CN107881123B - 一株利用甲醇生产丙酮酸的基因工程菌及其构建方法与应用 - Google Patents
一株利用甲醇生产丙酮酸的基因工程菌及其构建方法与应用 Download PDFInfo
- Publication number
- CN107881123B CN107881123B CN201710669661.7A CN201710669661A CN107881123B CN 107881123 B CN107881123 B CN 107881123B CN 201710669661 A CN201710669661 A CN 201710669661A CN 107881123 B CN107881123 B CN 107881123B
- Authority
- CN
- China
- Prior art keywords
- gene
- methanol
- pyruvic acid
- seed culture
- cta
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 title claims abstract description 105
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 title claims abstract description 81
- 229940107700 pyruvic acid Drugs 0.000 title claims abstract description 40
- 241000894006 Bacteria Species 0.000 title claims abstract description 26
- 238000010276 construction Methods 0.000 title description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 23
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 16
- 108010025188 Alcohol oxidase Proteins 0.000 claims abstract description 12
- 108010067193 Formaldehyde transketolase Proteins 0.000 claims abstract description 11
- 241001052560 Thallis Species 0.000 claims abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 26
- 238000011218 seed culture Methods 0.000 claims description 23
- 238000000855 fermentation Methods 0.000 claims description 18
- 230000004151 fermentation Effects 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 17
- 230000014509 gene expression Effects 0.000 claims description 13
- 238000012360 testing method Methods 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 6
- 239000010802 sludge Substances 0.000 claims description 6
- 238000010353 genetic engineering Methods 0.000 claims description 5
- 239000003550 marker Substances 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 2
- 102100028652 Gamma-enolase Human genes 0.000 claims description 2
- 108700007698 Genetic Terminator Regions Proteins 0.000 claims description 2
- 101001058231 Homo sapiens Gamma-enolase Proteins 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004327 boric acid Substances 0.000 claims description 2
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 2
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 2
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 claims description 2
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 2
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 2
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims description 2
- 229940035893 uracil Drugs 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 2
- UOQFZGVGGMHGEE-UHFFFAOYSA-N 1,1-dihydroxypropan-2-one Chemical compound CC(=O)C(O)O UOQFZGVGGMHGEE-UHFFFAOYSA-N 0.000 claims 4
- 108091000080 Phosphotransferase Proteins 0.000 claims 4
- 102000020233 phosphotransferase Human genes 0.000 claims 4
- 101100434663 Bacillus subtilis (strain 168) fbaA gene Proteins 0.000 claims 1
- 101150095274 FBA1 gene Proteins 0.000 claims 1
- 238000009472 formulation Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000008223 sterile water Substances 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 239000007222 ypd medium Substances 0.000 claims 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 7
- 108010053835 Catalase Proteins 0.000 abstract description 7
- 108010015895 Glycerone kinase Proteins 0.000 abstract description 7
- 229910052799 carbon Inorganic materials 0.000 abstract description 7
- 150000007524 organic acids Chemical class 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 238000003786 synthesis reaction Methods 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 230000004060 metabolic process Effects 0.000 description 5
- 229940076788 pyruvate Drugs 0.000 description 5
- 235000000346 sugar Nutrition 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 101000579123 Homo sapiens Phosphoglycerate kinase 1 Proteins 0.000 description 2
- KJWZYMMLVHIVSU-IYCNHOCDSA-N PGK1 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](CCCCCCC(O)=O)C(=O)CC1=O KJWZYMMLVHIVSU-IYCNHOCDSA-N 0.000 description 2
- 102100028251 Phosphoglycerate kinase 1 Human genes 0.000 description 2
- 150000004716 alpha keto acids Chemical class 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229940120503 dihydroxyacetone Drugs 0.000 description 2
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 2
- GSXOAOHZAIYLCY-UHFFFAOYSA-N D-F6P Natural products OCC(=O)C(O)C(O)C(O)COP(O)(O)=O GSXOAOHZAIYLCY-UHFFFAOYSA-N 0.000 description 1
- FNZLKVNUWIIPSJ-RFZPGFLSSA-N D-xylulose 5-phosphate Chemical compound OCC(=O)[C@@H](O)[C@H](O)COP(O)(O)=O FNZLKVNUWIIPSJ-RFZPGFLSSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 238000010564 aerobic fermentation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- BGWGXPAPYGQALX-ARQDHWQXSA-N beta-D-fructofuranose 6-phosphate Chemical compound OC[C@@]1(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BGWGXPAPYGQALX-ARQDHWQXSA-N 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 101150003389 tdh2 gene Proteins 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- 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.)
- C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
-
- 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- 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)
- C12Y101/01244—Methanol dehydrogenase (1.1.1.244)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01006—Catalase (1.11.1.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
- C12Y202/01—Transketolases and transaldolases (2.2.1)
- C12Y202/01003—Formaldehyde transketolase (2.2.1.3)
-
- 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)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01029—Glycerone kinase (2.7.1.29), i.e. dihydroxyacetone kinase
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一株可利用甲醇生产丙酮酸的基因工程菌,它是在宿主菌中导入甲醇氧化酶基因aox、过氧化氢酶基因cta、二羟基丙酮合酶基因das、二羟基丙酮激酶基因dak。利用本发明的方法构建的重组酿酒酵母在以甲醇为唯一碳源的条件下,菌体细胞量明显增加,且能生成丙酮酸。本发明的方法为利用廉价碳资源生产有机酸的合成的研究奠定了基础。
Description
技术领域
本发明属于生物工程领域,特别涉及一株利用甲醇生产丙酮酸的基因工程菌及其构建方法与应用。
背景技术
丙酮酸(Pyruvic acid),又称α-氧代丙酸,是一种重要的有机小分子,也是一种酸性较弱的有机酸,分子中同时具有羰基和羧基两个官能团,它除具有羧酸和酮的性质外,还具有α-酮酸的性质,是最简单的α-酮酸,属于羰基酸。丙酮酸也是体内产生的三碳酮酸,它是糖酵解途径的最终产物,在细胞浆中还原成乳酸供能,或进入线粒体内氧化生成乙酰辅酶A,进入三羧酸循环,被氧化成二氧化碳和水,完成葡萄糖的有氧氧化供能过程。因此,丙酮酸是糖代谢中具有关键作用的中间产物。丙酮酸可通过乙酰辅酶A和三羧酸循环实现体内糖、脂肪和氨基酸间的互相转化。因此丙酮酸在三大营养物质的代谢联系中起着重要的枢纽作用,是所有生物细胞糖代谢及体内多种物质相互转化的重要中间体,因分子中包含活化酮和羧基基团,所以作为一种基本化工原料广泛应用于化学、制药、食品、农业及环保等各个领域中,可通过化学合成和生物技术多种方法制备。同时有研究已表明,丙酮酸可作为一种抗氧化剂抑制鼠体内氧自由基的氧化作用。
与化学合成法和酶转化法相比,微生物发酵法因原料来源广,能耗低,污染少,成本低而更具有优越性,因而被广泛研究。在丙酮酸生产菌株中,酿酒酵母由于其遗传背景清楚,易操作易调控,培养基要求简单,菌株耐受性好等优点,近年来被广泛用于研究以获得丙酮酸生产菌株。微生物代谢过程中,主要利用糖类积累丙酮酸,但这些糖类物质的成本较高,限制了微生物法制备丁二酸的工业化。因此,若能以廉价的还原性底物为原料,可以在一定程度上降低成本。
甲醇是煤化工产业中的重要产品,近年来,随着甲醇生产工艺的发展,使得甲醇的价格持续走低,因而利用甲醇作为发酵原料成为生物转化过程降低成本的重要突破口。因此,若能通过合成生物学手段,将甲醇代谢模块引入酿酒酵母中,以甲醇作为唯一碳源生产丙酮酸,为利用廉价碳资源生产有机酸的合成的研究奠定了基础。
技术方案
本发明要解决的技术问题是提供一种利用合成生物学方法构建可以利用甲醇作为唯一碳源进行代谢的菌株,并利用该菌株好氧发酵生产丙酮酸,解决了甲醇利用型菌株不能生产有机酸的技术问题。
为解决上述技术问题,本发明采用如下技术方案:
一株利用甲醇生产丙酮酸的基因工程菌,它是在宿主菌种导入甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta、二羟基丙酮激酶基因dak。
其中,甲醇氧化酶aox1将甲醇氧化成甲醛,消耗大量氧气伴随生成过氧化氢;过氧化氢在过氧化氢酶cta的催化下转化成氧气和水;甲醛和木酮糖-5磷酸在二羟基丙酮合酶das的催化下转化成二羟基丙酮;随后二羟基丙酮在二羟基丙酮激酶dak的催化下转化成磷酸二羟丙酮,然后进一步代谢成果糖-6-磷酸,进而进入糖酵解途径参与物质循环和有机酸代谢。
其中,所述宿主菌为酿酒酵母,所述酿酒酵母优选酿酒酵母Saccharomycescerevisiae TAM ura3△Pdc-菌株,该菌株为荷兰Delft大学Antonius J.A.van Maris教授馈赠,以该菌株为出发菌可生产135g/L丙酮酸,发表论文题目为Directed Evolution ofPyruvate Decarboxylase-Negative Saccharomyces cerevisiae,Yielding a C2-Independent,Glucose-Tolerant,and Pyruvate-Hyperproducing Yeast,其具体信息已经在论文中详细公开。
其中,所述甲醇氧化酶基因aox1的GenBank登记号为XM_002494226.1;
所述二羟基丙酮合酶基因das的GenBank登记号为FJ752551.1;
所述过氧化氢酶基因cta的GenBank登记号为AB472085.1;
所述二羟基丙酮激酶基因的GenBank登记号为XM_002493026.1。
其中,所述甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta、二羟基丙酮激酶基因的启动子序列分别选自PGK1p、TDH3p、PDC1p、FBA1p中的一种,但上述基因的启动子不仅可以使用上述四种启动子,也可以使用其他类型的启动子;
甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta、二羟基丙酮激酶基因的终止子序列分别选自CYC1t、PGK1t、TDH2t、ENO2t中的一种,以上四种基因的终止子不仅可以使用上述四种终止子,也可以使用其他类型的终止子。
上述利用甲醇生产丙酮酸的基因工程菌的构建方法如下:
(1)构建如下四种表达框:PGK1p-aox1-CYC1t、TDH3p-das-PGK1t、PDC1p-cta-TDH2t、FBA1p-dak-ENO2t,在使用重叠PCR将上述四种表达框、上游同源臂δ1、下游同源臂δ2、G418抗生素筛选标记基因组合成重组基因片段;所述上游同源臂δ1的基因序列如SEQ IDNo.33所示,下游同源臂δ2的基因序列如SEQ ID No.34所示,G418抗生素筛选标记基因的基因序列如SEQ ID No.35所示;
(2)将步骤(1)得到的重组基因片段转化至宿主细胞,并整合至宿主细胞基因组δ位点中,使用G418抗生素筛选重组菌,得到利用甲醇生产丙酮酸的基因工程菌。通过提取转化子的基因组进行PCR来筛选得到重组菌。
上述利用甲醇生产丙酮酸的基因工程菌在发酵产丙酮酸中的应用在本发明的保护范围之内。
其中,利用该菌株发酵产丙酮酸的方法如下:
(1a)试管种子培养:将利用甲醇生产丙酮酸的基因工程菌接种到试管的种子培养基中,30℃培养18~22h;
(2a)摇瓶种子培养:将试管种子培养基接种到摇瓶的种子培养基中,30℃培养22~26h;
(3a)发酵产丙酮酸:将摇瓶种子培养液进行离心,去除上清种子培养基,并用灭菌水清洗菌体2次,并离心获得菌泥。将菌泥重悬于发酵培养基中,30℃培养72~74h。
其中,所述种子培养基为YPD培养基,其配方如下:20g/L葡萄糖,10g/L酵母粉20g/L蛋白胨,溶剂为水。
其中,所述发酵培养基的配方如下:5g/L硫酸铵,3g/L磷酸二氢钾,0.5g/L七水硫酸镁,15mg/L乙二胺四乙酸,4.5mg/L七水硫酸锌,0.3mg/L六水氯化钴,1mg/L四水氯化锰,0.3mg/L五水硫酸铜,4.5mg/L二水氯化钙,3mg/L七水硫酸亚铁,0.4mg/L二水钼酸钠,1mg/L硼酸,0.1mg/L碘化钾,0.15g/L尿嘧啶,10g/L甲醇,溶剂为水。有益效果:
通过利用合成生物学的方法将甲醇的代谢途径引入酿酒酵母中,从而实现酿酒酵母以非食品级原料甲醇作为唯一碳源生产丙酮酸,为实现甲醇替代葡萄糖工业化发酵生产丙酮酸提供了思路,在一定程度上降低了生产成本,具有重大的社会意义和经济价值。
附图说明
图1重组表达框连接示意图。
图2提基因组PCR验证,泳道1、2、3、4分别为基因aox、cta、das、dak PCR验证。
图3原始菌和重组菌在摇瓶中甲醇消耗趋势图。
图4原始菌和重组菌在摇瓶中细胞量变化趋势图。
图5原始菌的丙酮酸液相检测图,9.033min为丙酮酸出峰时间。
图6重组菌的丙酮酸液相检测图,9.033min为丙酮酸出峰时间。
具体实施方式
下述实施例中所用的材料、试剂等,如无特殊情况说明,均可从商业途径中获得。
pMD-19T载体:本实验自主保存。
酿酒酵母Saccharomyces cerevisiae TAM ura3△Pdc-菌株为荷兰Delft大学Antonius J.A.van Maris馈赠,以该菌株为出发菌可生产135g/L丙酮酸,发表论文题目为Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomycescerevisiae,Yielding a C2-Independent,Glucose-Tolerant,and Pyruvate-Hyperproducing Yeast,其具体信息已经在论文中详细公开。
实施例1:表达基因与启动子、终止子的获得
(1)以毕赤酵母Pichia pastoris基因组为模板,设计引物扩增甲醇氧化酶aox1、二羟基丙酮合酶das、过氧化氢酶cta、二羟基丙酮激酶dak。
(2)以酿酒酵母Saccharomyces cerevisiae基因组为模板,设计引物扩增启动子PGK1p、TDH3p、PDC1p、FBA1p,终止子CYC1t、PGK1t、TDH2t、ENO2t、上游同源臂δ1、下游同源臂δ2。
实施例2:利用合成生物学方法构建重组酿酒酵母S.c-aox-das-cta-dak
为了能够快速有效的实现多基因的共同表达并保证基因表达的稳定性,利用DNA集合的方法将基因表达框整合进酿酒酵母基因组中。
1、设计引物进行扩增,分别在每个基因两端加上启动子、终止子的同源臂,设计上下游引物,基因与引物序列见表1。
表1基因与引物序列对照表
2、设计引物进行扩增,在启动子、终止子上加上所连基因同源臂,设计上下游引物,基因名称与扩增引物编号如下:
3、进行重叠PCR,组成启动子-基因-终止子的表达框PGK1p-aox1-CYC1t、TDH3p-das-PGK1t、PDC1p-cta-TDH2t、FBA1p-dak-ENO2t,上述启动子-基因-终止子的表达框就是将所示的启动子、基因、终止子使用重叠PCR的方法依次组合连接组成表达片段,例如启动子PGK1p序列之后连接甲醇氧化酶基因aox1基因序列,然后再连接终止子CYC1t序列,在不影响启动子、基因、终止子功能的情况下,其中间可以***其他碱基序列。
4、将四个表达框与pMD-19T载体做连接,转化至E.coli DH5α中,经质粒酶切和菌落PCR验证,将验证正确的质粒送至测序公司进行测序。
5、将测序正确的质粒,设计引物扩增出表达框,将上游同源臂δ1、G418抗生素筛选标记基因、PGK1p-aox1-CYC1t、TDH3p-das-PGK1t、PDC1p-cta-TDH2t、FBA1p-dak-ENO2t、下游同源臂δ2用重叠PCR的方法整合成一个基因重组片段,将该基因重组片段电转化进入酿酒酵母Saccharomyces cerevisiae TAM ura3△Pdc-中,1g/LG418抗生素筛选得到重组酿酒酵母S.c-aox-das-cta-dak,以实现基因的同时表达,进而实现甲醇的代谢,以酿酒酵母Saccharomyces cerevisiae TAM ura3△Pdc-作为对照菌株进行发酵性能考察。
实施例3:重组菌株的发酵实验。
(1)试管种子培养:将重组酿酒酵母S.c-aox-das-cta-dak按1%(v/v)接种量从冻存管接种到试管种子培养基中,试管装液量5mL,30℃有氧培养20h,获得试管种子培养液。
(2)摇瓶种子培养:将重组酿酒酵母S.c-aox-das-cta-dak按1%(v/v)接种量从试管培养基接种到摇瓶种子培养基中,250mL三角瓶装液量50mL,30℃有氧培养24h,获得摇瓶种子培养液。
(3)发酵产丙酮酸:将摇瓶种子培养液倒入100mL离心管中,使用4℃离心机8000rpm/min离心10min,离心后将上清去除,并使用缓冲液重悬菌泥,再次使用4℃离心机8000rpm/min离心10min,重复以上操作1次,后以发酵培养基重悬菌泥,250mL三角瓶装液量50mL,发酵温度为30℃,发酵时间为72h,发酵结果如图2和图3所示。结果表明,当以甲醇为唯一碳源时,重组酿酒酵母共消耗甲醇1.04g/L,菌体细胞提高了3.13%,并检测到0.26g/L丙酮酸,液相结果如图4。
SEQUENCE LISTING
<110> 南京工业大学
<120> 一株利用甲醇生产丙酮酸的基因工程菌及其构建方法与应用
<130> SG20170728
<160> 41
<170> PatentIn version 3.5
<210> 1
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> P1
<400> 1
ctacttttta caacaaatat aaaaacaatg gctatccccg aagagtttga tatcc 55
<210> 2
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> P2
<400> 2
gtaagcgtga cataactaat tacatgattt agaatctagc aagaccggtc ttctcg 56
<210> 3
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> P3
<400> 3
aataaacaca cataaacaaa caaaatggct agaattccaa aagcagtatc gacac 55
<210> 4
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> P4
<400> 4
gatctatcga tttcaattca attcaatttt tacaacttgt catgctttgg ttttccc 57
<210> 5
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> P6
<400> 5
aaataacaca gtcaaatcaa tcaaaatgtc tcaaccacct aaatggacaa catc 54
<210> 6
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> P6
<400> 6
cattaaagta acttaaggag ttaaatctac aatcttgctg cagagtcacc tc 52
<210> 7
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> P7
<400> 7
ccataaccaa gtaatacata ttcaaaatgt ctagtaaaca ttgggattac aag 53
<210> 8
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> P8
<400> 8
gactaataat tcttagttaa aagcactcta caacttggtt tcagatttga agtatgc 57
<210> 9
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> P9
<400> 9
aggtgatatc agatccacta gtggcctatt attttagatt cctgacttca actcaag 57
<210> 10
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> P10
<400> 10
ggatatcaaa ctcttcgggg atagccattg tttttatatt tgttgtaaaa agtag 55
<210> 11
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> P11
<400> 11
cgagaagacc ggtcttgcta gattctaaat catgtaatta gttatgtcac gcttac 56
<210> 12
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> P12
<400> 12
ctcgaactga aaaagcgtgt tttttatgca aattaaagcc ttcgagcgtc ccaa 54
<210> 13
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> P13
<400> 13
ttgggacgct cgaaggcttt aatttgcata aaaaacacgc tttttcagtt cgag 54
<210> 14
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> P14
<400> 14
gtgtcgatac tgcttttgga attctagcca ttttgtttgt ttatgtgtgt ttattcg 57
<210> 15
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> P15
<400> 15
gggaaaacca aagcatgaca agttgtaaaa attgaattga attgaaatcg ataga 55
<210> 16
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> P16
<400> 16
gagatattac tttgaatagg ttacttaggt ttaacgaacg cagaattttc gag 53
<210> 17
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> P17
<400> 17
ctcgaaaatt ctgcgttcgt taaacctaag taacctattc aaagtaatat ctc 53
<210> 18
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> P18
<400> 18
tgttgtccat ttaggtggtt gagacatttt gattgatttg actgtgttat tttgcg 56
<210> 19
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> P19
<400> 19
gaggtgactc tgcagcaaga ttgtagattt aactccttaa gttactttaa tg 52
<210> 20
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> P20
<400> 20
caattattta gtactgtcag tattgttatg cgaaaagcca attagtgtga tactaag 57
<210> 21
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> P21
<400> 21
tagtatcaca ctaattggct tttcgcataa caatactgac agtactaaat aattgcc 57
<210> 22
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> P22
<400> 22
cttgtaatcc caatgtttac tagacatttt gaatatgtat tacttggtta tgg 53
<210> 23
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> P23
<400> 23
gcatacttca aatctgaaac caagttgtag agtgctttta actaagaatt attagtc 57
<210> 24
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> P24
<400> 24
caattacatc aaaatccaca ttctctttaa ggtatcatct ccatctccca tatgc 55
<210> 25
<211> 781
<212> DNA
<213> Artificial Sequence
<220>
<223> PGK1启动子
<400> 25
tattttagat tcctgacttc aactcaagac gcacagatat tataacatct gcacaatagg 60
catttgcaag aattactcgt gagtaaggaa agagtgagga actatcgcat acctgcattt 120
aaagatgccg atttgggcgc gaatccttta ttttggcttc accctcatac tattatcagg 180
gccagaaaaa ggaagtgttt ccctccttct tgaattgatg ttaccctcat aaagcacgtg 240
gcctcttatc gagaaagaaa ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact 300
gaaaaaaccc agacacgctc gacttcctgt cttcctattg attgcagctt ccaatttcgt 360
cacacaacaa ggtcctagcg acggctcaca ggttttgtaa caagcaatcg aaggttctgg 420
aatggcggga aagggtttag taccacatgc tatgatgccc actgtgatct ccagagcaaa 480
gttcgttcga tcgtactgtt actctctctc tttcaaacag aattgtccga atcgtgtgac 540
aacaacagcc tgttctcaca cactcttttc ttctaaccaa gggggtggtt tagtttagta 600
gaacctcgtg aaacttacat ttacatatat ataaacttgc ataaattggt caatgcaaga 660
aatacatatt tggtcttttc taattcgtag ttttttcaag ttcttagatg ctttcttttt 720
ctcttttttt acagatcatc aaggaagtaa ttatctactt tttacaacaa atataaaaac 780
a 781
<210> 26
<211> 700
<212> DNA
<213> Artificial Sequence
<220>
<223> 启动子TDH3p
<400> 26
ataaaaaaca cgctttttca gttcgagttt atcattatca atactgccat ttcaaagaat 60
acgtaaataa ttaatagtag tgattttcct aactttattt agtcaaaaaa ttagcctttt 120
aattctgctg taacccgtac atgcccaaaa tagggggcgg gttacacaga atatataaca 180
tcgtaggtgt ctgggtgaac agtttattcc tggcatccac taaatataat ggagcccgct 240
ttttaagctg gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc 300
aaccatcagt tcataggtcc attctcttag cgcaactaca gagaacaggg gcacaaacag 360
gcaaaaaacg ggcacaacct caatggagtg atgcaacctg cctggagtaa atgatgacac 420
aaggcaattg acccacgcat gtatctatct cattttctta caccttctat taccttctgc 480
tctctctgat ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattattcc 540
cctacttgac taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat 600
ttcttaaact tcttaaattc tacttttata gttagtcttt tttttagttt crtaaaacac 660
caagaactta gtttcgaata aacacacata aacaaacaaa 700
<210> 27
<211> 850
<212> DNA
<213> Artificial Sequence
<220>
<223> 启动子PDC1p
<400> 27
aagtaaccta ttcaaagtaa tatctcatac atgtttcatg agggtaacaa catgcgactg 60
ggtgagcata tgttccgctg atgtgatgtg caagataaac aagcaaggca gaaactaact 120
tcttcttcat gtaataaaca caccccgcgt ttatttacct atctctaaac ttcaacacct 180
tatatcataa ctaatatttc ttgagataag cacactgcac ccataccttc cttaaaaacg 240
tagcttccag tttttggtgg ttccggcttc cttcccgatt ccgcccgcta aacgcatatt 300
tttgttgcct ggtggcattt gcaaaatgca taacctatgc atttaaaaga ttatgtatgc 360
tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat gaatttgaga 420
acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga ggattttatg 480
caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa ttgcataata 540
ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat cgttcaacac 600
caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg gtgatggcac 660
atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat ataaacaagg 720
ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca tatttcttgt 780
catattcctt tctcaattat tattttctac tcataacctc acgcaaaata acacagtcaa 840
atcaatcaaa 850
<210> 28
<211> 630
<212> DNA
<213> Artificial Sequence
<220>
<223> 启动子FBA1p
<400> 28
ataacaatac tgacagtact aaataattgc ctacttggct tcacatacgt tgcatacgtc 60
gatatagata ataatgataa tgacagcagg attatcgtaa tacgtaatag ttgaaaatct 120
caaaaatgtg tgggtcatta cgtaaataat gataggaatg ggattcttct atttttcctt 180
tttccattct agcagccgtc gggaaaacgt ggcatcctct ctttcgggct caattggagt 240
cacgctgccg tgagcatcct ctctttccat atctaacaac tgagcacgta accaatggaa 300
aagcatgagc ttagcgttgc tccaaaaaag tattggatgg ttaataccat ttgtctgttc 360
tcttctgact ttgactcctc aaaaaaaaaa aatctacaat caacagatcg cttcaattac 420
gccctcacaa aaactttttt ccttcttctt cgcccacgtt aaattttatc cctcatgttg 480
tctaacggat ttctgcactt gatttattat aaaaagacaa agacataata cttctctatc 540
aatttcagtt attgttcttc cttgcgttat tcttctgttc ttctttttct tttgtcatat 600
ataaccataa ccaagtaata catattcaaa 630
<210> 29
<211> 249
<212> DNA
<213> Artificial Sequence
<220>
<223> 终止子CYC1
<400> 29
atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 60
gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 120
ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg 180
tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 240
ttaatttgc 249
<210> 30
<211> 283
<212> DNA
<213> Artificial Sequence
<220>
<223> 终止子PGK1
<400> 30
aaattgaatt gaattgaaat cgatagatca atttttttct tttctctttc cccatccttt 60
acgctaaaat aatagtttat tttatttttt gaatattttt tatttatata cgtatatata 120
gactattatt tatcttttaa tgattattaa gatttttatt aaaaaaaaat tcgctcctct 180
tttaatgcct ttatgcagtt tttttttccc attcgatatt tctatgttcg ggttcagcgt 240
attttaagtt taataactcg aaaattctgc gttcgttaaa cct 283
<210> 31
<211> 400
<212> DNA
<213> Artificial Sequence
<220>
<223> 终止子TDH2
<400> 31
atttaactcc ttaagttact ttaatgattt agtttttatt attaataatt catgctcatg 60
acatctcata tacacgttta taaaacttaa atagattgaa aatgtattaa agattcctca 120
gggattcgat ttttttggaa gtttttgttt ttttttcctt gagatgctgt agtatttggg 180
aacaattata caatcgaaag atatatgctt acattcgacc gttttagccg tgatcattat 240
cctatagtaa cataacctga agcataactg acactactat catcaatact tgtcacatga 300
gaactctgtg aataattagg ccactgaaat ttgatgcctg aaggaccggc atcacggatt 360
ttcgataaag cacttagtat cacactaatt ggcttttcgc 400
<210> 32
<211> 400
<212> DNA
<213> Artificial Sequence
<220>
<223> 终止子ENO2
<400> 32
agtgctttta actaagaatt attagtcttt tctgcttatt ttttcatcat agtttagaac 60
actttatatt aacgaatagt ttatgaatct atttaggttt aaaaattgat acagttttat 120
aagttacttt ttcaaagact cgtgctgtct attgcataat gcactggaag gggaaaaaaa 180
aggtgcacac gcgtggcttt ttcttgaatt tgcagtttga aaaataacta catggatgat 240
aagaaaacat ggagtacagt cactttgaga accttcaatc agctggtaac gtcttcgtta 300
attggatact caaaaaagat ggatagcatg aatcacaaga tggaaggaaa tgcgggccac 360
gaccacagtg atatgcatat gggagatgga gatgatacct 400
<210> 33
<211> 320
<212> DNA
<213> Artificial Sequence
<220>
<223> 上游同源臂δ1
<400> 33
aaaaatcaac tatcggctgg caactaatag ggacactacc aatatattat catatacggt 60
gttagacgat gacataagat acgaggaact gtcatcgaag ttagaggaag ctgaaatgca 120
aggattgata atgtaatagg ataatgaaac atataaaacg gaatgaggaa taatcgtaat 180
attagtatat agagataaag attccatttt gaggattcct atatcctcga ggagaacttc 240
tagtatattc tgtatacctg atattatagc ctttaccaac aatagaatcc caccaattat 300
ctcaaaattc accagtatct 320
<210> 34
<211> 330
<212> DNA
<213> Artificial Sequence
<220>
<223> 下游同源臂δ2
<400> 34
taaagagaat gtggattttg atgtaattgt tgggattcca ttgtgattaa ggctataata 60
ttaggtatgt agaaagtact agaagttctc ctccaggatt taggaatcca taaaagggaa 120
tctgcaattc tacacaattc tataaatatt attatcatca ttttatatgt taatattcat 180
tgatcctatt acattatcaa tccttgcgtt tcagcttcca ctaatttaga tgactatttc 240
tcatcatttg cgtcatcttc taacaccgta tatgataata tactagtaac gtaaatacta 300
gttagtagat gatagttgat ttttattcca 330
<210> 35
<211> 1600
<212> DNA
<213> Artificial Sequence
<220>
<223> 筛选标记基因
<400> 35
tcgtacgctg caggtcgaca acccttaata taacttcgta taatgtatgc tatacgaagt 60
tattaggtct agagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac 120
atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt 180
gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat 240
tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag 300
ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat 360
ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg 420
ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaag gaaaagactc 480
acgtttcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg 540
ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg 600
cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga 660
tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc 720
gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggcaaaaca gcattccagg 780
tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc 840
gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc 900
tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg 960
agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct 1020
caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg 1080
ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc 1140
ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc 1200
aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg 1260
agtttttcta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt 1320
atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt 1380
ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg 1440
cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca 1500
tccagtgtcg aaaacgagct ctcgagaacc cttaatataa cttcgtataa tgtatgctat 1560
acgaagttat taggtgatat cagatccact agtggcctat 1600
<210> 36
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> P25
<400> 36
tgttggaata aaaatcaact atcggctggc aactaatagg gacactacca 50
<210> 37
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> P26
<400> 37
taagggttgt cgacctgcag cgtacgaaga tactggtgaa ttttgagata attggt 56
<210> 38
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> P27
<400> 38
gcatatggga gatggagatg ataccttaaa gagaatgtgg attttgatgt aattg 55
<210> 39
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> P28
<400> 39
aaaaaaaagt tccgagtaat taatgttgag atatgttgga ataaaaatca ac 52
<210> 40
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> P29
<400> 40
accaattatc tcaaaattca ccagtatctt cgtacgctgc aggtcgacaa ccctta 56
<210> 41
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> P30
<400> 41
cttgagttga agtcaggaat ctaaaataat aggccactag tggatctgat atcacct 57
Claims (7)
1.一株利用甲醇生产丙酮酸的基因工程菌,其特征在于,它是在宿主菌中导入甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta、二羟基丙酮激酶基因dak;
所述甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta、二羟基丙酮激酶基因dak的GenBank登记号分别为XM_002494226.1、FJ752551.1、AB472085.1、XM_002493026.1;
其中,所述宿主菌为酿酒酵母。
2.根据权利要求1所述的利用甲醇生产丙酮酸的基因工程菌,其特征在于,所述甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta、二羟基丙酮激酶基因dak的启动子序列分别选自PGK1p、TDH3p、PDC1p、FBA1p中的一种;
甲醇氧化酶基因aox1、二羟基丙酮合酶基因das、过氧化氢酶基因cta、二羟基丙酮激酶基因dak的终止子序列分别选自CYC1t、PGK1t、TDH2t、ENO2t中的一种。
3.权利要求1~2任一所述的利用甲醇生产丙酮酸的基因工程菌的构建方法,其特征在于,包括如下步骤:
(1) 构建如下四种表达框:PGK1p- aox1-CYC1t、TDH3p-das- PGK1t、PDC1p-cta-TDH2t、FBA1p-dak-ENO2t,再 使用重叠PCR将上述四种表达框、上游同源臂δ1、下游同源臂δ2、筛选标记基因组合成重组基因片段;
(2) 将步骤(1)得到的重组基因片段转化至宿主细胞,筛选重组菌,得到利用甲醇生产丙酮酸的基因工程菌。
4.权利要求1~2任一所述利用甲醇生产丙酮酸的基因工程菌在发酵产丙酮酸中的应用。
5.根据权利要求4所述的应用,包括以下步骤:
(1a) 试管种子培养:将利用甲醇生产丙酮酸的基因工程菌接种到试管的种子培养基中,30 ℃培养18~22 h;
(2a) 摇瓶种子培养:将试管种子培养基接种到摇瓶的种子培养基中,30 ℃培养22~26h;
(3a) 发酵产丙酮酸:将摇瓶种子培养液进行离心,去除上清种子培养基,并用灭菌水清洗菌体2次,并离心获得菌泥,将菌泥重悬于发酵培养基中,30 ℃培养72~74h。
6.根据权利要求5所述的应用,其特征在于,所述种子培养基为YPD培养基,其配方如下:20 g/L葡萄糖,10 g/L酵母粉 20 g/L蛋白胨,溶剂为水。
7.根据权利要求5所述的应用,其特征在于,所述发酵培养基的配方如下:5 g/L硫酸铵,3 g/L磷酸二氢钾,0.5 g/L七水硫酸镁,15 mg/L乙二胺四乙酸,4.5 mg/L七水硫酸锌,0.3 mg/L六水氯化钴,1 mg/L四水氯化锰,0.3 mg/L五水硫酸铜,4.5 mg/L二水氯化钙,3mg/L七水硫酸亚铁,0.4 mg/L二水钼酸钠,1 mg/L硼酸,0.1 mg/L碘化钾,0.15 g/L尿嘧啶,10 g/L甲醇,溶剂为水。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710669661.7A CN107881123B (zh) | 2017-08-08 | 2017-08-08 | 一株利用甲醇生产丙酮酸的基因工程菌及其构建方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710669661.7A CN107881123B (zh) | 2017-08-08 | 2017-08-08 | 一株利用甲醇生产丙酮酸的基因工程菌及其构建方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107881123A CN107881123A (zh) | 2018-04-06 |
CN107881123B true CN107881123B (zh) | 2020-11-27 |
Family
ID=61780505
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710669661.7A Active CN107881123B (zh) | 2017-08-08 | 2017-08-08 | 一株利用甲醇生产丙酮酸的基因工程菌及其构建方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107881123B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111304105B (zh) * | 2020-02-27 | 2022-05-03 | 南京工业大学 | 利用甲醇和木糖共底物产脂肪酶的基因工程菌及其应用 |
CN114107081B (zh) * | 2021-11-30 | 2023-05-05 | 南京工业大学 | 一种利用甲醇生物转化的重组解脂耶氏酵母基因工程菌及其构建方法和应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102220251A (zh) * | 2011-05-18 | 2011-10-19 | 江南大学 | 一种产丙酮酸的酿酒酵母基因工程菌及其构建方法和应用 |
CN103882045A (zh) * | 2013-12-04 | 2014-06-25 | 合肥百迈生物技术有限公司 | 生产丙酮酸的菌株及其构建方法 |
-
2017
- 2017-08-08 CN CN201710669661.7A patent/CN107881123B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102220251A (zh) * | 2011-05-18 | 2011-10-19 | 江南大学 | 一种产丙酮酸的酿酒酵母基因工程菌及其构建方法和应用 |
CN103882045A (zh) * | 2013-12-04 | 2014-06-25 | 合肥百迈生物技术有限公司 | 生产丙酮酸的菌株及其构建方法 |
Non-Patent Citations (2)
Title |
---|
Regulation of methanol utilisation pathway genes in yeasts;Franz S Hartner et al.;《Microbial Cell Factories》;20061231;第5卷(第39期);第1-21页 * |
甲基营养微生物的甲醛代谢途径及其在环境生物技术中的应用;张韦等;《生命科学》;20120331;第24卷(第3期);第268页左栏以及图2 * |
Also Published As
Publication number | Publication date |
---|---|
CN107881123A (zh) | 2018-04-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10584359B2 (en) | Genetically recombinant Saccharomyces cerevisiae for degrading kitchen waste | |
CN101948794A (zh) | 产植物类黄酮合成相关酶的工程乳酸菌及其构建和应用 | |
CN107881123B (zh) | 一株利用甲醇生产丙酮酸的基因工程菌及其构建方法与应用 | |
CN114807206B (zh) | 合成聚(3-羟基丁酸酯-co-4-羟基丁酸酯)的菌株及其构建方法和应用 | |
CN102517303B (zh) | 一种产乳酸的重组蓝藻及其制备方法与应用 | |
CN106929459A (zh) | 一种重组大肠杆菌及其构建方法与通过代谢工程生产葡萄糖二酸的方法 | |
CN114107078A (zh) | 一种高产瓦伦烯基因工程菌及其构建方法与应用 | |
CN107227286B (zh) | 一株高产琥珀酸的基因工程菌及其构建方法与应用 | |
CN107937296A (zh) | 一种具有乙酸糠醛香草醛耐受性重组酿酒酵母及制备方法、应用 | |
CN101130782B (zh) | 以葡萄糖为底物产1,3-丙二醇重组酿酒酵母的构建方法 | |
CN103451201A (zh) | 高效利用碳源生产生物塑料phbv的极端嗜盐古菌工程菌 | |
CN111484942A (zh) | 一种利用酿酒酵母生产己二酸的方法 | |
CN115851569B (zh) | 利用非粮生物质联产乳酸和乙醇的运动发酵单胞菌及应用 | |
CN103849639A (zh) | 一种提高半胱氨酸利用率生物合成谷胱甘肽的方法 | |
CN114277068B (zh) | 一种r-3-羟基丁酸乙酯微生物发酵制备方法 | |
CN102816780A (zh) | 山梨糖脱氢酶基因、山梨酮脱氢酶基因与吡咯喹啉醌合成基因簇abcden的组合基因 | |
CN114317307B (zh) | 一种可提高虾青素生物合成产量的基因工程菌及其构建方法与应用 | |
CN116083332A (zh) | 一株产己二酸的重组大肠杆菌的构建及其应用 | |
CN104531747A (zh) | 一种通过引入聚-β-羟基丁酸酯代谢途径提高钝齿棒杆菌L-精氨酸产量的方法 | |
CN110951794B (zh) | 一种提高酿酒酵母工程菌生产葡萄糖二酸的发酵方法 | |
Wang et al. | Carbon-economic biosynthesis of poly-2-hydrobutanedioic acid driven by nonfermentable substrate ethanol | |
CN111304138B (zh) | 一种生产β-胡萝卜素的重组大肠杆菌及构建方法与应用 | |
CN111304105A (zh) | 利用甲醇和木糖共底物产脂肪酶的基因工程菌及其应用 | |
CN104232553A (zh) | 一株在低pH值下产丁二酸工程菌株及其发酵生产丁二酸的方法 | |
CN107723302A (zh) | 一种过表达产甘油假丝酵母CgGAD1提高渗透压耐受性的方法 |
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 | ||
CB02 | Change of applicant information |
Address after: 210000, 5 new model street, Gulou District, Jiangsu, Nanjing Applicant after: NANJING TECH University Address before: 210009 Nanjing City, Jiangsu Province, the new model road No. 5 Applicant before: NANJING TECH University |
|
CB02 | Change of applicant information | ||
GR01 | Patent grant | ||
GR01 | Patent grant |