CN112592843A - 一种生产α-蛇麻烯的重组解脂耶氏酵母菌及其构建方法和应用 - Google Patents
一种生产α-蛇麻烯的重组解脂耶氏酵母菌及其构建方法和应用 Download PDFInfo
- Publication number
- CN112592843A CN112592843A CN202011574323.3A CN202011574323A CN112592843A CN 112592843 A CN112592843 A CN 112592843A CN 202011574323 A CN202011574323 A CN 202011574323A CN 112592843 A CN112592843 A CN 112592843A
- Authority
- CN
- China
- Prior art keywords
- yarrowia lipolytica
- lupinene
- recombinant
- thmgr
- achs2
- 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.)
- Granted
Links
- 241000235015 Yarrowia lipolytica Species 0.000 title claims abstract description 80
- 238000010276 construction Methods 0.000 title claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000013612 plasmid Substances 0.000 claims description 50
- 230000014509 gene expression Effects 0.000 claims description 38
- 241000894006 Bacteria Species 0.000 claims description 25
- 101100342585 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) mus-51 gene Proteins 0.000 claims description 22
- 101100342589 Schizosaccharomyces pombe (strain 972 / ATCC 24843) pku70 gene Proteins 0.000 claims description 22
- 101150085005 ku70 gene Proteins 0.000 claims description 22
- 238000011144 upstream manufacturing Methods 0.000 claims description 20
- 238000004519 manufacturing process Methods 0.000 claims description 17
- 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 8
- 241000235013 Yarrowia Species 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 241000201640 Yarrowia lipolytica PO1f Species 0.000 claims description 4
- 238000012262 fermentative production Methods 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 abstract description 10
- 230000015572 biosynthetic process Effects 0.000 abstract description 8
- 238000000605 extraction Methods 0.000 abstract description 8
- 239000012074 organic phase Substances 0.000 abstract description 5
- 239000000592 Artificial Cell Substances 0.000 abstract 1
- 150000003505 terpenes Chemical class 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 12
- 230000010354 integration Effects 0.000 description 11
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 9
- 235000007586 terpenes Nutrition 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 101150032598 hisG gene Proteins 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- GVVPGTZRZFNKDS-JXMROGBWSA-N geranyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O GVVPGTZRZFNKDS-JXMROGBWSA-N 0.000 description 6
- 230000006801 homologous recombination Effects 0.000 description 6
- 238000002744 homologous recombination Methods 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- VWFJDQUYCIWHTN-YFVJMOTDSA-N 2-trans,6-trans-farnesyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-YFVJMOTDSA-N 0.000 description 5
- GVVPGTZRZFNKDS-YFHOEESVSA-N Geranyl diphosphate Natural products CC(C)=CCC\C(C)=C/COP(O)(=O)OP(O)(O)=O GVVPGTZRZFNKDS-YFHOEESVSA-N 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- KJTLQQUUPVSXIM-ZCFIWIBFSA-M (R)-mevalonate Chemical compound OCC[C@](O)(C)CC([O-])=O KJTLQQUUPVSXIM-ZCFIWIBFSA-M 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 4
- 101710088194 Dehydrogenase Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- VWFJDQUYCIWHTN-FBXUGWQNSA-N Farnesyl diphosphate Natural products CC(C)=CCC\C(C)=C/CC\C(C)=C/COP(O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-FBXUGWQNSA-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
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 108091033409 CRISPR Proteins 0.000 description 3
- 102000010911 Enzyme Precursors Human genes 0.000 description 3
- 108010062466 Enzyme Precursors Proteins 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- FAMPSKZZVDUYOS-UHFFFAOYSA-N alpha-Caryophyllene Natural products CC1=CCC(C)(C)C=CCC(C)=CCC1 FAMPSKZZVDUYOS-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- CBIDRCWHNCKSTO-UHFFFAOYSA-N prenyl diphosphate Chemical compound CC(C)=CCO[P@](O)(=O)OP(O)(O)=O CBIDRCWHNCKSTO-UHFFFAOYSA-N 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 238000010354 CRISPR gene editing Methods 0.000 description 2
- 101100126180 Escherichia coli (strain K12) intS gene Proteins 0.000 description 2
- 101710125754 Farnesyl pyrophosphate synthase Proteins 0.000 description 2
- 102100035111 Farnesyl pyrophosphate synthase Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- DIOQZVSQGTUSAI-UHFFFAOYSA-N decane Chemical compound CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000012269 metabolic engineering Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229930001457 monocyclic sesquiterpene Natural products 0.000 description 2
- 150000001374 monocyclic sesquiterpene derivatives Chemical class 0.000 description 2
- 229930003658 monoterpene Natural products 0.000 description 2
- 150000002773 monoterpene derivatives Chemical class 0.000 description 2
- 235000002577 monoterpenes Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- PXQPEWDEAKTCGB-UHFFFAOYSA-N orotic acid Chemical compound OC(=O)C1=CC(=O)NC(=O)N1 PXQPEWDEAKTCGB-UHFFFAOYSA-N 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 229930004725 sesquiterpene Natural products 0.000 description 2
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 2
- 230000001360 synchronised effect Effects 0.000 description 2
- -1 terpene compounds Chemical class 0.000 description 2
- FAMPSKZZVDUYOS-PGPZXUPKSA-N (1Z,4E,8Z)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene Chemical compound C\C1=C\CC(C)(C)\C=C\C\C(C)=C/CC1 FAMPSKZZVDUYOS-PGPZXUPKSA-N 0.000 description 1
- 101710165761 (2E,6E)-farnesyl diphosphate synthase Proteins 0.000 description 1
- OINNEUNVOZHBOX-QIRCYJPOSA-K 2-trans,6-trans,10-trans-geranylgeranyl diphosphate(3-) Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\COP([O-])(=O)OP([O-])([O-])=O OINNEUNVOZHBOX-QIRCYJPOSA-K 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 238000007702 DNA assembly Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101710156207 Farnesyl diphosphate synthase Proteins 0.000 description 1
- 101710089428 Farnesyl pyrophosphate synthase erg20 Proteins 0.000 description 1
- OINNEUNVOZHBOX-XBQSVVNOSA-N Geranylgeranyl diphosphate Natural products [P@](=O)(OP(=O)(O)O)(OC/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)O OINNEUNVOZHBOX-XBQSVVNOSA-N 0.000 description 1
- 244000025221 Humulus lupulus Species 0.000 description 1
- 235000008694 Humulus lupulus Nutrition 0.000 description 1
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical group CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 101710150389 Probable farnesyl diphosphate synthase Proteins 0.000 description 1
- 101150014136 SUC2 gene Proteins 0.000 description 1
- 101100348089 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) BUR6 gene Proteins 0.000 description 1
- 241000202758 Scirpus Species 0.000 description 1
- 241000207929 Scutellaria Species 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 101100215634 Yarrowia lipolytica (strain CLIB 122 / E 150) XPR2 gene Proteins 0.000 description 1
- VDFOMVRWDSKWSL-UHFFFAOYSA-N Zerumbone Natural products CC1=C2CC(C)(C)C=C2C(=O)C(=CCC1)C VDFOMVRWDSKWSL-UHFFFAOYSA-N 0.000 description 1
- 241000234314 Zingiber Species 0.000 description 1
- 240000000451 Zingiber zerumbet Species 0.000 description 1
- 235000014687 Zingiber zerumbet Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- PSVBPLKYDMHILE-UHFFFAOYSA-N alpha-humulene Natural products CC1=C/CC(C)(C)C=CCC=CCC1 PSVBPLKYDMHILE-UHFFFAOYSA-N 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011960 computer-aided design Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 150000004141 diterpene derivatives Chemical class 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- QBNFBHXQESNSNP-UHFFFAOYSA-N humulene Natural products CC1=CC=CC(C)(C)CC=C(/C)CCC1 QBNFBHXQESNSNP-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940094933 n-dodecane Drugs 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229930001119 polyketide Natural products 0.000 description 1
- 125000000830 polyketide group Chemical group 0.000 description 1
- 150000003097 polyterpenes Chemical class 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000007261 regionalization Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229930002368 sesterterpene Natural products 0.000 description 1
- 150000002653 sesterterpene derivatives Chemical class 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 108010087432 terpene synthase Proteins 0.000 description 1
- 150000003535 tetraterpenes Chemical class 0.000 description 1
- 235000009657 tetraterpenes Nutrition 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- GIHNTRQPEMKFKO-SKTNYSRSSA-N zerumbone Chemical compound C\C1=C/CC(C)(C)\C=C\C(=O)\C(C)=C\CC1 GIHNTRQPEMKFKO-SKTNYSRSSA-N 0.000 description 1
- GIHNTRQPEMKFKO-UHFFFAOYSA-N zurembone Natural products CC1=CCC(C)(C)C=CC(=O)C(C)=CCC1 GIHNTRQPEMKFKO-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—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
- C12N15/79—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
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- 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
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/002—Preparation of hydrocarbons or halogenated hydrocarbons cyclic
-
- 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
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
-
- 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)
- C12Y101/01034—Hydroxymethylglutaryl-CoA reductase (NADPH) (1.1.1.34)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
- C12Y402/03104—Alpha-humulene synthase (4.2.3.104)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (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)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种生产α‑蛇麻烯的重组解脂耶氏酵母菌及其构建方法和应用,所述重组解脂耶氏酵母菌是在解脂耶氏酵母基因组中插入优化的α‑蛇麻烯合酶编码基因ACHS2和tHMGR编码基因后得到。本发明重组解脂耶氏酵母菌能够高效合成α‑蛇麻烯,构建方法高效,操作简单,此外本发明提供的收集产物的方法采用边发酵边萃取的方法,及时的将生产的α‑蛇麻烯萃取至有机相中,缓解了胞内溶解α‑蛇麻烯的压力,有效提高α‑蛇麻烯的产量,为人工细胞合成α‑蛇麻烯奠定了基础。
Description
技术领域
本发明属于生物工程领域,具体涉及一种生产α-蛇麻烯的重组解脂耶氏酵母及其构建方法和应用。
背景技术
萜类化合物,又称类异戊二烯,是普遍存在于植物界的一类化合物,包含了超过50000个分子大小、结构和生物功能各异的分子。根据分子中异戊二烯单位的数目将萜类可分为:半萜、单萜、倍半萜、二萜、二倍半萜、三萜、四萜、多萜等。萜类化合物在生命活动中扮演着重要角色,除此之外,萜类化合物在医药、能源、食品等领域具有重要的应用价值。因此在自然界中挖掘这些数目繁多、结构多样的萜类,并实现其广泛的应用价值是非常有必要的。
传统萜类化合物的规模化制备主要为植物提取法以及化学合成法。然而,植物提取法受严格的地理和气候条件的限制;而对于大多数结构复杂的萜类化合物,化学合成法往往步骤繁琐,收率低。因此,寻找新的萜类生产途径,是今后萜类工业化生产的必然发展趋势。近年来随着合成生物学的快速发展,以微生物作为底盘细胞来实现萜类化合物的绿色高效转化引起越来越多的关注。
α-蛇麻烯(也称为α-石竹烯,α-Humulene)是一种单环倍半萜,其分子式为C15H24。近年来,由于单环倍半萜类α-蛇麻烯具有抗炎和潜在的抗癌特性,受到了越来越多的关注。α-蛇麻烯是由shampoo ginger plant,Zingiber zerumbet和Humulus lupulus产生的。另外,它是生物合成zerumbone的前体,其本身具有惊人的抗炎和抗癌作用。迄今为止,植物提取是萜烯生产的主要方式,这是一种不可持续的过程,还伴随着高昂的成本和废物的产生。Hu和他的同事们使用昂贵且对环境有害的催化剂,通过许多步骤实现了α-蛇麻烯的化学合成。因此,微生物生产为α-蛇麻烯的可持续性和大规模生产提供了一种有吸引力的方法。
解脂耶氏酵母又是被FDA认证的非常规安全产油酵母,其优势是胞内乙酰CoA/辅因子供应充足,细胞耐受性好,生成的油脂还可以包裹亲脂性萜类增强萜类存储能力,另外已经在解脂耶氏酵母中建立了包括Gibson、Gateway、BioBricks、体内一步组装法;以及基因组操作工具包括TALEN、CRISPR/Cas9、CRISPRa、CRISPRi、Cpf1、碱基编辑器、Cre/Loxp、URA/TRP Blaster、以及代谢工程技术手段包括推拉策略、多拷贝整合策略、启动子/终止子工程、区域化策略、随机整合/piggyBac转座筛选系统、基于计算机辅助的基因组规模代谢网络的设计等多种DNA组装工具;目前还没有在解脂耶氏酵母异源高产α-蛇麻烯的相关报道
发明内容
发明目的:针对现有技术存在的问题,本发明提供一种生产α-蛇麻烯的重组解脂耶氏酵母菌,本发明通过基因工程手段构建出生产α-蛇麻烯的重组解脂耶氏酵母菌株,为解脂耶氏酵母的代谢工程改造提供了有效的思路。
本发明还提供生产α-蛇麻烯的重组解脂耶氏酵母菌的构建方法与应用。
技术方案:为了实现上述目的,本发明所述一种生产α-蛇麻烯的重组解脂耶氏酵母菌,所述重组解脂耶氏酵母菌是在解脂耶氏酵母基因组中插入优化的α-蛇麻烯合酶编码基因ACHS2、tHMGR编码基因后得到。
具体地,本发明所述的重组解脂耶氏酵母是在解脂耶氏酵母基因组插入了α-蛇麻烯合酶编码基因ACHS2的表达盒和tHMGR表达盒得到。
进一步地,所述的α-蛇麻烯合酶表达盒和tHMGR表达盒通过质粒形式导入所述解脂耶氏酵母,然后整合到解脂耶氏酵母基因组上的步骤。
其中,所述优化的α-蛇麻烯合酶编码基因如SEQ ID NO.1所示;所述tHMGR编码基因如SEQ ID NO.2所示。
作为优选,所述解脂耶氏酵母为Yarrowia lipolytica Po1fΔku70。
其中,所述ACHS2表达盒的启动子为解脂耶氏酵母的PTEFin启动子、PTEF启动子或PEXP启动子;所述ACHS2终止子为解脂耶氏酵母的TXPR2T终止子、TCYC1t终止子或Tlip1t终止子。
其中,所述tHMGR表达盒的启动子为解脂耶氏酵母的PTEFin启动子、PTEF启动子或PEXP启动子;所述tHMGR终止子为解脂耶氏酵母的TXPR2T终止子、TCYC1t终止子或Tlip1t终止子。
本发明所述生产α-蛇麻烯的重组解脂耶氏酵母菌的构建方法,其特征在在于,包括如下步骤:
(1)重组质粒PUC-leu-A08-ACHS2的构建:
重组质粒PUC-Ieu-A08-ACHS2是以PUC-leu为骨架,插入了Yarrowia lipolyticaPolf-Δku70中A08位点处上下游同源臂,在该上下游同源臂之间插入了ACHS2基因表达盒;
(2)重组质粒PUC-HUH-IntC-tHMGR的构建:
重组质粒PUC-HUH-IntC-tHMGR是以PUC-his-ura-hisG为骨架,插入了Yarrowialipolytica Polf-Δku70中IntC位点处上下游同源臂,在该上下游同源臂之间插入了tHMGR表达盒;
(3)将质粒PUC-leu-A08-ACHS2导入Yarrowia lipolytica Polf-Δku70中得到重组菌1即为生产α-蛇麻烯的重组解脂耶氏酵母菌;重组质粒PUC-HUH-IntC-tHMGR导入重组菌1,tHMGR表达盒整合至基因组IntC位点,得到重组菌2即为高产α-蛇麻烯的重组解脂耶氏酵母菌。
本发明所述生产α-蛇麻烯的重组解脂耶氏酵母菌在发酵生产α-蛇麻烯的中的应用。
其中,所述发酵条件为28-30℃,200-240rpm,发酵培养基中葡萄糖浓度为40-80g/L。
作为优选,发酵条件为30℃,220rpm,60g/L葡萄糖。
其中,采用发酵培养基培养所述重组菌,得到发酵产物;有机溶液萃取所述发酵产物,收集有机相。
进一步地,所述生产α-蛇麻烯时进行同步萃取法,加入10-25%的有机溶剂同步萃取,有机溶剂可以为癸烷,十二烷等。
解脂耶氏酵母能够合成大量乙酰辅酶A作为甲羟戊酸(MVA)途径的起始底物,使其成为合成乙酰辅酶A衍生产物(如萜类或聚酮)的理想细胞工厂,为多种萜类化合物的积累提供更多的香叶基二磷酸(GPP)、法尼基二磷酸(FPP)和香叶基二磷酸(GGPP)。另一方面,大量的油积累在脂滴中,可以储存亲脂性的萜类化合物,成为生产萜类化合物的有价值的宿主。在甲羟戊酸途径(MVA)中,IPP与DMAPP是所有萜类的共同前体,DMAPP通过与不同数量的IPP相缩合,可以相继生成不同的异戊烯基二磷酸前体。DMAPP与一分子IPP在香叶基焦磷酸合酶(GPP synthase)催化下,生成单萜前体GPP(C10)。GPP与一分子IPP在法尼基焦磷酸合酶(FPP synthase)催化下,生成倍半萜前体FPP(C15)。FPP在不同的萜烯合酶作用下可以生产得到不同的萜烯化合物。
本发明首次发现优化的α-蛇麻烯合酶编码基因ACHS2插入解脂耶氏酵母配合tHMGR表达盒可以高效生产α-蛇麻烯,本发明发现ACHS2能在解脂耶氏酵母中表达使其能够生产蛇麻烯,并首次在解脂耶氏酵母中实现了蛇麻烯的异源生产,宿主的变化可能会导致无法生产或者产量过低,同时本发明中只需要配合一个tHMGR表达盒就可以达到α-蛇麻烯的大量生产,对此菌株继续改造具有广阔的工业化应用前景。
本发明的重组解脂耶氏酵母能够表达α-蛇麻烯合酶和tHMGR,实验证明该重组解脂耶氏酵母能够高效生产α-蛇麻烯,实现了天然产物α-蛇麻烯在解脂耶氏酵母中的异源合成。
有益效果:与现有技术相比,本发明具有如下优点:
(1)本发明构建的重组解脂耶氏酵母,是基于敲除了负责编码非同源重组基因ku70的解脂耶氏酵母菌株,使其同源重组能力增强,通过解脂耶氏酵母自身的同源重组功能实现基因的整合,能大幅度提高导入的基因的遗传稳定性,该重组解脂耶氏酵母菌株的构建方法高效,操作简单。
(2)解脂耶氏酵母是一种安全认证的产油酵母,其本身具有很强的油脂合成能力,目标产物α-蛇麻烯易溶于脂溶性环境,这为α-蛇麻烯在解脂耶氏酵母体内生产提供良好的胞内环境。
(3)重组解脂耶氏酵母菌株通过过表达tHMGR,实现了α-蛇麻烯的高效合成。
(4)重组解脂耶氏酵母菌株通过引入α-蛇麻烯合酶,实现了α-蛇麻烯的异源合成。
(5)本发明提供的收集产物的方法采用边发酵边萃取的方法,及时的将生产的α-蛇麻烯萃取至有机相中,缓解了胞内溶解α-蛇麻烯的压力,有效提高α-蛇麻烯的产量。
附图说明
图1为重组质粒PUC-leu-A08-ACHS2的结构图,其中A08-up表示A08位点上游序列,A08-dn表示A08位点下游序列,xpr2t表示终止子,TEFin表示启动子。
图2为重组质粒PUC-intC-HUH-tHMGR的的结构图,其中intC-up表示intC位点上游序列,intC-dn表示intC位点下游序列,tHMGRt表示终止子,TEFin表示启动子。
图3是重组菌生产的α-蛇麻烯的GC-MS检测图。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
本发明实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂家建议的条件。
Yarrowia lipolytica Po1fΔku70(MatA,ura3-302,leu2-270,xpr2-322,axp2-delta,NU49,XPR2::SUC2 MYA2613Δku70::hisG)为解脂耶氏酵母,原始菌株MYA2613购自于美国菌种保藏中心(ATCC),并敲除了负责非同源重组的编码基因ku70,得到Yarrowialipolytica Po1fΔku70(MYA2613Δku70)。(可参考文献Gao S,Tong Y,Zhu L,etal.Iterative integration of multiple-copy pathway genes in Yarrowialipolytica for heterologousβ-carotene production[J].Metabolic Engineering,2017:192.)
A08位点整合质粒是将Yarrowia lipolytica PolfΔku70基因组中染色体A上A08位点上游2521bp、下游2031bp的序列插入PUC-leu载体(构建方法见实施例1)中所得,leu表达盒在A08位点上下游序列之间。
IntC位点整合质粒是将Yarrowia lipolytica Polf-Δku70基因组中染色体C上IntC位点上游1402bp和下游1396bp的序列插入PUC-hisG-ura-hisG(构建方法见实施例1)载体中所得,两个hisG标签编码基因在IntC位点上,下游序列之间。
实施例1
一、基因元件的扩增与目标质粒的制备
1、目标基因的制备
根据NCBI上提供的α-蛇麻烯合酶编码基因ACHS2的核苷酸序列(KT893310),经过特定的密码子优化后,委托擎科生物有限公司合成优化后的α-蛇麻烯合酶编码基因ACHS2,并插入质粒PUC57中,得到质粒PUC-ACHS2。优化后的ACHS2的核苷酸序列如SEQ IDNO.1所示。
根据NCB1上提供的Yarrowia lipolytica中的3(B)-异丙基苹果酸脱氢酶编码基因leu的核苷酸序列(M37309.1),委托擎科生物科技有限公司合成leu,将3(B)-异丙基苹果酸脱氢酶编码基因插入质粒PUC中,得到质粒PUC-leu。根据NCBI上提供的Yarrowialipolytica中的乳清酸核普-5'-磷酸脱浚酶编码基因ura的核苷酸序列(genebank登录号AJ306421.1)和hisG标签(genebank登录号AF324729.1),委托擎科生物科技有限公司合成,将两个hisG标签编码基因序列插入质粒PUC中,具体使用PUC57作为骨架,使用ECORI和HindIII酶切后回收骨架,将hisG标签编码基因采用ClonExpress MultiS One StepCloning Kit实现一步克隆,构建得到质粒PUC-hisG-hisG,再以此为骨架,采用HindIII酶切后回收骨架,将ura编码基因采用ClonExpress MultiS One Step Cloning Kit实现一步克隆,构建得到两个hisG标签编码基因序列中,使得在两个hisG标签编码基因序列中插入乳清酸核-5'-磷酸脱羚酶编码基因,以便实现ura标记回收,得到质粒PUC-hisG-ura-hisG。
以Yarrowia lipolytica Polf-Δku70基因组DNA为模板,以tHMG-F(SEQ IDNO.6)和tHMG-R(SEQ ID NO.7)为引物扩增3-基-3-甲基戊二酰CoA还原酶编码基因tHMGR。tHMGR的核苷酸序列如SEQ ID NO.2所示。
启动子PTEFin的核甘酸序列如SEQ ID NO.3所示,终止子Txpr2t核酸序列如SEQIDNO.4所示,终止子TtHMGR核酸序列如SEQ IDNO.5所示。
二、重组质粒的构建
1、重组质粒PUC-leu-A08-ACHS2的构建
重组质粒PUC-Ieu-A08-ACHS2是以PUC-leu为骨架,插入了Yarrowia lipolyticaPolf-Δku70中A08位点处上下游同源臂,在该上下游同源臂之间插入了ACHS2基因表达盒,ACHS2编码基因表达盒在该上下游同源臂之间,结构如图1。具体是将A08位点上游2521bp、下游2031b构建到PUC-leu骨架质粒中,再将ACHS2表达盒构建在这个质粒上,然后将这个质粒线性化之后,转入到解脂耶氏酵母感受态中,由于质粒上带有A08的同源臂,所以构建的ACHS2表达盒会定点整合到解脂耶氏酵母基因组中A08这个位点。
以TEFin-ACHS2-F(SEQ ID NO.8)和TEFin-ACHS2-R(SEQ ID NO.9)为引物,以Yarrowia lipolytica Polf-Δku70基因组DNA为模板,扩增ACHS2表达盒启动子PTEFin-ACHS2。以xpr2t-ACHS2-F(SEQ ID NO.10)和xpr2t-ACHS2-R(SEQ ID N.11)为引物,以Yarrowialipolytica Polf-Δku70基因组DNA为模板扩增ACHS2表达盒终止子Txpr2t-ACHS2。
以质粒PUC57-ACHS2为模板,以ACHS2-F(SEQID NO.12)和ACHS2-R(SEQ ID NO.13)为引物,扩增两端分别带有启动子PTEFin-ACHS2和终止子Txpr2t-ACHS2同源臂的ACHS2基因。
上述PCR反应中所用PCR酶为TAKARA的PrimeSTAR MaxDNA聚合酶。上述PCR扩增体系如下表1:
表1
试剂 | 使用量 | 终浓度 |
PrimeSTAR Max(2×) | 25ul | 1× |
Primer 1 | 10-15pmol | 0.2-0.3uM |
Primer 2 | 10-15pmol | 0.2-0.3uM |
Template | <200ng | |
灭菌蒸馏水 | Up to 50ul |
上述PCR的程序如下:98℃变性10s,55℃退火10s,72℃延伸(延伸时间=目标片段长度/1kb,单位min),重复35个循环。
用AxyPrepTM DNA Gel Extraction Kit(购自康宁生命科学(吴江)有限公司)纯化回收各片段。
将A08位点整合质粒用NEB公司的限制性内切酶SnaB1酶切后,再用琼脂糖凝胶电泳胶回收线性化A08位点整合质粒。
将线性化A08位点整合质粒和构建的ACHS2基因表达盒(包括启动子PTEFin-ACHS2、终止子Txpr2t-ACHS2、带有启动子PTEFin-ACHS2和终止子Txpr2t-ACHS2同源臂的ACHS2基因)利用南京诺唯赞生物科技有限公司的ClonExpress MultiS One Step Cloning Kit实现一步克隆,反应体系见下表。将反应体系在50℃孵育15-30min后,得到环状的重组载体,在A08位点的上下同源臂之间成功插入了ACHS2基因表达盒和3(B)-异丙基苹果酸脱氢酶编码基因表达盒(筛选标记)。
一步克隆的体系如下表2:
表2
组分 | 重组反应 |
线性化载体 | X ul |
N个插入片段 | Y<sub>1</sub>+Y<sub>2</sub>+…Y<sub>n</sub> ul |
2×ClonExpress Mix | 5ul |
ddH<sub>2</sub>O | To 10ul |
X=(0.02×克隆载体碱基对数)ng(0.03pmol)
Y=(0.02×每个片段碱基对数)ng(0.03pmol)
将环状的重组载体转化大肠杆菌DH5a感受态细胞,通过氨苄抗性的平板筛选并通过菌落PCR及测序验证,得到阳性重组质粒PUC-leu-A08-ACHS2,构建成功。
2、重组质粒PUC-HUH-IntC-tHMGR的构建
重组质粒PUC-HUH-IntC-tHMGR是以PUC-his-ura-hisG为骨架,插入了Yarrowialipolytica Polf-Δku70中IntC位点处上下游同源臂,在该上下游同源臂之间插入了tHMGR表达盒,乳清酸核-5’-磷酸脱酶编码基因表达盒也在上下游同源臂之间,具体结构见图2。
以TEFin-tHMGR-F(SEQ ID No.14)和TEFin-tHMGR-R(SEQ ID NO.15)为引物,以Yarrowia lipolytica Polf-Δku70基因组DNA为模板,扩增tHMGR表达盒启动子PTEFin-tHMGR。以tHMGRt-tHMGR-F(SEQ ID NO.16)和tHMGRt-tHMGR-R(SEQ ID N0.17)为引物,以Yarrowialipolytica Polf-Δku70基因组DNA为模板,扩增tHMGR表达盒终止子TtHMGRt-tHMGR。
以Yarrowia lipolytica Polf-Δku70基因组DNA为模板,以tHMGR-F2(SEQIDNO.18)和tHMGR-R2(SEQ ID N0.19)为引物扩增两端分别带有启动子PTEFin-tHMGR和终止子TtHMGRt-tHMGR同源臂的tHMGR基因。
用NEB公司的限制性内切酶Pacl对IntC位点整合质粒进行酶切后,琼脂糖凝胶电泳胶回收线性化IntC位点整合质粒。
将线性化IntC位点整合质粒和构建的tHMG基因表达盒(包括启动子PTEFin-tHMGR、终止子TtHMGRt-tHMGR、带有启动子PTEFin-tHMGR和终止子TtHMGRt-tHMGR同源臂的tHMGR基因)利用南京诺唯赞生物科技有限公司的ClonExpress MultiS One Step Cloning Kit实现一步克隆,得到重组质粒PUC-HUH-IntC-tHMGR,具体步骤同重组质粒PUC-leu-A08-ACHS2的构建。
本实施例中构建的重组质粒如表3所示,使用的引物序列如表4所示。
表3各重组质粒中插入序列
质粒名称 | 插入序列 |
PUC-leu-A08-ACHS2 | ACHS2表达盒(P<sub>TEFin</sub>-ACHS2-T<sub>xpr2t</sub>) |
PUC-HUH-intC-tHMGR | tHMGR表达盒(P<sub>TEFin</sub>-tHMGR-T<sub>xpr2t</sub>) |
表4引物序列
实施例2
重组菌的构建
一、重组菌1的构建
1、将含有ACHS2基因表达盒的质粒PUC-leu-A08-ACHS2导入Yarrowia lipolyticaPolf-Δku70中,ACHS2表达盒整合到基因组A08位点处,得到重组菌1。具体方法如下:(1)Yarrowia lipolytica Polf-Δku70于YPD液体培养基(含有2%蛋白胨、1%酵母提取物和2%葡萄糖)中过夜培养,待OD600长到0.8-1.0之间时,制备感受态细胞(试剂盒:ZymogenFrozen EZ Yeast Transformation Kit II,厂家:Zymo Research Corporation)。(2)利用Zymo Research Corporation的Zymogen Frozen EZ Yeast Transformation Kit II将PUC-leu-A08-ACHS2转化Yarrowia lipolytica Polf-Δku70,进行同源重组。(3)采用筛选培养基SD-Leu筛选,将PCR鉴定正确的阳性克隆,命名为重组菌1。其中筛选培养基SD-Leu含有:葡萄糖20g/L,Yeast Nitrogen Base(YNB,无氨基酵母氮源)6.7g/L,CSM-Leu 0.67g/L,琼脂粉23g/L。
二、重组菌2的构建
将重组质粒PUC-HUH-IntC-tHMGR导入重组菌1,tHMGR表达盒整合至基因组IntC位点,得到重组菌2。具体方法如下:将重组菌1于YPD液体培养基(含有2%蛋白膝,1%酵母提取物,2%葡萄糖)中过夜培养,待OD600长到0.8-1.0之间时,制备感受态细胞,利用ZymoResearch Corporation的Zymogen Frozen EZ Yeast Transformation Kit II将重组质粒PUC-HUH-IntC-tHMGR转化入重组菌1中,进行同源重组,采用筛选培养基SD-Leu-Ura筛选,将PCR鉴定正确的阳性克隆,命名为重组菌2。其中筛选培养基SD-Leu-Ura的成分为:葡萄糖20g/L,YNB 6.7g/L,CSM-Leu-Ura 0.67g/L,琼脂粉23g/L。
实施例3
重组菌1和2在生产α-蛇麻烯中的应用
1、工程菌的培养及产物提取
分别采用实施例2中重组菌1和2生产α-蛇麻烯。具体方法如下:活化重组菌,于YPD液体培养基中30℃、220rpm条件下培养16h,得到种子液。将种子液以1%体积比的接种量接种于50ml发酵培养基中,在30℃、220rpm震荡培养1天,然后加入发酵液体积25%的正十二烷,继续震荡培养3天。发酵结束后,将发酵液转移至50ml离心管,5000rpm离心15min,收集上层有机相备用。
其中发酵培养基含有60g/L葡萄糖、10g/L酵母粉和20g/L胰蛋白陈。
2、α-蛇麻烯的定性定量分析
将各重组菌发酵液中的有机相过有机尼龙滤膜(0.22um),用GC-MS检测。检测条件:进样口温度250℃,进样体积1ul,分流比为20:1;色谱柱:HP-5ms(30m*0.25mM);色谱条件:初始温度为60℃,按照10℃/min的速度上升到150℃,然后20℃/min上升到280℃,保持2min。用α-蛇麻烯的标准品进行定性定量。图3为重组菌2生产的α-蛇麻烯的GC-MS检测图。发酵4天后,重组菌2的α-蛇麻烯产量最高,达到了50mg/L,即每升发酵液产50mg的α-蛇麻烯。重组菌1、重组菌2分别为24mg/L、50mg/L。
序列表
<110> 南京师范大学
<120> 一种生产α-蛇麻烯的重组解脂耶氏酵母菌及其构建方法和应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1665
<212> DNA
<213> 人工序列(Artificial Sequence ACHS2)
<400> 1
atgtctcctg cacaggcgcc ccaggtgtct gccccaaccc agaaggctgc agacgaagag 60
gccaaccgac gatctgccgg ttaccatcct agtttctggg gcgagttctt cctcactcac 120
tccagcgggt acaccaagag cgacacgaag atccagcaga agcacgagga gctcaagcaa 180
caggtgcgag gaatgatcct tgatgctgcc gccgacactt cccagaagct ggaactcatc 240
gacgccgctc tccggcttgg tgttggttat cactttgagg ctgaaattca gagccaactg 300
caaaagattc atggccaagg atcgttccac agtgatcttt atactgcctg catatggttc 360
cgagtgctta gaggccaggg cttcacggtt tcagcagatg tcttcaacat catgaaaaac 420
aaggacggag gattcgaggc tcgtgatgcg agaaccctcc tgtgtctcta tgagactacc 480
catctgcgaa tccagggtga acaggtcctt gaagaggcgc tggagttttc cagaaaacag 540
ctgggagact tattagccga actgtctagc cctctggccg agtacgtcaa caactccttg 600
gagctcccgt accacaaggg catgcagagg ctcgaggcac gacagtacat tcccatctac 660
gagtcgtacg caaacaagaa cgacacacta ctgcagtttg ctaaactcga ttttaatctg 720
ctccaggccc tacaccagtc ggagattcga gaaatcaccc gatggtggaa ggacttggac 780
ttcaaggcac gtctcccgta tgctcgcgac cggttggtcg agtgctactt ttggatcctg 840
ggtgtccagt atgagcctca atactccata tcgagagtct ttctgacaaa ggtcatttca 900
cttgcatccg tatttgacga tacatacgac atttacggca ccttcgacga gctcaaactg 960
ttgactgatg ccgttgaaag atgggagccc gaggccactg atagtcttcc cggatacatg 1020
cagattctat atggagctct tctcaaggtg tttgaagagt acaaggatga gcttatcaat 1080
gctggaggcc gggattactg cctctattac gccaaggagg ccatgaaggg tctagtacgg 1140
tcctaccaca ccgaggccgt gagtttccat acaggctacg tgcagaattt tgaggaatat 1200
ctggacaact ctgctgtgtc ttcgggatac cccatgctga cggtggaagc attgattggt 1260
atgggggctc cttacgccac tcgagagtca ctggactggg ccctgaaggt tcccaagatc 1320
atcaaggctt cttctgacat ttgtcggctg gtcgacgacc ttaggacgta caaggtggag 1380
gaggagcgag gcgacgctcc gtccggcgtg cactgttaca tgcgagacta caacgtctcg 1440
gaggaagaag cttgcaccaa aattgaggag atgatcgatc tcgcctggaa ggccatcaac 1500
gaggagatcc aaaaacccaa ccaccttcca ctgcccattc tcctgcctgc gctcaacttc 1560
gctcgaatga tggaggttct gtaccagaac attgacggct acaccaattc gggtggacgt 1620
accaaggaac gcatttcatc cttactggtt catcccttca ccatc 1665
<210> 2
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence tHMGR)
<400> 2
atgctatgac cgtatgcaaa tattcgaacc gttttgtaga cgctgcagat cggcctgaga 60
ctgcttggcc ggagtaggag cctgggaccg gttgtgggtc atatgacttt gcacaagatg 120
gccggcagca agagcagaac acagcgaaag ctccgctgca agaactccag aagcaatgat 180
gcgagcaagc tgttgggcgt tggcaccggg ggtctcgatg tgaggacctc gcacgccaag 240
catctccagc atagccccct ggggctccaa aatagtacct ccaccaatgg taccgacctc 300
gatagaaggc atggaaacgg agatgagcag gttaccgtcg acgttgctca tcagcgtgat 360
gcagttggaa gactcgacat tctgagcagg atcctggcca gtggcaaggt agatggcggt 420
caccaggttt gcggcgtgtg cattgaaacc tcccacagag ccagccatgg cactaccgat 480
cagattcttg ctgatgttga gctcaacaag agcgtcaacc tcacttttga gaacagactt 540
gacaatgtga gcagggatgg tggcttcggc aacaacactc ttgcctcggc cttcgatcca 600
gttgatcgct gcgggcttct tgtcagtgca gtagttaccc gagacagaca caatgtccat 660
atcagggaag ccgtactcct tgaccatgac ggccagagag tgttcgacgc ccttggagat 720
catgttcatg cccatggcat caccagtggt ggttcggaat cgaataaaca gcaggttacc 780
agcaagggta gagtgaagag actggagacg agcaaatcga gaggtggagt tgaaggcctt 840
tcgcatggac ttgagaccct cctcggaatc aagccagatc ttagcggctc cagcccgctt 900
gagagagggg aaggaaacac aaggacctcg tgtcataccg tcctgagtaa gcacagtggt 960
aacaccgcca ccggcgttga tggccttgca acctcgcatg gttgaggcaa caagacaacc 1020
ctcagtggtg gccataggaa tgtggtagtt cttgccatca atgttcatgg ggccagcaac 1080
accaacgggg agaggcatgt aaccaataac gttctcgcaa caggctccaa aaacacggtc 1140
gtagtcgtag tgcaggtaag gaagctttga ggtctctaaa gtcttggtat tagactgctg 1200
ggagatgata gatcgtcgga tgccaacagc tcgggtgttg tcaccaagct gcttctcaag 1260
agcatacaaa ggaagcttgc cctcgagaga gagcttgaca acctcgtggt cctccagaag 1320
cttggtctta cctgccttca tgatagcctc taggtcgtcc agagaacggg tctcggtcac 1380
gggcttgggc tgctttccga cagtcagctc aatggagtct tcagaagagg tgtcctcctc 1440
cttctcgctg ggcttctcaa tgacgatagg aacgtgcttc tcaaccacct tcacagactg 1500
ggt 1503
<210> 3
<211> 531
<212> DNA
<213> 人工序列(Artificial Sequence PTEFin)
<400> 3
agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc cccaattgac 60
cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttcacc ccacatatca 120
aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 180
cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 240
gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 300
aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 360
cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaaaatgg tgagtttcag 420
aggcagcagc aattgccacg ggctttgagc acacggccgg gtgtggtccc attcccatcg 480
acacaagacg ccacgtcatc cgaccagcac tttttgcagt actaaccgca g 531
<210> 4
<211> 516
<212> DNA
<213> 人工序列(Artificial Sequence Txpr2t)
<400> 4
gatccaacta cggaacttgt gttgatgtct ttgcccccgg ctccgatatc atctctgcct 60
cttaccagtc cgactctggt actttggtct actccggtac ctccatggcc tgtccccacg 120
ttgccggtct tgcctcctac tacctgtcca tcaatgacga ggttctcacc cctgcccagg 180
tcgaggctct tattactgag tccaacaccg gtgttcttcc caccaccaac ctcaagggct 240
ctcccaacgc tgttgcctac aacggtgttg gcatttaggc aattaacaga tagtttgccg 300
gtgataattc tcttaacctc ccacactcct ttgacataac gatttatgta acgaaactga 360
aatttgacca gatattgttg taaatagaaa atctggcttg taggtggcaa aatcccgtct 420
ttgttcatca attccctctg tgactactcg tcatcccttt atgttcgact gtcgtatttt 480
tattttccat acatacgcaa gtgagatgcc cgtgtc 516
<210> 5
<211> 617
<212> DNA
<213> 人工序列(Artificial Sequence TtHMGRt)
<400> 5
gtgtacaata gtgaaggaaa cagagttaat aaatatatac aattagtgta cgtacatctg 60
gtagaacaat gtgttggcat aattagggac ggtatgaact gagtcctgcc aacctgatgc 120
taactaatgt gtggttgtaa ttggactggg tcgattttcg agaaataaac caataaacga 180
acacgttgtt cctagcccct ttaaagagac agtgcaagta ctgtacatac tgtacttaga 240
agcatactgg tatgtactgt atgtactgta catgcttcat ctggctggat caaagacgat 300
gatcaataca cagggaccca acgggaatag aaatcacata ggtgtacaag tattttataa 360
taacaagtaa ccaaagaagc tgtcttagtt ggtcggttct agtactccag attggtatgt 420
cccctatgta atacttacgt cgattttaat aaagttcatg cacaccacca tctaacatct 480
cagccaagtt tactccacac cattatcatt acacacgcag cacttctggt atggcatctc 540
tagcaacatt tcaggtggag aacgacattg tggacgtcga cagcacgccc caacaaggat 600
ttgatcgaga cgacctg 617
Claims (9)
1.一种生产α-蛇麻烯的重组解脂耶氏酵母菌,其特征在于,所述重组解脂耶氏酵母菌是在解脂耶氏酵母基因组中插入优化的α-蛇麻烯合酶编码基因ACHS2和tHMGR编码基因后得到。
2.根据权利要求1所述的生产α-蛇麻烯的重组解脂耶氏酵母菌,其特征在于,所述重组解脂耶氏酵母菌是在解脂耶氏酵母基因组中插入优化的α-蛇麻烯合酶酶编码基因ACHS2的表达盒和tHMGR编码基因表达盒后得到。
3.根据权利要求1所述的生产α-蛇麻烯的重组解脂耶氏酵母菌,其特征在于,所述优化的α-蛇麻烯合酶编码基因如SEQ ID NO.1所示;所述tHMGR编码基因如SEQ ID NO.2所示。
4.根据权利要求1所述的生产α-蛇麻烯的重组解脂耶氏酵母菌,其特征在于,所述解脂耶氏酵母为Yarrowia lipolytica Po1fΔku70。
5.根据权利要求2所述的生产α-蛇麻烯的重组解脂耶氏酵母菌,其特征在于,所述ACHS2表达盒的启动子优选为解脂耶氏酵母的PTEFin启动子、PTEF启动子或PEXP启动子;所述ACHS2终止子为解脂耶氏酵母的TXPR2T终止子、TCYC1t终止子或Tlip1t终止子。
6.根据权利要求2所述的生产α-蛇麻烯的重组解脂耶氏酵母菌,其特征在于,所述tHMGR表达盒的启动子为解脂耶氏酵母的PTEFin启动子、PTEF启动子或PEXP启动子;所述tHMGR终止子为解脂耶氏酵母的TXPR2T终止子、TCYC1t终止子或Tlip1t终止子。
7.一种权利要求1所述生产α-蛇麻烯的重组解脂耶氏酵母菌的构建方法,其特征在在于,包括如下步骤:
(1)重组质粒PUC-leu-A08-ACHS2的构建:
重组质粒PUC-Ieu-A08-ACHS2是以PUC-leu为骨架,插入了Yarrowia lipolyticaPolf-Δku70中A08位点处上下游同源臂,在该上下游同源臂之间插入了ACHS2基因表达盒;
(2)重组质粒PUC-HUH-IntC-tHMGR的构建:
重组质粒PUC-HUH-IntC-tHMGR是以PUC-his-ura-hisG为骨架,插入了Yarrowialipolytica Polf-Δku70中IntC位点处上下游同源臂,在该上下游同源臂之间插入了tHMGR表达盒;
(3)将质粒PUC-leu-A08-ACHS2导入Yarrowia lipolytica Polf-Δku70中得到重组菌1为生产α-蛇麻烯的重组解脂耶氏酵母菌;质粒PUC-HUH-IntC-tHMGR导入重组菌1,tHMGR表达盒整合至基因组IntC位点,得到重组菌2为高产α-蛇麻烯的重组解脂耶氏酵母菌。
8.一种权利要求1所述生产α-蛇麻烯的重组解脂耶氏酵母菌在发酵生产α-蛇麻烯的中的应用。
9.根据权利要求8所述的应用,其特征在于,所述发酵条件为28-30℃,200-240rpm,发酵培养基中葡萄糖浓度为40-80g/L。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011574323.3A CN112592843B (zh) | 2020-12-25 | 2020-12-25 | 一种生产α-蛇麻烯的重组解脂耶氏酵母菌及其构建方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011574323.3A CN112592843B (zh) | 2020-12-25 | 2020-12-25 | 一种生产α-蛇麻烯的重组解脂耶氏酵母菌及其构建方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112592843A true CN112592843A (zh) | 2021-04-02 |
CN112592843B CN112592843B (zh) | 2022-06-14 |
Family
ID=75203399
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011574323.3A Active CN112592843B (zh) | 2020-12-25 | 2020-12-25 | 一种生产α-蛇麻烯的重组解脂耶氏酵母菌及其构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112592843B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113549562A (zh) * | 2021-07-14 | 2021-10-26 | 南京师范大学 | 一种高效生产广藿香醇的工程菌及其构建方法和应用 |
CN113717871A (zh) * | 2021-09-15 | 2021-11-30 | 南京师范大学 | 重组解脂耶氏酵母菌及其构建方法和应用 |
CN114196689A (zh) * | 2021-12-24 | 2022-03-18 | 江南大学 | 一种高产α-蛇麻烯的酵母重组菌株的构建方法及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100112672A1 (en) * | 2006-09-26 | 2010-05-06 | Keasling Jay D | Production of isoprenoids and isoprenoid precursors |
WO2016142503A1 (de) * | 2015-03-11 | 2016-09-15 | Basf Se | Verfahren zur mikrobiellen de novo synthese von terpenen |
CN111041041A (zh) * | 2019-12-28 | 2020-04-21 | 天津大学 | 产α-蛇麻烯、8-羟基-α-蛇麻烯及花姜酮的酿酒酵母重组菌及构建方法 |
-
2020
- 2020-12-25 CN CN202011574323.3A patent/CN112592843B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100112672A1 (en) * | 2006-09-26 | 2010-05-06 | Keasling Jay D | Production of isoprenoids and isoprenoid precursors |
WO2016142503A1 (de) * | 2015-03-11 | 2016-09-15 | Basf Se | Verfahren zur mikrobiellen de novo synthese von terpenen |
CN111041041A (zh) * | 2019-12-28 | 2020-04-21 | 天津大学 | 产α-蛇麻烯、8-羟基-α-蛇麻烯及花姜酮的酿酒酵母重组菌及构建方法 |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113549562A (zh) * | 2021-07-14 | 2021-10-26 | 南京师范大学 | 一种高效生产广藿香醇的工程菌及其构建方法和应用 |
CN113549562B (zh) * | 2021-07-14 | 2023-10-24 | 南京师范大学 | 一种高效生产广藿香醇的工程菌及其构建方法和应用 |
CN113717871A (zh) * | 2021-09-15 | 2021-11-30 | 南京师范大学 | 重组解脂耶氏酵母菌及其构建方法和应用 |
CN114196689A (zh) * | 2021-12-24 | 2022-03-18 | 江南大学 | 一种高产α-蛇麻烯的酵母重组菌株的构建方法及其应用 |
CN114196689B (zh) * | 2021-12-24 | 2023-07-25 | 江南大学 | 一种高产α-蛇麻烯的酵母重组菌株的构建方法及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN112592843B (zh) | 2022-06-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112592843B (zh) | 一种生产α-蛇麻烯的重组解脂耶氏酵母菌及其构建方法和应用 | |
US20110287476A1 (en) | Production of isoprenoids | |
CN106635853B (zh) | 产甘草次酸的重组酿酒酵母、其构建方法以及用途 | |
US20100178679A1 (en) | Production of isoprenoids | |
CN111778167B (zh) | 高产桦木酸的酿酒酵母工程菌及其构建方法与应用 | |
BRPI0712508B1 (pt) | Sistemas e métodos para produzir composto isoprenoide, sistema de produção de composição de combustível, e, método para fabricar uma composição de combustível | |
CN109415672A (zh) | 气体搅拌式发酵装置 | |
CN112063647A (zh) | 酿酒酵母重组菌Cuol01的构建方法、酿酒酵母重组菌Cuol02及应用 | |
CN111088175A (zh) | 一种产红没药烯的解脂耶氏酵母及其构建方法与用途 | |
CN111527203A (zh) | 细胞色素p450单加氧酶催化的倍半萜的氧化 | |
CN111154665B (zh) | 一株重组解脂耶罗维亚酵母及其构建方法和应用 | |
CN112608936A (zh) | 调控酵母外源基因表达的启动子,调控方法及其应用 | |
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 | |
CN111849846A (zh) | 一种高产单鼠李糖脂的基因改造方法及其应用 | |
CN103820506B (zh) | 一种基因重组菌发酵生产辅酶q10的方法 | |
CN114736918A (zh) | 一种整合表达生产红景天苷的重组大肠杆菌及其应用 | |
CN115161208A (zh) | 酿酒酵母基因工程菌及其生产葫芦素中间体的应用 | |
CN107903227B (zh) | 琥珀酸酐类化合物、与其相关的基因和蛋白及其制备方法 | |
CN114806914B (zh) | 一种高产β-胡萝卜素的解脂耶氏酵母菌及其应用 | |
LU505601B1 (en) | ß-ELEMENE-PRODUCING GENETICALLY ENGINEERED BACTERIUM OF YARROWIA LIPOLYTICA, CONSTRUCTION METHOD THEREFOR AND PREPARATION METHOD FOR ß-ELEMENE | |
Bianchi et al. | Selection of microbial biocatalysts for the reduction of cyclic and heterocyclic ketones | |
KR102346076B1 (ko) | α-bisabolene 생산용 형질전환 메탄자화균 및 이의 용도 | |
CN109777745B (zh) | 一种合成桧烯的基因工程菌及其构建方法与应用 | |
CN117587045B (zh) | 一种藜芦胆固醇22(R)-羟化酶VnCYP90B27基因及应用 | |
CN110106129B (zh) | 谷氨酸脱氢酶基因受抑制的类球红细菌及其制备方法和法尼醇的生产方法 |
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 |