CN109251938B - 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 - Google Patents
一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 Download PDFInfo
- Publication number
- CN109251938B CN109251938B CN201710571717.5A CN201710571717A CN109251938B CN 109251938 B CN109251938 B CN 109251938B CN 201710571717 A CN201710571717 A CN 201710571717A CN 109251938 B CN109251938 B CN 109251938B
- Authority
- CN
- China
- Prior art keywords
- xylose
- acidilactici
- strain
- xylab
- lactic acid
- 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.)
- Expired - Fee Related
Links
- 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 162
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 title claims abstract description 97
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims abstract description 81
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 title claims abstract description 81
- 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 title claims abstract description 28
- 239000008103 glucose Substances 0.000 title claims abstract description 28
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 title claims abstract description 21
- 238000010276 construction Methods 0.000 title claims abstract description 19
- 241000191998 Pediococcus acidilactici Species 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 108010004621 phosphoketolase Proteins 0.000 claims abstract description 11
- 108010092060 Acetate kinase Proteins 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 108010043652 Transketolase Proteins 0.000 claims abstract description 3
- 108700040099 Xylose isomerases Proteins 0.000 claims abstract description 3
- 108091022915 xylulokinase Proteins 0.000 claims abstract description 3
- 108020004530 Transaldolase Proteins 0.000 claims abstract 2
- 102100029089 Xylulose kinase Human genes 0.000 claims abstract 2
- 238000000855 fermentation Methods 0.000 claims description 36
- 230000004151 fermentation Effects 0.000 claims description 29
- 101150005016 ackA2 gene Proteins 0.000 claims description 27
- 239000001963 growth medium Substances 0.000 claims description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 230000037361 pathway Effects 0.000 claims description 21
- 238000004519 manufacturing process Methods 0.000 claims description 17
- 230000010354 integration Effects 0.000 claims description 16
- 230000010261 cell growth Effects 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 239000013613 expression plasmid Substances 0.000 claims description 9
- 230000004127 xylose metabolism Effects 0.000 claims description 7
- 239000006227 byproduct Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 230000002779 inactivation Effects 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 3
- 238000002744 homologous recombination Methods 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- 230000006801 homologous recombination Effects 0.000 claims description 2
- 230000004108 pentose phosphate pathway Effects 0.000 claims description 2
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims 1
- 101100157012 Thermoanaerobacterium saccharolyticum (strain DSM 8691 / JW/SL-YS485) xynB gene Proteins 0.000 claims 1
- 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 1
- 238000003780 insertion Methods 0.000 claims 1
- 230000037431 insertion Effects 0.000 claims 1
- 101150110790 xylB gene Proteins 0.000 claims 1
- 230000003044 adaptive effect Effects 0.000 abstract description 11
- 230000003287 optical effect Effects 0.000 abstract description 6
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 102000014701 Transketolase Human genes 0.000 abstract description 2
- 102100028601 Transaldolase Human genes 0.000 abstract 1
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 38
- 108020004414 DNA Proteins 0.000 description 21
- 229960003276 erythromycin Drugs 0.000 description 19
- 239000013612 plasmid Substances 0.000 description 19
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 18
- 241000894006 Bacteria Species 0.000 description 15
- 239000007788 liquid Substances 0.000 description 13
- 238000012258 culturing Methods 0.000 description 11
- 239000002994 raw material Substances 0.000 description 11
- 238000009630 liquid culture Methods 0.000 description 10
- 239000004310 lactic acid Substances 0.000 description 9
- 235000014655 lactic acid Nutrition 0.000 description 9
- 239000002609 medium Substances 0.000 description 7
- 239000012634 fragment Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 5
- 235000005822 corn Nutrition 0.000 description 5
- 238000003209 gene knockout Methods 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 101150109073 ldhD gene Proteins 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000010902 straw Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108010001539 D-lactate dehydrogenase Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241001643582 Pediococcus acidilactici DSM 20284 Species 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229940116871 l-lactate Drugs 0.000 description 3
- 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 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 3
- 238000012269 metabolic engineering Methods 0.000 description 3
- 230000037353 metabolic pathway Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000010979 pH adjustment Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 229930182843 D-Lactic acid Natural products 0.000 description 2
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 2
- AUTALUGDOGWPQH-UBLOVXTBSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;(2r,3s,4r)-2,3,4,5-tetrahydroxypentanal Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O AUTALUGDOGWPQH-UBLOVXTBSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 101100433987 Latilactobacillus sakei subsp. sakei (strain 23K) ackA1 gene Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 241000192001 Pediococcus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 244000057717 Streptococcus lactis Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 101150006213 ackA gene Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229940022769 d- lactic acid Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000003317 industrial substance Substances 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000037323 metabolic rate Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 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
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- 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
- 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)
- C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
-
- 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
- C12N9/90—Isomerases (5.)
- C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
-
- 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)
- C12Y202/01001—Transketolase (2.2.1.1)
-
- 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)
- C12Y202/01002—Transaldolase (2.2.1.2)
-
- 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)
- C12Y207/01017—Xylulokinase (2.7.1.17)
-
- 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)
- C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
- C12Y207/02001—Acetate kinase (2.7.2.1)
-
- 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)
- C12Y503/01005—Xylose isomerase (5.3.1.5)
-
- 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
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种共发酵葡萄糖和木糖生产L‑乳酸的乳酸片球菌构建方法,属于基因工程技术领域。构建步骤包括,利用热敏型敲除***在L‑乳酸生产菌株乳酸片球菌Pediococcusacidilactici TY112(保藏号为CGMCC NO.8664)基因组上整合异源木糖异构酶,木酮糖激酶,转酮醇酶以及转醛醇酶编码基因;敲除磷酸转酮酶和乙酸激酶编码基因;适应性进化提高共发酵葡萄糖和木糖的能力。本发明成功得到了一株高效共发酵葡萄糖和木糖生产光学纯L‑乳酸的工程菌株,并命名为P.acidilactici ZY271,其保藏号为CGMCC NO.13611。
Description
技术领域
本发明属于基因工程领域,特别涉及一种基于同源重组原理,利用热敏型敲除***的共发酵葡萄糖和木糖生产L-乳酸的乳酸片球菌构建方法。
背景技术
乳酸是一种重要的工业化学品,它广泛运用于食品、医药、皮革以及纺织业。近年来,以光学纯L-乳酸为前体生产生物可降解性塑料聚乳酸,使得光学纯L-乳酸的需求大大提升。目前,乳酸生产主要包括两种路径:化学合成和微生物发酵。化学合成主要缺点在于其生产的乳酸是D/L混合型乳酸,而微生物发酵优点在于可生产光学纯乳酸、低的生产温度以及低能耗,使得微生物发酵成为主要的乳酸生产方式。世界上90%的商用L-乳酸都是由玉米淀粉类原料进行发酵得到的,而玉米淀粉原料的成本约占总发酵成本的70%。因此,亟需找到一种廉价的原料替代淀粉类原料进行L-乳酸生产。木质纤维素原料是一种价格低廉、来源广泛的可再生生物质能源,利用廉价、含量丰富的木质纤维素原料将会大大减少L-乳酸生产成本。
木质纤维素主要包括纤维素(30-60%干重)、半纤维素(20-40%)和木质素(15-25%)。其中纤维素来源的葡萄糖可以被微生物利用。半纤维来源的木糖约占木质纤维素来源总糖的30%,但大部分生物炼制菌株都不能利用木糖,木糖的不可利用是木质纤维素原料进行高指标生物基化学品生产的一个重要难点。本实验室先前工作中,从玉米秸秆乙醇发酵醪中筛选得到一株乳酸片球菌P. acidilatici DQ2,该菌可以很好的适应木质纤维素体系,并可以利用玉米秸秆原料获得103g/L的D/L混合型乳酸。之后通过基因工程改造敲除了P. acidilatici DQ2基因组上D-乳酸脱氢酶基因ldhD,得到的工程菌株P. acidilaticiTY112可以利用30%(w/w)固含量的玉米秸秆原料产生104.5 g/L的光学纯L-乳酸,但其不能利用木糖。如果将木糖代谢路径构建至P. acidilatici TY112中,共发酵玉米秸秆中的葡萄糖和木糖,L-乳酸的产量必将大大提高。
据文献报道,乳酸菌中木糖代谢路径构建以生产L-乳酸只在Lactococcus lactis中实现过。Shinkawa等(Shinkawa S, Okano K, Yoshida S, Tanaka T, Ogino C, FukudaH, Kondo A. Improved homo L-lactic acid fermentation from xylose byabolishment of the phosphoketolase pathway and enhancement of the pentosephosphate pathway in genetically modified xylose-assimilating Lactococcuslactis. Appl Microbiol Biotechnol, 2011, 91:1537-1544.)敲除了PK路径的磷酸转酮酶编码基因pkt,并将异源的转酮醇酶编码基因(tkt)整合至pkt基因位点处,并携带了xylRAB的表达质粒,得到的工程菌株可以通过PP路径利用木糖进行同型L-乳酸生产。但是该文献中的xylRAB是在质粒中进行表达的,并没有整合到基因组上,得到的工程菌株并不是一株稳定的工程菌株,在发酵过程中需要添加抗生素维持重组菌的正常生长,抗生素的添加将会增加发酵成本,对于大规模工业运用来说是一个重要缺陷。另外该文献及作者后期的研究中并没有使用木质纤维素原料进行共发酵生产L-乳酸,没有实际证明该工程菌株具备共发酵葡萄糖和木糖生产L-乳酸的能力。
目前没有乳酸片球菌共发酵葡萄糖和木糖生产L-乳酸的报道,因此在乳酸片球菌中构建木糖代谢路径生产L-乳酸,对于共发酵木质纤维素原料中的葡萄糖和木糖进行高浓度L-乳酸生产具有重要现实意义。
发明内容
本发明目的在于构建一株能够高效共发酵葡萄糖和木糖进行L-乳酸生产的P.acidilactici工程菌株。
本发明实现P. acidilactici共发酵葡萄糖和木糖生产L-乳酸所采用的技术方案是:首先,敲除PK路径中的磷酸转酮酶基因pkt以阻断PK路径,从而减少副产物乙酸生成。然后,将异源的磷酸转酮酶(tkt)和转酮醇酶(tal)表达框整合至已敲除的pkt基因位点处,从而引入PP路径。接着,将xylAB表达框整合至乙酸激酶ackA2处,在引入木糖代谢路径的同时,***失活乙酸激酶基因ackA2。最后,将得到的工程菌株在木糖为唯一碳源的合成培养基中进行适应性进化,直至细胞生长、发酵液中的木糖残留以及L-乳酸生产稳定为止。
上述基因敲除以及基因整合具体方法是:通过电穿孔技术,将构建得到的敲除质粒或整合质粒转化至P. acidilactici中,涂布于含红霉素的MRS平板,28℃静置3天,然后将平板上长出的单菌落接种至含红霉素的液体MRS培养基中,42℃培养16h后,取1微升菌液稀释106倍后,取200微升涂布含红霉素的MRS平板上,42℃静置培养24h。将长出的单菌落接种至不含红霉素的液体MRS培养基中,28℃培养36h后,取1微升菌液稀释106倍后,取200微升涂布于无抗性的MRS平板上,42℃静置培养24h。将长出的单菌落尽可能多的分别点板至不含红霉素的MRS平板和含红霉素的MRS平板上,42℃静置培养24h,将在无红霉素的平板上生长、而含红霉素的平板上不生长的菌落进行进一步的基因组PCR验证,以确认是否完成了基因敲除或基因整合。
上述适应性进化具体方法是:将整合了木糖代谢路径的工程菌株接种至MRS液体培养基中,42℃培养12h后,以10%(v/v)的接种量接种至含40g/L木糖为唯一碳源的MRS液体培养基中,培养24h后,以10%接种量转接至新鲜的木糖MRS液体培养基中继续培养24h(其中进行木糖,L-乳酸含量测定的摇瓶中需添加CaCO3进行pH调节,而进行细胞生长监测的摇瓶中不添加CaCO3)。长期驯化不断进行,直至发酵液中残留的木糖、L-乳酸生成以及细胞生长稳定为止,对得到的木糖发酵性能稳定的驯化菌株进行甘油保种。
本发明结合代谢工程以及适应性进化,首次在P. acidilactici中实现共发酵葡萄糖和木糖生产L-乳酸。
本发明结合代谢工程和适应性进化,得到了一株高效共发酵葡萄糖和木糖进行L-乳酸生产的工程菌株,并命名为P. acidilactici ZY271,该菌株已于2017年01月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),保藏登记号为CGMCC NO.13611,其分类命名为乳酸片球菌(Pediococcus acidilactici)。
附图说明
图1:P. acidilactici中木糖代谢路径构建示意图。
图2:P. acidilactici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB的基因组PCR验证图
图中:M,DL5,000 DNA Marker;1,利用引物ldhD-F和ldhD-R扩增D-乳酸脱氢酶基因ldhD;2,利用引物ldh-F和ldh-R扩增已敲除的L-乳酸脱氢酶基因ldh;3,利用引物pkt-F和pkt-R扩增已敲除的磷酸转酮酶基因pkt;4,利用引物tkt_tal-F和tkt_tal-R扩增整合至基因组上的异源基因tkt_tal;5,利用引物Em-F和Em-R扩增红霉素抗性基因Em;6,利用引物xylAB_2911-F和xylAB_2911-R扩增异源木糖异构酶和木酮糖激酶基因。
图3:适应性进化过程中的木糖残留、L-乳酸生成以及细胞生长监测。
图4:驯化前后菌株发酵木糖生产L-乳酸性能比较。
图5:驯化前后菌株共发酵葡萄糖和木糖生产L-乳酸性能比较。
图6:木糖利用工程菌株P. acidilactici ZY271与母本菌株P. acidilacticiTY112共发酵葡萄糖和木糖生产L-乳酸性能比较。
具体实施方案
一、本发明所使用的菌株与质粒
L-乳酸生产菌株P. acidilactici TY112是将野生型D/L-乳酸生产菌株P.acidilactici DQ2的D-乳酸脱氢酶基因ldhD敲除得到,已于2013年12月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),保藏登记号为CGMCC NO.8664。质粒构建所用的宿主菌Escherichia coli XLI-blue保存于本实验室。基因xylAB_2911,tkt和tal来源于乳酸片球菌P. acidilactici DSM20284,购自DSM (Scheiwg,Germany)。所用表达质粒pMG36e来源见Van de Guchte M, Van der Vossen JM, Kok J, Venema G. Construction of alactococcal expression vector: expression of hen egg white lysozyme inLactococcus lactis subsp. lactis. Applied Environment and Microbiology, 1989,55(1):224-228。所用热敏型敲除质粒pSET4E来源见Yi X, Zhang P, Sun J, Tu Y, GaoQ, Zhang J, Bao J. Engineering wild-type robust Pediococcus acidilacticistrain for high titer l- and d-lactic acid production from corn stoverfeedstock. Journal of Biotechnology, 2016, 217:112-121。
二、试剂与培养基
DNA聚合酶购自Takara生物工程公司;限制性内切酶和T4 DNA ligase购自Fermentas公司;基因组抽提、质粒抽提、PCR产物回收、DNA凝胶回收试剂盒均购自上海捷瑞生物工程有限公司;乳酸光学纯度检测试剂盒购自Megazyme公司;红霉素购自Biosharp生物科技有限公司;酵母粉和蛋白胨购买于Oxoid, Hampshire, UK;基因测序由上海美吉生物医药科技有限公司完成;引物合成由上海捷瑞生物工程有限公司完成。
MRS培养基:1L去离子水中含有20.0g葡萄糖,10.0g蛋白胨,4.0g酵母粉,8.0g牛肉粉,2.0g柠檬酸氢二铵,3.0g无水乙酸钠,2.0g磷酸氢二钾,0.2g七水硫酸镁,0.05g一水硫酸锰和1mL的Tween 80;
简化MRS液体培养基:1L去离子水中含有20.0g葡萄糖,10.0g蛋白胨,10.0g酵母粉,5.0g无水乙酸钠,2.0g柠檬酸氢二铵,2.0g磷酸氢二钾,0.58g七水硫酸镁和0.25g一水硫酸锰;
适应性进化所用培养基:1L去离子水中含有40.0g木糖,10.0g蛋白胨,10.0g酵母粉,5.0g乙酸钠,2.0g柠檬酸氢二铵,2.0g磷酸氢二钾,0.58g七水硫酸镁,0.25一水硫酸锰,12g CaCO3(进行细胞生长的摇瓶不加CaCO3);
LB培养基:
液体:1L去离子水中含有5.0g酵母粉,10.0g蛋白胨,10.0g氯化钠;固体:在液体培养基中额外添加15.0g/L的琼脂;
以上培养基均需115℃高压蒸汽灭菌20min。
三、所用主要仪器
Mastercycler型PCR仪(Eppendorf公司);Gene Pulser Xcell型电穿孔仪(Bio-Rad公司);EPS-100型DNA电泳***(上海天能公司);Tanon 1600凝胶成像***(上海天能公司);DU-800型核酸蛋白质分析仪(Beckman公司); LC-20AD型高效液相色谱(岛津公司)。
实施案例1:木糖代谢在P. acidilactici TY112中的实现。
(1)表达质粒pMG36e-PldhD-xylAB_2911的构建
首先以P. acidilactici DSM20284基因组为模板,以xylAB_2911-F(SEQ ID NO:1)和xylAB_2911-R(SEQ ID NO:2)为引物扩增得到xylAB_2911,以P. acidilactici TY112为模板,以PldhD-F(SEQ ID NO:3)和PldhD-R(SEQ ID NO:4)为引物扩增得到ldhD起始密码子上游约300bp的启动子序列PldhD,然后通过融合PCR技术得到表达框PldhD_xylAB_2911,然后通过EcoR I和Xba I双酶切,用表达框PldhD_xylAB_2911替换pMG36e自身的P32启动子,得到表达质粒pMG36e-PldhD_xylAB_2911。
(2)重组菌P. acidilactici TY112 (pMG36e-PldhD_xylAB_2911)的木糖发酵能力验证
将(1)中得到的表达质粒pMG36e-PldhD_xylAB_2911通过电穿孔技术转化至P.acidilactici TY112中,得到重组菌P. acidilactici TY112 (pMG36e-PldhD_xylAB_2911)。该重组菌可利用木糖进行生长,48h可以消耗12.61g/L木糖,产生5.62g/L的L-乳酸,但同时产生了6.67g/L的副产物乙酸。这说明重组菌可以通过PK路径代谢木糖生产L-乳酸,并伴随着大量副产物乙酸生成(见表1)。
实施案例2:磷酸转酮酶路径(PK路径)的阻断
(1)敲除质粒pSET4E-Δpkt的构建
以P. acidilactici TY112基因组为模板,以up-pkt-F(SEQ ID NO:5)和up-pkt-R(SEQ ID NO:6)为引物扩增得到pkt基因的上游约1,000bp同源臂片段(up-pkt),以down-pkt-F(SEQ ID NO:7)和down-pkt-R(SEQ ID NO:8)为引物扩增得到pkt基因的下游约1,000bp同源臂片段序列(down-pkt)。将down-pkt片段***至pSET4E的BamH I和Sac I位点之间,然后将up-pkt片段***至Pst I和Sal I位点之间,得到用于pkt基因敲除的质粒pSET4E-Δpkt。
(2)磷酸转酮酶基因pkt的敲除
将敲除质粒pSET4E-Δpkt电转化至P. acidilactici TY112中,得到重组菌P.acidilactici TY112 (pSET4E-Δpkt),将重组菌接种至含红霉素的液体MRS培养液中,42℃培养16h后,取1微升菌液稀释106倍后,取200微升涂布含红霉素的MRS平板上,42℃静置培养24h。将长出的单菌落接种至不含红霉素的液体MRS培养液中,28℃培养36h后,取1微升菌液稀释106倍后,取200微升涂布于无抗性的MRS平板上,42℃静置培养24h。将长出的单菌落尽可能多的分别点板至不含红霉素的MRS平板和含红霉素的MRS平板上,42℃静置培养24h,将在无红霉素的平板上生长、而含红霉素的平板上不生长的菌落进行进一步的基因组PCR验证。以pkt-F(SEQ ID NO:9)和pkt-R(SEQ ID NO:10)为引物扩增pkt,若扩增不出磷酸转酮酶基因pkt,则基因pkt被敲除,得到的工程菌株命名为P. acidilactici TY112-Δpkt。
(3)工程菌P. acidilactici TY112-Δpkt (pMG36e-PldhD-xylAB_2911)的木糖发酵
将表达质粒pMG36e-PldhD_xylAB_2911电转化至工程菌株P. acidilacticiTY112-Δpkt中,得到重组菌P. acidilactici TY112-Δpkt (pMG36e-PldhD_xylAB_2911),将重组菌培养在含35g/L木糖的MRS液体培养基中。该重组菌的木糖利用能力显著下降,48h内只消耗了4.90g/L的木糖,只产生了0.19g/L的L-乳酸,而乙酸的生成也降至0.20g/L(见表1)。这说明pkt的敲除成功阻断了PK路径,使得菌株基本不能生成副产物乙酸,但不生成L-乳酸说明P. acidilactici TY112中只存在PK路径进行L-乳酸生产。
实施案例3:戊糖磷酸路径(PP路径)的构建
(1)整合质粒pSET4E-Δpkt::(tkt_tal)的构建
以P. acidilactici TY112基因组为模板,以PldhD-F(SEQ ID NO:3)和PldhD-R(SEQ ID NO:4)为引物扩增得到启动子PldhD;以P. acidilactici DSM20284为模板,设计引物tkt_tal-F(SEQ ID NO:11)和tkt_tal-R(SEQ ID NO:12)扩增得到tkt_tal。接着将克隆得到的PldhD与tkt_tal通过融合PCR得到表达框PldhD_tkt_tal,并***至案例2得到的敲除质粒pSET4E-Δpkt的Sal I和BamH I之间,得到整合质粒pSET4E-Δpkt::(tkt_tal)。
(2)tkt_tal的基因组整合
将整合质粒pSET4E-Δpkt::(tkt_tal)电转化至案例2得到的工程菌株P.acidilactici TY112-Δpkt中,得到重组菌P. acidilactici TY112-Δpkt (pSET4E-Δpkt::(tkt_tal))。通过与上述类似的基因敲除与整合方法,筛选得到的单菌落为模板,若能扩增出tkt_tal,而扩增不出红霉素抗性基因Em,则基因tkt_tal基因组整合成功,得到的工程菌株命名为P. acidilatici TY112-Δpkt::(tkt_tal)。
(3)重组菌P. acidilatici TY112-Δpkt::(tkt_tal) (pMG36e-PldhD_xylAB_2911)的木糖发酵
将表达质粒pMG36e-PldhD_xylAB_2911通过电穿孔技术转化至P. acidilaticiTY112-Δpkt::(tkt_tal),将得到的重组菌P. acidilatici TY112-Δpkt::(tkt_tal)(pMG36e-PldhD_xylAB_2911)接种于简化MRS液体培养基中,培养12h后,以10%接种量转接至含35g/L木糖的MRS液体培养基中进行木糖发酵。工程菌株在48h内可以消耗9.64g/L木糖,产生了5.86g/L的L-乳酸,但同时产生了1.73g/L的乙酸(见表1)。这一结果说明异源的tkt_tal成功在该菌株中表达,并且tkt_tal的整合,成功将PP路径引入到工程菌株中,使得菌株又恢复了代谢木糖产L-乳酸的能力,但仍有少量乙酸产生。
实施案例4:xylAB表达框的整合以及ackA2基因的***失活
(1)整合质粒pSET4E-ΔackA2::xylAB的构建
首先以P. acidilactici TY112基因组为模板,以up-ackA2-F(SEQ ID NO:13)和up-ackA-R(SEQ ID NO:14)为引物扩增得到ackA2上游约700bp的同源臂序列up-ackA2,以down-ackA2-F(SEQ ID NO:15)和down-ackA-R(SEQ ID NO:16)为引物扩增扩增得到ackA2下游约700bp的同源臂序列down-ackA2。以表达质粒pMG36e-PldhD_xylAB_2911为模板,以PldhD_xylAB_2911-F(SEQ ID NO:17)和PldhD_xylAB_2911-R(SEQ ID NO:18)为引物扩增得到PldhD_xylAB_2911。然后通过酶切连接,将up-ackA2片段***至pSET4E的Hind和BamH I位点之间,将down-ackA2片段***至BamH I和EcoR I位点之间,得到用于ackA2基因缺失的质粒pSET4E-ΔackA2,然后将表达框PldhD_xylAB_2911***至pSET4E-ΔackA2的Xho I和BamH I之间,得到用于将ackA2失活,同时将xylAB_2911整合至ackA2基因内部的质粒pSET4E-ΔackA2::xylAB。
(2)xylAB的基因组整合以及ackA2基因的***失活
将整合质粒pSET4E-ΔackA2::xylAB电转化至P. acidilatici TY112-Δpkt::(tkt_tal)中,得到重组菌P. acidilatici TY112-Δpkt::(tkt_tal) (pSET4E-ΔackA2::xylAB),通过与上述类似的基因敲除与整合方法,筛选得到的单菌落为模板,若能够扩增出xylAB,而扩增不出红霉素抗性基因Em,则xylAB整合成功,得到的整合菌株命名为P.acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB。
(3)工程菌P. acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB的木糖发酵
将工程菌株P. acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB接种于简化MRS液体培养基中,培养12h后,以10%接种量转接至含35g/L木糖的MRS液体培养基中进行木糖发酵。工程菌株P. acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB可以代谢木糖生产L-乳酸,但木糖代谢速率较慢(见表1)。
表1:P. acidilatici TY112相关工程菌株的木糖发酵性能比较
实施案例5:工程菌株P. acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB的适应性进化
案例4中得到的工程菌株P. acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB木糖代谢速率很慢。本发明采取了适应性进化策略提高该工程菌株的木糖代谢速率。首先,将工程菌株P. acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB接种至MRS液体培养基中,培养12h后,以10%的接种量接种至以木糖为唯一碳源的MRS液体培养基中(添加CaCO3调节pH),培养24h后,以10%接种量转接至新鲜含木糖为唯一碳源的MRS液体培养基中继续培养24h(用于检测细胞生长的摇瓶中不添加CaCO3调节pH,其余条件一样),期间不断进行转接,直至细胞生长、发酵液中残留的木糖以及L-乳酸生成稳定为止。连续转接66次(66天)后,驯化菌株的木糖发酵性能有了显著提高,且保持稳定(见图3),对得到的木糖发酵性能稳定的驯化菌株进行保种,并命名为P. acidilactici ZY271。
实施案例6:驯化前后木糖发酵性能比较
(1)木糖为唯一碳源的发酵性能比较
将驯化菌株P. acidilactici ZY271与未驯化菌株P. acidilatici TY112-Δpkt::(tkt_tal)-ΔackA2::xylAB进行木糖为唯一碳源的发酵性能比较。将-80℃冰箱保存的两株菌株分别接种至简化MRS液体培养基中活化12h后,以10%接种量分别接种至含35g/L木糖为唯一碳源的MRS培养基中进行发酵性能比较(添加CaCO3进行pH调节),用于检测细胞生长的摇瓶中不添加CaCO3。
就生长而言,在培养16h后,驯化菌株P. acidilactici ZY271的OD600达到了4.50,远高于未驯化菌株的1.74。就木糖发酵能力而言,驯化菌株的木糖消耗速率,L-乳酸生产速率均高于未驯化菌株(见图4)。长期驯化大大提升了工程菌株利用木糖的能力。
(2)葡萄糖和木糖的共发酵性能比较
接着又比较了驯化菌株和未驯化菌株的葡萄糖木糖共发酵能力。使用的培养基是含有40g/L葡萄糖和40g/L木糖的MRS培养基。两菌株的细胞生长以及葡萄糖消耗没有什么差别,但驯化菌株P. acidilactici ZY271在48h内90.0%的木糖被消耗,而未驯化菌株只消耗了58.0%的木糖(见图5)。这说明,适应性进化也大大提升了工程菌株的共发酵能力。依据Megazyme D-/L-Lactic acid Kit试剂盒方法,测得L-乳酸光学纯度为99.6%。
实施案例7:木糖利用工程菌株与母本菌株的共发酵性能比较
将本发明构建的木糖利用工程菌株P. acidilactici ZY271和不能利用木糖的母本菌株P. acidilactici TY112分别接种至简化MRS液体培养基中活化12h后,以10%接种量分别接种至含40g/L葡萄糖和40g/L木糖的MRS培养基中进行共发酵性能比较(添加CaCO3进行pH调节)。工程菌株P. acidilactici ZY15在48h已基本耗完所有葡萄糖和木糖;而母本菌株只消耗了葡萄糖,木糖并没有消耗(见图6)。以上结果说明,本发明通过代谢工程和适应性进化成功构建了一株具备共发酵葡萄糖和木糖能力的L-乳酸生产菌株。
以上具体描述了本发明技术方案的操作实例,不视为对本发明的应用限制。凡操作条件的等同替换,均在本发明的保护范围之内。
序列表
<110> 华东理工大学
<120> 一种共发酵葡萄糖和木糖生产L-乳酸的乳酸片球菌构建方法
<130> 2017-06-01
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213> xylAB_2911-F
<400> 1
aaaagaaagg ggtaatatta caatgtcatt atttgatcgc aaaaaaatgg 50
<210> 2
<211> 35
<212> DNA
<213> xylAB_2911-R
<400> 2
ctagtctaga ttacaacatt tcacgccggt aattc 35
<210> 3
<211> 31
<212> DNA
<213> PldhD-F
<400> 3
ccggaattct gctctggtgt gcagaccaga c 31
<210> 4
<211> 49
<212> DNA
<213> PldhD-R
<400> 4
ttttttgcga tcaaataatg acattgtaat attacccctt tctttttta 49
<210> 5
<211> 31
<212> DNA
<213> up-pkt-F
<400> 5
aactgcagcc aagtggttcg tgaatttgtt g 31
<210> 6
<211> 37
<212> DNA
<213> up-pkt-R
<400> 6
gcgtcgactt aaagcttaaa gattaaacaa taaaagc 37
<210> 7
<211> 31
<212> DNA
<213> down-pkt-F
<400> 7
cgcggatccc gtaatcttcc tttcgtgagc g 31
<210> 8
<211> 32
<212> DNA
<213> down-pkt-R
<400> 8
cgagctcagc aatgtatttt agcacattga gc 32
<210> 9
<211> 29
<212> DNA
<213> pkt-F
<400> 9
atgacagact attcatctaa agcttactt 29
<210> 10
<211> 25
<212> DNA
<213> pkt-R
<400> 10
ttatttaacg tctttccata cccag 25
<210> 11
<211> 46
<212> DNA
<213> tkt_tal-F
<400> 11
aaagaaaggg gtaatattac agtgtttact aaaaaagata ttgacg 46
<210> 12
<211> 32
<212> DNA
<213> tkt_tal-R
<400> 12
aactgcagct ataaaattga caatcccgat tc 32
<210> 13
<211> 29
<212> DNA
<213> up-ackA2-F
<400> 13
cccaagcttt tcgcgaattt gttaacgct 29
<210> 14
<211> 42
<212> DNA
<213> up-ackA2-R
<400> 14
cgcggatccg cgccgctcga gccaaatggc aggtgattaa tt 42
<210> 15
<211> 32
<212> DNA
<213> down-ackA2-F
<400> 15
cgcggatcct tagcgtagaa gaagtcgttg ac 32
<210> 16
<211> 30
<212> DNA
<213> down-ackA2-R
<400> 16
ccggaattca gacaaaacca aaagagcgtg 30
<210> 17
<211> 31
<212> DNA
<213> PldhD-xylAB_2911-F
<400> 17
ccgctcgagt gctctggtgt gcagaccaga c 31
<210> 18
<211> 34
<212> DNA
<213> PldhD-xylAB_2911-R
<400> 18
cgcggatcct tacaacattt cacgccggta attc 34
Claims (2)
1.一种共发酵葡萄糖和木糖生产L-乳酸的乳酸片球菌Pediococcus acidilactici构建方法,步骤包括:
(1)木糖代谢在P. acidilacticiTY112中的实现:将携带木糖异构酶xylA和木酮糖激酶xylB的表达质粒pMG36e-PldhD_xylAB_2911电转化至P. acidilacticiTY112,其菌种保藏号为CGMCC NO.8664;得到的重组菌P. acidilactici TY112-pMG36e-PldhD_xylAB_2911可以利用木糖进行生长和L-乳酸生产,但同时产生了大量副产物乙酸;
(2)磷酸转酮酶路径(PK路径)的阻断:通过同源重组敲除P. acidilacticiTY112基因组上的磷酸转酮酶基因pkt,以阻断PK路径从而减少副产物乙酸生成,得到的工程菌株为P. acidilacticiTY112-Δpkt;
(3)戊糖磷酸路径(PP路径)的构建:在步骤(2)得到的菌株基础上,通过同源重组将异源的转酮醇酶基因tkt和转醛醇酶基因tal整合至步骤(2)中敲除的pkt基因位点处,使菌株通过PP路径代谢木糖,得到的工程菌株为P. acidilactici TY112-Δpkt::tkt_tal;
(4)xylAB的整合以及乙酸激酶ackA2的***失活:在步骤(3)得到的菌株基础上,将步骤(1)中的xylAB表达框整合至乙酸激酶ackA2位点处,同时将ackA2进行***失活,得到的工程菌株为P. acidilacticiTY112-Δpkt::tkt_tal-ΔackA2::xylAB;
(5)驯化加快木糖代谢:将步骤(4)得到的工程菌株,在木糖为唯一碳源的培养基中进行驯化以提高木糖代谢能力;所述驯化方法是:将步骤(4)得到的工程菌株P. acidilactici TY112-Δpkt::tkt_tal-ΔackA2::xylAB在简化MRS培养基中培养12h后,以体积比为10%的接种量接种至以40g/L木糖为唯一碳源的MRS培养基中,之后,每24h以10%接种量转接至新鲜的木糖MRS培养基中进行发酵,直至细胞生长、发酵液中残留的木糖以及生成的L-乳酸稳定时停止驯化;其中,启动子PldhD是以SEQ ID NO:3和SEQ ID NO:4所示的引物序列从P. acidilacticiTY112基因组上扩增得到,基因xylAB_2911是以SEQ ID NO:1和SEQ ID NO:2所示的引物序列从P. acidilactici DSM20284基因组上扩增得到,基因tkt_tal是以SEQ ID NO:11和SEQ ID NO:12所示的引物序列从P. acidilacticiDSM20284基因组上扩增得到。
2.如权利要求1所述的构建方法,其特征在于,所述驯化经过66天共计66次的连续转接,得到的木糖发酵性能稳定的驯化菌株命名为P. acidilactici ZY271,其保藏编号为CGMCC NO.13611。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710571717.5A CN109251938B (zh) | 2017-07-13 | 2017-07-13 | 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710571717.5A CN109251938B (zh) | 2017-07-13 | 2017-07-13 | 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109251938A CN109251938A (zh) | 2019-01-22 |
CN109251938B true CN109251938B (zh) | 2022-08-23 |
Family
ID=65051717
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710571717.5A Expired - Fee Related CN109251938B (zh) | 2017-07-13 | 2017-07-13 | 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109251938B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108866090B (zh) * | 2017-05-11 | 2022-09-06 | 华东理工大学 | 一种共发酵葡萄糖和木糖生产d-乳酸的乳酸片球菌构建方法 |
CN109251938B (zh) * | 2017-07-13 | 2022-08-23 | 华东理工大学 | 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 |
CN112941117A (zh) * | 2020-12-23 | 2021-06-11 | 华东理工大学 | 一种使用木质纤维素生物质为原料生产的手性l-乳酸合成l-丙交酯的方法 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006109632A1 (ja) * | 2005-04-06 | 2006-10-19 | Kaneka Corporation | 新規α-ケト酸還元酵素、その遺伝子、およびその利用法 |
CN103124783A (zh) * | 2010-06-03 | 2013-05-29 | 马斯科马公司 | 表达用于使用淀粉和纤维素进行联合生物加工的糖分解酶的酵母 |
CN104126011A (zh) * | 2011-11-30 | 2014-10-29 | 帝斯曼知识产权资产有限公司 | 由乙酸和甘油生产乙醇的工程化酵母菌株 |
CN105814191A (zh) * | 2013-08-15 | 2016-07-27 | 拉勒曼德匈牙利流动管理有限责任公司 | 通过甘油回收改进微生物中的产物产率和生产的方法 |
CN105821071A (zh) * | 2015-01-06 | 2016-08-03 | 华东理工大学 | 一种基于同源重组的乳酸片球菌dq2的无标记基因敲除方法 |
CN106811489A (zh) * | 2015-11-28 | 2017-06-09 | 华东理工大学 | 一种基于产乳酸片球菌素的乳酸菌的开放式乳酸生产方法 |
CN108866090A (zh) * | 2017-05-11 | 2018-11-23 | 华东理工大学 | 一种共发酵葡萄糖和木糖生产d-乳酸的乳酸片球菌构建方法 |
CN109251938A (zh) * | 2017-07-13 | 2019-01-22 | 华东理工大学 | 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7083955B2 (en) * | 2003-01-13 | 2006-08-01 | Purac Biochem Bv | Preparation of lactic acid from a pentose-containing substrate |
WO2010095600A1 (ja) * | 2009-02-18 | 2010-08-26 | 関西化学機械製作株式会社 | ペントースからのホモ乳酸発酵 |
-
2017
- 2017-07-13 CN CN201710571717.5A patent/CN109251938B/zh not_active Expired - Fee Related
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006109632A1 (ja) * | 2005-04-06 | 2006-10-19 | Kaneka Corporation | 新規α-ケト酸還元酵素、その遺伝子、およびその利用法 |
CN103124783A (zh) * | 2010-06-03 | 2013-05-29 | 马斯科马公司 | 表达用于使用淀粉和纤维素进行联合生物加工的糖分解酶的酵母 |
CN104126011A (zh) * | 2011-11-30 | 2014-10-29 | 帝斯曼知识产权资产有限公司 | 由乙酸和甘油生产乙醇的工程化酵母菌株 |
CN105814191A (zh) * | 2013-08-15 | 2016-07-27 | 拉勒曼德匈牙利流动管理有限责任公司 | 通过甘油回收改进微生物中的产物产率和生产的方法 |
CN105821071A (zh) * | 2015-01-06 | 2016-08-03 | 华东理工大学 | 一种基于同源重组的乳酸片球菌dq2的无标记基因敲除方法 |
CN106811489A (zh) * | 2015-11-28 | 2017-06-09 | 华东理工大学 | 一种基于产乳酸片球菌素的乳酸菌的开放式乳酸生产方法 |
CN108866090A (zh) * | 2017-05-11 | 2018-11-23 | 华东理工大学 | 一种共发酵葡萄糖和木糖生产d-乳酸的乳酸片球菌构建方法 |
CN109251938A (zh) * | 2017-07-13 | 2019-01-22 | 华东理工大学 | 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 |
Non-Patent Citations (10)
Title |
---|
Engineering Pediococcus acidilactici with xylose assimilation pathway for high titer cellulosic l-lactic acid fermentation;Qiu Z等;《Bioresour Technol》;20170921;第249卷;9-15 * |
Engineering wild-type robust Pediococcus acidilactici strain for high titer L- and D-lactic acid production from corn stover feedstock;Yi X等;《J Biotechnol》;20160110;第217卷;摘要 * |
High titer L-lactic acid production from corn stover with minimum wastewater generation and techno-economic evaluation based on Aspen plus modeling;Liu G等;《Bioresour Technol》;20151009;第198卷;803-810 * |
Improved homo L-lactic acid fermentation from xylose by abolishment of the phosphoketolase pathway and enhancement of the pentose phosphate pathway in genetically modified xylose-assimilating Lactococcus lactis;Shinkawa S等;《Appl Microbiol Biotechnol》;20110603;第91卷(第6期);摘要,第1539页右栏第2段,第1542页左栏第3段 * |
Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1;Tanaka K等;《Appl Microbiol Biotechnol》;20020817;第60卷(第1期);第166页左栏第2段,图1,表5 * |
乳酸片球菌L-乳酸脱氢酶基因的克隆及在重组大肠杆菌JH12中的过量表达;罗璇等;《生物技术通报》;20140605(第4期);121-126 * |
产L-乳酸的大肠杆菌基因工程菌的构建;陈五九等;《广西农业生物科学》;20070813;第26卷(第1期);第20页第3段 * |
微生物利用木糖发酵L―乳酸代谢途径的研究;付晓芬等;《食品工业科技》;20090825(第08期);359-362 * |
木质纤维素依赖型Pediococcus acidilactici DQ2中外源功能基因的表达及基因敲除;涂毅;《中国优秀硕士学位论文全文数据库(电子期刊) 基础科学辑》;20131015;摘要 * |
高产木质纤维素手性乳酸的乳酸片球菌全糖代谢路径构建与抗逆性强化改造;邱忠洋;《中国博士学位论文全文数据库(电子期刊)基础科学辑》;20200115(第01期);A006-211 * |
Also Published As
Publication number | Publication date |
---|---|
CN109251938A (zh) | 2019-01-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Dien et al. | Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars | |
JP5605597B2 (ja) | D(−)−乳酸生成のための耐熱性Bacilluscoagulansの遺伝子操作 | |
KR101576186B1 (ko) | 에탄올 생산 경로가 봉쇄된 클루이베로마이세스 막시아누스 균주 및 이의 용도 | |
CN109251938B (zh) | 一种共发酵葡萄糖和木糖生产l-乳酸的乳酸片球菌构建方法 | |
US9944957B2 (en) | Recombinant Escherichia coli for producing D-lactate and use thereof | |
CN105492613B (zh) | 用于使用代谢工程化丙酸杆菌产生正丙醇和丙酸的方法 | |
CN108866090B (zh) | 一种共发酵葡萄糖和木糖生产d-乳酸的乳酸片球菌构建方法 | |
JP6404575B2 (ja) | 遺伝子改変クロストリジウム・サッカロパーブチルアセトニカム | |
EP3194592B1 (en) | Genetically modified (r)-lactic acid producing thermophilic bacteria | |
CN102864116B (zh) | 产丁二酸基因工程菌及其构建及应用 | |
CN105062938A (zh) | 一种可同步利用五碳糖和六碳糖发酵产d-乳酸的工程菌及其构建和应用 | |
WO2018010698A1 (zh) | 一种菌及其获取方法和应用 | |
WO2007110592A2 (en) | Fermentation process for the production of ethanol | |
CN115927148A (zh) | 一种无乳糖添加的高效生产乳酰-n-新四糖的基因工程菌及其应用 | |
JP7158107B2 (ja) | 有機化合物の生産方法 | |
EP2774986A1 (en) | Improvement of clostridial butanol production by gene overexpression | |
US10273509B2 (en) | Genetic modification of (S)-lactic acid producing thermophilic bacteria | |
CN106884001B (zh) | 一种重组嗜碱芽孢杆菌及其制备方法和应用以及制备d-乳酸的方法 | |
JP2007174947A (ja) | 酵母株および乳酸の製造方法 | |
JP5713317B2 (ja) | ペントースからのホモ乳酸発酵 | |
CN115725486A (zh) | 一种利用甲醇为碳源的苏云金芽孢杆菌及其应用 | |
CN118185974A (zh) | 提升梭菌气体发酵性能的方法 | |
JP2007130015A (ja) | 有機酸生産能が向上した酵母株およびそれを用いた有機酸の製造法 | |
JP2015136339A (ja) | 2,3−ブタンジオールの製造方法 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220823 |