CN107815436A - 一株利用d‑木糖生产d‑1,2,4‑丁三醇的基因工程菌及其构建方法与应用 - Google Patents
一株利用d‑木糖生产d‑1,2,4‑丁三醇的基因工程菌及其构建方法与应用 Download PDFInfo
- Publication number
- CN107815436A CN107815436A CN201710963451.9A CN201710963451A CN107815436A CN 107815436 A CN107815436 A CN 107815436A CN 201710963451 A CN201710963451 A CN 201710963451A CN 107815436 A CN107815436 A CN 107815436A
- Authority
- CN
- China
- Prior art keywords
- xylose
- butantriols
- genetic engineering
- productions
- engineering bacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 29
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 title claims abstract description 26
- 238000010353 genetic engineering Methods 0.000 title claims abstract description 17
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 title claims abstract description 14
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims abstract description 13
- 238000010276 construction Methods 0.000 title claims description 7
- 239000013612 plasmid Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 11
- PYMYPHUHKUWMLA-VPENINKCSA-N aldehydo-D-xylose Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-VPENINKCSA-N 0.000 claims description 25
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 10
- 108090000489 Carboxy-Lyases Proteins 0.000 claims description 10
- 101710088194 Dehydrogenase Proteins 0.000 claims description 10
- 108030005697 Xylonate dehydratases Proteins 0.000 claims description 10
- 108010053754 Aldehyde reductase Proteins 0.000 claims description 9
- 102000004031 Carboxy-Lyases Human genes 0.000 claims description 9
- 102100027265 Aldo-keto reductase family 1 member B1 Human genes 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 5
- 239000003054 catalyst Substances 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 210000001082 somatic cell Anatomy 0.000 claims description 4
- 229910019626 (NH4)6Mo7O24 Inorganic materials 0.000 claims description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 238000006555 catalytic reaction Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 239000011565 manganese chloride Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 239000011686 zinc sulphate Substances 0.000 claims description 3
- 239000007993 MOPS buffer Substances 0.000 claims description 2
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 239000013613 expression plasmid Substances 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- 150000001299 aldehydes Chemical class 0.000 claims 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 10
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 230000003197 catalytic effect Effects 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 230000001580 bacterial effect Effects 0.000 abstract description 3
- 238000000855 fermentation Methods 0.000 abstract description 3
- 230000004151 fermentation Effects 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 abstract 1
- 230000007812 deficiency Effects 0.000 abstract 1
- 238000005265 energy consumption Methods 0.000 abstract 1
- 238000000605 extraction Methods 0.000 abstract 1
- 239000002207 metabolite Substances 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 8
- 229960003487 xylose Drugs 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 6
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- ARXKVVRQIIOZGF-UHFFFAOYSA-N 1,2,4-butanetriol Substances OCCC(O)CO ARXKVVRQIIOZGF-UHFFFAOYSA-N 0.000 description 3
- 241000589776 Pseudomonas putida Species 0.000 description 3
- 101100157012 Thermoanaerobacterium saccharolyticum (strain DSM 8691 / JW/SL-YS485) xynB gene Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 3
- 239000012267 brine Substances 0.000 description 3
- 239000001630 malic acid Substances 0.000 description 3
- 235000011090 malic acid Nutrition 0.000 description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 101150110790 xylB gene Proteins 0.000 description 3
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 2
- RDLIBIDNLZPAQD-UHFFFAOYSA-N 1,2,4-butanetriol trinitrate Chemical compound [O-][N+](=O)OCCC(O[N+]([O-])=O)CO[N+]([O-])=O RDLIBIDNLZPAQD-UHFFFAOYSA-N 0.000 description 2
- 101100108235 Escherichia coli (strain K12) adhP gene Proteins 0.000 description 2
- 101100106372 Escherichia coli (strain K12) yjhG gene Proteins 0.000 description 2
- 101100297542 Streptomyces viridochromogenes (strain DSM 40736 / JCM 4977 / BCRC 1201 / Tue 494) phpC gene Proteins 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- -1 carbon polyols Chemical class 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 150000003742 xyloses Chemical class 0.000 description 2
- CQSYGAZTCJHVFE-UHFFFAOYSA-N 3,4-dihydroxybutanal Chemical class OCC(O)CC=O CQSYGAZTCJHVFE-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 108010071778 Benzoylformate decarboxylase Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000010804 Caulobacter vibrioides Species 0.000 description 1
- 108010002731 D-xylo-aldonate dehydratase Proteins 0.000 description 1
- 108010044520 D-xylose dehydrogenase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229940122440 HIV protease inhibitor Drugs 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 1
- 229960001830 amprenavir Drugs 0.000 description 1
- YMARZQAQMVYCKC-OEMFJLHTSA-N amprenavir Chemical compound C([C@@H]([C@H](O)CN(CC(C)C)S(=O)(=O)C=1C=CC(N)=CC=1)NC(=O)O[C@@H]1COCC1)C1=CC=CC=C1 YMARZQAQMVYCKC-OEMFJLHTSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- CLQZEZFINZCXFG-UHFFFAOYSA-N butane-1,1,4-triol Chemical compound OCCCC(O)O CLQZEZFINZCXFG-UHFFFAOYSA-N 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000004030 hiv protease inhibitor Substances 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- JWQNPBVSDJHOBD-UHFFFAOYSA-N pentan-2-one;propane-1,2,3-triol Chemical compound CCCC(C)=O.OCC(O)CO JWQNPBVSDJHOBD-UHFFFAOYSA-N 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229910052701 rubidium Inorganic materials 0.000 description 1
- IGLNJRXAVVLDKE-UHFFFAOYSA-N rubidium atom Chemical compound [Rb] IGLNJRXAVVLDKE-UHFFFAOYSA-N 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- 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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
-
- 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)
- C12Y101/01002—Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01072—Branched-chain-2-oxoacid decarboxylase (4.1.1.72)
-
- 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)
- C12Y402/01082—Xylonate dehydratase (4.2.1.82)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一株利用D‑木糖生产D‑1,2,4‑丁三醇的基因工程菌，先构建两种质粒PMB和PGP，再将已构建好的双质粒导入到大肠杆菌E.coliBL21(DE3)中构建好重组菌株E.coliBL21‑PMB‑PGP，全细胞催化生产D‑1,2,4丁三醇。本发明方法解决了发酵法生产周期长，代谢产物复杂，底物转化率低，产物分离提取困难，及能耗高的问题，也解决了酶法催中催化过程不易实现的不足，提高催化效率，同时省了酶纯化过程。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一株利用D-木糖生产D-1,2,4-丁三醇的基因工程菌及其构建方法与应用。
背景技术
D-1,2,4-丁三醇(D-1,2,4-Butanetriol，简称BT)是一种重要的四碳多元醇，无色、无味、透明的溶于水是粘稠糖浆类液体。其性质和丙三醇(甘油)类似，具有一定的吸湿性和稳定性。D-1,2,4-丁三醇及其衍生物是许多化合物的重要的底物及合成的前体。在军事上丁三醇可以用来合成丁三醇三硝酸酯(BTTN)，它是一种高效的推进剂和增塑剂。在医药上，能合成降胆固醇的药物Movinolin、抗癌药物、还可以用于治疗艾滋病药物Agenerase(一种HIV蛋白酶抑制剂)。在烟草行业，可以作为卷烟添加剂，可以降低焦油对人体的伤害。另外，D-1,2,4-丁三醇还可以用于高级服装的处理，以及抑菌剂和高分子交联剂等。
目前丁三醇的生产主要采用NaBH4还原苹果酸二酯或铷和碳催化苹果酸加氢等相关方法使用不同的催化剂在19.7-34.0MPa的H2压力下和60-160℃的条件下，可将苹果酸以60％-80％的转化率转化为1,2,4-丁三醇。然后由于化学合成法存在原料成本高、产物转化率低、反应条件严苛、生产危险大、副产物多、环境污染严重等问题，随着近年来生物技术的发展以及1,2,4-丁三醇市场的快速增长，人们开始关注原料成本低廉、反应条件温和、对环境友好且安全高效的生物合成1,2,4-丁三醇的技术途径。
Wei Niu等首次报道了在大肠杆菌(Escherichia coli)中构建了异源代谢途径，随后由E.coli DH5α/p WN6.186A(表达了苯甲酰甲酸脱羧酶基因的工程菌)经三步催化反应生成D-BT，摩尔转化率为25％，D-木糖酸和L-阿拉伯糖酸为底物合成1,2,4-丁三醇对映体，产量为2.4g/L。这种方法耗时长，发酵液中其它物质大大增加了生产成本，另外发酵法生产丁三醇摩尔收率较低，耗时较长，反应体系较复杂，产物分离较困难。
发明内容
本发明要解决的技术问题是提供一株利用D-木糖生产D-1,2,4-丁三醇的基因工程菌，以解决现有技术中D-1,2,4-丁三醇生产成本高、转化率低的问题。
本发明还要解决的技术问题是提供上述利用D-木糖生产D-1,2,4-丁三醇的基因工程菌的构建方法。
本发明最后要解决的技术问题是提供上述利用D-木糖生产D-1,2,4-丁三醇的基因工程菌在催化生产D-1,2,4-丁三醇中的应用。
为解决上述技术问题，本发明采用如下技术方案：
一株利用D-木糖生产D-1,2,4-丁三醇的基因工程菌，其特征在于，在宿主菌中导入了木糖脱氢酶、2-酮酸脱羧酶、木糖酸脱水酶、醛还原酶。D-木糖在D-木糖脱氢酶作用下生成D-木糖酸，之后在D-木糖酸脱水酶作用下生成3-脱氧-D甘油戊酮糖酸，在2-酮酸脱羧酶作用下生成3，4二羟基丁醛，最后在醇脱氢酶作用下生成D-1，2，4丁三醇。
作为优选，所述的木糖脱氢酶的核苷酸序列如SEQ ID NO.1所示，所述的2-酮酸脱羧酶核苷酸序列如SEQ ID NO.2所示、所述的木糖酸脱水酶的核苷酸序列如SEQ IDNO.3所示，所述的醛还原酶的核苷酸序列如SEQ ID NO.4所示。
上述利用D-木糖生产D-1,2,4-丁三醇的基因工程菌的构建方法，包括如下步骤：
(1)将木糖脱氢酶、2-酮酸脱羧酶、木糖酸脱水酶、醛还原酶克隆到表达质粒中，得到重组质粒；
(2)将重组质粒转化宿主菌，既得到利用D-木糖生产D-1,2,4-丁三醇的基因工程菌E.coliBL-PMB-PGP。
作为优选，将木糖脱氢酶、2-酮酸脱羧酶同时构建到pTRC-99a质粒上，得到重组质粒A；将木糖酸脱水酶、醛还原酶克隆到pCWJ-1质粒上，得到重组质粒B，将重组质粒A和重组质粒B同时转化宿主菌，既得到利用D-木糖生产D-1,2,4-丁三醇的基因工程菌E.coliBL-PMB-PGP。
作为优选，所述的宿主菌为大肠杆菌BL21(DE3)。
上述利用D-木糖生产D-1,2,4-丁三醇的基因工程菌在生产D-1,2,4-丁三醇中的应用。
在利用基因工程菌E.coliBL-PMB-PGP催化生产D-1,2,4-丁三醇的方法如下：
(a1)将培养基因工程菌E.coliBL-PMB-PGP，收集E.coliBL-PMB-PGP菌体细胞；
(a2)将E.coliBL-PMB-PGP菌体细胞转接到催化体系中，反应，得到D-1,2,4-丁三醇。
步骤(a2)中，所述的催化体系包括如下组分：
NaCl 0.5g/L，NH4Cl 10g/L，Na2HPO4·12H2O 17.1g/L，KH2PO4 3g/L，MgSO4 0.12g/L，CaCl2 1.1×10-2g/L，(NH4)6Mo7O24 3.71×10-3g/L，H3BO3 2.47×10-2g/L，CoCl2·6H2O7.14×10-3g/L，MnCl2·4H2O 1.60×10-2g/L，ZnSO4·7H2O 2.8×10-3g/L，CuSO4·5H2O2.51×10-3g/L，D-木糖10g/L-40g/L，碳酸钙1g/L，MOPS 100mmol/L，Triton X-1000.5g/L，Tween-20 0.5g/L，Tween-800.5g/L，SDS 0.5g/L。
步骤(a2)中，催化反应条件为：温度37℃，搅拌转速200rpm。
有益效果
本发明公开了一株利用D-木糖生产D-1,2,4-丁三醇的基因工程菌，通过在宿主菌中过表达了木糖脱氢酶(xylB)、2-酮酸脱羧酶(mdlc)、木糖酸脱水酶(yjhG)、醛还原酶(adhP)，实现了D-木糖为底物生产高价值的D-1,2,4丁三醇，解决了体外酶催化D-木糖转化为丁三醇过程中，需要外源添加昂贵辅酶带来的经济问题和发酵法时间长问题，同时，通过调节底物D-木糖浓度来提高产量，实现底物更有效的利用。另外通过调节重组菌E.coliBL-PMB-PGP OD600比值提高丁三醇的摩尔收率。
附图说明
图1为不同糖浓度对产物产量的影响。1号10g/L木糖，2号20g/L木糖，3号30g/L木糖，4号40g/L木糖。
图2为不同细胞OD600对产物产量的影响。1号OD600为10，2号为OD60020，3号OD60030，4号OD60040，5号是OD60060。
图3不同细胞通透剂对产物产量的影响。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：
将来源于大肠杆菌MG1655的木糖酸脱水酶基因(yjhG)和来源于大肠杆菌MG1655醛还原酶基因(adhP)分别插入pCWJ质粒，到重组质粒pCWJ-yjhG和pCWJ-adhp。以pCWJ-adhp质粒为模板扩增出带有TRC启动子的adhp片段，将该片段插入质粒pCWJ-yjhG，得到质粒pCWJ-yjhG-TRC-adhp。
将来源于恶臭假单胞杆菌的2-酮酸脱羧酶基因(mdlc)插入质粒pTRC99a的NcoⅠ和SacⅠ位点之间，从而获得重组质粒pTRC-mdlC。将来源于新月柄杆菌木糖脱氢酶基因(xylB)基因插入质粒pTRC99a的NcoⅠ和BamHⅠ位点之间，从而获得重组质粒pTRC-xylB。然后通过PCR得到带有TRC启动子的xylB片段，将该片段插入质粒pTRC-mdlC的SacⅠ和BamHⅠ位点之间，得到新的重组质粒pTRC-mdlC-TRC-xylB。
实施例2：不同糖浓度对产量影响
从平板上挑取重组菌株E.coliBL-PMB-PGP的单菌落接种到含有100mg/L氨苄青霉素抗性和68mg/L氯霉素抗性的5ml LB摇管里，培养10-12h后转接到含有100mg/L氨苄青霉素抗性和68mg/L氯霉素抗性的100MLLB培养基里，再次转接放大培养12-18h离心集菌，得到重组菌株E.coliBL-PMB-PGP的细胞。
将细胞收菌用生理盐水洗涤，用M9溶液(NaCl 0.5g/L，NH4Cl 10g/L，Na2HPO4·12H2O17.1g/L，KH2PO4 3g/L，MgSO4 0.12g/L，CaCl2 1.1×10-2g/L，(NH4)6Mo7O24 3.71×10- 3g/L，H3BO3 2.47×10-2g/L，CoCl2·6H2O 7.14×10-3g/L，MnCl2·4H2O 1.60×10-2g/L，ZnSO4·7H2O2.8×10-3g/L，CuSO4·5H2O 2.51×10-3g/L)重悬细胞到OD60030，底物D-木糖浓度分别是1号10g/L，2号20g/L，3号30g/L，4号40g/L。并加入10g/L碳酸钙和100mmolMOPS。在200rpm37℃条件下转化。最终底物20g/L产物最高，如图1。
实施例3：
将细胞收菌用生理盐水洗涤，用M9溶液重悬细胞到OD600分别为10、20、30、40、60，底物D-木糖浓度20g/L，并加入10g/L碳酸钙和100mmolMOPS。在200rpm37℃条件下转化，如图2。
实施例4：
将细胞收菌用生理盐水洗涤，用M9溶液重悬细胞到OD600 30，底物D-木糖浓度20g/L，并加入10g/L碳酸钙和100mmolMOPS分别。加入表面活性剂Triton X-100，Tween-20，Tween-80,SDS在200rpm37℃条件下转化，如图3。
液相检测方法：
每隔一定时间取样，液相检测丁三醇的积累情况，其中检测方法为色谱柱：Bio-RadAminex HPX-87H(300mm*7.8mm)，柱温：55℃，流动相：0.005M H2SO4，流速：0.5mL/min，检测器：紫外，RID。
序列表
<110> 南京工业大学
<120> 一株利用D-木糖生产D-1,2,4-丁三醇的基因工程菌及其构建方法与应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1968
<212> DNA
<213> 大肠杆菌MG1655(Escherichia coli MG1655)
<400> 1
atgtctgttc gcaatatttt tgctgacgag agccacgata tttacaccgt cagaacgcac 60
gccgatggcc cggacggcga actcccatta accgcagaga tgcttatcaa ccgcccgagc 120
ggggatctgt tcggtatgac catgaatgcc ggaatgggtt ggtctccgga cgagctggat 180
cgggacggta ttttactgct cagtacactc ggtggcttac gcggcgcaga cggtaaaccc 240
gtggcgctgg cgttgcacca ggggcattac gaactggaca tccagatgaa agcggcggcc 300
gaggttatta aagccaacca tgccctgccc tatgccgtgt acgtctccga tccttgtgac 360
gggcgtactc agggtacaac ggggatgttt gattcgctac cataccgaaa tgacgcatcg 420
atggtaatgc gccgccttat tcgctctctg cccgacgcga aagcagttat tggtgtggcg 480
agttgcgata aggggcttcc ggccaccatg atggcactcg ccgcgcagca caacatcgca 540
accgtgctgg tccccggcgg cgcgacgctg cccgcaaagg atggagaaga caacggcaag 600
gtgcaaacca ttggcgcacg cttcgccaat ggcgaattat ctctacagga cgcacgccgt 660
gcgggctgta aagcctgtgc ctcttccggc ggcggctgtc aatttttggg cactgccggg 720
acatctcagg tggtggccga aggattggga ctggcaatcc cacattcagc cctggcccct 780
tccggtgagc ctgtgtggcg ggagatcgcc agagcttccg cgcgagctgc gctgaacctg 840
agtcaaaaag gcatcaccac ccgggaaatt ctcaccgata aagcgataga gaatgcgatg 900
acggtccatg ccgcgttcgg tggttcaaca aacctgctgt tacacatccc ggcaattgct 960
caccaggcag gttgccatat cccgaccgtt gatgactgga tccgcatcaa caagcgcgtg 1020
ccccgactgg tgagcgtact gcctaatggc ccggtttatc atccaacggt caatgccttt 1080
atggcaggtg gtgtgccgga agtcatgttg catctgcgca gcctcggatt gttgcatgaa 1140
gacgttatga cggttaccgg cagcacgctg aaagaaaacc tcgactggtg ggagcactcc 1200
gaacggcgtc agcggttcaa gcaactcctg ctcgatcagg aacaaatcaa cgctgacgaa 1260
gtgatcatgt ctccgcagca agcaaaagcg cgcggattaa cctcaactat caccttcccg 1320
gtgggcaata ttgcgccaga aggttcggtg atcaaatcca ccgccattga cccctcgatg 1380
attgatgagc aaggtatcta ttaccataaa ggtgtggcga aggtttatct gtccgagaaa 1440
agtgcgattt acgatatcaa acatgacaag atcaaggcgg gcgatattct ggtcattatt 1500
ggcgttggac cttcaggtac agggatggaa gaaacctacc aggttaccag tgccctgaag 1560
catctgtcat acggtaagca tgtttcgtta atcaccgatg cacgtttctc gggcgtttct 1620
actggcgcgt gcatcggcca tgtggggcca gaagcgctgg ccggaggccc catcggtaaa 1680
ttacgcaccg gggatttaat tgaaattaaa attgattgtc gcgagcttca cggcgaagtc 1740
aatttcctcg gaacccgtag cgatgaacaa ttaccttcac aggaggaggc aactgcaata 1800
ttaaatgcca gacccagcca tcaggattta cttcccgatc ctgaattgcc agatgatacc 1860
cggctatggg caatgcttca ggccgtgagt ggtgggacat ggaccggttg tatttatgat 1920
gtaaacaaaa ttggcgcggc tttgcgcgat tttatgaata aaaactga 1968
<210> 2
<211> 1011
<212> DNA
<213> 大肠杆菌MG1655(Escherichia coli MG1655)
<400> 2
atgaaggctg cagttgttac gaaggatcat catgttgacg ttacgtataa aacactgcgc 60
tcactgaaac atggcgaagc cctgctgaaa atggagtgtt gtggtgtatg tcataccgat 120
cttcatgtta agaatggcga ttttggtgac aaaaccggcg taattctggg ccatgaaggc 180
atcggtgtgg tggcagaagt gggtccaggt gtcacctcat taaaaccagg cgatcgtgcc 240
agcgtggcgt ggttctacga aggatgcggt cattgcgaat actgtaacag tggtaacgaa 300
acgctctgcc gttcagttaa aaatgccgga tacagcgttg atggcgggat ggcggaagag 360
tgcatcgtgg tcgccgatta cgcggtaaaa gtgccagatg gtctggactc ggcggcggcc 420
agcagcatta cctgtgcggg agtcaccacc tacaaagccg ttaagctgtc aaaaattcgt 480
ccagggcagt ggattgctat ctacggtctt ggcggtctgg gtaacctcgc cctgcaatac 540
gcgaagaatg tctttaacgc caaagtgatc gccattgatg tcaatgatga gcagttaaaa 600
ctggcaaccg aaatgggcgc agatttagcg attaactcac acaccgaaga cgccgccaaa 660
attgtgcagg agaaaactgg tggcgctcac gctgcggtgg taacagcggt agctaaagct 720
gcgtttaact cggcagttga tgctgtccgt gcaggcggtc gtgttgtggc tgtcggtcta 780
ccgccggagt ctatgagcct ggatatccca cgtcttgtgc tggatggtat tgaagtggtc 840
ggttcgctgg tcggcacgcg ccaggattta actgaagcct tccagtttgc cgccgaaggt 900
aaagtggtgc cgaaagtcgc cctgcgtccg ttagcggaca tcaacaccat ctttactgag 960
atggaagaag gcaaaatccg tggccgcatg gtgattgatt tccgtcacta a 1011
<210> 3
<211> 1587
<212> DNA
<213> 恶臭假单胞杆菌(Pseudomonas putida)
<400> 3
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgcttggaa ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctctgctg accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
caccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtgct 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 4
<211> 747
<212> DNA
<213> 新月柄杆菌(Caulobacter crescentus)
<400> 4
atgtcttctg ctatctaccc gtctctgaaa ggtaaacgtg ttgttatcac cggtggtggt 60
tctggtatcg gtgctggtct gaccgctggt ttcgctcgtc agggtgctga agttatcttc 120
ctggacatcg ctgacgaaga ctctcgtgct ctggaagctg aactggctgg ttctccgatc 180
ccgccggttt acaaacgttg cgacctgatg aacctggaag ctatcaaagc tgttttcgct 240
gaaatcggtg acgttgacgt tctggttaac aacgctggta acgacgaccg tcacaaactg 300
gctgacgtta ccggtgctta ctgggacgaa cgtatcaacg ttaacctgcg tcacatgctg 360
ttctgcaccc aggctgttgc tccgggtatg aaaaaacgtg gtggtggtgc tgttatcaac 420
ttcggttcta tctcttggca cctgggtctg gaagacctgg ttctgtacga aaccgctaaa 480
gctggtatcg aaggtatgac ccgtgctctg gctcgtgaac tgggtccgga cgacatccgt 540
gttacctgcg ttgttccggg taacgttaaa accaaacgtc aggaaaaatg gtacaccccg 600
gaaggtgaag ctcagatcgt tgctgctcag tgcctgaaag gtcgtatcgt tccggaaaac 660
gttgctgctc tggttctgtt cctggcttct gacgacgctt ctctgtgcac cggtcacgaa 720
tactggatcg acgctggttg gcgttaa 747
<210> 5
<211> 3580
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
catgcagcgc tcttccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg 60
gcgagcggtg tcagctcact caaaagcggt aatacggtta tccacagaat caggggataa 120
agccggaaag aacatgtgag caaaaagcaa agcaccggaa gaagccaacg ccgcaggcgt 180
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agccagaggt 240
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360
gcgtggcgct ttctcatagc tcacgctgtt ggtatctcag ttcggtgtag gtcgttcgct 420
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccattg 540
gtaactgatt tagaggactt tgtcttgaag ttatgcacct gttaaggcta aactgaaaga 600
acagattttg gtgagtgcgg tcctccaacc cacttacctt ggttcaaaga gttggtagct 660
cagcgaacct tgagaaaacc accgttggta gcggtggttt ttctttattt atgagatgat 720
gaatcaatcg gtctatcaag tcaacgaaca gctattccgt tactctagat ttcagtgcaa 780
tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca 840
tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca 900
tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac 960
tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 1020
cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg 1080
ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg 1140
ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat 1200
gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg 1260
gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca 1320
gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc 1380
cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag 1440
ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc 1500
tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa 1560
ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg 1620
caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca 1680
ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt 1740
tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg 1800
acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac 1860
tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc 1920
gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa 1980
acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca 2040
ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg 2100
cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa 2160
atactagttt gacaattaat catccggctc gtataatgtg tggaattgtg agcggataac 2220
aatttcacac aggaaacaga ccatggaatt cgagctcggt acccggggat cctctagagt 2280
cgacctgcag gcatgcaagc ttggctgttt tggcggatga gagaagattt tcagcctgat 2340
acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag 2400
cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg 2460
tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg 2520
ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga 2580
gtaggacaaa tagatctctg aaacctcagg catttgagaa gcacacggtc acactgcttc 2640
cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc 2700
ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg ccattcatcc gcttattatc 2760
acttattcag gcgtagcacc aggcgtttaa gggcaccaat aactgcctta aaaaaattac 2820
gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg 2880
aagccatcac agacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct 2940
tgcgtataat atttgcccat agtgaaaacg ggggcgaaga agttgtccat attggccacg 3000
tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca 3060
ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat 3120
atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca 3180
gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg 3240
tctttcattg ccatacggaa ctccggatga gcattcatca ggcgggcaag aatgtgaata 3300
aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc 3360
agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct 3420
ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta 3480
gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt 3540
tcattatggt gaaagttgga acctcttacg tgccgatcag 3580
Claims (9)
1.一株利用D-木糖生产D-1,2,4-丁三醇的基因工程菌，其特征在于，在宿主菌中导入了木糖脱氢酶、2-酮酸脱羧酶、木糖酸脱水酶、醛还原酶。
2.根据权利要求1所述的利用D-木糖生产D-1,2,4-丁三醇的基因工程菌，其特征在于，所述的木糖脱氢酶的核苷酸序列如SEQ ID NO.1所示，所述的2-酮酸脱羧酶的核苷酸序列如SEQ ID NO.2所示、所述的木糖酸脱水酶的核苷酸序列如SEQ ID NO.3所示，所述的醛还原酶的核苷酸序列如SEQ ID NO.4所示。
3.权利要求1～2所述利用D-木糖生产D-1,2,4-丁三醇的基因工程菌的构建方法，其特征在于，包括如下步骤：
(1)将木糖脱氢酶、2-酮酸脱羧酶、木糖酸脱水酶、醛还原酶克隆到表达质粒中，得到重组质粒；
(2)将重组质粒转化宿主菌，既得到利用D-木糖生产D-1,2,4-丁三醇的基因工程菌E.coliBL-PMB-PGP。
4.根据权利要求3所述的利用D-木糖生产D-1,2,4-丁三醇的基因工程菌的构建方法，其特征在于，将木糖脱氢酶、2-酮酸脱羧酶同时构建到pTRC-99a质粒上，得到重组质粒A；将木糖酸脱水酶、醛还原酶克隆到pCWJ-1质粒上，得到重组质粒B，将重组质粒A和重组质粒B同时转化宿主菌，既得到利用D-木糖生产D-1,2,4-丁三醇的基因工程菌E.coliBL-PMB-PGP。
5.根据权利要求3或4所述的利用D-木糖生产D-1,2,4-丁三醇的基因工程菌的构建方法，其特征在于，所述的宿主菌为大肠杆菌BL21(DE3)。
6.权利要求1或2所述的利用D-木糖生产D-1,2,4-丁三醇的基因工程菌在生产D-1,2,4-丁三醇中的应用。
7.根据权利要求6所述的应用，其特征在于，包括如下步骤：
(a1)将培养基因工程菌E.coliBL-PMB-PGP，收集E.coliBL-PMB-PGP菌体细胞；
(a2)将E.coliBL-PMB-PGP菌体细胞转接到催化体系中，反应，得到D-1,2,4-丁三醇。
8.根据权利要求7所述的应用，其特征在于，步骤(a2)中，所述的催化体系包括如下组分：
NaCl 0.5g/L，NH4Cl 10g/L，Na2HPO4·12H2O 17.1g/L，KH2PO4 3g/L，MgSO4 0.12g/L，CaCl2 1.1×10-2g/L，(NH4)6Mo7O24 3.71×10-3g/L，H3BO3 2.47×10-2g/L，CoCl2·6H2O 7.14×10-3g/L，MnCl2·4H2O 1.60×10-2g/L，ZnSO4·7H2O 2.8×10-3g/L，CuSO4·5H2O 2.51×10-3g/L，D-木糖10g/L-40g/L，碳酸钙3g/L，MOPS 100mmol/L，Triton X-1000.5g/L，Tween-20 0.5g/L，Tween-80 0.5g/L，SDS 0.5g/L。
9.根据权利要求7所述的应用，其特征在于，步骤(a2)中，催化反应条件为：温度37℃，搅拌转速200rpm。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710963451.9A CN107815436A (zh) | 2017-10-17 | 2017-10-17 | 一株利用d‑木糖生产d‑1,2,4‑丁三醇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 |
|---|---|---|---|
| CN201710963451.9A CN107815436A (zh) | 2017-10-17 | 2017-10-17 | 一株利用d‑木糖生产d‑1,2,4‑丁三醇的基因工程菌及其构建方法与应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107815436A true CN107815436A (zh) | 2018-03-20 |
Family
ID=61608318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710963451.9A Pending CN107815436A (zh) | 2017-10-17 | 2017-10-17 | 一株利用d‑木糖生产d‑1,2,4‑丁三醇的基因工程菌及其构建方法与应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107815436A (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450798A (zh) * | 2014-11-24 | 2015-03-25 | 中国科学院青岛生物能源与过程研究所 | 一种体外酶反应生成1,2,4-丁三醇的方法及应用 |
| CN106148429A (zh) * | 2016-08-25 | 2016-11-23 | 南京工业大学 | 一种生物转化纤维素水解液生产d‑1,2,4‑丁三醇的方法 |
-
2017
- 2017-10-17 CN CN201710963451.9A patent/CN107815436A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450798A (zh) * | 2014-11-24 | 2015-03-25 | 中国科学院青岛生物能源与过程研究所 | 一种体外酶反应生成1,2,4-丁三醇的方法及应用 |
| CN106148429A (zh) * | 2016-08-25 | 2016-11-23 | 南京工业大学 | 一种生物转化纤维素水解液生产d‑1,2,4‑丁三醇的方法 |
Non-Patent Citations (1)
| Title |
|---|
| WEI NIU等: "Microbial Synthesis of the Energetic Material Precursor 1,2,4-Butanetriol", 《J. AM. CHEM. SOC.》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101883861B (zh) | 利用hima表达减少的重组发酵单胞菌属菌株从包含木糖的培养基生产乙醇的方法 | |
| Chow et al. | Using recombinant cyanobacterium (Synechococcus elongatus) with increased carbohydrate productivity as feedstock for bioethanol production via separate hydrolysis and fermentation process | |
| CN112280726B (zh) | 一种高产四氢嘧啶工程菌株的构建方法与应用 | |
| JP2011515107A5 (zh) | ||
| CN101880696B (zh) | 发酵生产l-乳酸的方法及该方法所用的菌株 | |
| CN103881954B (zh) | 一株产γ-聚谷氨酸基因工程菌及其高产γ-聚谷氨酸方法 | |
| CN107574176A (zh) | 一种改进的白藜芦醇生物生产方法 | |
| CN112063572B (zh) | 一种高产o-乙酰-l-高丝氨酸的重组大肠杆菌及其应用 | |
| CN110358720A (zh) | 一种生产异丁醇的运动发酵单胞菌重组菌株、构建方法及其应用 | |
| CN105754925B (zh) | 一种提高库德毕赤酵母高温耐性的方法 | |
| US20240102055A1 (en) | Method for producing a fermentation product | |
| CN112080452B (zh) | 一种高产苯乳酸地衣芽孢杆菌基因工程菌、生产苯乳酸的方法和应用 | |
| CN106801064B (zh) | 一种脱氢异构酶基因敲除的裂殖壶菌工程菌的构建方法及其应用 | |
| CN108531434A (zh) | 一种提高拉乌尔菌2,5-呋喃二甲酸产量的方法 | |
| CN107674889B (zh) | 一种酶反应合成1,2,4-丁三醇的方法 | |
| CN107815436A (zh) | 一株利用d‑木糖生产d‑1,2,4‑丁三醇的基因工程菌及其构建方法与应用 | |
| CN102533870A (zh) | 一种生物法合成异戊二烯的方法、酶系统和重组细胞及其应用 | |
| CN106967662A (zh) | 一种固定二氧化碳合成丁二酸的重组菌及其构建方法和应用 | |
| JP5713333B2 (ja) | ジヒドロキシアセトンの製造方法 | |
| CN114806996B (zh) | 一株高产戊二胺基因工程菌及构建方法与应用 | |
| CN107699534A (zh) | 一种利用d‑阿拉伯糖生产d‑1,2,4‑丁三醇的基因工程菌及其构建方法与应用 | |
| CN116875521A (zh) | 一种敲除大肠杆菌maa基因和lacA基因提高β-熊果苷产量的方法 | |
| CN114540404B (zh) | 一种乙醇发酵过程中二氧化碳原位固定的方法 | |
| Choonut et al. | The production of polyhydroxybutyrate from liquid stillage and its application | |
| CN117844837A (zh) | 产琥珀酸的大肠杆菌基因工程菌的构建方法与应用 |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180320 |