于合成供体的前体分子例如L-岩藻糖和10~15g/L的寡糖受体例如乳糖;和/或,所述发酵培养的条件为:25~27℃、220r/min。
本发明的第二方面提供一种基因工程菌,所述基因工程菌异源表达岩藻糖基转移酶,所述岩藻糖基转移酶具有α-1,2-岩藻糖基转移酶活性;所述岩藻糖基转移酶将供体的岩藻糖基转移至寡糖受体上,所述供体为核苷酸活化的供体;
其中,所述岩藻糖基转移酶为NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的酶中的一种或多种。
所述寡糖受体、所述岩藻糖基化寡糖和所述供体优选如第一方面限定。
在本发明一些实施方案中,所述基因工程菌为工程化改造的大肠杆菌或酵母;较佳地,所述基因工程菌为工程化改造的大肠杆菌(E.coli)BL21(DE3)菌株。
在本发明一些实施方案中,所述基因工程菌表达兼具L-岩藻糖激酶/岩藻糖-1-磷酸酯鸟嘌呤基转移酶的双功能酶;较佳地,所述双功能酶为NCBI登录号WP_010993080.1对应的酶。
和/或,所述基因工程菌中,所述寡糖受体的旁路代谢途径受到抑制;较佳地,所述寡糖受体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述寡糖受体为乳糖时,所述基因工程菌中编码β-半乳糖苷酶的基因例如lacZ基因敲除而失活,乳糖降解为半乳糖的代谢途径受到抑制。
和/或,所述基因工程菌中,所述供体的前体的旁路代谢途径受到抑制;较佳地,所述前体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述供体为鸟苷二磷酸岩藻糖时,所述前体为L-岩藻糖,所述基因工程菌中编码L-岩藻糖基异构酶和/或L-墨角藻糖激酶的基因例如FucI和/或FucK敲除而失活,L-岩藻糖的旁路代谢途径受到抑制。
和/或,所述基因工程菌中,所述供体的旁路代谢途径受到抑制;较佳地,所述供体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述供体为鸟苷二磷酸岩藻糖时,所述基因工程菌中编码UDP-葡萄糖脂质载体转移酶的基因例如wacJ敲除而失活,鸟苷二磷酸岩藻糖降解为克拉酸的竞争性利用途径被阻断。
本发明的第三方面提供一种制备岩藻糖基化寡糖的方法,所述方法包括:
在反应体系中提供具有α-1,2-岩藻糖基转移酶活性的岩藻糖基转移酶,所述岩藻糖基转移酶将核苷酸活化的供体的岩藻糖基转移至寡糖受体上;
其中,所述岩藻糖基转移酶为NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的酶中的一种或多种。
在本发明一些实施方案中,在所述反应体系中还提供兼具L-岩藻糖激酶和岩藻糖-1-磷酸酯鸟嘌呤基转移酶活性的双功能酶,例如NCBI登录号WP_010993080.1对应的酶。
本发明的第四方面提供一种酶组合,所述酶组合包括选自NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的岩藻糖基转移酶的两种或多种。
或者,所述酶组合包括选自NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的岩藻糖基转移酶的一种或多种,还包括L-岩藻糖激酶/岩藻糖-1-磷酸酯鸟嘌呤基转移酶双功能酶优选NCBI登录号WP_010993080.1对应的酶。
本发明中,编码NCBI登录号WP_109047124.1对应的酶的核苷酸序列优选如SEQ IDNO:1所示;编码NCBI登录号RTL12957.1对应的酶的核苷酸序列优选如SEQ ID NO:2所示;编码NCBI登录号MBP7103497.1对应的酶的核苷酸序列优选如SEQ ID NO:3所示;编码NCBI登录号RYE22506.1对应的酶的核苷酸序列优选如SEQ ID NO:4所示;编码NCBI登录号WP_120175093.1对应的酶的核苷酸序列优选如SEQ ID NO:5所示;编码NCBI登录号WP_140393075.1对应的酶的核苷酸序列优选如SEQ ID NO:6所示;编码NCBI登录号HJB91111.1对应的酶的核苷酸序列优选如SEQ ID NO:7所示;编码NCBI登录号WP_010993080.1对应的酶的核苷酸序列优选如SEQ ID NO:10所示。
本发明的第五方面提供一种岩藻糖基转移酶或如第四方面所述的酶组合在制备岩藻糖基化寡糖中的应用,所述岩藻糖基转移酶为NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1、HJB91111.1或MBE2189475.1对应的酶。
本发明中,所述寡糖受体和所述岩藻糖基化寡糖优选如下表1所示:
表1寡糖受体和岩藻糖基化寡糖
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明的基因工程菌即利用其制备岩藻糖基化寡糖的方法产率高、底物转化率和产物转化率得到了较大提高,具备应用于工业化生产的潜力。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
本发明中的实验方法如无特别说明均为常规方法,基因克隆操作具体可参加J.萨姆布鲁克等编的《分子克隆实验指南》。
pET28a/pCDFduet-1购买自Novagen公司;E.coli BL21(DE3)感受态细胞均购买自Thermo Fisher公司,E.coli DH5α感受态细胞购买自北京鼎国昌盛生物技术有限责任公司,内切酶市售可得,乳糖购自国药试剂,L-岩藻糖购买自Carbosynth,无缝克隆试剂盒ClonExpress II One Step Cloning Kit购买自诺唯赞。
实施例中采用高效液相色谱(HPLC)系统(SHIMADZULC-20ADXR)定量检测重组大肠杆菌发酵液中2'-FL的合成,通过HP-Amide柱(Sepax,4.6×250mm5μm)测定发酵液中2'-FL和底物乳糖的浓度。HPLC检测器为示差检测器,色谱柱的检测温度设置为35℃,流动相通过乙腈:水=68:32洗脱,检测流速为1.4mL/min。
实施例1FucT基因获取及FucT粗酶液的制备
全合成NCBI上公开的α-1,2-岩藻糖基转移酶基因FucT序列,并将其在酶切位点NcoI和HindIII插入到载体pCDFduet-1上,构建重组质粒pCDFduet-1-FucT。全合成序列见表2,基因合成公司为苏州金唯智生物科技有限公司(苏州工业园区星湖街218号生物纳米科技园C3楼)。
表2合成的基因序列及相关信息
将上述基因载体,分别转化至宿主大肠杆菌BL21(DE3)感受态细胞;含pCDFduet-1-FucT载体的重组细胞接种至含30μg/mL壮观霉素的LB液体培养基,于37℃下,200rpm摇床培养,待OD600至0.8-1.0时,加IPTG至终浓度0.05mM,降温至30℃过夜诱导。发酵结束,5000rpm离心20min,去发酵液,留菌体。
取菌体5g,加50mL磷酸盐缓冲液(pH7.0,25mM)重悬菌体,4℃800mbar,均质破碎3min,之后于5000rpm,15℃离心30min,留上清液制得粗酶液,置于4℃,待纯化。
LB液体培养基组成:蛋白胨10g/L,酵母粉5g/L,NaCl 10g/L,用去离子水溶解后定容,121℃灭菌20min,待用。
实施例2FucT酶的纯化和酶活分析
酶的纯化
纯化步骤如下:取4℃保存的Ni柱,打开封闭的柱头,流尽原柱液。使用50mL去离子水冲洗Ni柱。使用10mL的1×Binding Buffer冲洗Ni柱。将实施例1制得的粗酶液上柱两次。使用10mL Binding Buffer(含20mM咪唑)冲洗Ni柱。使用10mL Wash Buffer(含40mM咪唑)冲洗Ni柱。使用5mL Elution Buffer(含80mM咪唑)洗脱杂蛋白,再使用5mL的ElutionBuffer(含250mM咪唑)洗脱纯蛋白。使用10kDa的Millipore超滤浓缩管浓缩除盐。蛋白纯化SDS PAGE后即可得纯FucT。
FucT酶活力测定
反应条件如下:反应总体积50μL,其中包括终浓度25mM磷酸盐缓冲液(pH5.6),5mM的GDP-岩藻糖,10mM的乳糖,1mg/mL的FucT纯酶,37℃反应20min。沸水浴10min终止反应,12000rpm高速离心5min,取上清HPLC分析,使用外标法确定产物终浓度,并计算酶活和比酶活。1U的酶活定义为,如上反应体系,每分钟产生1μmol 2'-FL所需要的酶量。比酶活实验数据如下表3所示。
表3比酶活数据
酶序号 | 比酶活U/mg |
GT062 | 615 |
GT065 | 532 |
GT072 | 413 |
GT083 | 397 |
GT093 | 459 |
GT104 | 411 |
GT107 | 566 |
GT059 | 113 |
HpFucT | 85 |
实施例3FucT和fkp基因共表达载体的制备
全合成NCBI上公开的双功能基因L-岩藻糖激酶/岩藻糖-1-磷酸酯鸟嘌呤基转移酶基因fkp序列(见表2),以酶切位点NdeI和HindIII连到到载体pET28a上,基因合成公司为苏州金唯智生物科技有限公司(苏州工业园区星湖街218号生物纳米科技园C3楼),得到fkp基因。
将fkp基因克隆至实施例1制备的各pCDFduet-1-FucT质粒的第二读码框位置,酶切位点为NdeI和XhoI,使用无缝克隆试剂盒,构建一系列共表达载体,如表4所示,引物列表如表5所示。将上述含有fkp和FucT的共表达质粒载体转化至宿主E.coli DH5α感受态细胞,得到重组基因工程菌株。其中载体构建的具体操作方法,参考ClonExpress II One StepCloning Kit试剂盒使用说明。
表4共表达载体列表
酶序号 | 载体名称 | GenBank No. |
GT062 | pCDF-AzoFucT-fkp | WP_109047124.1 |
GT065 | pCDF-NeiFucT-fkp | RTL12957.1 |
GT072 | pCDF-BacFucT-fkp | MBP7103497.1 |
GT083 | pCDF-SphFucT-fkp | RYE22506.1 |
GT093 | pCDF-PreFucT-fkp | WP_120175093.1 |
GT104 | pCDF-LacFucT-fkp | WP_140393075.1 |
GT107 | pCDF-CeiFucT-fkp | HJB91111.1 |
GT059 | pCDF-CkaFucT-fkp | MBE2189475.1 |
HpFucT(对照) | pCDF-HpFucT-fkp | AAC99764.1 |
表5fkp引物序列表
fkp引物 | 引物序列 | SEQ ID NO: |
fkp正向 | ctttaataaggagatataccatgcaaaaactactatctttaccgtccaatc | 11 |
fkp反向 | gcattatgcggccgcaagcttatgatcgtgatacttggaatcccttatc | 12 |
实施例4大肠杆菌BL21(DE3)菌株的改造
本实施例使用大肠杆菌BL21(DE3)作为亲本宿主,构建了一个用于全细胞生物合成2'-岩藻糖基乳糖的菌株,所述的基因组改造,包括基因断裂和缺失等。
2'-岩藻糖基乳糖的生物合成是以乳糖为受体底物、L-岩藻糖为糖基供体的前体,GDP-L-岩藻糖为糖基供体,因此本实施例首先失活了宿主细胞内编码β-半乳糖苷酶的lacZ基因(Qi Li,Bingbing Sun,Jun Chen,Yiwen Zhang,Yu Jiang,Sheng Yang,A modifiedpCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichiacoli,Acta Biochimica et Biophysica Sinica,Volume 53,Issue 5,May 2021,Pages620-627),以阻止底物乳糖的降解;其次使用相同的方法,使编码L-岩藻糖基异构酶和L-墨角藻糖-激酶的FucI基因和fucK基因缺失,以阻止L-岩藻糖的降解;第三步缺失了编码UDP-葡萄糖脂质载体转移酶的wacJ基因,用于阻断GDP-岩藻糖降解为克拉酸的竞争性利用途径(Dumon,C.,Priem,B.,Martin,S.L.et al.In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacterpyloriα-1,3fucosyltransferase in engineered Escherichia coli.Glycoconj J 18,465–474(2001))。最终得到BL21(DE3)lacZ(ΔM15)ΔfucK-fucIΔwacJ的菌株。
实施例5发酵制备2'-岩藻糖基乳糖
将实施例3中表4所描述的一系列共表达载体质粒分别转入实施例4所述的BL21(DE3)lacZ(ΔM15)ΔfucK-fucIΔwacJ的菌株,37℃复苏1h涂布终浓度为25μg/mL的壮观霉素抗性LB平板,37℃培养10-12h,以获得含有fkp和FucT基因且发酵重组菌。
挑取单菌落到终浓度为25μg/mL壮观霉素的LB培养基中培养8-10h,作为摇瓶发酵的种子液。
然后将种子液按1%的接种量接入到装有100mL发酵培养基的250mL三角瓶中,同时添加终浓度为25μg/mL的壮观霉素,发酵培养基的配方是:甘油20g/L、蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L;用去离子水定容。随后将三角瓶置于25℃、220r/min条件下培养至OD600=0.6-0.8时,添加终浓度为0.1mM的IPTG,5g/L的L-岩藻糖,10g/L的乳糖,持续发酵72h。
发酵结束,采用高效液相色谱仪(HPLC)测定菌体胞外2'-岩藻糖基乳糖(2'-FL)的产量及乳糖、岩藻糖的余量。
首先将2mL发酵液在12000rpm下离心10min后,收集上清液,过0.22μm滤膜,HPLC检测细胞外2'-岩藻糖基乳糖、乳糖、L-岩藻糖浓度。结果如下表6所示。
表6发酵实验结果
如上表所述,重组菌株中除GT059外,其它菌株发酵所得2'-FL的产量远高于对照组。
SEQUENCE LISTING
<110> 弈柯莱生物科技(上海)股份有限公司
<120> 基因工程菌及利用其制备岩藻糖基化寡糖的方法
<130> P21019395C
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 849
<212> DNA
<213> Artificial Sequence
<220>
<223> GT062
<400> 1
atgatcatcg ttcgtctgtc ggatggcctg ggtaaccaga tgttccagta cgcgttcggt 60
cgtgcgctga gcacccgccg tggtgttccg ctgcgtctgg acgtttccgc ataccgcgta 120
gaacgtaaac gtcgttacga actgcaccac tttctgaccg aagaaacctt cgttaccgat 180
gaggaagcgc accgtgttat cacccgtccg cattccccgg acgaaccgtg gtggtcccag 240
ccggttgttc gtgaaccgca cttccactat agcccggatg ttgttcaggt ttcgtccgcg 300
ggttacttcg cgggctactg gcagtctgaa cgtcacttcg atgatgttgc gccgctgatc 360
cgcctggaat ttaccccgaa acagccgctg accggcgcga acctggaagt tgcgcgtgcg 420
atcgcggcgc gtaacgcggt tagcctgcac gttcgtcgtg gtgattacat ctgcgatccg 480
aaagttaaca tcctgcacgg cgtttgctct ctggaatact accgtgcggc ggttgcgtac 540
gttgcggcgc gtgttgaaaa accggaattt ttcgttttca ccgatgatcc ggattggacc 600
cgtaccaaac tgaaactgga tttcccggcg tacctggtga cccagaacca ggatgcgccg 660
gttgaagacc tgcgcctgat gaccctgtgc cgtcaccaca tcatcgcgaa ctcttctttc 720
tcctggtggg gtgcttggct gggtgaaaaa ccgggtcaga tcgtttgcgc gccgcagcgt 780
tggttcggtg cgtacccgca cgatacccgt gatctggttc cggatcgttg gacccgtctg 840
gatggttaa 849
<210> 2
<211> 858
<212> DNA
<213> Artificial Sequence
<220>
<223> GT065
<400> 2
atgcagatca tctggtgcag cggcggcctg ggcaaccaga tgttccagta cgcgttctac 60
cgtcgtctgc aactggacgg caaaagcgtg accctggaca tcagcggttt caacgactac 120
ggcctgcaca acggcttcga actggataaa atcttcccgg tgaaaatcaa cctggcggac 180
gaagtgctga tcaacaacat caaacagaaa atctctcacc tgagcctgct gaaaaagatc 240
tggtggaaag tgttcaccaa cttccgtccg gtgatcgtgc agaaaaactt cggctacagt 300
agccgtctgt ctaacttgca gggtctgaaa tacctggaag gctactggca gagcgaaaaa 360
tacttcggca cccacagcga caccatccgt aacgacttca aattcccgct gctggacatt 420
aaaaacaaag actacgccga caaaattagc cagggcgaag cggtgagcat ccacatccgt 480
atgggcgact acgtgaacca cccgctgcac ggtggcatct gcaccctgga atactacaaa 540
aaagcgctga gcctgattga agaaaaagta gaaagcccgc tgttcttcat ctttagcaac 600
gacattgaat ggtgccagaa caacctgaaa ctggacaaag cgatctacgt gaccggtaac 660
gaaggcaaaa acagcttccg tgatatgcac ctgatgtcca tgtgcaaaca caacatcatc 720
gcgaacagct ccttctcttg gtggggcgcg tggctgaaca acaacccgga caaagttgtt 780
gtggcgccga gcaaatggtt caacgacaaa accatcaaca ccaaagatct gctgccggac 840
agctggatcc agatctaa 858
<210> 3
<211> 882
<212> DNA
<213> Artificial Sequence
<220>
<223> GT072
<400> 3
atgatcatcg ttaacctgat gggcggcctg ggcaaccaga tgttccagta cgcgctgggc 60
cgccgtctgg cgcaggataa gggcgttgcg ctgaaactgg acacccagtg gttcggcaaa 120
cagaacctgc gcaaattcga gctggacaaa ttcaacatca ccttcaaaat cgcatccgaa 180
gaagaaatct acagcacccg tcacttcttc cgtaaccgta tcatccgcaa agtctacagc 240
atctaccaga accagctgcc gtacttcaaa cgttctttca tcaacgaacc gaacttcggt 300
ttcttcgatc accacatcct ggaagtgccg aaaaactgct acctgaccgg ctactggcag 360
tccgaaaaat acttcagcag catggaagat accatccgca aagagttcac cctgaaagaa 420
atcgcggact ccaacttcat cgagttgagc aaagaaatgc agaacatcaa ctccgtgagc 480
ctgcacgtgc gccgcggcga ctacgttacc aacccgcaga ccaacaaatt ccacggcgtg 540
ctgagcaccg attactacaa actggcggtg aaactgatcc agaacaaaat cgacaccccg 600
cacttctacg tgttcagcga cgacctggaa tgggttaagg aacgcctgaa cttcgtgacc 660
ccgtgcacct acatcgaagg caaaaaagaa ggccgtgatt gcgaagaaat gtggctgatg 720
agccagtgca aacaccacat catcgcgaac agcagcttct cctggtgggg cgcgtggctg 780
ggcaacaaac cggacaaaat cgttatcagc ccgaaccagt ggttcgcgga taaaaactat 840
aaagttccgg acctgatccc ggaaaaatgg atccgtatct aa 882
<210> 4
<211> 909
<212> DNA
<213> Artificial Sequence
<220>
<223> GT083
<400> 4
atgcaggtgg ttgttaaaat caacggcggc ctgggtaacc agatgttcca gtacgcggcg 60
ggtcgtgcta cctctctgcg tttcaacagc gttctgcaaa tcgaaaccat cttcttcaaa 120
gacatcctga acgaaggtga acacaaacgt cagtaccagc tgaacatctt cccgaacatc 180
gcggcgctgg atttacagga aatcagcccg aaaaaccgcc acaaacagaa aaaatacatc 240
aactcttcta tctacaaagc ggaaaactct ctgcgtggca aactgggtat caaactggct 300
taccagcaca tctgggaaaa gaacctgctg acctacgacc cgtctttcca gcagtctaac 360
aaaaaagcgc acctgaccta cctgatcggt gactggcaga acgaacagta cttcgaaagc 420
gttgcggcga tcatccgcaa cgatttcagc ttcccgacca tcgaaagcgg cagcctgaac 480
gcggacatcc tgtctcagat ctacgcgagc gaagcggtgg cggttcacgt gcgtcgcggt 540
gattacctgc tgccgggtat ccactccccg gtgagcccgg cttactacca ggaagctctg 600
agcctgatcc gttcgaaagt tgcgagcccg aaattcttcg ttttctctga tgacatcaac 660
tggtgccgtg ctaacctggg tctggcggac gcgtgcttcg ttgaacacaa caccggcacc 720
aacaactatc gtgatatgca gctgatgtcc tcttgcaaac acaacatcat cgcgaactcc 780
agcttctctt ggtggggcgc gtggctgaac aacaacccga ccaaaatcgt tatcgcgccg 840
agcatgtgga tgccgaccca ggcggttgaa tctagccgtg ttgttccgct gagctggatc 900
accctgtaa 909
<210> 5
<211> 867
<212> DNA
<213> Artificial Sequence
<220>
<223> GT093
<400> 5
atgaaaatcg ttaaaatcct gggcggcctg ggtaaccaga tgttccagta cgcgctgtac 60
ctgtccttac aggaaacctt cccggaagaa cgtgtgatgc tggatctgag ctgcttccag 120
ggctaccacc tgcacaacgg tttcgaactg gaaaaaatct tcagcatcaa aggcgaaaaa 180
gcgagcgcga gcgacatcat gcgtgttgcg tactactacc cgaactacct gctgtggcgt 240
atcggtaaac gtctgctgcc gtgccgtaaa ggcatgtgcc tggaaagcag caccctgcgt 300
ttcgatgaaa ccgttctgac caaagaaggc aaccgttact tcgatggtta ctggcaggat 360
gaacgttact tcgcggcgtg ccgtgaaaaa gttctgaaag cgttcacctt cccggcgttc 420
aaacgtgctg aaaacctgag cctgctggaa aaactggatg aaaactctgt tagcctgcac 480
gttcgtcgtg gtgattacat cggtaacagc ctgtaccagg gtatctgcga tctggactac 540
taccgtaccg cgatcgaaaa aatgtgctct tacgttaccc cgagcctgtt ctgcgttttc 600
agcaacgaca tcgaatggtg ccgtgaacac ctggaacagt acatcaacgc gccggttgtt 660
tatgttacct ggaacaccgg cgcggaaagc taccgtgata tgcagctgat gtcttgctgc 720
gcgcacaaca tcatcgcgaa ctctagcttc tcctggtggg gcgcgtggct gaaccagaac 780
tctgataaag ttgttatcgc gccgaaaaaa tggctgaaca tggaagaatg ccatttcgcg 840
ctgccgagca gctggatcaa aatctaa 867
<210> 6
<211> 885
<212> DNA
<213> Artificial Sequence
<220>
<223> GT104
<400> 6
atgaaaagcg gtaaatacaa agacaaactg atcatccgtt tcaaaggtgg cctgggcaac 60
cagatgttcc agtacgcgat gtactgcaaa cagaaacatc tgggcaaaca ggtttgcgcg 120
gatgtgagcg cgtacactga acgtgaaggc tgtatgccgt tcgttctgtg cgacgttttc 180
ccgcagatca gcctccagct ggtgaaagat gaagaagcgg cgtattacct ggcggcgcag 240
aacaagaaaa acatcctgga taaagtgatc gcggcgttct ggtggcagga acgtgactac 300
acctctgaaa aagaaaacgg cgtgttcgat aaacgtgtgt tcagcctgaa aaaaggcttc 360
ctggatggct attggcagac cgaaaaatac ttctccgaca tccgtgaaga actgctgaaa 420
gatttccagt tcgaagttgc cgatagctcg ctgaaaaaat acgcggataa aatccgtgac 480
aacagcgtta gcgttcacgt tcgtcgtggt gattacctga acttcccgga catctacggt 540
ggtatctgcg gcatggacta ctacaaaaaa gcgatggact tcttctgcga gaaaaacccg 600
gaaaccgttt tctacgtttt ctctgatgac aaagaatggg ttcagaaagc gttccgtgaa 660
tacaacgctg tggtggttga aaaagacttc ttcagcgact acgaagattg gtacgacatg 720
tacctgatga gccagtgcaa ccacaacatc atcgcgaact ccagcttctc ctggtggggt 780
gcgtggctga accagaacaa aaacaaaaaa gttatctctc cgggcaaatg gttcaacggc 840
gaaaaaacca gcgacatctg gtgcccggaa tggatccgta tgtaa 885
<210> 7
<211> 858
<212> DNA
<213> Artificial Sequence
<220>
<223> GT107
<400> 7
atggttatcg ttcagctgtc tggtggcctg ggtaaccaga tgttcgaata cgcgctgtac 60
ctgcgtctga aatctatggg taaagaagtt ctggttgatg ataccacctg ctacggtccg 120
ggccagcgta ccaaacagct ggatgttttc ggcgtttctt acggtgcggc ggatgaacgt 180
cagctgcgtc gtatgaccga tagcgcgatg gacccgctgt ctcgtgcgcg tcgtaaactg 240
tctggtcgtc gtgatctgtc ttaccgtgaa gcgggttgcg atttcgatcc gctggttctg 300
gaaaaagatc cggcgctgtt gcagggttgc ttccagtctg aacgttactt cggcgaaatc 360
cgtgatcagg ttcgtgaagc gtaccgtttc cgtaacctgg tgaccaaccg tcgtgttgaa 420
gaataccgtc tgcgtatcct ggagaaaaaa ggtgcgtccg ttgcggttca cctgcgtcgt 480
ggtgattacc tggacccgaa atacgcgggt ctgtaccagg gtatctgcac cgatgcgtgg 540
tacggtgaag cgatccgtct gatgaaacag aaagttccgg gtgcggcgtt cttcttcttc 600
tctaacgatc cggattgggt taaagaacgt tacggtggtg cgggcaacgt taccgttgaa 660
ggtggttctg aagatgcggg ttacgaagat ctgtacctga tgagcctgtg cggtcaccag 720
atcatcgcga acagcagctt ctcttggtgg ggcgcttggc tgaacgaaaa cccggataaa 780
accgttatcg cgccgaaacg ttggctgaac ggccgtagct gccgtgacat ctacaccaaa 840
gaaatgaccc tgctgtaa 858
<210> 8
<211> 913
<212> DNA
<213> Artificial Sequence
<220>
<223> GT059
<400> 8
atgatcatcg ttaaaatgat gggcggtctg ggtaaccaga tgttccagtg ggcgctgggc 60
cgtgcgctgg cgatcaaaaa cagcagcgaa tttaaaatcg acgtgtactt cctgatcgag 120
cgccagccgc gtaaaaactt caccatccgt acctacgatc tggacgtttt caaactgaac 180
gcggagttcg cgaccaaaaa agaaatcgcg tactacccga tcccgaaatt cggcaaatac 240
ggcattttcc tggtgcacct gaaacagatg tggcgccgta gcatcaacac caacggctac 300
aactacctga tccagacccg ctttgattac gacgaacaga tcgacaacgc tccggtcaac 360
agctatctgg aaggctattt ccagaccgag cgctacttcg aaccgtactc cgacatcatc 420
cgcaaagact tcgagttccg cgacgaactg agcgggaaag cgctggaaat cgcccagctg 480
atcaacaaaa cccagtctgt ggcggtgcac atccgtcgcg gcgattacgt taccaaccgc 540
cgcgccaaca aaacgcacgg cgtactgggc aaagaatact acgacaaagc gatggaaacc 600
atcgcgagca aagttgaaag cccgcactac ttcatcttca gcgatgataa cgaatggtgc 660
cgtgaaaact tcgcgttcgg cgaaaacatg accatcatcg aagatgacat caaaggtaac 720
aaattccagt tctctctgaa cctgatgtct cagtgcaaac acgcgatcat cgcgaacagc 780
agcttctctt ggtggggcgc gtggctgagc gcgaacccga acaaaatcgt tatcggcccg 840
cagaactggt tcaaaaacac cgacctgaac gttaaagaca tcatcccgga aaaatggctg 900
cgcatctaag ctt 913
<210> 9
<211> 903
<212> DNA
<213> Artificial Sequence
<220>
<223> HpFucT
<400> 9
atggctttta aagtggtgca aatttgtggg gggcttggga atcaaatgtt tcaatacgct 60
ttcgctaaaa gtttgcaaaa acaccttaat acgcccgtgc tattagacac tacttctttt 120
gattggagca ataggaaaat gcaattagag cttttcccta ttgatttgcc ctatgcgaat 180
gcaaaagaaa tcgctatagc taaaatgcaa catctcccca agttagtaag agatgcactc 240
aaatacatag gatttgatag ggtgagtcaa gaaatcgttt ttgaatacga gcctaaattg 300
ttaaagccaa gccgtttgac ttattttttt ggctatttcc aagatccacg atattttgat 360
gctatatcct ctttaatcaa gcaaaccttc actctacccc ccccccccga aaataataaa 420
aataataata aaaaagagga agaataccag cgcaagcttt ctttgatttt agccgctaaa 480
aacagcgtat ttgtgcatat aagaagaggg gattatgtgg ggattggctg tcagcttggt 540
attgattatc aaaaaaaggc gcttgagtat atggcaaagc gcgtgccaaa catggagctt 600
tttgtgtttt gcgaagactt aaaattcacg caaaatcttg atcttggcta ccctttcacg 660
gacatgacca ctagggataa agaagaagag gcgtattggg atatgctgct catgcaatct 720
tgcaagcatg gcattatcgc taatagcact tatagctggt gggcggctta tttgatggaa 780
aatccagaaa aaatcattat tggccccaaa cactggcttt ttgggcatga aaatattctt 840
tgtaaggaat gggtgaaaat agaatcccat tttgaggtaa aatcccaaaa atataacgct 900
taa 903
<210> 10
<211> 2850
<212> DNA
<213> Artificial Sequence
<220>
<223> Fkp
<400> 10
atgcaaaaac tactatcttt accgtccaat ctggttcagt cttttcatga actggagagg 60
gtgaatcgta ccgattggtt ttgtacttcc gacccggtag gtaagaaact tggttccggt 120
ggtggaacat cctggctgct tgaagaatgt tataatgaat attcagatgg tgctactttt 180
ggagagtggc ttgaaaaaga aaaaagaatt cttcttcatg cgggtgggca aagccgtcgt 240
ttacccggct atgcaccttc tggaaagatt ctcactccgg ttcctgtgtt ccggtgggag 300
agagggcaac atctgggaca aaatctgctt tctctgcaac ttcccctata tgaaaaaatc 360
atgtctttgg ctccggataa actccataca ctgattgcga gtggtgatgt ctatattcgt 420
tcggagaaac ctttgcagag tattcccgaa gcggatgtgg tttgttatgg actgtgggta 480
gatccgtctc tggctaccca tcatggcgtg tttgcttccg atcgcaaaca tcccgaacaa 540
ctcgacttta tgcttcagaa gccttcgttg gcagaattgg aatctttatc gaagacccat 600
ttgttcctga tggacatcgg tatatggctt ttgagtgacc gtgccgtaga aatcttgatg 660
aaacgttctc ataaagaaag ctctgaagaa ctaaagtatt atgatcttta ttccgatttt 720
ggattagctt tgggaactca tccccgtatt gaagacgaag aggtcaatac gctatccgtt 780
gctattctgc ctttgccggg aggagagttc tatcattacg ggaccagtaa agaactgatt 840
tcttcaactc tttccgtaca gaataaggtt tacgatcagc gtcgtatcat gcaccgtaaa 900
gtaaagccca atccggctat gtttgtccaa aatgctgtcg tgcggatacc tctttgtgcc 960
gagaatgctg atttatggat cgagaacagt catatcggac caaagtggaa gattgcttca 1020
cgacatatta ttaccggggt tccggaaaat gactggtcat tggctgtgcc tgccggagtg 1080
tgtgtagatg tggttccgat gggtgataag ggctttgttg cccgtccata cggtctggac 1140
gatgttttca aaggagattt gagagattcc aaaacaaccc tgacgggtat tccttttggt 1200
gaatggatgt ccaaacgcgg tttgtcatat acagatttga aaggacgtac ggacgattta 1260
caggcagttt ccgtattccc tatggttaat tctgtagaag agttgggatt ggtgttgagg 1320
tggatgttgt ccgaacccga actggaggaa ggaaagaata tctggttacg ttccgaacat 1380
ttttctgcgg acgaaatttc ggcaggtgcc aatctgaagc gtttgtatgc acaacgtgaa 1440
gagttcagaa aaggaaactg gaaagcattg gccgttaatc atgaaaaaag tgttttttat 1500
caacttgatt tggccgatgc agctgaagat tttgtacgtc ttggtttgga tatgcctgaa 1560
ttattgcctg aggatgctct gcagatgtca cgcatccata accggatgtt gcgtgcgcgt 1620
attttgaaat tagacgggaa agattatcgt ccggaagaac aggctgcttt tgatttgctt 1680
cgtgacggct tgctggacgg gatcagtaat cgtaagagta ccccaaaatt ggatgtatat 1740
tccgatcaga ttgtttgggg acgtagcccc gtgcgcatcg atatggcagg gggatggacc 1800
gatactcctc cttattcact ttattcggga ggaaatgtgg tgaatctagc cattgagttg 1860
aacggacaac ctcccttaca ggtctatgtg aagccgtgta aagacttcca tatcgtcctg 1920
cgttctatcg atatgggtgc tatggaaata gtatctacgt ttgatgaatt gcaagattat 1980
aagaagatcg gttcaccttt ctctattccg aaagccgctc tgtcattggc aggctttgca 2040
cctgcgtttt ctgctgtatc ttatgcttca ttagaggaac agcttaaaga tttcggtgca 2100
ggtattgaag tgactttatt ggctgctatt cctgccggtt ccggtttggg caccagttcc 2160
attctggctt ctaccgtact tggtgccatt aacgatttct gtggtttagc ctgggataaa 2220
aatgagattt gtcaacgtac tcttgttctt gaacaattgc tgactaccgg aggtggatgg 2280
caggatcagt atggaggtgt gttgcagggt atgaagcttc ttcagaccga ggccggcttt 2340
gctcaaagtc cattggtgcg ttggctaccc gatcatttat ttacgcatcc tgaatacaaa 2400
gactgtcact tgctttatta taccggtata actcgtacgg caaaagggat cttggcagaa 2460
atagtcagtt ccatgttcct caattcatcg ttgcatctca atttactctc ggaaatgaag 2520
gcgcatgcat tggatatgaa tgaagctata cagcgtggaa gttttgttga gtttggccgt 2580
ttggtaggaa aaacctggga acaaaacaaa gcattggata gcggaacaaa tcctccggct 2640
gtggaggcaa ttatcgatct gataaaagat tataccttgg gatataaatt gccgggagcc 2700
ggtggtggcg ggtacttata tatggtagcg aaagatccgc aagctgctgt tcgtattcgt 2760
aagatactga cagaaaacgc tccgaatccg cgggcacgtt ttgtcgaaat gacgttatct 2820
gataagggat tccaagtatc acgatcataa 2850
<210> 11
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> fkp
<400> 11
ctttaataag gagatatacc atgcaaaaac tactatcttt accgtccaat c 51
<210> 12
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> fkp
<400> 12
gcattatgcg gccgcaagct tatgatcgtg atacttggaa tcccttatc 49
Claims (10)
1.一种制备岩藻糖基化寡糖的方法,其特征在于,所述方法包括:通过在基因工程菌中异源表达的岩藻糖基转移酶将供体的岩藻糖基转移至寡糖受体上;所述供体为核苷酸活化的供体,所述岩藻糖基转移酶具有α-1,2-岩藻糖基转移酶活性;
其中,所述岩藻糖基转移酶选自NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的酶中的一种或多种;
优选地,所述岩藻糖基转移酶为NCBI登录号为RTL12957.1或WP_120175093.1对应的酶。
2.如权利要求1所述的方法,其特征在于,所述寡糖受体选自乳糖、3-岩藻糖基乳糖、乳-N-四糖、乳-N-新四糖、乳-N-岩藻戊糖Ⅱ、乳-N-己糖和唾液酸乳-N-四糖b;
和/或,所述岩藻糖基化寡糖选自2'-岩藻糖基乳糖、2',3-二岩藻糖基乳糖、乳-N-岩藻戊糖I、乳-N-新岩藻戊糖I、乳-N-二岩藻糖己糖I、乳-N-岩藻糖基庚糖I和岩藻糖基乳-N-唾液酸戊糖b;
和/或,所述供体为鸟苷二磷酸岩藻糖;
和/或,所述基因工程菌为工程化改造的大肠杆菌或酵母;较佳地,所述基因工程菌为工程化改造的大肠杆菌(E.coli)BL21(DE3)菌株。
3.如权利要求1或2所述的方法,其特征在于,所述基因工程菌还表达兼具L-岩藻糖激酶和岩藻糖-1-磷酸酯鸟嘌呤基转移酶活性的双功能酶;较佳地,所述双功能酶为NCBI登录号WP_010993080.1对应的酶;
和/或,所述基因工程菌中,所述寡糖受体的旁路代谢途径受到抑制;较佳地,所述寡糖受体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述寡糖受体为乳糖时,所述基因工程菌中编码β-半乳糖苷酶的基因例如lacZ基因敲除而失活,乳糖降解为半乳糖的代谢途径受到抑制;
和/或,所述基因工程菌中,所述供体的前体的旁路代谢途径受到抑制;较佳地,所述前体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述供体为鸟苷二磷酸岩藻糖时,所述前体为L-岩藻糖,所述基因工程菌中编码L-岩藻糖基异构酶和/或L-墨角藻糖激酶的基因例如FucI和/或FucK敲除而失活,L-岩藻糖的旁路代谢途径受到抑制;
和/或,所述基因工程菌中,所述供体的旁路代谢途径受到抑制;较佳地,所述供体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述供体为鸟苷二磷酸岩藻糖时,所述基因工程菌中编码UDP-葡萄糖脂质载体转移酶的基因例如wacJ敲除而失活,鸟苷二磷酸岩藻糖降解为克拉酸的竞争性利用途径被阻断。
4.如权利要求1~3任一项所述的方法,其特征在于,所述方法还包括将所述基因工程菌在发酵培养基中进行发酵培养;
较佳地,所述发酵培养基包含:20~25g/L的甘油、10~12g/L的蛋白胨、5~6g/L的酵母粉、10~12g/L的NaCl,在所述发酵培养基的OD600=0.6-0.8时加入0.1~0.2mM的IPTG、5~6g/L的用于合成供体的前体分子例如L-岩藻糖和10~15g/L的寡糖受体例如乳糖;和/或,所述发酵培养的条件为:25~27℃、220r/min。
5.一种基因工程菌,其特征在于,所述基因工程菌异源表达岩藻糖基转移酶,所述岩藻糖基转移酶具有α-1,2-岩藻糖基转移酶活性;所述岩藻糖基转移酶将供体的岩藻糖基转移至寡糖受体上,所述供体为核苷酸活化的供体;
其中,所述岩藻糖基转移酶为NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的酶中的一种或多种;
较佳地,编码所述岩藻糖基转移酶的核苷酸序列如SEQ ID NO:1~7任一所示。
6.如权利要求5所述的基因工程菌,其特征在于,所述寡糖受体选自乳糖、3-岩藻糖基乳糖、乳-N-四糖、乳-N-新四糖、乳-N-岩藻戊糖Ⅱ、乳-N-己糖和唾液酸乳-N-四糖b;
和/或,所述岩藻糖基化寡糖选自2'-岩藻糖基乳糖、2',3-二岩藻糖基乳糖、乳-N-岩藻戊糖I、乳-N-新岩藻戊糖I、乳-N-二岩藻糖己糖I、乳-N-岩藻糖基庚糖I和岩藻糖基乳-N-唾液酸戊糖b;
和/或,所述供体为鸟苷二磷酸岩藻糖;
和/或,所述基因工程菌为工程化改造的大肠杆菌或酵母;较佳地,所述基因工程菌为工程化改造的大肠杆菌(E.coli)BL21(DE3)菌株。
7.如权利要求5所述的基因工程菌,其特征在于,所述基因工程菌表达兼具L-岩藻糖激酶和岩藻糖-1-磷酸酯鸟嘌呤基转移酶活性的双功能酶;较佳地,所述双功能酶为NCBI登录号WP_010993080.1对应的酶,优选编码所述双功能酶的核苷酸序列如SEQ ID NO:10所示;
和/或,所述基因工程菌中,所述寡糖受体的旁路代谢途径受到抑制;较佳地,所述寡糖受体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述寡糖受体为乳糖时,所述基因工程菌中编码β-半乳糖苷酶的基因例如lacZ基因敲除而失活,乳糖降解为半乳糖的代谢途径受到抑制;
和/或,所述基因工程菌中,所述供体的前体的旁路代谢途径受到抑制;较佳地,所述前体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述供体为鸟苷二磷酸岩藻糖时,所述前体为L-岩藻糖,所述基因工程菌中编码L-岩藻糖基异构酶和/或L-墨角藻糖激酶的基因例如FucI和/或FucK敲除而失活,L-岩藻糖的旁路代谢途径受到抑制;
和/或,所述基因工程菌中,所述供体的旁路代谢途径受到抑制;较佳地,所述供体的旁路代谢途径通过敲除或突变基因而受到抑制;更佳地,所述供体为鸟苷二磷酸岩藻糖时,所述基因工程菌中编码UDP-葡萄糖脂质载体转移酶的基因例如wacJ敲除而失活,鸟苷二磷酸岩藻糖降解为克拉酸的竞争性利用途径被阻断。
8.一种制备岩藻糖基化寡糖的方法,其特征在于,所述方法包括:
在反应体系中提供具有α-1,2-岩藻糖基转移酶活性的岩藻糖基转移酶,所述岩藻糖基转移酶将核苷酸活化的供体的岩藻糖基转移至寡糖受体上;
其中,所述岩藻糖基转移酶选自NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的酶中的一种或多种;
较佳地,在所述反应体系中还提供兼具L-岩藻糖激酶和岩藻糖-1-磷酸酯鸟嘌呤基转移酶活性的双功能酶,例如NCBI登录号WP_010993080.1对应的酶。
9.一种酶组合,其特征在于,所述酶组合包括选自NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的岩藻糖基转移酶的两种或多种;或,
所述酶组合包括选自NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1和HJB91111.1对应的岩藻糖基转移酶的一种或多种,还包括兼具L-岩藻糖激酶和岩藻糖-1-磷酸酯鸟嘌呤基转移酶活性的双功能酶,优选NCBI登录号WP_010993080.1对应的酶。
10.一种岩藻糖基转移酶或酶组合在制备岩藻糖基化寡糖中的应用,其特征在于,所述岩藻糖基转移酶为NCBI登录号WP_109047124.1、RTL12957.1、MBP7103497.1、WP_120175093.1、RYE22506.1、WP_140393075.1、HJB91111.1或MBE2189475.1对应的酶;所述酶组合如权利要求9所定义。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111468092.2A CN116286919A (zh) | 2021-12-03 | 2021-12-03 | 基因工程菌及利用其制备岩藻糖基化寡糖的方法 |
PCT/CN2022/124634 WO2023098299A1 (en) | 2021-12-03 | 2022-10-11 | Genetically engineered bacteria and methods for preparing a fucosylated oligosaccharide using the same |
AU2022399640A AU2022399640A1 (en) | 2021-12-03 | 2022-10-11 | Genetically engineered bacteria and methods for preparing a fucosylated oligosaccharide using the same |
EP22793653.1A EP4344436A1 (en) | 2021-12-03 | 2022-10-11 | Genetically engineered bacteria and methods for preparing a fucosylated oligosaccharide using the same |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111468092.2A CN116286919A (zh) | 2021-12-03 | 2021-12-03 | 基因工程菌及利用其制备岩藻糖基化寡糖的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116286919A true CN116286919A (zh) | 2023-06-23 |
Family
ID=83995481
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111468092.2A Pending CN116286919A (zh) | 2021-12-03 | 2021-12-03 | 基因工程菌及利用其制备岩藻糖基化寡糖的方法 |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP4344436A1 (zh) |
CN (1) | CN116286919A (zh) |
AU (1) | AU2022399640A1 (zh) |
WO (1) | WO2023098299A1 (zh) |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9029136B2 (en) * | 2012-07-25 | 2015-05-12 | Glycosyn LLC | Alpha (1,2) fucosyltransferases suitable for use in the production of fucosylated oligosaccharides |
JP2017515455A (ja) * | 2014-05-15 | 2017-06-15 | グリコシン リミテッド ライアビリティー カンパニー | フコシル化オリゴ糖の生産に使用するためのアルファ(1,2)フコシルトランスフェラーゼ・シンジーン |
EP3425052A1 (en) * | 2017-07-07 | 2019-01-09 | Jennewein Biotechnologie GmbH | Fucosyltransferases and their use in producing fucosylated oligosaccharides |
-
2021
- 2021-12-03 CN CN202111468092.2A patent/CN116286919A/zh active Pending
-
2022
- 2022-10-11 WO PCT/CN2022/124634 patent/WO2023098299A1/en active Application Filing
- 2022-10-11 AU AU2022399640A patent/AU2022399640A1/en active Pending
- 2022-10-11 EP EP22793653.1A patent/EP4344436A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2023098299A1 (en) | 2023-06-08 |
AU2022399640A1 (en) | 2024-01-25 |
EP4344436A1 (en) | 2024-04-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111712570A (zh) | 一种生产阿洛酮糖及其衍生物的工程菌株及其构建方法和应用 | |
AU2009329543B2 (en) | Synthesis of fucosylated compounds | |
KR20200027496A (ko) | 푸코실트랜스퍼라제 및 푸코실화된 올리고사카라이드의 제조에서 그의 용도 | |
TW201819636A (zh) | 用於生產岩藻醣化寡醣的改良方法 | |
BR112021010116A2 (pt) | Síntese do oligossacarídeo fucosilado lnfp-v | |
WO2021227363A1 (zh) | Haloferula sp.β-N-乙酰氨基己糖苷酶在合成人乳寡糖中的应用 | |
JP2021525522A (ja) | シアリル化糖の発酵産生 | |
JP2022522366A (ja) | 混合原料を利用する微生物細胞による炭水化物の発酵生産 | |
EP3847239A1 (en) | Fermentative production of oligosaccharides by total fermentation utilizing a mixed feedstock | |
JP2022546825A (ja) | バチルス属細胞におけるシアリル化オリゴ糖の生産 | |
WO1995034642A1 (fr) | Nouvelles transferase et amylase, procede de production de ces enzymes, utilisation, et genes les codant | |
WO2023099680A1 (en) | Cells with tri-, tetra- or pentasaccharide importers useful in oligosaccharide production | |
EP3088520A1 (en) | IMPROVED ß-FRUCTOFURANOSIDASE | |
Xu et al. | Improved production of 2′-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus | |
CN117321217A (zh) | 用于体内生产纯LNFP-I的α-1,2-岩藻糖基转移酶的识别 | |
CN114480465A (zh) | 一种产生2’-岩藻糖基乳糖的枯草芽孢杆菌及其应用 | |
WO2024042235A1 (en) | Hybrid method for producing complex hmos | |
CN116286919A (zh) | 基因工程菌及利用其制备岩藻糖基化寡糖的方法 | |
CN113832092B (zh) | 一种提高乳酰-n-岩藻五糖产量的基因工程菌及其生产方法 | |
WO2024021455A1 (zh) | 一种β-半乳糖苷酶基因及其编码酶的应用 | |
WO2023169200A1 (zh) | 重组酵母菌及其应用 | |
WO2023093337A1 (en) | A genetically engineered bacterium with lacz inactivation and its use in producing human milk oligosaccharides | |
Ito | Catalysis, structures, and applications of carbohydrate epimerases | |
WO2024110667A1 (en) | Two-strain system for producing oligosaccharides | |
EP4119655A1 (en) | Yarrowia sp. variant and method for preparing fat by using same |
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 |