CN116334025A - 一种RNA聚合酶β亚单位突变体及应用 - Google Patents
一种RNA聚合酶β亚单位突变体及应用 Download PDFInfo
- Publication number
- CN116334025A CN116334025A CN202310064132.XA CN202310064132A CN116334025A CN 116334025 A CN116334025 A CN 116334025A CN 202310064132 A CN202310064132 A CN 202310064132A CN 116334025 A CN116334025 A CN 116334025A
- Authority
- CN
- China
- Prior art keywords
- gene
- fucosyllactose
- rna polymerase
- trc
- beta subunit
- 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
- 108010064250 RNA polymerase beta subunit Proteins 0.000 title claims abstract description 32
- 229940062827 2'-fucosyllactose Drugs 0.000 claims abstract description 38
- 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 abstract description 38
- 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 abstract description 38
- 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 abstract description 38
- 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 abstract description 36
- 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 abstract description 35
- 241000588724 Escherichia coli Species 0.000 claims abstract description 27
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 64
- 239000013612 plasmid Substances 0.000 claims description 47
- 101150049349 setA gene Proteins 0.000 claims description 25
- 101100156625 Escherichia coli (strain K12) wcaJ gene Proteins 0.000 claims description 16
- 101150066555 lacZ gene Proteins 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 241000894006 Bacteria Species 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 101150032120 manC gene Proteins 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 101100075927 Aspergillus aculeatus mndA gene Proteins 0.000 claims description 9
- 101100280818 Escherichia coli (strain K12) fcl gene Proteins 0.000 claims description 9
- 101100022282 Escherichia coli O157:H7 manC2 gene Proteins 0.000 claims description 9
- 101150014383 adhE gene Proteins 0.000 claims description 9
- 101150109249 lacI gene Proteins 0.000 claims description 9
- 101150001899 lacY gene Proteins 0.000 claims description 9
- 101150088678 manB gene Proteins 0.000 claims description 9
- 101150042391 rpoC gene Proteins 0.000 claims description 9
- 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 claims description 8
- 239000008101 lactose Substances 0.000 claims description 8
- 230000002018 overexpression Effects 0.000 claims description 7
- 101150018163 wcaJ gene Proteins 0.000 claims description 7
- 108700005075 Regulator Genes Proteins 0.000 claims description 6
- 101150106565 gmd gene Proteins 0.000 claims description 6
- 101710088194 Dehydrogenase Proteins 0.000 claims description 5
- 238000000855 fermentation Methods 0.000 abstract description 27
- 230000004151 fermentation Effects 0.000 abstract description 27
- 150000001413 amino acids Chemical class 0.000 abstract description 9
- 238000010353 genetic engineering Methods 0.000 abstract description 6
- 230000001965 increasing effect Effects 0.000 abstract description 5
- 239000012634 fragment Substances 0.000 description 36
- 230000006801 homologous recombination Effects 0.000 description 25
- 238000002744 homologous recombination Methods 0.000 description 25
- 238000010276 construction Methods 0.000 description 20
- 238000012795 verification Methods 0.000 description 14
- 238000012408 PCR amplification Methods 0.000 description 11
- 108091033409 CRISPR Proteins 0.000 description 10
- 229960000723 ampicillin Drugs 0.000 description 10
- 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 10
- 239000007788 liquid Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 238000010354 CRISPR gene editing Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000010362 genome editing Methods 0.000 description 5
- 235000020256 human milk Nutrition 0.000 description 5
- 210000004251 human milk Anatomy 0.000 description 5
- 229920001542 oligosaccharide Polymers 0.000 description 5
- 150000002482 oligosaccharides Chemical class 0.000 description 5
- 230000006798 recombination Effects 0.000 description 5
- 238000005215 recombination Methods 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 101000819503 Homo sapiens 4-galactosyl-N-acetylglucosaminide 3-alpha-L-fucosyltransferase 9 Proteins 0.000 description 4
- 101001022183 Homo sapiens 4-galactosyl-N-acetylglucosaminide 3-alpha-L-fucosyltransferase FUT5 Proteins 0.000 description 4
- 101001022175 Homo sapiens 4-galactosyl-N-acetylglucosaminide 3-alpha-L-fucosyltransferase FUT6 Proteins 0.000 description 4
- 101000862183 Homo sapiens Alpha-(1,3)-fucosyltransferase 10 Proteins 0.000 description 4
- 101000862213 Homo sapiens Alpha-(1,3)-fucosyltransferase 11 Proteins 0.000 description 4
- 101000819497 Homo sapiens Alpha-(1,3)-fucosyltransferase 7 Proteins 0.000 description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 102100021333 Alpha-(1,3)-fucosyltransferase 7 Human genes 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- 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 3
- 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 3
- 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 3
- 101710161145 Sugar efflux transporter Proteins 0.000 description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-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
- 210000004748 cultured cell Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 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 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 101100494773 Caenorhabditis elegans ctl-2 gene Proteins 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
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 101100112369 Fasciola hepatica Cat-1 gene Proteins 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 241000590002 Helicobacter pylori Species 0.000 description 2
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-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
- 101100005271 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cat-1 gene Proteins 0.000 description 2
- 101100145480 Prochlorococcus marinus (strain SARG / CCMP1375 / SS120) rpoC2 gene Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- -1 UDP-glucose lipid Chemical class 0.000 description 2
- 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 2
- 101150035354 araA gene Proteins 0.000 description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 101150111848 fucA gene Proteins 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 229940037467 helicobacter pylori Drugs 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 101150091078 rhaA gene Proteins 0.000 description 2
- 101150109946 rpo1C gene Proteins 0.000 description 2
- 101150103066 rpoC1 gene Proteins 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 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 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- 241000644323 Escherichia coli C Species 0.000 description 1
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Natural products O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 238000004977 Hueckel calculation Methods 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
- 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
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- 241000080590 Niso Species 0.000 description 1
- 101710112075 Para-Rep C7 Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010091086 Recombinases Proteins 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
- OIRDTQYFTABQOQ-UHTZMRCNSA-N Vidarabine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1O OIRDTQYFTABQOQ-UHTZMRCNSA-N 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
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- OIRDTQYFTABQOQ-UHFFFAOYSA-N ara-adenosine Natural products Nc1ncnc2n(cnc12)C1OC(CO)C(O)C1O OIRDTQYFTABQOQ-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical compound C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 101150015731 fucI gene Proteins 0.000 description 1
- 101150025078 fucK gene Proteins 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009776 industrial production Methods 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
- 238000011081 inoculation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000001308 synthesis method Methods 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
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
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
- 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)
- C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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.)
- C12N9/1048—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
- 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)
- C12N9/1288—Transferases for other substituted phosphate groups (2.7.8)
-
- 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
- C12N9/88—Lyases (4.)
-
- 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
- C12N9/90—Isomerases (5.)
-
- 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
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01271—GDP-L-fucose synthase (1.1.1.271)
-
- 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)
- C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
-
- 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)
- C12Y207/07022—Mannose-1-phosphate guanylyltransferase (GDP) (2.7.7.22)
-
- 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)
- C12Y207/08—Transferases for other substituted phosphate groups (2.7.8)
- C12Y207/08031—Undecaprenyl-phosphate glucose phosphotransferase (2.7.8.31)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01047—GDP-mannose 4,6-dehydratase (4.2.1.47), i.e. GMD
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
- C12Y503/01008—Mannose-6-phosphate isomerase (5.3.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y504/00—Intramolecular transferases (5.4)
- C12Y504/02—Phosphotransferases (phosphomutases) (5.4.2)
- C12Y504/02008—Phosphomannomutase (5.4.2.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- 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)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于基因工程技术领域,具体涉及一种RNA聚合酶β亚单位突变体及其应用。所述RNA聚合酶β亚单位突变体,是在野生型RNA聚合酶β亚单位(RpoC,NCBI Reference Sequences:NP_418415.1)的基础上,缺失第215位至第220位的6个氨基酸获得的,具有SEQ ID NO.1所示的氨基酸序列。将其应用于生产2'‑岩藻糖基乳糖和3‑岩藻糖基乳糖的大肠杆菌中,能够大幅提高菌株在发酵时的活细胞数,提高了菌株在发酵环境中的耐受性,并且2'‑岩藻糖基乳糖或3‑岩藻糖基乳糖产量也大幅度提高。
Description
技术领域:
本发明属于基因工程技术领域,具体涉及一种RNA聚合酶β亚单位突变体及其应用。
背景技术:
2'-岩藻糖基乳糖(2'-fucosyllactose,2'-FL)和3-岩藻糖基乳糖(3-fucosyllactose,3-FL)是母乳低聚糖(human milk oligosaccharides,HMOs)的重要成分,在调节肠道菌群、免疫力等方面发挥重要作用,具有广阔的市场前景。
目前2'-岩藻糖基乳糖和3-岩藻糖基乳糖的生产方法包括化学合成法、酶合成法、微生物发酵法等,其中微生物发酵法具有环境友好、成本低等优势。大肠杆菌遗传背景清晰、代谢旺盛且增殖迅速,是微生物发酵法中常用的工程菌株。大肠杆菌发酵制备各种母乳低聚糖涉及到多种酶和转运体。其中2'-岩藻糖基乳糖和3-岩藻糖基乳糖的生物合成路径相似。为提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵产率,对二者生物合成路径所涉及的相关基因的编辑手段也相似(Huang D,Yang KX,Liu J,et al.Metabolic engineeringof Escherichia coli for the production of 2’-fucosyllactose and 3-fucosyllactose through modular pathway enhancement[J].Metab Eng,2017,41:23-38;徐铮,李娜,陈盈利,等.人乳寡糖2'-FL和3-FL的生物制备研究进展[J].生物工程学报,2020,36(12):12.)。
本领域对2'-岩藻糖基乳糖的从头合成途径和补救途径的关键酶、糖外排转运体等已有深入了解(Zhu,Y.Y.,et al.Recent advances on 2'-fucosyllactose:physiological properties,applications,and production approaches.CriticalReviews in Food Science and Nutrition,DOI:10.1080/10408398.2020.1850413),并通过对相关酶或转运体编码基因的基因编辑,考察了其对2'-岩藻糖基乳糖、3-岩藻糖基乳糖发酵产率的影响。根据报道,敲除以下基因中的一种或多种:β-半乳糖苷酶编码基因lacZ、UDP-葡萄糖脂质载体转移酶编码基因wcaJ、乳糖lac操纵子序列中的调节基因lacI、L-岩藻糖异构酶编码基因fucI、L-墨角藻糖激酶编码基因fucK、L-墨角藻糖-1-磷酸醛缩酶编码基因fucA(Ni ZJ,et al.Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41(DE3)Derivative[J].Frontiers in Bioengineering and Biotechnology,2020,8);和/或过表达以下基因的一种或多种:GDP-岩藻糖合成酶编码基因wcaG、GDP-甘露糖-4,6-脱水酶编码基因gmd、β-半乳糖苷透性酶编码基因lacY、磷酸甘露糖异构酶编码基因manA、磷酸甘露糖变位酶编码基因manB、糖外排转运体A编码基因setA、甘露糖-1-磷酸鸟嘌呤转移酶编码基因manC、L-阿拉伯糖异构酶编码基因araA、鼠李糖异构酶编码基因rhaA、2'-岩藻糖基乳糖合成酶编码基因futC、L-岩藻糖激酶/GDP-L-岩藻糖焦磷酸化酶编码基因fkp有利于提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵产率。其中fucI、fucK、fucA、araA、rhaA、fkp等参与2'-岩藻糖基乳糖的补救合成途径。由于α-(1,3)-岩藻糖基转移酶(FutA)的低活性和不溶性表达,与2'-岩藻糖基乳糖相比,3-岩藻糖基乳糖的发酵产率较低。因此,进一步过表达或插入α-(1,3)-岩藻糖基转移酶编码基因futA或对α-(1,3)-岩藻糖基转移酶编码基因futA进行有益突变,更有利于提高3-岩藻糖基乳糖的发酵产率(Yun HC,Park BS,Seo J,et al.Biosynthesisof the human milk oligosaccharide 3-fucosyllactose in metabolicallyengineered Escherichia coli via the salvage pathway through increasing GiPsynthesis andβ-galactosidase modification[J].Biotechnology andBioengineering,2019.)。
目前,工业生产2'-岩藻糖基乳糖和3-岩藻糖基乳糖时,由于发酵环境中高浓度的2'-岩藻糖基乳糖和3-岩藻糖基乳糖会对菌株的生产性能造成抑制,因此菌株的环境耐受性对其高效生产来说至关重要。进一步对菌株进行改良,以提高其环境耐受性,进而提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖产率,仍是目前需要解决的问题。
rpoC基因编码大肠杆菌RNA聚合酶β亚单位,其突变体可以通过提高部分代谢物产量,引起细胞膜的变化以及增强菌株对特殊代谢物的利用能力,进而增强菌株的环境耐受性。工业生产2'-岩藻糖基乳糖和3-岩藻糖基乳糖时,由于发酵环境中高浓度的2'-岩藻糖基乳糖和3-岩藻糖基乳糖会对菌株的生产性能造成抑制,因此通过引入RpoC突变体,期望能够提升菌株对发酵环境的耐受性,进而实现2'-岩藻糖基乳糖和3-岩藻糖基乳糖产量的进一步提升。本发明将通过提供一种RNA聚合酶β亚单位突变体以提高菌株耐受性,并将其用于2'-岩藻糖基乳糖和3-岩藻糖基乳糖的生产。
发明内容:
为解决上述技术问题,本发明利用基因编辑技术,对大肠杆菌多种酶或转运体的编码基因进行编辑。并通过基因编辑,获得了一种可提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖产率的RNA聚合酶β亚单位突变体并将其应用于2'-岩藻糖基乳糖和3-岩藻糖基乳糖的生产。
本发明提供的技术方案之一,是一种RNA聚合酶β亚单位突变体,是在野生型RNA聚合酶β亚单位(RpoC,NCBI Reference Sequences:NP_418415.1)的基础上,缺失第215位至第220位的6个氨基酸获得的,具有SEQ ID NO.1所示的氨基酸序列。
本发明提供的技术方案之二,是上述RNA聚合酶β亚单位突变体的应用,特别是在生产2'-岩藻糖基乳糖和3-岩藻糖基乳糖中的应用。
本发明提供的技术方案之三,是一种生产2'-岩藻糖基乳糖的基因工程菌,所述工程菌以大肠杆菌K12 MG1655为出发菌株,敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ,在原lacZ位点之后以Ptrc启动子过表达wcaG、gmd和lacY基因,进而将乙醇脱氢酶编码基因adhE替换为Ptrc启动子过表达的manA和manB,之后敲除基因组上的wcaJ基因,将基因组上的RNA聚合酶β亚单位基因rpoC突变为SEQ ID NO.2所示的RNA聚合酶β亚单位突变体基因,并通过质粒pirc99a过表达futC、manC以及setA基因所得。
本发明提供的技术方案之四,是一种生产3-岩藻糖基乳糖的基因工程菌,所述工程菌以大肠杆菌K12 MG1655为出发菌株,敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ,在原lacZ位点之后以Ptrc启动子过表达wcaG、gmd和lacY基因,进而将乙醇脱氢酶编码基因adhE替换为Ptrc启动子过表达的manA和manB,之后敲除基因组上的wcaJ基因,将基因组上的RNA聚合酶β亚单位基因rpoC突变为SEQ ID NO.2所示的RNA聚合酶β亚单位突变体基因,并通过质粒pirc99a过表达futA、manC以及setA基因所得。
进一步地,lacI的Gene ID为945007;lacZ的Gene ID为945006;wcaG的Gene ID为946563;gmd的Gene ID为946562;lacY的Gene ID为949083;adhE的Gene ID为945837;manA的Gene ID为944840;manB的Gene ID为946574;wcaJ的Gene ID为946583;manC的Gene ID为946580;futC的核苷酸序列如SEQ ID NO:8所示;setA的Gene ID为944793;futA基因的核苷酸序列如SEQ ID NO:6所示。
本发明提供的技术方案之五,是上述生产2'-岩藻糖基乳糖的基因工程菌的应用,或生产3-岩藻糖基乳糖的基因工程菌的应用。
有益效果:
本发明通过基因编辑技术获得一种RNA聚合酶β亚单位突变体,将其应用于生产2'-岩藻糖基乳糖和3-岩藻糖基乳糖的大肠杆菌中,能够大幅提高菌株在发酵时的活细胞数,提高了菌株在发酵罐后期环境中的耐受性,并且2'-岩藻糖基乳糖或3-岩藻糖基乳糖产量也大幅度提高。
附图说明:
图1菌株W2△wcaJ第一步同源重组验证;
图2菌株W2△wcaJ第二步同源重组验证;
图3菌株W3第一步同源重组验证;
图4菌株W3第二步同源重组验证。
具体实施方式:
下面通过具体的实施方案进一步叙述本发明。除非特别说明,以下实施方案所涉及的技术手段、材料等均可以是本领域技术人员所公知的,可以在已知的能解决相应技术问题的手段和材料中选择合适的。另外,实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
本发明所涉及的RNA聚合酶β亚单位突变体,是在野生型RNA聚合酶β亚单位(RpoC,NP_418415.1)的基础上,缺失第215位至第220位的6个氨基酸获得的,具有SEQ ID NO.1所示的氨基酸序列:
MKDLLKFLKAQiKiEEFDAIKIALASPDMIRSWSFGEVKKPEiINYRiFKPERDGLFCARIF
GPVKDYECLCGKYKRLKHRGVICEKCGVEViQiKVRRERMGHIELASPiAHIWFLKSLPS
RIGLLLDMPLRDIERVLYFESYVVIEGGMiNLERQQILiEEQYLDALEEFGDEFDAKMGAE
AIQALLKSMDLEQECEQLREELNEiNSEiKRIKLLEAFVQSGNKPEWMILiVLPVLPPDLR
PLVPLDGGRFAiSDLNDLYRRVINRNNRLKRLLDLAAPDIIVRNEKRMLQEAVDALLDNGR
RGRAIiGSNKRPLKSLADMIKGKQGRFRQNLLGKRVDYSGRSVIiVGPYLRLHQCGLPKK
MALELFKPFIYGKLELRGLAiiIKAAKKMVEREEAVVWDILDEVIREHPVLLNRAPiLHRL
GIQAFEPVLIEGKAIQLHPLVCAAYNADFDGDQMAVHVPLiLEAQLEARALMMSiNNILSP
ANGEPIIVPSQDVVLGLYYMiRDCVNAKGEGMVLiGPKEAERLYRSGLASLHARVKVRIi
EYEKDANGELVAKiSLKDiiVGRAILWMIVPKGLPYSIVNQALGKKAISKMLNiCYRILG
LKPiVIFADQIMYiGFAYAARSGASVGIDDMVIPEKKHEIISEAEAEVAEIQEQFQSGLViAG
ERYNKVIDIWAAANDRVSKAMMDNLQiEiVINRDGQEEKQVSFNSIYMMADSGARGSA
AQIRQLAGMRGLMAKPDGSIIEiPIiANFREGLNVLQYFISiHGARKGLADiALKiANSGY
LiRRLVDVAQDLVViEDDCGiHEGIMMiPVIEGGDVKEPLRDRVLGRViAEDVLKPGiAD
ILVPRNiLLHEQWCDLLEENSVDAVKVRSVVSCDiDFGVCAHCYGRDLARGHIINKGEAI
GVIAAQSIGEPGiQLiMRiFHIGGAASRAAAESSIQVKNKGSIKLSNVKSVVNSSGKLVIiS
RNiELKLIDEFGRiKESYKVPYGAVLAKGDGEQVAGGEiVANWDPHiMPVIiEVSGFVRF
iDMIDGQiIiRQiDELiGLSSLVVLDSAERiAGGKDLRPALKIVDAQGNDVLIPGiDMPAQ
YFLPGKAIVQLEDGVQISSGDiLARIPQESGGiKDIiGGLPRVADLFEARRPKEPAILAEISGI
VSFGKEiKGKRRLVIiPVDGSDPYEEMIPKWRQLNVFEGERVERGDVISDGPEAPHDILRL
RGVHAViRYIVNEVQDVYRLQGVKINDKHIEVIVRQMLRKAiIVNAGSSDFLEGEQVEYS
RVKIANRELEANGKVGAiYSRDLLGIiKASLAiESFISAASFQEiiRVLiEAAVAGKRDELR
GLKENVIVGRLIPAGiGYAYHQDRMRRRAAGEAPAAPQViAEDASASLAELLNAGLGGS
DNE
所述RNA聚合酶β亚单位突变体的核苷酸序列,如SEQ ID NO.2所示:atgaaagatttattaaagtttctgaaagcgcagactaaaaccgaagagtttgatgcgatcaaaattgctctggcttcgccagacatgatccgttcatggtctttcggtgaagttaaaaagccggaaaccatcaactaccgtacgttcaaaccagaacgtgacggccttttctgcgcccgtatctttgggccggtaaaagattacgagtgcctgtgcggtaagtacaagcgcctgaaacaccgtggcgtcatctgtgagaagtgcggcgttgaagtgacccagactaaagtacgccgtgagcgtatgggccacatcgaactggcttccccgactgcgcacatctggttcctgaaatcgctgccgtcccgtatcggtctgctgctcgatatgccgctgcgcgatatcgaacgcgtactgtactttgaatcctatgtggttatcgaaggcggtatgaccaacctggaacgtcagcagatcctgactgaagagcagtatctggacgcgctggaagagttcggtgacgaattcgacgcgaagatgggggcggaagcaatccaggctctgctgaagagcatggatctggagcaagagtgcgaacagctgcgtgaagagctgaacgaaaccaactccgaaaccaagcgtatcaaactgctggaagcgttcgttcagtctggtaacaaaccagagtggatgatcctgaccgttctgccggtactgccgccagatctgcgtccgctggttccgctggatggtggtcgtttcgcgacttctgacctgaacgatctgtatcgtcgcgtcattaaccgtaacaaccgtctgaaacgtctgctggatctggctgcgccggacatcatcgtacgtaacgaaaaacgtatgctgcaggaagcggtagacgccctgctggataacggtcgtcgcggtcgtgcgatcaccggttctaacaagcgtcctctgaaatctttggccgacatgatcaaaggtaaacagggtcgtttccgtcagaacctgctcggtaagcgtgttgactactccggtcgttctgtaatcaccgtaggtccatacctgcgtctgcatcagtgcggtctgccgaagaaaatggcactggagctgttcaaaccgttcatctacggcaagctggaactgcgtggtcttgctaccaccattaaagctgcgaagaaaatggttgagcgcgaagaagctgtcgtttgggatatcctggacgaagttatccgcgaacacccggtactgctgaaccgtgcaccgactctgcaccgtctgggtatccaggcatttgaaccggtactgatcgaaggtaaagctatccagctgcacccgctggtttgtgcggcatataacgccgacttcgatggtgaccagatggctgttcacgtaccgctgacgctggaagcccagctggaagcgcgtgcgctgatgatgtctaccaacaacatcctgtccccggcgaacggcgaaccaatcatcgttccgtctcaggacgttgtactgggtctgtactacatgacccgtgactgtgttaacgccaaaggcgaaggcatggtgctgactggcccgaaagaagcagaacgtctgtatcgctctggtctggcttctctgcatgcgcgcgttaaagtgcgtatcaccgagtatgaaaaagatgctaacggtgaattagtagcgaaaaccagcctgaaagacacgactgttggccgtgccattctgtggatgattgtaccgaaaggtctgccttactccatcgtcaaccaggcgctgggtaaaaaagcaatctccaaaatgctgaacacctgctaccgcattctcggtctgaaaccgaccgttatttttgcggaccagatcatgtacaccggcttcgcctatgcagcgcgttctggtgcatctgttggtatcgatgacatggtcatcccggagaagaaacacgaaatcatctccgaggcagaagcagaagttgctgaaattcaggagcagttccagtctggtctggtaactgcgggcgaacgctacaacaaagttatcgatatctgggctgcggcgaacgatcgtgtatccaaagcgatgatggataacctgcaaactgaaaccgtgattaaccgtgacggtcaggaagagaagcaggtttccttcaacagcatctacatgatggccgactccggtgcgcgtggttctgcggcacagattcgtcagcttgctggtatgcgtggtctgatggcgaagccggatggctccatcatcgaaacgccaatcaccgcgaacttccgtgaaggtctgaacgtactccagtacttcatctccacccacggtgctcgtaaaggtctggcggataccgcactgaaaactgcgaactccggttacctgactcgtcgtctggttgacgtggcgcaggacctggtggttaccgaagacgattgtggtacccatgaaggtatcatgatgactccggttatcgagggtggtgacgttaaagagccgctgcgcgatcgcgtactgggtcgtgtaactgctgaagacgttctgaagccgggtactgctgatatcctcgttccgcgcaacacgctgctgcacgaacagtggtgtgacctgctggaagagaactctgtcgacgcggttaaagtacgttctgttgtatcttgtgacaccgactttggtgtatgtgcgcactgctacggtcgtgacctggcgcgtggccacatcatcaacaagggtgaagcaatcggtgttatcgcggcacagtccatcggtgaaccgggtacacagctgaccatgcgtacgttccacatcggtggtgcggcatctcgtgcggctgctgaatccagcatccaagtgaaaaacaaaggtagcatcaagctcagcaacgtgaagtcggttgtgaactccagcggtaaactggttatcacttcccgtaatactgaactgaaactgatcgacgaattcggtcgtactaaagaaagctacaaagtaccttacggtgcggtactggcgaaaggcgatggcgaacaggttgctggcggcgaaaccgttgcaaactgggacccgcacaccatgccggttatcaccgaagtaagcggttttgtacgctttactgacatgatcgacggccagaccattacgcgtcagaccgacgaactgaccggtctgtcttcgctggtggttctggattccgcagaacgtaccgcaggtggtaaagatctgcgtccggcactgaaaatcgttgatgctcagggtaacgacgttctgatcccaggtaccgatatgccagcgcagtacttcctgccgggtaaagcgattgttcagctggaagatggcgtacagatcagctctggtgacaccctggcgcgtattccgcaggaatccggcggtaccaaggacatcaccggtggtctgccgcgcgttgcggacctgttcgaagcacgtcgtccgaaagagccggcaatcctggctgaaatcagcggtatcgtttccttcggtaaagaaaccaaaggtaaacgtcgtctggttatcaccccggtagacggtagcgatccgtacgaagagatgattccgaaatggcgtcagctcaacgtgttcgaaggtgaacgtgtagaacgtggtgacgtaatttccgacggtccggaagcgccgcacgacattctgcgtctgcgtggtgttcatgctgttactcgttacatcgttaacgaagtacaggacgtataccgtctgcagggcgttaagattaacgataaacacatcgaagttatcgttcgtcagatgctgcgtaaagctaccatcgttaacgcgggtagctccgacttcctggaaggcgaacaggttgaatactctcgcgtcaagatcgcaaaccgcgaactggaagcgaacggcaaagtgggtgcaacttactcccgcgatctgctgggtatcaccaaagcgtctctggcaaccgagtccttcatctccgcggcatcgttccaggagaccactcgcgtgctgaccgaagcagccgttgcgggcaaacgcgacgaactgcgcggcctgaaagagaacgttatcgtgggtcgtctgatcccggcaggtaccggttacgcgtaccaccaggatcgtatgcgtcgccgtgctgcgggtgaagctccggctgcaccgcaggtgactgcagaagacgcatctgccagcctggcagaactgctgaacgcaggtctgggcggttctgataacgagtaa
以下将通过具体实施方式对本发明作进一步地解释说明。
实施例1构建菌株W2△wcaJ
在大肠杆菌W2(E.coli K12 MG1655△lacIZ::Ptrc-wcaG-gmd-lacY,△adhE::Ptrc-manB-manA)的基础上,敲除基因组上的UDP-葡萄糖脂质载体转移酶编码基因wcaJ,构建出菌株W2△wcaJ。
其中,大肠杆菌W2是以大肠杆菌K12 MG1655(Escherichia coli K12 MG1655)为出发菌株构建,敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ,在原lacZ位点上以Ptrc启动子过表达wcaG、gmd和lacY得到W1菌株,进而将乙醇脱氢酶编码基因adhE替换为Ptrc启动子过表达的manA和manB,得到W2菌株。W2菌株的具体构建过程参见CN112501106A的实施例1和实施例2。
大肠杆菌W2构建涉及的Plac启动子序列为SEQ ID NO.3:caccatcgaatggcgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaat;lacI的Gene ID为945007;lacZ的Gene ID为945006;Ptrc启动子序列为SEQ ID NO.4:gcgcaacgcaattaatgtgagttagcgcgaattgatctggtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatcggaagctgt ggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggcgcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatattc tgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatctcacacaggaaacagacc;wcaG的Gene ID为946563;gmd的Gene ID为946562;lacY的Gene ID为949083;adhE的Gene ID为945837;manA的Gene ID为944840;manB的Gene ID为946574。
使用菌株W2作为出发菌株,利用CRISPR/Cas9技术敲除wcaJ(Gene ID为946583)。实验中所用的CRISPR/Cas9技术参考前期的研究报道【Zhao D,et al.CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations andimproved targeting efficiency.Sci Rep 7,16624】。首先,构建第一步同源重组片段,包含上下游同源臂、氯霉素抗性基因cat和通用的N20序列(iAGiCCAiCGAACCGAAGiAAGG),将第一步同源重组片段通过电转化导入含有pCAGO质粒的W2菌株中,进行第一步重组,pCAGO质粒含有重组酶基因,以及cas9和gRNA基因等【Zhao D,et al.CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improvedtargeting efficiency.Sci Rep7,16624】。挑选正确克隆,进行第二次同源重组。挑取第二次同源重组后的正确克隆,传代丢失pCAGO质粒,从而获得敲除wcaJ基因的W2△wcaJ菌株。
以下详细描述具体方法:
(1)同源重组片段的构建。以大肠杆菌菌株E.coli K12 MG1655基因组(GeneBankaccession NO.NC_000913.3)为模板,分别利用表1中的引物up-1和up-2,以及引物down-1和down-2,PCR扩增得到同源重组的上、下游同源臂。以人工合成的含有氯霉素抗性基因cat、cat自身启动子和N20序列的片段(cat-N20序列,核苷酸序列如SEQ ID NO:5所示)的载体为模板,利用引物cat-1和cat20-2进行PCR扩增,获得带有cat-N20序列的片段。以上、下游同源臂,带有cat-N20序列的片段,这3个片段为模板,利用引物up-1和down-2进行重叠PCR扩增,得到同源重组片段。
(2)第一步同源重组。利用常规的质粒转化法将pCAGO质粒转化到菌株W2中,获得菌株W2(pCAGO)。利用含有1%葡萄糖以及浓度为0.1mM的IPiG(异丙基-β-D-硫代半乳糖苷)的LB培养基制备W2(pCAGO)感受态,利用电转化方法导入同源重组片段,转化后的菌液涂布于含100mg/L氨苄霉素和25mg/L氯霉素,以及1%葡萄糖的LB平板上,30℃培养。挑取转化子进行菌落PCR鉴定,如果重组正确,条带大小为3400bp,验证结果如图1所示,条带正确,获得了第一步同源重组菌株。
(3)第二步同源重组。将第一步同源重组菌株接种到含有100mg/L氨苄霉素、0.1mM的IPiG以及2g/L阿拉伯糖的LB液体培养基中,在30℃培养6h以上,平板划线分离单菌落,挑选出在含有100mg/L氨苄霉素的LB平板上生长,但在含有25mg/L氯霉素的LB平板上不生长的克隆,进行菌落PCR验证,如果重组正确,条带大小为1300bp,验证结果如图2所示,条带正确,并将该条带的PCR产物进行测序,测序结果正确,获得了第二步同源重组菌株。将第二步同源重组菌株,进一步在37℃条件下培养,丢失其中的pCAGO质粒,从而获得菌株W2△wcaJ。
表1敲除wcaJ基因所用引物
引物名称 | 引物序列 |
up-1 | tcaccactttgtcgttctccatcactttc |
up-2 | aacgatgacaaatctaaaaaagcgcg |
cat-1 | tttttagatttgtcatcgttattaattaatctcgagtgtgacg |
cat20-2 | gcgccataaggtgaaaccggccttacttcggttcgatggactattacgccccgccctgccac |
down-1 | ccggtttcaccttatggcgcagcatgtagccttcaatgaggttcctgttattagccccttaccc |
down-2 | aacgcggtcgctatcagcaaatcaacctg |
实施例2构建菌株W3
大肠杆菌K12 MG1655的野生型RNA聚合酶β亚单位氨基酸序列为NP_418415.1。在菌株W2△wcaJ的基础上,利用与上述CRISPR/Cas9技术同样的方法,对基因组上的RNA聚合酶β亚单位基因rpoC进行突变,将其翻译蛋白的第215位至第220位氨基酸共6个氨基酸进行缺失,对应RNA聚合酶β亚单位突变的氨基酸序列如SEQ ID NO:1所示,构建出的菌株命名为W3。具体构建方法如下:
(1)同源重组片段的构建
以实验室保存的MG1655野生型菌株为模板,分别利用表2中的引物对RP-up-F/R和RP-down-F/R为引物,PCR扩增得到同源重组的上、下游同源臂。以构建菌株W2△wcaJ时PCR获得的带有cat-N20序列的片段为模板,利用引物对RP-cat-F/R为引物进行PCR扩增,获得新的带有cat-N20序列的片段。以上、下游同源臂,新的带有cat-N20序列的片段,这3个片段为模板,利用引物RP-up-F和RP-down-R进行重叠PCR,得到同源重组片段,该片段含有rpoC基因的突变,即野生型RpoC蛋白的第215位至第220位氨基酸共6个氨基酸缺失。
(2)第一步同源重组
利用常规的质粒转化法将pCAGO质粒转化到菌株W2△wcaJ中,获得菌株W2△wcaJ(pCAGO)。利用含有1%葡萄糖和浓度为0.1mM的IPiG的LB培养基制备W2△wcaJ(pCAGO)感受态,利用电转化方法导入同源重组片段,转化后的菌液涂布于含100mg/L氨苄霉素和25mg/L氯霉素,以及1%葡萄糖的LB平板上,30℃培养。挑取转化子进行菌落PCR验证,如果重组正确,条带大小为2200bp左右,验证结果如图3所示,条带正确,获得了第一步同源重组菌株。
(3)第二步同源重组
与上述敲除wcaJ基因时第二步同源重组的步骤相同,将长出的单克隆进行菌落PCR验证。如果重组正确,条带大小为1200bp左右,验证结果如图4所示,条带正确,并将该条带的PCR产物进行测序,测序结果正确,获得了第二步同源重组菌株。将第二步同源重组菌株进一步在37℃条件下培养,丢失其中的pCAGO质粒,从而获得带有rpoC突变(SEQ IDNO.2)的菌株,命名为W3。
表2构建rpoC基因突变菌株所用引物
实施例3构建质粒pTrc99a-Ptrc-futC-manC
在质粒pirc99a-futC-manC的基础上(在质粒pirc99a上使用Ptrc启动子过表达futC基因,使用阿拉伯糖诱导型启动子Para过表达manC基因),构建质粒pirc99a-Ptrc-futC-manC。质粒pirc99a-futC-manC的具体构建过程参考CN112501106A实施例3。
质粒pirc99a-Ptrc-futC-manC构建涉及的Ptrc启动子核苷酸序列为SEQ ID NO.4;甘露糖-1-磷酸鸟嘌呤转移酶编码基因manC的Gene ID为946580;2'-岩藻糖基乳糖合成酶编码基因futC核苷酸序列如SEQ ID NO:8所示:
atggcttttaaagtggtgcaaatttgcggagggcttgggaatcaaatgtttcaatacgctttcgctaaaagtttgcaaaaacactctaatacgcctg
tgctgttagatattacttcttttgattggagcaataggaaaatgcaattagagcttttccctattgatttaccctatgcgaatgcaaaagaaatcgctat
agctaaaatgcaacacctccccaagctagtaagagatacgctcaaatacatgggatttgatagggtgagtcaagaaatcgtgtttgaatacgag
cctaaattgttaaagccaagccgcttgacttatttttatggctattttcaagatccacgatattttgatgctatatcccctttaatcaagcaaactttcac
cctaccccacccccccccccccgaaaatggaaataataaaaaaaaagaggaagaataccaccgcaaacttgctttgattttagccgctcaaaa
cagcgtgtttgtgcatataagaagaggggattatgtggggattggctgtcagcttggcattgactatcaaaaaaaggcgcttgagtatatggcaa
aacgcgtgccaaacatggaacttttcgtgttttgcgaagacttagaattcacgcaaaatcttgatcttggctacccttttatggacatgaccactag
ggatagagaagaagaggcgtattgggatatgctgctcatgcaatcctgtcagcatggcattatcgctaatagcacttatagctggtgggcggct
tatttgatagaaaatccagaaaaaatcattattggccccaaacactggctttttgggcatgagaatatcctttgtgaggaatgggtgaaaatagaatcccattttgaggtaaaatcccaaaagtataacgcttaa。
以质粒pirc99a-futC-manC为模板,以表3中的Darac-F和Darac-R为引物进行PCR扩增,以去除pirc99a-futC-manC质粒中manC基因上游的阿拉伯糖启动子,只用Ptrc启动子过表达futC和manC基因,扩增后获得线性片段pirc99a-Ptrc-futC-manC。将PCR获得的线性基因片段纯化、回收,使用Ⅱ重组连接试剂盒(诺唯赞生物技术有限公司)进行自连,转化到大肠杆菌DH5α感受态细胞中,在含有100mg/L氨苄霉素的LB平板上培养,挑取转化子进行菌落PCR以及测序验证,获得正确的重组质粒,命名为质粒pirc99a-Ptrc-futC-manC。
表3构建质粒pirc99a-Ptrc-futC-manC所用引物
实施例4构建质粒pTrc99a-Ptrc-futA-manC
利用幽门螺杆菌(Helicobacter pylori)NCiC 11637来源的α-(1,3)-岩藻糖基转移酶基因futA替换质粒pirc99a-Ptrc-futC-manC中的futC,构建出质粒pirc99a-Ptrc-futA-manC。FutA能够以GDP-岩藻糖与乳糖为底物催化产生3-岩藻糖基乳糖。
以质粒pirc99a-Ptrc-futC-manC为模板,以表4中的FUiA-Zi-F和FUiA-Zi-R为引物进行PCR扩增,得到去除futC的线性载体片段。以人工合成的含有futA基因片段(核苷酸序列如SEQ ID NO:6所示atgttccagcccctattagacgcctttatagaaagcgcttccattgaaaaaatggcctctaaatctccccccctaaaaatcgctgtggcgaattggtggggagatgaagaaattaaagaatttaaaaagagcgttctttattttatcctaagccaacgctacacaatcaccctccaccgaaaccctgataaacctgcggacatcgtttttggtaacccccttggatcagccagaaaaatcctatcctatcaaaacactaaaagggtgttttacaccggtgaaaacgaatcgcctaatttcaacctctttgattacgccataggctttgatgaattggattttaatgatcgttatttgagaatgcctttgtattatgataggctacaccataaagccgagagcgtgaatgacaccacttcgccctacaaactcaaagataatagcctttatgctttaaaaaaacccacccatcattttaaagaaaaccaccctaatttatgcgcagtagtgaataatgagagcgatcctttgaaaagagggtttgcgagctttgtcgcgagcaaccctaacgctcctataaggaacgctttctatgacgctttaaacgctattgagccagttactgggggagggagcgtgaaaaacactttaggctataacgtcaaaaacaaaaacgagtttttaagccaatacaagttcaatctgtgttttgaaaactcgcaaggctatggctatgtaactgaaaaaatcattgacgcttatttcagccataccattcctatttattgggggagtcctagcgtggcgaaagactttaaccctaagagttttgtgaacatttgtgattttaaaaactttgatgaagcgattgattacgtgagatacttgcacacgcacccaaacgcctatttagacatgctctatgaaaaccctttaaacacccttgatgggaaagcttacttttaccaaaatttgagttttaaaaaaatcctagatttttttaaaacgattttagaaaacgatacgatttatcacaataacccttttgttttctatcgtgatttgaatgagcctttagtatctattgatgatttgagggttaattatgatgatttgagggttaattatgatgatttgagggttaattatgatgatttgagggttaattatgatgatttgagggttaattatgatgatttgagggttaattatgatgatttgagggttaattattaaaggtcagccgtgaacgcgtcaccttcaacg)的载体为模板,以FUiA-F和FUiA-R为引物进行PCR扩增,获得带有futA基因的片段。将PCR获得的产物futA基因片段、去除futC的线性载体片段进行纯化、回收,使用Ⅱ重组连接试剂盒(诺唯赞生物技术有限公司)将回收的上述PCR产物进行连接,转化到大肠杆菌DH5α感受态细胞中,在含有100mg/L氨苄霉素的LB平板上培养,挑取转化子进行菌落PCR以及测序验证,获得正确的重组质粒,命名为质粒pirc99a-Ptrc-futA-manC。
表4构建质粒pirc99a-Ptrc-futA-manC所用引物
实施例5在质粒pTrc99a-Ptrc-futC-manC上过表达setA基因的质粒构建
进一步在质粒过表达糖流出转运蛋白基因setA(Gene ID为944793),在质粒pirc99a-Ptrc-futC-manC的基础上,利用启动子PJ23110(核苷酸序列为SEQ ID NO:7所示:GAAiiCGCGGCCGCiiCiAGAGiiiACGGCiAGCiCAGiCCiAGGiACAAiGCiAGCiA C)过表达setA,构建出质粒pirc99a-Ptrc-futC-manC-PJ23110-setA。
以质粒pirc99a-Ptrc-futC-manC为模板,以表5中的pirc-F和pirc-110-R为引物进行PCR扩增,获得pirc99a-Ptrc-futC-manC线性载体片段。以大肠杆菌MG1655基因组为模板,以setA-110-F和setA-R为引物进行PCR扩增,获得带有PJ23110、setA基因的片段。将带有PJ23110、setA基因的片段、和经过PCR获得的pirc99a-Ptrc-futC-manC线性载体片段纯化、回收,使用Ⅱ重组连接试剂盒(诺唯赞生物技术有限公司)将回收的PJ23110、setA基因片段与pirc99a-Ptrc-futC-manC线性载体片段进行连接,转化到大肠杆菌DH5α感受态细胞中,在含有100mg/L氨苄霉素的LB平板上培养,挑取转化子进行菌落PCR以及测序验证,获得正确的重组质粒,命名为质粒pirc99a-Ptrc-futC-manC-PJ23110-setA。
表5构建质粒pirc99a-Ptrc-futC-manC-PJ23110-setA所用引物
引物名称 | 引物序列 |
pirc-F | tcaaacgtctttaacctttgc |
pirc-110-R | caaaggttaaagacgtttgagtagaacaactgttcaccgttac |
setA-110-F | atcagggttgtagctagcattgtacctaggactgagctagccgtaaatgcgtttctacaaactcttt |
setA-R | atgctagctacaaccctgataaatgcttctagagaaagaggagaaatactagatgatctggataatgacgatg |
实施例6在质粒pTrc99a-Ptrc-futA-manC上过表达setA基因的质粒构建
在质粒pirc99a-Ptrc-futA-manC的基础上,利用启动子PJ23110过表达setA,构建出质粒pirc99a-Ptrc-futA-manC-PJ23110-setA。
以质粒pirc99a-Ptrc-futA-manC为模板,以表5中的pirc-F和pirc-110-R为引物进行PCR扩增获得线性载体片段。以大肠杆菌MG1655基因组为模板,以setA-110-F和setA-R为引物进行PCR扩增,获得带有PJ23110、setA基因的片段。将带有PJ23110、setA基因的片段、pirc99a-Ptrc-futA-manC线性载体片段纯化、回收,使用Ⅱ重组连接试剂盒(诺唯赞生物技术有限公司)将回收的PJ23110、setA基因片段与pirc99a-Ptrc-futA-manC线性载体片段进行连接,转化到大肠杆菌DH5α感受态细胞中,在含有100mg/L氨苄霉素的LB平板上培养,挑取转化子进行菌落PCR以及测序验证,获得正确的重组质粒,命名为质粒pirc99a-Ptrc-futA-manC-PJ23110-setA。
实施例7 2'-岩藻糖基乳糖生产菌种的构建和发酵测试
利用电转化的方法,将质粒pirc99a-Ptrc-futC-manC-PJ23110-setA和pirc99a-Ptrc-futA-manC-PJ23110-setA分别导入W2△wcaJ和W3,分别构建出2'-岩藻糖基乳糖生产菌株W2△wcaJ(pirc99a-Ptrc-futC-manC-PJ23110-setA)和W3(pirc99a-Ptrc-futC-manC-PJ23110-setA),以及3-岩藻糖基乳糖生产菌株W2△wcaJ(pirc99a-Ptrc-futA-manC-PJ23110-setA)和W3(pirc99a-Ptrc-futA-manC-PJ23110-setA)。测试上述菌株发酵生产水平。
所用培养基为:
LB培养基:NaCl 10g/L,酵母粉5g/L,蛋白胨10g/L,pH为7.0。
发酵培养基:KH2PO4 3g/L,酵母粉8g/L,(NH4)2SO4 4g/L,柠檬酸1.7g/L,MgSO4·7H2O 2g/L,硫胺素10mg/L,甘油10g/L,乳糖5g/L,1ml/L微量元素(FeCl3·6H2O25g/L,MnCl2·4H2O 9.8g/L,CoCl2·6H2O 1.6g/L,CuCl2·H2O 1g/L,H3BO3 1.9g/L,ZnCl22.6g/L,Na2MOO4·2H2O 1.1g/L,Na2SeO3 1.5g/L,NiSO4·6H2O 1.5g/L,利用氨水调pH为7.2。
发酵测试过程为:
分别挑取2'-岩藻糖基乳糖生产菌株和3-岩藻糖基乳糖生产菌株单菌落,在含有50mg/L氨苄霉素的LB液体培养基中,37℃、220rpm/min,培养过夜。取过夜培养的菌液作为种子液,以1%的接种量将菌液转接到含有2mL发酵培养基的24孔板中,发酵培养基中含有50mg/L氨苄霉素以及0.1mmol/L的IPiG,37℃,800rpm/min条件下进行发酵。每个菌株平行培养3个样品。发酵过程中,测定菌体的生长(OD600)、2'-岩藻糖基乳糖或3-岩藻糖基乳糖含量,并计算活细胞数(通过用LB液体培养基适当稀释菌液,涂布LB固体平板后,计数在平板上生长的单菌落数量)。利用HPLC检测样品中2'-岩藻糖基乳糖或3-岩藻糖基乳糖的浓度,HPLC分析所用色谱柱为Carbohydrate ES 5u 250mm*4.6mm,检测器为蒸发光检测器,流动相为70%乙腈(乙腈:水),流速为0.8mL/min,柱温为30℃,进样量为5μL。利用2'-岩藻糖基乳糖或3-岩藻糖基乳糖标准品对样品浓度进行定量。结果如表6、表7所示:
表6 2'-岩藻糖基乳糖生产菌株发酵测试结果
表7 3-岩藻糖基乳糖生产菌株发酵测试结果
由表6和表7可以看出,RpoC蛋白第215位至第220位氨基酸6个氨基酸的缺失,大幅提高了菌株在发酵48h时的活细胞数,提高了其在发酵罐后期环境中的耐受性,并且2'-岩藻糖基乳糖或3-岩藻糖基乳糖产量也大幅度提高。
虽然本发明已经以较佳实施例公开如上,但其并非用以限定本发明,任何本领域技术人员,在不脱离本发明的精神和原理的情况下,可以对这些实施例进行各种形式和细节的变化、修改、替换和变型,本发明的范围由权利要求及其等同物所限定。
Claims (8)
1.一种RNA聚合酶β亚单位突变体,其特征在在于,具有SEQ ID NO.1所示的氨基酸序列。
2.权利要求1所述RNA聚合酶β亚单位突变体的应用。
3.如权利要求2所述的应用,其特征在于,是所述RNA聚合酶β亚单位突变体在生产2'-岩藻糖基乳糖或3-岩藻糖基乳糖中的应用。
4.一种生产2'-岩藻糖基乳糖的基因工程菌,其特征在于,所述工程菌以大肠杆菌K12MG1655为出发菌株,敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ,在原lacZ位点之后以Ptrc启动子过表达wcaG、gmd和lacY基因,进而将乙醇脱氢酶编码基因adhE替换为Ptrc启动子过表达的manA和manB,之后敲除基因组上的wcaJ基因,将基因组上的RNA聚合酶β亚单位基因rpoC突变为SEQ ID NO.2所示的RNA聚合酶β亚单位突变体基因,并通过质粒pirc99a过表达futC、manC以及setA基因所得。
5.一种生产3-岩藻糖基乳糖的基因工程菌,其特征在于,所述工程菌以大肠杆菌K12MG1655为出发菌株,敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ,在原lacZ位点之后以Ptrc启动子过表达wcaG、gmd和lacY基因,进而将乙醇脱氢酶编码基因adhE替换为Ptrc启动子过表达的manA和manB,之后敲除基因组上的wcaJ基因,将基因组上的RNA聚合酶β亚单位基因rpoC突变为SEQ ID NO.2所示的RNA聚合酶β亚单位突变体基因,并通过质粒pirc99a过表达futA、manC以及setA基因所得。
6.如权利要求4或5所述的工程菌,其特征在于,lacI的Gene ID为945007;lacZ的GeneID为945006;wcaG的Gene ID为946563;gmd的Gene ID为946562;lacY的Gene ID为949083;adhE的Gene ID为945837;manA的Gene ID为944840;manB的Gene ID为946574;wcaJ的GeneID为946583;manC的Gene ID为946580;futC的核苷酸序列如SEQ ID NO:8所示;setA的GeneID为944793;futA基因的核苷酸序列如SEQ ID NO:6所示。
7.权利要求4所述基因工程菌在生产2'-岩藻糖基乳糖中的应用。
8.权利要求5所述基因工程菌在生产3-岩藻糖基乳糖中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310064132.XA CN116334025A (zh) | 2023-01-17 | 2023-01-17 | 一种RNA聚合酶β亚单位突变体及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310064132.XA CN116334025A (zh) | 2023-01-17 | 2023-01-17 | 一种RNA聚合酶β亚单位突变体及应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116334025A true CN116334025A (zh) | 2023-06-27 |
Family
ID=86875435
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310064132.XA Pending CN116334025A (zh) | 2023-01-17 | 2023-01-17 | 一种RNA聚合酶β亚单位突变体及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116334025A (zh) |
-
2023
- 2023-01-17 CN CN202310064132.XA patent/CN116334025A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106190937B9 (zh) | 一种构建重组大肠杆菌生物合成2’-岩藻乳糖的方法 | |
CN114480240B (zh) | 一种产岩藻糖基乳糖的基因工程菌及生产方法 | |
CN109536428B (zh) | 一种产l-异亮氨酸的基因工程菌及其构建方法和应用 | |
CN113201535B (zh) | 谷氨酸脱氢酶基因启动子的突变体及其应用 | |
CN115786220B (zh) | 一种生产2`-岩藻糖基乳糖的重组菌株及构建方法和应用 | |
CN112481179A (zh) | 产l-苏氨酸的基因工程菌及其构建方法与应用 | |
CN114874964A (zh) | 一种高产2′-岩藻糖基乳糖的重组大肠杆菌的构建方法及应用 | |
CN108588108B (zh) | 一种高效代谢甘油的芽胞杆菌的制备方法和应用 | |
CN116676243A (zh) | 产2'-岩藻糖基乳糖的重组大肠杆菌的构建方法及其应用 | |
CN116334025A (zh) | 一种RNA聚合酶β亚单位突变体及应用 | |
CN114806991A (zh) | 一种提高岩藻糖基乳糖产量的工程大肠杆菌及生产方法 | |
CN117737060A (zh) | 一种非编码RNA CsrC突变体、基因工程菌及应用 | |
CN110872595B (zh) | 抗酸表达盒及其在发酵产有机酸中的应用 | |
CN117736280A (zh) | 一种SecY蛋白突变体、基因工程菌及应用 | |
CN117737061A (zh) | 一种非编码RNA CsrB突变体、基因工程菌及应用 | |
CN116042684B (zh) | 大肠杆菌及其在催化合成阿洛酮糖中的应用 | |
CN116925993B (zh) | 用于酶催化生产胞苷酸的基因工程改造菌株和方法 | |
US20240060056A1 (en) | Modified beta-1,3-n-acetylglucosaminyltransferase polypeptides | |
CN109136297B (zh) | 生产1,5-戊二胺的方法 | |
CN116949005A (zh) | 应用岩藻糖基转移酶生产2’-岩藻糖基乳糖的方法 | |
CN116004489A (zh) | 一种生产nmn的重组大肠杆菌及其应用 | |
CN115029289A (zh) | 高产l-苏氨酸的基因工程菌及其构建方法与应用 | |
CN116478894A (zh) | 一种提高唾液酸乳糖产量的基因工程菌及其生产方法 | |
CN117511837A (zh) | 一种高产o-乙酰-l-高丝氨酸的重组大肠杆菌及其应用 | |
CN117660277A (zh) | 代谢工程改造大肠杆菌及其在发酵制备红景天苷中的应用 |
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 |