CN115074302A - 一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途 - Google Patents
一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途 Download PDFInfo
- Publication number
- CN115074302A CN115074302A CN202210535851.0A CN202210535851A CN115074302A CN 115074302 A CN115074302 A CN 115074302A CN 202210535851 A CN202210535851 A CN 202210535851A CN 115074302 A CN115074302 A CN 115074302A
- Authority
- CN
- China
- Prior art keywords
- gene
- seq
- nucleotide sequence
- bisabolol
- alpha
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 148
- RGZSQWQPBWRIAQ-CABCVRRESA-N (-)-alpha-Bisabolol Chemical compound CC(C)=CCC[C@](C)(O)[C@H]1CCC(C)=CC1 RGZSQWQPBWRIAQ-CABCVRRESA-N 0.000 title claims abstract description 140
- RGZSQWQPBWRIAQ-LSDHHAIUSA-N alpha-Bisabolol Natural products CC(C)=CCC[C@@](C)(O)[C@@H]1CCC(C)=CC1 RGZSQWQPBWRIAQ-LSDHHAIUSA-N 0.000 title claims abstract description 85
- 229940036350 bisabolol Drugs 0.000 title claims abstract description 82
- 239000001500 (2R)-6-methyl-2-[(1R)-4-methyl-1-cyclohex-3-enyl]hept-5-en-2-ol Substances 0.000 title claims abstract description 81
- 241000894006 Bacteria Species 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims description 15
- 239000002773 nucleotide Substances 0.000 claims abstract description 77
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 77
- 241000588724 Escherichia coli Species 0.000 claims abstract description 66
- 101150064873 ispA gene Proteins 0.000 claims abstract description 39
- 108090000364 Ligases Proteins 0.000 claims abstract description 28
- 102100035111 Farnesyl pyrophosphate synthase Human genes 0.000 claims abstract description 25
- 108010026318 Geranyltranstransferase Proteins 0.000 claims abstract description 25
- 102000003960 Ligases Human genes 0.000 claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 230000037361 pathway Effects 0.000 claims abstract description 11
- 239000013612 plasmid Substances 0.000 claims description 57
- 238000000855 fermentation Methods 0.000 claims description 42
- 230000004151 fermentation Effects 0.000 claims description 42
- 239000001963 growth medium Substances 0.000 claims description 33
- 230000004927 fusion Effects 0.000 claims description 30
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 28
- 101150075592 idi gene Proteins 0.000 claims description 28
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 27
- 108700040132 Mevalonate kinases Proteins 0.000 claims description 25
- 102000002678 mevalonate kinase Human genes 0.000 claims description 23
- 229960000723 ampicillin Drugs 0.000 claims description 22
- 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 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 239000013604 expression vector Substances 0.000 claims description 20
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 claims description 19
- 239000000843 powder Substances 0.000 claims description 19
- 101150096918 mvaD gene Proteins 0.000 claims description 17
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 16
- 238000011218 seed culture Methods 0.000 claims description 16
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 claims description 15
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 claims description 15
- 108010065958 Isopentenyl-diphosphate Delta-isomerase Proteins 0.000 claims description 15
- 101150063369 mvaS gene Proteins 0.000 claims description 15
- 108091000116 phosphomevalonate kinase Proteins 0.000 claims description 14
- CABVTRNMFUVUDM-VRHQGPGLSA-N (3S)-3-hydroxy-3-methylglutaryl-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[C@@](O)(CC(O)=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 CABVTRNMFUVUDM-VRHQGPGLSA-N 0.000 claims description 13
- 108010006229 Acetyl-CoA C-acetyltransferase Proteins 0.000 claims description 13
- 102100037768 Acetyl-CoA acetyltransferase, mitochondrial Human genes 0.000 claims description 13
- 102000057412 Diphosphomevalonate decarboxylases Human genes 0.000 claims description 13
- 108090000895 Hydroxymethylglutaryl CoA Reductases Proteins 0.000 claims description 13
- 102100027665 Isopentenyl-diphosphate Delta-isomerase 1 Human genes 0.000 claims description 13
- 101000958906 Panax ginseng Diphosphomevalonate decarboxylase 2 Proteins 0.000 claims description 13
- 108010049285 dephospho-CoA kinase Proteins 0.000 claims description 13
- 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 12
- 102100024279 Phosphomevalonate kinase Human genes 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 238000003259 recombinant expression Methods 0.000 claims description 12
- 102100037458 Dephospho-CoA kinase Human genes 0.000 claims description 11
- 102000004286 Hydroxymethylglutaryl CoA Reductases Human genes 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 9
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- 229940094933 n-dodecane Drugs 0.000 claims description 9
- 239000012137 tryptone Substances 0.000 claims description 9
- 241000193998 Streptococcus pneumoniae Species 0.000 claims description 8
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 8
- 229940031000 streptococcus pneumoniae Drugs 0.000 claims description 8
- 108700026220 vif Genes Proteins 0.000 claims description 6
- 108091081024 Start codon Proteins 0.000 claims description 5
- 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 claims description 5
- 229960005091 chloramphenicol Drugs 0.000 claims description 5
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 235000007866 Chamaemelum nobile Nutrition 0.000 claims description 3
- 108020004705 Codon Proteins 0.000 claims description 3
- 235000007232 Matricaria chamomilla Nutrition 0.000 claims description 3
- WLMRFQRPGSAEDX-UHFFFAOYSA-N 3-methylbut-1-ene phosphono dihydrogen phosphate Chemical compound OP(O)(=O)OP(=O)(O)O.C=CC(C)C WLMRFQRPGSAEDX-UHFFFAOYSA-N 0.000 claims description 2
- 102000004031 Carboxy-Lyases Human genes 0.000 claims description 2
- 108090000489 Carboxy-Lyases Proteins 0.000 claims description 2
- 241000194032 Enterococcus faecalis Species 0.000 claims description 2
- 108090000769 Isomerases Proteins 0.000 claims description 2
- 102000004195 Isomerases Human genes 0.000 claims description 2
- 241000205274 Methanosarcina mazei Species 0.000 claims description 2
- 108020005038 Terminator Codon Proteins 0.000 claims description 2
- 229940032049 enterococcus faecalis Drugs 0.000 claims description 2
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 229940005657 pyrophosphoric acid Drugs 0.000 claims description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims 2
- 240000003538 Chamaemelum nobile Species 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 20
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 108091026890 Coding region Proteins 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 62
- 239000000047 product Substances 0.000 description 60
- 239000012634 fragment Substances 0.000 description 39
- 238000000246 agarose gel electrophoresis Methods 0.000 description 14
- 238000012795 verification Methods 0.000 description 12
- 206010072219 Mevalonic aciduria Diseases 0.000 description 10
- 241001183012 Modified Vaccinia Ankara virus Species 0.000 description 10
- 239000007787 solid Substances 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 8
- 229910052799 carbon Inorganic materials 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000002194 synthesizing effect Effects 0.000 description 8
- 230000009466 transformation Effects 0.000 description 8
- 101100397224 Bacillus subtilis (strain 168) isp gene Proteins 0.000 description 7
- 101100052502 Shigella flexneri yciB gene Proteins 0.000 description 7
- 230000001131 transforming effect Effects 0.000 description 7
- 238000010367 cloning Methods 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 238000012807 shake-flask culturing Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- WTVHAMTYZJGJLJ-UHFFFAOYSA-N (+)-(4S,8R)-8-epi-beta-bisabolol Natural products CC(C)=CCCC(C)C1(O)CCC(C)=CC1 WTVHAMTYZJGJLJ-UHFFFAOYSA-N 0.000 description 5
- 101100286286 Dictyostelium discoideum ipi gene Proteins 0.000 description 4
- 101100520453 Haloferax volcanii (strain ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2) mvaD gene Proteins 0.000 description 4
- HHGZABIIYIWLGA-UHFFFAOYSA-N bisabolol Natural products CC1CCC(C(C)(O)CCC=C(C)C)CC1 HHGZABIIYIWLGA-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 101150014423 fni gene Proteins 0.000 description 4
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 4
- 239000010413 mother solution Substances 0.000 description 4
- 239000000341 volatile oil Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 244000042664 Matricaria chamomilla Species 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 101710204829 Alpha-bisabolol synthase Proteins 0.000 description 2
- 235000007516 Chrysanthemum Nutrition 0.000 description 2
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 2
- 241000588914 Enterobacter Species 0.000 description 2
- 101100507308 Enterococcus faecalis mvaS gene Proteins 0.000 description 2
- 241000205276 Methanosarcina Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000013329 compounding Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- WTVHAMTYZJGJLJ-LSDHHAIUSA-N β-bisabolol Chemical compound CC(C)=CCC[C@H](C)[C@]1(O)CCC(C)=CC1 WTVHAMTYZJGJLJ-LSDHHAIUSA-N 0.000 description 2
- 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 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000219496 Alnus Species 0.000 description 1
- 108010075254 C-Peptide Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- VWFJDQUYCIWHTN-UHFFFAOYSA-N Farnesyl pyrophosphate Natural products CC(C)=CCCC(C)=CCCC(C)=CCOP(O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-UHFFFAOYSA-N 0.000 description 1
- 235000004429 Matricaria chamomilla var recutita Nutrition 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940074775 beta-bisabolol Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000037307 sensitive skin Effects 0.000 description 1
- 229930004725 sesquiterpene Natural products 0.000 description 1
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229930000053 β-bisabolol Natural products 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
- C12N15/70—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
- 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.)
- C12N9/1025—Acyltransferases (2.3)
-
- 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.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
-
- 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)
- C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
-
- 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)
- C12Y101/01088—Hydroxymethylglutaryl-CoA reductase (1.1.1.88)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01009—Acetyl-CoA C-acetyltransferase (2.3.1.9)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
- C12Y203/0301—Hydroxymethylglutaryl-CoA synthase (2.3.3.10)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/0101—(2E,6E)-Farnesyl diphosphate synthase (2.5.1.10), i.e. geranyltranstransferase
-
- 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)
- C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
- C12Y207/04002—Phosphomevalonate kinase (2.7.4.2)
-
- 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)
- C12Y401/01033—Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
-
- 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)
- C12Y402/03138—(+)-Epi-alpha-bisabolol synthase (4.2.3.138)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/03—Intramolecular oxidoreductases (5.3) transposing C=C bonds (5.3.3)
- C12Y503/03002—Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种产(‑)‑α‑红没药醇的重组基因工程菌，它是包含(‑)‑α‑红没药醇合成酶MrBBS基因、法尼基二磷酸合酶ispA基因、MVA途径基因的重组大肠杆菌；所述(‑)‑α‑红没药醇合成酶MrBBS基因与法尼基二磷酸合酶ispA基因之间通过SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列连接。本发明通过将特定的基因重组入大肠杆菌，同时在重组大肠杆菌中将MrBBS基因、ispA基因之间通过短肽编码序列连接，并过表达相关基因，使之在生产过程的摇瓶阶段产量就高达6.8g/L，适宜实际推广应用。
Description
技术领域
本发明属于基因重组发酵技术领域，具体涉及一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途。
背景技术
红没药醇是一种有机化合物，分子式为C15H26O，是一种存在天然精油中无毒的倍半萜烯醇；在自然界中以两种结构形式存在：β和α。β-红没药醇是从玉米和棉花中收获的，主要用作调味剂。(-)-α-红没药醇主要存在于春黄菊精油中，含量达17％，其右旋体存在于胶杨精油以及某些苦槛蓝属和鼠尾草属的精油中，具有消炎、灭菌、愈合溃疡、溶解胆石等药效，故在医药行业中的用途较广。
(-)-α-红没药醇能够保护和治愈皮肤，使其免受日常张力的影响，能够加速皮肤的治愈过程，尤其适用于作为敏感皮肤和身体，被广泛应用于个人护肤(皮肤和身体的护理液、须后水和晒后护理产品)的配方中，加上其抗炎、天然、安全特性，使其成为一种用于皮肤护理的常用活性成分。国际上仅个人护理产品上的应用年需求量达到400t以上，逐渐成为个人护肤品原料新宠；此外，(-)-α-红没药醇香气清淡愉快，也是一种稳定性较好的定香剂，在香料香精中的应用也日益受到重视；然而，(-)-α-红没药醇作为次级代谢产物，纯天然从植物提取的量俨然无法满足市场的需求，因其受制于生物生长周期，环境等各种因素，扩大生产规模会导致生态环境的破坏甚至物种灭绝；若采用化学合成方法，α-红没药醇复杂手性化学结构，使得直接化学合成难度高，生物活性低以及纯度低等弊端。因此，利用合成生物学构建工程菌实现以廉价的碳源和培养基生产具有高附加值的(-)-α-红没药醇是一条最具有潜力的途径。
目前文献报道中，利用基因工程菌生产(-)-α-红没药醇，发酵罐取得一定成果，但摇瓶产量并不理想；2016年Gui Hwan Han等人以大肠杆菌为宿主细胞，通过质粒过表达德国洋甘菊来源的(-)-α-红没药醇合成酶MrBBS、大肠杆菌来源的ispA以及外源性MVA途径，从而获得最终产(-)-α-红没药醇基因工程菌，发酵48h后产量仅为0.08g/L；CN110016458A公开了一种利用基因工程菌生产(-)-α-红没药醇的方法，其虽然摇瓶产量达到4.15g/L，但还有进一步提高的空间，使发酵罐放大生产后的产量呈倍数级提高。
发明内容
为解决上述问题，本发明提供了一种产(-)-α-红没药醇的重组基因工程菌，它是包含(-)-α-红没药醇合成酶MrBBS基因、法尼基二磷酸合酶ispA基因、MVA途径基因的重组大肠杆菌；所述(-)-α-红没药醇合成酶MrBBS基因与法尼基二磷酸合酶ispA基因通过SEQID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列连接。
进一步地，所述(-)-α-红没药醇合成酶MrBBS基因中的终止密码子TAA被SEQ IDNO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列代替，并与无起始密码子ATG的法尼基二磷酸合酶ispA基因连接。
进一步地，所述(-)-α-红没药醇合成酶MrBBS基因5’端带有核苷酸序列SEQ IDNO.1。
更进一步地，所述(-)-α-红没药醇合成酶MrBBS基因来自春黄菊花；所述法尼基二磷酸合酶ispA基因来自大肠杆菌。
更进一步地，所述(-)-α-红没药醇合成酶MrBBS基因的核苷酸序列如SEQ IDNO.12所示；所述法尼基二磷酸合酶ispA基因的核苷酸序列如SEQ ID NO.13所示。
更进一步地，所述(-)-α-红没药醇合成酶MrBBS基因与法尼基二磷酸合酶ispA基因连接后的核苷酸序列如SEQ ID NO.52、SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55或SEQ ID NO.56所示。
进一步地，所述MVA途径基因包括甲羟戊酸激酶mvaKmm基因、甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因、异戊烯基二磷酸δ-异构酶idi基因、3-羟基-3-甲基戊二酰CoA合酶mvaS基因、乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因和/或甲羟戊酸激酶mvaK1基因。
更进一步地，所述甲羟戊酸激酶mvaKmm基因来自甲烷八叠球古菌Methanosarcinamazei；
所述甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因和甲羟戊酸激酶mvaK1基因来自肺炎链球菌Streptococcus pneumoniae；
所述异戊烯二磷酸δ异构酶idi基因来自大肠杆菌Escherichia coli；
所述3-羟基-3-甲基戊二酰CoA合酶mvaS基因和乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因来自粪肠球菌Enterococcus faecalis。
更进一步地，所述甲羟戊酸激酶mvaKmm基因的核苷酸序列如SEQ ID NO.14所示，甲羟戊酸5-焦磷酸脱羧酶mvaD基因的核苷酸序列如SEQ ID NO.15所示，磷酸甲羟戊酸激酶mvaK2基因的核苷酸序列如SEQ ID NO.16所示，异戊烯二磷酸δ异构酶idi基因的核苷酸序列如SEQ ID NO.17所示，3-羟基-3-甲基戊二酰CoA合酶mvaS基因的核苷酸序列如SEQ IDNO.18所示，乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因的核苷酸序列如SEQ ID NO.19所示，甲羟戊酸激酶mvaK1基因的核苷酸序列如SEQ ID NO.20所示。
更进一步地，所述异戊烯基二磷酸δ-异构酶idi基因5’端带有核苷酸序列SEQ IDNO.3，乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因5’端带有核苷酸序列SEQ ID NO.4，3-羟基-3-甲基戊二酰CoA合酶mvaS基因5’端带有核苷酸序列SEQ ID NO.5，甲羟戊酸激酶mvaKmm基因5’端带有核苷酸序列SEQ ID NO.6。
更进一步地，所述MVA途径基因与(-)-α-红没药醇合成酶MrBBS基因和法尼基二磷酸合酶ispA基因连接在两个质粒上，其中一个质粒连接的核苷酸序列包括SEQ ID NO.52、SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55或SEQ ID NO.56，以及SEQ ID NO.50，另一个质粒连接的核苷酸序列包括SEQ ID NO.51。
更进一步地，所述质粒优选为质粒pSTV28和质粒pTrc99A。
进一步地，所述重组大肠杆菌为重组大肠杆菌E.coli DH5α或E.coli W3110。
本发明还提供了一种前述重组基因工程菌的制备方法，它包括如下步骤：
1)取(-)-α-红没药醇合成酶MrBBS基因和法尼基二磷酸合酶ispA基因融合，融合产物与线性化表达载体连接，再导入大肠杆菌，提取重组表达载体；
所述(-)-α-红没药醇合成酶MrBBS基因中的终止密码子TAA被如SEQ ID NO.7、SEQID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列代替；
所述法尼基二磷酸合酶ispA基因中的起始密码子ATG被如SEQ ID NO.7、SEQ IDNO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列代替；
2)取MVA途径基因融合，融合产物与酶切后的步骤1)所得重组表达载体连接，再导入大肠杆菌，提取重组表达载体；
3)取甲羟戊酸激酶mvaK1基因，与包含甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因和异戊烯基二磷酸δ-异构酶idi基因的基因片段融合，融合产物与线性化表达载体连接，再导入大肠杆菌，提取重组表达载体；
4)将步骤2)所得重组表达载体和步骤3)所得重组表达载体，导入大肠杆菌中，即得重组基因工程菌。
进一步地，步骤1)所述表达载体为质粒pSTV28。
进一步地，步骤2)所述取MVA途径基因融合是取包含甲羟戊酸激酶mvaKmm基因、甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因和异戊烯基二磷酸δ-异构酶idi基因的基因片段，与包含3-羟基-3-甲基戊二酰CoA合酶mvaS基因和乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因的基因片段融合。
进一步地，步骤3)所述表达载体为质粒pTrc99A。
本发明还提供了一种前述的重组基因工程菌在制备(-)-α-红没药醇及其制剂中的用途。
本发明最后提供了一种产(-)-α-红没药醇的方法，它包括如下步骤：
取前述的重组基因工程菌，接种于种子培养基，培养8～10h，取种子液，接种于发酵培养基，加正十二烷，发酵培养30～60h，分离纯化即得；
所述种子培养基的配方为：胰蛋白胨5～15g/L、酵母粉2～8g/L、氯化钠5～15g/L、氨苄青霉素终浓度50～150mg/L和氯霉素终浓度30～40mg/L；
所述发酵培养基的配方为:葡萄糖或甘油5～15g/L、磷酸二氢钾2～3g/L、磷酸氢二钾2.5～3.0g/L、酵母粉20～28g/L、酵母蛋白胨10～20g/L、IPTG0.1～0.2mM、氨苄青霉素终浓度50～150mg/L和氯霉素终浓度为30～38mg/L。
进一步地，所述种子液、发酵培养基、正十二烷的体积比为2：25：5；所述培养为振荡培养，温度30℃，转速200rpm；所述发酵培养到3h添加培养容器容量4～8×10-4的0.25MIPTG。
进一步地，所述种子培养基的配方为：胰蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L、氨苄青霉素终浓度100mg/L和氯霉素终浓度34mg/L；
所述发酵培养基的配方为葡萄糖或甘油10g/L、磷酸二氢钾2.2g/L、磷酸氢二钾2.9g/L、酵母粉24g/L、酵母蛋白胨12g/L、IPTG 0.1mM、氨苄青霉素终浓度100mg/L和氯霉素终浓度为34mg/L。
本发明一种产(-)-α-红没药醇的基因工程菌，通过将特定的基因如mvaKmm等MVA途径基因重组入大肠杆菌，同时在重组大肠杆菌中将MrBBS基因、ispA基因之间通过短肽编码序列连接，并过表达mvaK2、mvaD、idi等基因，使之在生产过程的摇瓶阶段产量就高达6.8g/L，再经发酵罐放大后的产量相对目前公开报道的产量进一步突破新高，适宜实际推广应用。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。
附图说明
图1pSTV28-2质粒图谱
图2pTrc99A-1质粒图谱
图3pSTV28-24质粒图谱
图4 E.coli中(-)-α-红没药醇合成代谢图
图5实施的大肠杆菌工程菌株合成(-)-α-红没药醇的GC-MS分析(A,GC-MS图，B,产量柱状图)
图6重组大肠杆菌在摇瓶上培养50h后(-)-α-红没药醇积累量
图7不同连接短肽对(-)-α-红没药醇合成量的影响
具体实施方式
本发明涉及的核苷酸序列信息
(一)RBS序列：
MrBBS基因5’端的RBS(SEQ ID NO.1)：GGTTAAACC
ispA基因5’端的RBS(SEQ ID NO.2)：aaggaggttacggaaa
idi基因5’端的RBS(SEQ ID NO.3)：aggagagaaatt
mvaE基因5’端的RBS(SEQ ID NO.4)：AGGAGCATTTAG
mvaS基因5’端的RBS(SEQ ID NO.5)：AGGAGAAACCTT
(二)启动子序列
mvaKmm基因5’端带有的pLac(SEQ ID NO.6)：
TAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGGGATCC
(三)连接短肽序列
分别表达(-)-α-红没药醇合成酶和法尼基二磷酸合酶融合酶连接肽的核苷酸序列：SEQ ID NO.7:CCAACGACGACGACGCCA
SEQ ID NO.8:GGAGGAGGAGGATCATCATCA
SEQ ID NO.9:GGAGGAGGAATC
SEQ ID NO.10:GGAAGCGGAGGA
SEQ ID NO.11:GGAGGAATCGGA
(四)功能基因序列
(-)-α-红没药醇合成酶MrBBS基因的核苷酸序列(SEQ ID NO.12)：
atgagcacactgagcgtcagcaccccgagctttagcagcagccctctgtcgagcgtgaataagaacagcaccaagcagcatgtcactcgtaacagcgtgatctttcacgactcgatttggggggaccagttcctggaatacaaagagaaattcaacgttgcaaccgagaaacagcttatagaagagctgaaagaagaagtgcgtaacgaactgatgattcgtgcatgtaatgaagcgagccggtatatcaaactgatccagctgatcgatgttgttgaacgtctggggctggcctatcattttgaaaaagagattgaggaaagcctccagcatatatatgtgacgtatggtcataaatggacgaattacaacaatattgagagcctgagtctgtggttccgcctgcttcgtcaaaatggctttaatgttagctcggatatatttgaaaatcacattgatgagaaaggaaattttcaggagagcctgtgcaatgatccgcaggggatgctggcgctgtatgaagcggcatatatgcgtgttgaaggagagatcattctggacaaagcactcgaatttaccaagctgcatctggggatcattagcaatgatcctagctgtgatagcagcctacgtacggaaatcaagcaggcactgaaacagccactgcgccggcggctgccaaggctggaagccgttcgttacattgccatttatcagcagaaggcgagccatagcgaggttctgctgaagctggccaaactggacttcaacgttctgcaggaaatgcacaaagacgaattgagccaaatatgcaaatggtggaaagatctggatatacgtaacaaactgccctatgttcgtgatcgtctgattgaaggctatttttggattctgggtatttatttcgaaccgcaacactcccgtacccgtatgttcctgatgaaaacctgtatgtggctgatcgtgctggacgatacgtttgataattacggcacctatgaagagttagagatctttacccaagcagtcgaacgttggagcattacctgtctggatgaactgccagagtatatgaagctgatatatcacgagcaatttcgcgtgcatcaggaaatggaggaaagcctggaaaaggagggtaaggcctaccagattcattatatcaaagaaatggccaaagaaggtactcgttcgctgctgctggaagcgaaatggctgaaggaaggctatatgcctaccctggatgagtacctgagcaacagcctggtcacctgcggctatgcactgatgaccgcacgcagctacgttgcccgtgacgacggcattgttaccgaagatgcattcaaatgggttgcaacgcacccgccgattgttaaagcagcatgcaaaattctgcgcctgatggacgacattgcaacccataaagaggaacaggagcggggacacattgcaagtagcattgagtgttacaggaaggaaaccggagctagcgaagaggaggcttgcatggactttctgaagcaggttgaagatggttggaaagttattaatcaagaaagcctgatgccgaccgatgttccgttccctctgctgattccggcaattaacctggcacgtgtgagcgacaccctgtacaaagacaacgatggttataatcatgccgataaagaggttataggttatattaaaagcctgtttgtacatccgatgatagtctaa
法尼基二磷酸合酶ispA基因的核苷酸序列(SEQ ID NO.13)：
atggactttccgcagcaactcgaagcctgcgttaagcaggccaaccaggcgctgagccgttttatcgccccactgccctttcagaacactcccgtggtcgaaaccatgcagtatggcgcattattaggtggtaagcgcctgcgacctttcctggtttatgccaccggtcatatgttcggcgttagcacaaacacgctggacgcacccgctgccgccgttgagtgtatccacgcttactcattaattcatgatgatttaccggcaatggatgatgacgatctgcgtcgcggtttgccaacctgccatgtgaagtttggcgaagcaaacgcgattctcgctggcgacgctttacaaacgctggcgttctcgattttaagcgatgccgatatgccggaagtgtcggaccgcgacagaatttcgatgatttctgaactggcgagcgccagtggtattgccggaatgtgcggtggtcaggcattagatttagacgcggaaggcaaacacgtacctctggacgcgcttgagcgtattcatcgtcataaaaccggcgcattgattcgcgccgccgttcgccttggtgcattaagcgccggagataaaggacgtcgtgctctgccggtactcgacaagtatgcagagagcatcggccttgccttccaggttcaggatgacatcctggatgtggtgggagatactgcaacgttgggaaaacgccagggtgccgaccagcaacttggtaaaagtacctaccctgcacttctgggtcttgagcaagcccggaagaaagcccgggatctgatcgacgatgcccgtcagtcgctgaaacaactggctgaacagtcactcgatacctcggcactggaagcgctagcggactacatcatccagcgtaataaataa
甲羟戊酸激酶mvaKmm基因的核苷酸序列(SEQ ID NO.14)：
atggtgagctgcagcgcgccgggcaaaatttatctgtttggcgaacatgcggtggtgtatggcgaaaccgcgattgcgtgcgcggtggaactgcgcacccgcgtgcgcgcggaactgaacgatagcattaccattcagagccagattggccgcaccggcctggattttgaaaaacatccgtatgtgagcgcggtgattgaaaaaatgcgcaaaagcattccgattaacggcgtgtttctgaccgtggatagcgatattccggtgggcagcggcctgggcagcagcgcggcggtgaccattgcgagcattggcgcgctgaacgaactgtttggctttggcctgagcctgcaggaaattgcgaaactgggccatgaaattgaaattaaagtgcagggcgcggcgagcccgaccgatacctatgtgagcacctttggcggcgtggtgaccattccggaacgccgcaaactgaaaaccccggattgcggcattgtgattggcgataccggcgtgtttagcagcaccaaagaactggtggcgaacgtgcgccagctgcgcgaaagctatccggatctgattgaaccgctgatgaccagcattggcaaaattagccgcattggcgaacagctggtgctgagcggcgattatgcgagcattggccgcctgatgaacgtgaaccagggcctgctggatgcgctgggcgtgaacattctggaactgagccagctgatttatagcgcgcgcgcggcgggcgcgtttggcgcgaaaattaccggcgcgggcggcggcggctgcatggtggcgctgaccgcgccggaaaaatgcaaccaggtggcggaagcggtggcgggcgcgggcggcaaagtgaccattaccaaaccgaccgaacagggcctgaaagtggattaa
甲羟戊酸5-焦磷酸脱羧酶mvaD基因的核苷酸序列(SEQ ID NO.15)：
atggatagagagcctgtaacagtacgttcctacgcaaatattgctattatcaaatattggggaaagaaaaaagaaaaagagatggtgcctgctactagcagtatttctctaactttggaaaatatgtatacagagacgaccttgtcgcctttaccagccaatgtaacagctgacgaattttacatcaatggtcagctacaaaatgaggtcgagcatgccaagatgagtaagattattgaccgttatcgtccagctggtgagggctttgtccgtatcgatactcaaaacaatatgcctactgcagcgggcctgtcctcaagttctagtggtttgtccgccctggtcaaggcttgtaatgcttatttcaagcttggattggatagaagtcagttggcacaggaagccaaatttgcctcaggctcttcttctcggagtttttatggaccactaggagcctgggataaggatagtggagaaatttaccctgtagagacagacttgaaactagctatgattatgttggtgctagaggacaagaaaaaaccaatctctagccgtgacgggatgaaactttgtgtggaaacctcgacgacttttgacgactgggttcgtcagtctgagaaggactatcaggatatgctgatttatctcaaggaaaatgattttgccaagattggagaattaacggagaaaaatgccctggctatgcatgctacgacaaagactgctagtccagccttttcttatctgacggatgcctcttatgaggctatggactttgttcgccagcttcgtgagaaaggagaggcctgctactttaccatggatgctggtcccaatgttaaggtcttctgtcaggagaaagacttggagcatttatcagaaattttcggtcatcgttatcgcttgattgtgtcaaaaacaaaggatttgagtcaagatgattgctgttaa
磷酸甲羟戊酸激酶mvaK2基因的核苷酸序列(SEQ ID NO.16)：
atgattgctgttaaaacttgcggaaaactctattgggcaggtgaatatgctattttagagccagggcagttagctttgataaaggatattcccatctatatgagggctgagattgctttttctgacagctaccgtatctattcagatatgtttgatttcgcagtggacttaaggcctaatcctgactacagcttgattcaagaaacgattgctttgatgggagacttcctcgctgttcgtggtcagaatttaagacctttttctctagaaatctgtggcaaaatggaacgagaagggaaaaagtttggtctaggttctagtggcagcgtcgttgtcttggttgtcaaggctttactggctctgtatgatgtttctgttgatcaggagctcttgttcaagctgactagcgctgtcttgctcaagcgaggagacaatggttccatgggcgaccttgcctgtattgtggcagaggatttggttctctaccagtcatttgatcgccagaaggtggctgcttggttagaagaagaaaacttggcgacagttctggagcgtgattggggcttttcaatttcacaagtgaaaccaactttagaatgtgatttcttagtgggatggaccaaggaagtggctgtatcgagtcacatggtccagcaaatcaagcaaaatatcaatcaaaattttttaagttcctcaaaagaaacggtggtttctttggtcgaagccttggaacaggggaaatcagaaaagattatcgagcaagtagaagtagccagcaagcttttagaaggcttgagtacagatatttacacgcctttgcttagacagttgaaagaagccagtcaagatttgcaggccgttgccaagagtagtggtgctggtggtggtgactgtggcatcgccctgagttttgatgcgcaatcaaccaaaaccttaaaaaatcgttgggccgatctggggattgagctcttatatcaagaaaggataggacatgacgacaaatcgtaa
异戊烯基二磷酸δ-异构酶idi基因的核苷酸序列(SEQ ID NO.17)：
atgcaaacggaacacgtcattttattgaatgcacagggagttcccacgggtacgctggaaaagtatgccgcacacacggcagacacccgcttacatctcgcgttctccagttggctgtttaatgccaaaggacaattattagttacccgccgcgcactgagcaaaaaagcatggcctggcgtgtggactaactcggtttgtgggcacccacaactgggagaaagcaacgaagacgcagtgatccgccgttgccgttatgagcttggcgtggaaattacgcctcctgaatctatctatcctgactttcgctaccgcgccaccgatccgagtggcattgtggaaaatgaagtgtgtccggtatttgccgcacgcaccactagtgcgttacagatcaatgatgatgaagtgatggattatcaatggtgtgatttagcagatgtattacacggtattgatgccacgccgtgggcgttcagtccgtggatggtgatgcaggcgacaaatcgcgaagccagaaaacgattatctgcatttacccagcttaaataa
3-羟基-3-甲基戊二酰CoA合酶mvaS基因的核苷酸序列(SEQ ID NO.18)：
atgacaattgggattgataaaattagtttttttgtgcccccttattatattgatatgacggcactggctgaagccagaaatgtagaccctggaaaatttcatattggtattgggcaagaccaaatggcggtgaacccaatcagccaagatattgtgacatttgcagccaatgccgcagaagcgatcttgaccaaagaagataaagaggccattgatatggtgattgtcgggactgagtccagtatcgatgagtcaaaagcggccgcagttgtcttacatcgtttaatggggattcaacctttcgctcgctctttcgaaatcaaggaagcttgttacggagcaactgcaggcttacagttagctaagaatcacgtagccttacatccagataaaaaagtcttggtcgtagcagcagatattgcaaaatatggcttaaattctggcggtgagcctacacaaggagctggggcggttgcaatgttagttgctagtgaaccgcgcattttggctttaaaagaggataatgtgatgctgacgcaagatatctatgacttttggcgtccaacaggccatccatatcctatggtcgatggtcctttgtcaaacgaaacctacatccaatcttttgcccaagtctgggatgaacataaaaaacgaaccggtcttgattttgcagattatgatgctttagcgttccatattccttacacaaaaatgggcaaaaaagccttattagcaaaaatctccgaccaaactgaagcagaacaggaacgaattttagcccgttatgaagaaagcatcatctatagtcgtcgcgtaggaaacttgtatacgggttcactttatctgggactcatttcccttttagaaaatgcaacgactttaaccgcaggcaatcaaattgggttattcagttatggttctggtgctgtcgctgaatttttcactggtgaattagtagctggttatcaaaatcatttacaaaaagaaactcatttagcactgctggataatcggacagaactttctatcgctgaatatgaagccatgtttgcagaaactttagacacagacattgatcaaacgttaaaagatgaattaaaatatagtatttctgctattaataataccgttcgttcttatcgaaactaa
乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因的核苷酸序列(SEQ ID NO.19)：
atgaaaacagtagttattattgatgcattacgaacaccaattggaaaatataaaggcagcttaagtcaagtaagtgccgtagacttaggaacacatgttacaacacaacttttaaaaagacattccactatttctgaagaaattgatcaagtaatctttggaaatgttttacaagctggaaatggccaaaatcccgcacgacaaatagcaataaacagcggtttatctcatgaaattcccgcaatgacagttaatgaggtctgcggatcaggaatgaaggccgttattttggcgaaacaattgattcaattaggagaagcggaagttttaattgctggcgggattgagaatatgtcccaagcacctaaattacaacgatttaattacgaaacagaaagctatgatgcgcctttttctagtatgatgtacgatgggttaacggatgcctttagtggtcaagcaatgggcttaactgctgaaaatgtggccgaaaagtatcatgtaactagagaagagcaagatcaattttctgtacattcacaattaaaagcagctcaagcacaagcagaagggatattcgctgacgaaatagccccattagaagtatcaggaacgcttgtggagaaagatgaagggattcgccctaattcgagcgttgagaagctaggaacgcttaaaacagtttttaaagaagacggtactgtaacagcagggaatgcatcaaccattaatgatggggcttctgctttgattattgcttcacaagaatatgccgaagcacacggtcttccttatttagctattattcgagacagtgtggaagtcggtattgatccagcctatatgggaatttcgccgattaaagccattcaaaaactgttagcgcgcaatcaacttactacggaagaaattgatctgtatgaaatcaacgaagcatttgcagcaacttcaatcgtggtccaaagagaactggctttaccagaggaaaaggtcaacatttatggtggcggtatttcattaggtcatgcgattggtgccacaggtgctcgtttattaacgagtttaagttatcaattaaatcaaaaagaaaagaaatatggagtggcttctttatgtatcggcggtggcttaggactcgctatgctactagagagacctcagcaaaaaaaaaacagccgattttatcaaatgagtcctgaggaacgcctggcttctcttcttaatgaaggccagatttctgctgatacaaaaaaagaatttgaaaatacggctttatcttcgcagattgccaatcatatgattgaaaatcaaatcagtgaaacagaagtgccgatgggcgttggcttacatttaacagtggacgaaactgattatttggtaccaatggcgacagaagagccctcagtgattgcggctttgagtaatggtgcaaaaatagcacaaggatttaaaacagtgaatcaacaacgtttaatgcgtggacaaatcgttttttacgatgttgcagacgccgagtcattgattgatgaactacaagtaagagaaacggaaatttttcaacaagcagagttaagttatccatctatcgttaaacgcggcggcggcttaagagatttgcaatatcgtgcttttgatgaatcatttgtatctgtcgactttttagtagatgttaaggatgcaatgggggcaaatatcgttaacgctatgttggaaggtgtggccgagttgttccgtgaatggtttgcggagcaaaagattttattcagtattttaagtaattatgccacggagtcggttgttacgatgaaaacggctattccagtttcacgtttaagtaaggggagcaatggccgggaaattgctgaaaaaattgttttagcttcacgctatgcttcattagatccttatcgggcagtcacgcataacaaagggatcatgaatggcattgaagctgtcgttttagctacaggaaatgatacacgcgctgttagcgcttcttgtcatgcttttgcggtgaaggaaggtcgctaccaaggtttgactagttggacgctggatggcgaacaactaattggtgaaatttcagttccgcttgcgttagccacggttggcggtgccacaaaagtcttacctaaatctcaagcagctgctgatttgttagcagtgacggatgcaaaagaactaagtcgagtagtagcggctgttggtttggcacaaaatttagcggcgttacgggccttagtctctgaaggaattcaaaaaggacacatggctctacaagcacgttctttagcgatgacggtcggagctactggtaaagaagttgaggcagtcgctcaacaattaaaacgtcaaaaaacgatgaaccaagaccgagccttggctattttaaatgatttaagaaaacaataa
甲戊酸激酶mvaK1基因的核苷酸序列(SEQ ID NO.20)：
ATGACAAAAAAAGTTGGTGTCGGTCAGGCACATAGTAAGATAATTTTAATAGGGGAACATGCGGTCGTTTACGGTTATCCTGCCATTTCCCTGCCTCTTTTGGAGGTGGAGGTGACCTGTAAGGTAGTTCCTGCAGAGAGTCCTTGGCGCCTTTATGAGGAGGATACCTTGTCCATGGCGGTTTATGCCTCACTGGAGTATTTGGATATCACAGAAGCCTGCATTCGTTGTGAGATTGACTCGGCTATCCCTGAGAAACGGGGGATGGGTTCGTCAGCGGCTATCAGCATAGCGGCCATTCGTGCGGTATTTGACTACTATCAGGCTGATCTGCCTCATGATGTACTAGAAATCTTGGTCAATCGAGCTGAAATGATTGCCCATATGAATCCTAGTGGTTTGGATGCTAAGACCTGTCTCAGTGACCAACCTATTCGCTTTATCAAGAACGTAGGATTTACAGAACTTGAGATGGATTTATCCGCCTATTTGGTGATTGCCGATACGGGTGTTTATGGTCATACTCGTGAAGCCATCCAAGTGGTTCAAAATAAGGGCAAGGATGCCCTACCGTTTTTGCATGCCTTGGGAGAATTAACCCAGCAAGCAGAAGTTGCGATTTCACAAAAAGATGCTGAAGGACTGGGACAAATCCTCAGTCAAGCGCATTTACATTTAAAAGAAATTGGAGTCAGTAGCCCTGAGGCAGACTTTTTGGTTGAAACGACTCTTAGCCATGGTGCTCTGGGTGCCAAGATGAGCGGTGGTGGGCTAGGAGGTTGTATCATAGCCTTGGTAACCAATTTGACACACGCACAAGAACTAGCAGAAAGATTAGAAGAGAAAGGAGCTGTTCAGACATGGATAGAGAGCCTGTAA
五、涉及到的引物信息
表1引物的核苷酸序列
| 名称 | 引物 | SEQ ID NO |
| MrBBs-F | ATTTCACACAGGAAACAGCTGGTTAAACCATGAGCACACTGAGCGTCAG | 21 |
| MrBBs-R | AGTTGCTGCGGAAAGTCCATTTTCCGTAACCTCCTTGGATCCTTAGACTATCATCGGATGTA | 22 |
| ispA-F | TACATCCGATGATAGTCTAAGGATCCAAGGAGGTTACGGAAAATGGACTTTCCGCAGCAACT | 23 |
| ispA-R | AACTCACATTACAGGTCGACTTATTTATTACGCTGGATGA | 24 |
| pSTV28-F | ACTGGCCGTCGTTTTACAAC | 25 |
| pSTV28-R | AGCTGTTTCCTGTGTGAAAT | 26 |
| mvaKmm-F | TCATCCAGCGTAATAAATAAGTCGACCTGTAATGTGAGTT | 27 |
| idi-R | ATAATAACTACTGTTTTCATCTAAATGCTCCTTTATTTAAGCTGGGTAAATG | 28 |
| mvaES-F | CATTTACCCAGCTTAAATAAAGGAGCATTTAGATGAAAACAGTAGTTATTAT | 29 |
| mvaES-R | GTTGTAAAACGACGGCCAGTTTAGTTTCGATAAGAACGAA | 30 |
| pSTV28-1--F | ACTGGCCGTCGTTTTACAAC | 31 |
| pSTV28-1-R | TTATTTAAGCTGGGTAAATG | 32 |
| mvaK1-F | TTTCACACAGGAAACAGACCATGACAAAAAAAGTTGGTGT | 33 |
| mvaK1-R | ATTTGCGTAGGAACGTACTGTTACAGGCTCTCTATCCATG | 34 |
| mvaDK2-idi-F | CATGGATAGAGAGCCTGTAACAGTACGTTCCTACGCAAAT | 35 |
| mvaDK2-idi-R | AAAACAGCCAAGCTTGCATGTTATTTAAGCTGGGTAAATG | 36 |
| pTrc99A-F | CATGCAAGCTTGGCTGTTTT | 37 |
| pTrc99A-R | GGTCTGTTTCCTGTGTGAAA | 38 |
| MrBBS-R1 | TGGCGTCGTCGTCGTTGGGACTATCATCGGATGTACAA | 39 |
| MrBBS-R2 | TGATGATGATCCTCCTCCTCCGACTATCATCGGATGTACAA | 40 |
| MrBBS-R3 | GGAAAGTCGATTCCTCCTCCGACTATCATCGGATGTACAA | 41 |
| MrBBS-R4 | GGAAAGTCTCCTCCGATTCCGACTATCATCGGATGTACAA | 42 |
| MrBBS-R5 | GGAAAGTCTCCGATTCCTCCGACTATCATCGGATGTACAA | 43 |
| ispA-F1 | CCAACGACGACGACGCCAGACTTTCCGCAGCAACTCGA | 44 |
| ispA-F2 | GGAGGAGGAGGATCATCATCAGACTTTCCGCAGCAACTCGA | 45 |
| ispA-F3 | TGATAGTCGGAGGAGGAATCGACTTTCCGCAGCAACTCGA | 46 |
| ispA-F4 | TGATAGTCGGAAGCGGAGGAGACTTTCCGCAGCAACTCGA | 47 |
| ispA-F5 | TGATAGTCGGAGGAATCGGAGACTTTCCGCAGCAACTCGA | 48 |
六、PCR融合产物
MrBBS基因和ispA基因融合产物的核苷酸序列(SEQ ID NO.49)：
注：大写加粗部分为MrBBS基因5’端RBS序列(SEQ ID NO.1)；小写正体部分为MrBBS基因序列(SEQ ID NO.12)；小写加粗部分为ispA基因5’端RBS序列(SEQ ID NO.2)；小写划线部分ispA基因序列(SEQ ID NO.13)。
mvaKmm基因、mvaD基因、mvaK2基因、idi基因、mvaS基因、mvaE基因融合产物的核苷酸序列(SEQ ID NO.50)：
注：大写正体部分为mvaKmm基因5’端pLac序列(SEQ ID NO.6)；小写正体部分为mvaKmm基因序列(SEQ ID NO.14)；小写斜体划线部分为mvaD基因序列(SEQ ID NO.15)；小写斜体部分为mvaK2基因序列(SEQ ID NO.16)；小写加粗部分为idi基因5’端RBS序列(SEQID NO.3)，大写正体划线部分为idi基因序列(SEQ ID NO.17)；大写正体加粗部分为mvaE基因5’端RBS序列(SEQ ID NO.4)，大写正体加粗划线部分为mvaE基因序列(SEQ ID NO.19)；大写斜体部分为mvaS基因5’端RBS序列(SEQ ID NO.5)，大写斜体划线部分为mvaS基因序列(SEQ ID NO.18)。
mvaK1基因、mvaD基因、mvaK2基因和idi基因融合产物的核苷酸序列(SEQ IDNO.51)：
注：大写正体部分为mvaK1基因序列(SEQ ID NO.20)；小写正体部分为mvaD基因序列(SEQ ID NO.15)；小写划线部分为mvaK2基因序列(SEQ ID NO.16)；小写加粗斜体部分为idi基因5’端RBS序列(SEQ ID NO.3)，小写斜体部分为idi基因序列(SEQ ID NO.17)。
连接有短肽序列的MrBBS基因和ispA基因融合产物的核苷酸序列：SEQ ID NO.52：
注：大写正体部分为MrBBS基因5’端RBS序列(SEQ ID NO.1)；小写正体部分为MrBBS基因序列(SEQ ID NO.12)；大写加粗部分为连接短肽序列(SEQ ID NO.7)；小写斜体部分为ispA基因序列(SEQ ID NO.13)。
SEQ ID NO.53：
注：大写正体部分为MrBBS基因5’端RBS序列(SEQ ID NO.1)；小写正体部分为MrBBS基因序列(SEQ ID NO.12)；大写加粗部分为连接短肽序列(SEQ ID NO.8)；小写斜体部分为ispA基因序列(SEQ ID NO.13)。
SEQ ID NO.54：
注：大写正体部分为MrBBS基因5’端RBS序列(SEQ ID NO.1)；小写正体部分为MrBBS基因序列(SEQ ID NO.12)；大写加粗部分为连接短肽序列(SEQ ID NO.9)；小写斜体部分为ispA基因序列(SEQ ID NO.13)。
SEQ ID NO.55
注：大写正体部分为MrBBS基因5’端RBS序列(SEQ ID NO.1)；小写正体部分为MrBBS基因序列(SEQ ID NO.12)；大写加粗部分为连接短肽序列(SEQ ID NO.10)；小写斜体部分为ispA基因序列(SEQ ID NO.13)。
SEQ ID NO.56
注：大写正体部分为MrBBS基因5’端RBS序列(SEQ ID NO.1)；小写正体部分为MrBBS基因序列(SEQ ID NO.12)；大写加粗部分为连接短肽序列(SEQ ID NO.11)；小写斜体部分为ispA基因序列(SEQ ID NO.13)。
实施例1本发明产(-)-α-红没药醇的重组基因工程菌的构建
(1)化合合成(苏州金唯智生物科技有限公司)编码春黄菊花来源的(-)-α-红没药醇合成酶基因MrBBS；分别以MrBBS-F/MrBBS-R1、MrBBS-F/MrBBS-R2、MrBBS-F/MrBBS-R3、MrBBS-F/MrBBS-R4、MrBBS-F/MrBBS-R5为引物，对(-)-α-红没药醇合成酶基因MrBBS进行PCR扩增，PCR产物是5’端端带有9bp RBS、末尾去除终止密码子TAA后，连接有不同长度短肽编码序列的(-)-α-红没药醇合成酶基因MrBBS(RBS的核苷酸序列如SEQ ID NO.1所示，短肽编码序列如SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示)，对PCR产物用1.0％琼脂糖凝胶电泳检测并用clean-up试剂盒纯化片段；
以E.coli DH5α/W3110基因组为模板，通过PCR分别使用引物ispA-F1/ispA-R、ispA-F2/ispA-R、ispA-F3/ispA-R、ispA-F4/ispA-R以及ispA-F5/ispA-R扩增出无起始密码子ATG，同时带有不同长度短肽编码序列的ispA片段(短肽编码序列如SEQ ID NO.7、SEQID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示)，并用clean-up试剂盒纯化片段；
将回收的两个DNA片段分别使用引物ispA-F1/MrBBS-R1、ispA-F2/MrBBS-R2、ispA-F3/MrBBS-R3、ispA-F4/MrBBS-R4、ispA-F5/MrBBS-R5进行融合PCR，PCR产物用1.0％琼脂糖凝胶电泳检测并切胶回收纯化该片段；然后采用诺唯赞的一步克隆试剂盒将纯化好的融合PCR产物(核苷酸序列如SEQ ID NO.52～56所示)与线性化质粒pSTV28进行连接(37℃30min)，质粒线性化获得使用引物pSTV28-F和pSTV28-R；将连接产物转化到E.coli DH5α，得到转化产物；将转化产物涂布在LB固体培养基(含终浓度34mg/L氯霉素)上，于37℃、220rpm的条件下摇瓶培养8～12h后提取质粒进行测序验证，验证正确即获得重组质粒pSTV28-11/12/13/14/15；
(2)化合合成(苏州金唯智生物科技有限公司)pUC57/mvaKmmDK2-idi质粒为模板，该合成质粒包含编码甲烷八叠球古菌来源的甲羟戊酸激酶基因mvaKmm、肺炎链球菌来源的甲羟戊酸5-焦磷酸脱羧酶mvaD、磷酸甲羟戊酸激酶基因mvaK2以及带有12bp RBS的大肠杆菌来源的异戊烯二磷酸δ异构酶基因idi(RBS核苷酸序列如SEQ ID NO.3所示)；以mvaKmm-F、idi-R为引物，对mvaKmmDK2-idi基因进行PCR扩增，扩增得到带有pLac的mvaK1DK2-idi片段(pLac核苷酸序列如SEQ ID NO.6所示，插入mvaKmm基因5’端)，PCR产物用1.0％琼脂糖凝胶电泳检测并用clean-up试剂盒纯化片段；
以化合合成(苏州金唯智生物科技有限公司)的pUC57/mvaES质粒为模板，该合成质粒包含粪肠杆菌来源的3-羟基-3-甲基戊二酰CoA合酶基因mvaS以及乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶基因mvaE；(mvaE基因5’端带有RBS，RBS核苷酸序列如SEQ ID NO.4所示；mvaS基因5’端带有RBS，RBS核苷酸序列如SEQ ID NO.5所示)通过PCR使用引物mvaES-F、mvaES-R进行扩增，PCR产物用clean-up试剂盒纯化片段；
将回收的两个DNA片段使用mvaKmm-F和mvaES-R进行融合PCR，PCR产物用1.0％琼脂糖凝胶电泳检测并切胶回收纯化该片段；然后采用诺唯赞的一步克隆试剂盒将纯化好的融合PCR产物(核苷酸序列如SEQ ID NO.50所示)与线性化质粒pSTV28-11/12/13/14/15进行连接(37℃30min)，质粒线性化获得使用引物pSTV28-1-F和pSTV28-1-R；将连接产物转化到E.coli DH5α，得到转化产物；将转化产物涂布在LB固体培养基(含终浓度34mg/L氯霉素)上，于37℃、220rpm的条件下摇瓶培养8～12h后提取质粒进行测序验证，验证正确即获得重组质粒pSTV28-21/22/23/24/25；
(3)化合合成(苏州金唯智生物科技有限公司)pUC57/mvaK1质粒为模板；以mvaK1-F、mvaK1-R为引物，对肺炎链球菌来源甲羟戊酸激酶mvaK1基因进行PCR扩增，扩增得到mvaK1片段，PCR产物用1.0％琼脂糖凝胶电泳检测并用clean-up试剂盒纯化该片段；
以pUC57/mvaKmmDK2-idi质粒为模板，使用引物mvaDK2-idi-F和mvaDK2-idi-R进行PCR扩增，扩增得到包含肺炎链球菌来源的甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因和异戊烯基二磷酸δ-异构酶idi基因的基因片段，即mvaDK2-idi片段(该片段的idi基因5’端带有12bp RBS，核苷酸序列如SEQ ID NO.3所示)，将PCR产物用1.0％琼脂糖凝胶电泳检测并用clean-up试剂盒纯化该片段；
将回收的两个DNA片段使用引物mvaK1-F和mvaDK2-idi-R进行融合PCR，PCR产物用1.0％琼脂糖凝胶电泳检测并切胶回收纯化该片段mvaK1DK2-idi；然后采用诺唯赞的一步克隆试剂盒将纯化好的PCR产物(核苷酸序列如SEQ ID NO.51所示)与线性化质粒pTrc99A进行连接(37℃30min)，质粒线性化获得使用引物pTrc99A-F和pTrc99A-R；将连接产物转化到E.coli DH5α，得到转化产物；将转化产物涂布在LB固体培养基(含终浓度100mg/L氨苄)上，于37℃、220rpm的条件下试管培养8～12h后提取质粒进行测序验证，验证正确即获得重组质粒pTrc99A-1；
(4)将上述构建的质粒-21/22/23/24/25和pTrc99A-1一起转化到大肠杆菌DH5α，得到转化产物；将转化产物涂布于LB固体培养基(含有终浓度为100mg/L氨苄以及34mg/L氯霉素抗性)上，于37℃恒温培养箱中倒置培养12h左右，得到转化子，此转化子即为重组大肠杆菌工程菌株E.coli DH5αpSTV28-21/22/23/24/25&pTrc99A-1。
实施例2(-)-α-红没药醇的制备
1)取实施例1构建的大肠杆菌工程菌株E.coli DH5αpSTV28-21/22/23/24/25&pTrc99A-1，接种于种子培养基，30℃、200rpm摇床振荡培养8～10h，取种子液，按照8％(v/v)接种到发酵培养基，再覆盖20％(v/v)正十二烷，30℃200rpm摇床振荡培养3h，添加10μL0.25M IPTG母液，再30℃200rpm摇床振荡培养47h，即得；
其中，种子培养基的配方为：胰蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L、氨苄青霉素终浓度100mg/L和氯霉素终浓度34mg/L；
发酵培养基的配方为甘油10g/L、磷酸二氢钾2.2g/L、磷酸氢二钾2.9g/L、酵母粉24g/L、酵母蛋白胨12g/L、IPTG 0.1mM、氨苄青霉素终浓度100mg/L和氯霉素终浓度为34mg/L。
实施例3(-)-α-红没药醇的制备
1)取实施例1构建的大肠杆菌工程菌株E.coli DH5αpSTV28-21/22/23/24/25&pTrc99A-1，接种于种子培养基，30℃、200rpm摇床振荡培养8～10h，取种子液，按照8％(v/v)接种到发酵培养基，再覆盖20％(v/v)正十二烷，30℃200rpm摇床振荡培养3h，添加10μL0.25M IPTG母液，再30℃200rpm摇床振荡培养47h，即得；
其中，种子培养基的配方为：胰蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L、氨苄青霉素终浓度100mg/L和氯霉素终浓度34mg/L；
发酵培养基的配方为葡萄糖10g/L、磷酸二氢钾2.2g/L、磷酸氢二钾2.9g/L、酵母粉24g/L、酵母蛋白胨12g/L、IPTG 0.1mM、氨苄青霉素终浓度100mg/L和氯霉素终浓度为34mg/L。
对比例1产(-)-α-红没药醇的重组基因工程菌的构建
(1)化合合成(苏州金唯智生物科技有限公司)编码春黄菊花来源的(-)-α-红没药醇合成酶基因MrBBS；以MrBBS-F、MrBBS-R为引物，对(-)-α-红没药醇合成酶基因MrBBS进行PCR扩增，PCR产物是带有9bp RBS的(-)-α-红没药醇合成酶基因MrBBS(RBS的核苷酸序列如SEQ ID NO.1所示)，对PCR产物用1.0％琼脂糖凝胶电泳检测并用clean-up试剂盒纯化片段；
以E.coli DH5α/W3110基因组为模板，通过PCR使用引物ispA-F、ispA-R扩增出带有16bp RBS的ispA片段(RBS的核苷酸序列如SEQ ID NO.2所示)，并用clean-up试剂盒纯化片段；
将回收的两个DNA片段使用引物MrBBS-F和ispA-R进行融合PCR，PCR产物用1.0％琼脂糖凝胶电泳检测并切胶回收纯化该片段；然后采用诺唯赞的一步克隆试剂盒将纯化好的融合PCR产物(核苷酸序列如SEQ ID NO.49所示)与线性化质粒pSTV28进行连接(37℃30min)，质粒线性化获得使用引物pSTV28-F和pSTV28-R；将连接产物转化到E.coli DH5α，得到转化产物；将转化产物涂布在LB固体培养基(含终浓度34mg/L氯霉素)上，于37℃、220rpm的条件下摇瓶培养8～12h后提取质粒进行测序验证，验证正确即获得重组质粒pSTV28-1；
(2)化合合成(苏州金唯智生物科技有限公司)pUC57/mvaKmmDK2-idi质粒，该合成质粒包含编码甲烷八叠球古菌来源的甲羟戊酸激酶基因mvaKmm、肺炎链球菌来源的甲羟戊酸5-焦磷酸脱羧酶mvaD、磷酸甲羟戊酸激酶基因mvaK2以及带有12bp RBS的大肠杆菌来源的异戊烯二磷酸δ异构酶基因idi(RBS核苷酸序列如SEQ ID NO.3所示)；以mvaKmm-F、idi-R为引物，对mvaKmmDK2-idi基因进行PCR扩增，扩增得到带有pLac的mvaK1DK2-idi片段(pLac核苷酸序列如SEQ ID NO.6所示，插入mvaKmm基因5’端)，PCR产物用1.0％琼脂糖凝胶电泳检测并用clean-up试剂盒纯化片段；
化合合成(苏州金唯智生物科技有限公司)的pUC57/mvaES质粒，该合成质粒包含粪肠杆菌来源的3-羟基-3-甲基戊二酰CoA合酶基因mvaS以及乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶基因mvaE；(mvaE基因5’端带有RBS，RBS核苷酸序列如SEQ ID NO.4所示；mvaS基因5’端带有RBS，RBS核苷酸序列如SEQ ID NO.5所示)通过PCR使用引物mvaES-F、mvaES-R进行扩增，PCR产物用clean-up试剂盒纯化片段；
将回收的两个DNA片段使用mvaKmm-F和mvaES-R进行融合PCR，PCR产物用1.0％琼脂糖凝胶电泳检测并切胶回收纯化该片段；然后采用诺唯赞的一步克隆试剂盒将纯化好的融合PCR产物(核苷酸序列如SEQ ID NO.50所示)与线性化质粒pSTV28-1进行连接(37℃30min)，质粒线性化获得使用引物pSTV28-1-F和pSTV28-1-R；将连接产物转化到E.coliDH5α，得到转化产物；将转化产物涂布在LB固体培养基(含终浓度34mg/L氯霉素)上，于37℃、220rpm的条件下摇瓶培养8～12h后提取质粒进行测序验证，验证正确即获得重组质粒pSTV28-2(图1)；
(3)化合合成(苏州金唯智生物科技有限公司)pUC57/mvaK1质粒；以mvaK1-F、mvaK1-R为引物，对肺炎链球菌来源甲羟戊酸激酶mvaK1基因进行PCR扩增，扩增得到mvaK1片段，PCR产物用1.0％琼脂糖凝胶电泳检测并用clean-up试剂盒纯化该片段；
以pUC57/mvaKmmDK2-idi质粒为模板，使用引物mvaDK2-idi-F和mvaDK2-idi-R进行PCR扩增，扩增得到包含肺炎链球菌来源的甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因和异戊烯基二磷酸δ-异构酶idi基因的基因片段，即mvaDK2-idi片段(该片段的idi基因5’端带有12bp RBS，核苷酸序列如SEQ ID NO.3所示)，将PCR产物用1.0％琼脂糖凝胶电泳检测并用clean-up试剂盒纯化该片段；
将回收的两个DNA片段使用引物mvaK1-F和mvaDK2-idi-R进行融合PCR，PCR产物用1.0％琼脂糖凝胶电泳检测并切胶回收纯化该片段mvaK1DK2-idi；然后采用诺唯赞的一步克隆试剂盒将纯化好的PCR产物(核苷酸序列如SEQ ID NO.51所示)与线性化质粒pTrc99A进行连接(37℃30min)，质粒线性化获得使用引物pTrc99A-F和pTrc99A-R；将连接产物转化到E.coli DH5α，得到转化产物；将转化产物涂布在LB固体培养基(含终浓度100mg/L氨苄)上，于37℃、220rpm的条件下试管培养8～12h后提取质粒进行测序验证，验证正确即获得重组质粒pTrc99A-1(图2)；
(4)将上述构建的质粒pSTV28-2和pTrc99A-1一起转化到大肠杆菌DH5α/W3110，得到转化产物；将转化产物涂布于LB固体培养基(含有终浓度为100mg/L氨苄以及34mg/L氯霉素抗性)上，于37℃恒温培养箱中倒置培养12h左右，得到转化子，此转化子即为重组大肠杆菌工程菌株DH5α/W3110 pSTV28-2&pTrc99A-1。
对比例2(-)-α-红没药醇的制备
1)取对比例1构建的大肠杆菌工程菌株DH5α/W3110 pSTV28-2&pTrc99A-1，接种于种子培养基，30℃、200rpm摇床振荡培养8～10h，取种子液，按照8％(v/v)接种到发酵培养基，再覆盖20％(v/v)正十二烷，30℃200rpm摇床振荡培养3h，添加10μL 0.25M IPTG母液，再30℃200rpm摇床振荡培养47h，即得；
其中，种子培养基的配方为：胰蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L、氨苄青霉素终浓度100mg/L和氯霉素终浓度34mg/L；
发酵培养基的配方为甘油10g/L、磷酸二氢钾2.2g/L、磷酸氢二钾2.9g/L、酵母粉24g/L、酵母蛋白胨12g/L、IPTG 0.1mM、氨苄青霉素终浓度100mg/L和氯霉素终浓度为34mg/L。
对比例3(-)-α-红没药醇的制备
1)取对比例1构建的大肠杆菌工程菌株DH5α/W3110 pSTV28-2&pTrc99A-1，接种于种子培养基，30℃、200rpm摇床振荡培养8～10h，取种子液，按照8％(v/v)接种到发酵培养基，再覆盖20％(v/v)正十二烷，30℃200rpm摇床振荡培养3h，添加10μL 0.25M IPTG母液，再30℃200rpm摇床振荡培养47h，即得；
其中，种子培养基的配方为：胰蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L、氨苄青霉素终浓度100mg/L和氯霉素终浓度34mg/L；
发酵培养基的配方为葡萄糖10g/L、磷酸二氢钾2.2g/L、磷酸氢二钾2.9g/L、酵母粉24g/L、酵母蛋白胨12g/L、IPTG 0.1mM、氨苄青霉素终浓度100mg/L和氯霉素终浓度为34mg/L。
以下通过试验例进一步说明本发明的有益效果：
试验例1重组大肠杆菌在合成(-)-α-红没药醇中的应用
1、培养基配制
LB培养基组成：蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L，溶剂为去离子水，pH值自然。LB平板是在LB液体培养基中添加终浓度2g/L琼脂。
种子培养基：胰蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L、氨苄青霉素终浓度100mg/L和氯霉素终浓度34mg/L；
发酵培养基：葡萄糖/甘油10g/L、磷酸二氢钾2.2g/L、磷酸氢二钾2.9g/L、酵母粉24g/L、酵母蛋白胨12g/L、IPTG 0.1mM、氨苄青霉素终浓度100mg/L和氯霉素终浓度为34mg/L。
(2)(-)-α-红没药醇生产
挑取对比例1构建的大肠杆菌工程菌株DH5α/W3110 pSTV28-2&pTrc99A-1在摇瓶进行发酵实验测试(发酵过程中(-)-α-红没药醇合成代谢过程见图4)，具体摇瓶发酵实验步骤如下：
挑取在LB固体培养基划线培养过夜的单菌落接种种子培养基，在30℃、200rpm摇床振荡培养8～10h。将培养好的种子按照8％(v/v)接种到发酵培养基，再添加20％(v/v)正十二烷，在30℃200rpm摇床振荡培养至50h，其中发酵3h后添加10μL 0.25M IPTG母液，发酵结束后发酵液取上清即得到(-)-α-红没药醇。
(3)(-)-α-红没药醇含量的测定：
标样准备：配置9g/L(-)-α-红没药醇标样，分别稀释成浓度为10、30、50、70、90mg/L的标样，1mL过膜待测；
样品准备：取1mL发酵液在转速12000rpm下离心5min，两相分离；分离得到的有机相过膜待测；
GC-MS检测方法:柱温控温程序为50℃保持3min；以20℃/min升温到280℃保持5min；进样口温度为200℃；进样模式分流比为10：1；分流流量为10mL/min；色谱柱为安捷伦HP-5MS UI(30m*250um*0.25um)；柱流量为：1mL/min；
检测重组E.coli DH5α/W3110 pSTV28-2&pTrc99A-1发酵获得的发酵液中(-)-α-红没药醇的含量、检测结果以及GC-MS图谱图5。由图5可知，将E.coli DH5α/W3110 pSTV28-2&pTrc99A-1分别接种至以葡萄糖为碳源的发酵培养基中发酵50h，可使E.coli DH5αpSTV28-2&pTrc99A-1发酵液中的(-)-α-红没药醇的产量达到5g/L；而E.coli W3110pSTV28-2&pTrc99A-1产量为3.9g/L。
发酵培养基中分别以甘油或葡萄糖为唯一碳源，考察重组E.coli DH5αpSTV28-2&pTrc99A-1在不同碳源下合成(-)-α-红没药醇的能力。(-)-α-红没药醇在摇瓶培养条件下合成产量如图6所示。当甘油为唯一碳源时，摇瓶发酵50h时，菌株E.coli DH5αpSTV28-2&pTrc99A-1合成(-)-α-红没药醇产量达到2.8g/L，比以葡萄糖为唯一碳源产量低，表明以葡萄糖为唯一碳源更有利于合成(-)-α-红没药醇。
试验例2：不同连接短肽对(-)-α-红没药醇产量的影响
挑取实施例1构建的5株分别表达由不同连接多肽连接的α-红没药醇合成酶MrBBS和法尼基二磷酸合酶ispA融合酶的大肠杆菌工程菌株E.coli DH5αpSTV28-21/22/23/24/25&pTrc99A-1在摇瓶进行发酵实验测试。发酵培养基中以葡萄糖为唯一碳源，按试验例1方法发酵生产。(-)-α-红没药醇在摇瓶培养条件下合成产量如图7所示。当摇瓶发酵50h时，菌株E.coli DH5αpSTV28-24&pTrc99A-1合成(-)-α-红没药醇产量达到6.8g/L,其中pSTV28-24质粒图谱见图3。
从图7发酵结果可见：α-红没药醇合成酶MrBBS和法尼基二磷酸合酶IspA之间的连接短肽对红没药醇产量具有较大的影响。其中SEQ ID NO.10编码的连接短肽(氨基酸序列相应为Gly-Ser-Gly-Gly)对红没药醇产量提升有较大促进作用，原因可能为将MrBBS和IspA通过连接短肽融合表达，使两种酶在空间结构上更加接近，便于法尼基二磷酸合酶ispA催化产物法尼基焦磷酸作为红没药醇合成酶MrBBS底物迅速被MrBBS获取进行反应，从而提高(-)-α-红没药醇的最终产量。
综上，本发明通过将特定的基因如mvaKmm等MVA途径基因重组入大肠杆菌，同时在重组大肠杆菌中将MrBBS基因、ispA基因之间通过短肽编码序列连接，并过表达mvaK2、mvaD、idi等基因，使之在生产过程的摇瓶阶段产量就高达6.8g/L，再经发酵罐放大后的产量相对目前公开报道的产量能突破新高，适宜实际推广应用。
SEQUENCE LISTING
<110> 上海锐康生物技术研发有限公司
<120> 一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途
<130> GY218-2022P0115014CCR3
<160> 56
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> DNA
<213> 人工序列
<400> 1
ggttaaacc 9
<210> 2
<211> 16
<212> DNA
<213> 人工序列
<400> 2
aaggaggtta cggaaa 16
<210> 3
<211> 12
<212> DNA
<213> 人工序列
<400> 3
aggagagaaa tt 12
<210> 4
<211> 12
<212> DNA
<213> 人工序列
<400> 4
aggagcattt ag 12
<210> 5
<211> 12
<212> DNA
<213> 人工序列
<400> 5
aggagaaacc tt 12
<210> 6
<211> 151
<212> DNA
<213> 人工序列
<400> 6
taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 60
tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 120
ttacgaattc gagctcggta cccggggatc c 151
<210> 7
<211> 18
<212> DNA
<213> 人工序列
<400> 7
ccaacgacga cgacgcca 18
<210> 8
<211> 21
<212> DNA
<213> 人工序列
<400> 8
ggaggaggag gatcatcatc a 21
<210> 9
<211> 12
<212> DNA
<213> 人工序列
<400> 9
ggaggaggaa tc 12
<210> 10
<211> 12
<212> DNA
<213> 人工序列
<400> 10
ggaagcggag ga 12
<210> 11
<211> 12
<212> DNA
<213> 人工序列
<400> 11
ggaggaatcg ga 12
<210> 12
<211> 1719
<212> DNA
<213> 人工序列
<400> 12
atgagcacac tgagcgtcag caccccgagc tttagcagca gccctctgtc gagcgtgaat 60
aagaacagca ccaagcagca tgtcactcgt aacagcgtga tctttcacga ctcgatttgg 120
ggggaccagt tcctggaata caaagagaaa ttcaacgttg caaccgagaa acagcttata 180
gaagagctga aagaagaagt gcgtaacgaa ctgatgattc gtgcatgtaa tgaagcgagc 240
cggtatatca aactgatcca gctgatcgat gttgttgaac gtctggggct ggcctatcat 300
tttgaaaaag agattgagga aagcctccag catatatatg tgacgtatgg tcataaatgg 360
acgaattaca acaatattga gagcctgagt ctgtggttcc gcctgcttcg tcaaaatggc 420
tttaatgtta gctcggatat atttgaaaat cacattgatg agaaaggaaa ttttcaggag 480
agcctgtgca atgatccgca ggggatgctg gcgctgtatg aagcggcata tatgcgtgtt 540
gaaggagaga tcattctgga caaagcactc gaatttacca agctgcatct ggggatcatt 600
agcaatgatc ctagctgtga tagcagccta cgtacggaaa tcaagcaggc actgaaacag 660
ccactgcgcc ggcggctgcc aaggctggaa gccgttcgtt acattgccat ttatcagcag 720
aaggcgagcc atagcgaggt tctgctgaag ctggccaaac tggacttcaa cgttctgcag 780
gaaatgcaca aagacgaatt gagccaaata tgcaaatggt ggaaagatct ggatatacgt 840
aacaaactgc cctatgttcg tgatcgtctg attgaaggct atttttggat tctgggtatt 900
tatttcgaac cgcaacactc ccgtacccgt atgttcctga tgaaaacctg tatgtggctg 960
atcgtgctgg acgatacgtt tgataattac ggcacctatg aagagttaga gatctttacc 1020
caagcagtcg aacgttggag cattacctgt ctggatgaac tgccagagta tatgaagctg 1080
atatatcacg agcaatttcg cgtgcatcag gaaatggagg aaagcctgga aaaggagggt 1140
aaggcctacc agattcatta tatcaaagaa atggccaaag aaggtactcg ttcgctgctg 1200
ctggaagcga aatggctgaa ggaaggctat atgcctaccc tggatgagta cctgagcaac 1260
agcctggtca cctgcggcta tgcactgatg accgcacgca gctacgttgc ccgtgacgac 1320
ggcattgtta ccgaagatgc attcaaatgg gttgcaacgc acccgccgat tgttaaagca 1380
gcatgcaaaa ttctgcgcct gatggacgac attgcaaccc ataaagagga acaggagcgg 1440
ggacacattg caagtagcat tgagtgttac aggaaggaaa ccggagctag cgaagaggag 1500
gcttgcatgg actttctgaa gcaggttgaa gatggttgga aagttattaa tcaagaaagc 1560
ctgatgccga ccgatgttcc gttccctctg ctgattccgg caattaacct ggcacgtgtg 1620
agcgacaccc tgtacaaaga caacgatggt tataatcatg ccgataaaga ggttataggt 1680
tatattaaaa gcctgtttgt acatccgatg atagtctaa 1719
<210> 13
<211> 900
<212> DNA
<213> 人工序列
<400> 13
atggactttc cgcagcaact cgaagcctgc gttaagcagg ccaaccaggc gctgagccgt 60
tttatcgccc cactgccctt tcagaacact cccgtggtcg aaaccatgca gtatggcgca 120
ttattaggtg gtaagcgcct gcgacctttc ctggtttatg ccaccggtca tatgttcggc 180
gttagcacaa acacgctgga cgcacccgct gccgccgttg agtgtatcca cgcttactca 240
ttaattcatg atgatttacc ggcaatggat gatgacgatc tgcgtcgcgg tttgccaacc 300
tgccatgtga agtttggcga agcaaacgcg attctcgctg gcgacgcttt acaaacgctg 360
gcgttctcga ttttaagcga tgccgatatg ccggaagtgt cggaccgcga cagaatttcg 420
atgatttctg aactggcgag cgccagtggt attgccggaa tgtgcggtgg tcaggcatta 480
gatttagacg cggaaggcaa acacgtacct ctggacgcgc ttgagcgtat tcatcgtcat 540
aaaaccggcg cattgattcg cgccgccgtt cgccttggtg cattaagcgc cggagataaa 600
ggacgtcgtg ctctgccggt actcgacaag tatgcagaga gcatcggcct tgccttccag 660
gttcaggatg acatcctgga tgtggtggga gatactgcaa cgttgggaaa acgccagggt 720
gccgaccagc aacttggtaa aagtacctac cctgcacttc tgggtcttga gcaagcccgg 780
aagaaagccc gggatctgat cgacgatgcc cgtcagtcgc tgaaacaact ggctgaacag 840
tcactcgata cctcggcact ggaagcgcta gcggactaca tcatccagcg taataaataa 900
<210> 14
<211> 906
<212> DNA
<213> 人工序列
<400> 14
atggtgagct gcagcgcgcc gggcaaaatt tatctgtttg gcgaacatgc ggtggtgtat 60
ggcgaaaccg cgattgcgtg cgcggtggaa ctgcgcaccc gcgtgcgcgc ggaactgaac 120
gatagcatta ccattcagag ccagattggc cgcaccggcc tggattttga aaaacatccg 180
tatgtgagcg cggtgattga aaaaatgcgc aaaagcattc cgattaacgg cgtgtttctg 240
accgtggata gcgatattcc ggtgggcagc ggcctgggca gcagcgcggc ggtgaccatt 300
gcgagcattg gcgcgctgaa cgaactgttt ggctttggcc tgagcctgca ggaaattgcg 360
aaactgggcc atgaaattga aattaaagtg cagggcgcgg cgagcccgac cgatacctat 420
gtgagcacct ttggcggcgt ggtgaccatt ccggaacgcc gcaaactgaa aaccccggat 480
tgcggcattg tgattggcga taccggcgtg tttagcagca ccaaagaact ggtggcgaac 540
gtgcgccagc tgcgcgaaag ctatccggat ctgattgaac cgctgatgac cagcattggc 600
aaaattagcc gcattggcga acagctggtg ctgagcggcg attatgcgag cattggccgc 660
ctgatgaacg tgaaccaggg cctgctggat gcgctgggcg tgaacattct ggaactgagc 720
cagctgattt atagcgcgcg cgcggcgggc gcgtttggcg cgaaaattac cggcgcgggc 780
ggcggcggct gcatggtggc gctgaccgcg ccggaaaaat gcaaccaggt ggcggaagcg 840
gtggcgggcg cgggcggcaa agtgaccatt accaaaccga ccgaacaggg cctgaaagtg 900
gattaa 906
<210> 15
<211> 954
<212> DNA
<213> 人工序列
<400> 15
atggatagag agcctgtaac agtacgttcc tacgcaaata ttgctattat caaatattgg 60
ggaaagaaaa aagaaaaaga gatggtgcct gctactagca gtatttctct aactttggaa 120
aatatgtata cagagacgac cttgtcgcct ttaccagcca atgtaacagc tgacgaattt 180
tacatcaatg gtcagctaca aaatgaggtc gagcatgcca agatgagtaa gattattgac 240
cgttatcgtc cagctggtga gggctttgtc cgtatcgata ctcaaaacaa tatgcctact 300
gcagcgggcc tgtcctcaag ttctagtggt ttgtccgccc tggtcaaggc ttgtaatgct 360
tatttcaagc ttggattgga tagaagtcag ttggcacagg aagccaaatt tgcctcaggc 420
tcttcttctc ggagttttta tggaccacta ggagcctggg ataaggatag tggagaaatt 480
taccctgtag agacagactt gaaactagct atgattatgt tggtgctaga ggacaagaaa 540
aaaccaatct ctagccgtga cgggatgaaa ctttgtgtgg aaacctcgac gacttttgac 600
gactgggttc gtcagtctga gaaggactat caggatatgc tgatttatct caaggaaaat 660
gattttgcca agattggaga attaacggag aaaaatgccc tggctatgca tgctacgaca 720
aagactgcta gtccagcctt ttcttatctg acggatgcct cttatgaggc tatggacttt 780
gttcgccagc ttcgtgagaa aggagaggcc tgctacttta ccatggatgc tggtcccaat 840
gttaaggtct tctgtcagga gaaagacttg gagcatttat cagaaatttt cggtcatcgt 900
tatcgcttga ttgtgtcaaa aacaaaggat ttgagtcaag atgattgctg ttaa 954
<210> 16
<211> 1008
<212> DNA
<213> 人工序列
<400> 16
atgattgctg ttaaaacttg cggaaaactc tattgggcag gtgaatatgc tattttagag 60
ccagggcagt tagctttgat aaaggatatt cccatctata tgagggctga gattgctttt 120
tctgacagct accgtatcta ttcagatatg tttgatttcg cagtggactt aaggcctaat 180
cctgactaca gcttgattca agaaacgatt gctttgatgg gagacttcct cgctgttcgt 240
ggtcagaatt taagaccttt ttctctagaa atctgtggca aaatggaacg agaagggaaa 300
aagtttggtc taggttctag tggcagcgtc gttgtcttgg ttgtcaaggc tttactggct 360
ctgtatgatg tttctgttga tcaggagctc ttgttcaagc tgactagcgc tgtcttgctc 420
aagcgaggag acaatggttc catgggcgac cttgcctgta ttgtggcaga ggatttggtt 480
ctctaccagt catttgatcg ccagaaggtg gctgcttggt tagaagaaga aaacttggcg 540
acagttctgg agcgtgattg gggcttttca atttcacaag tgaaaccaac tttagaatgt 600
gatttcttag tgggatggac caaggaagtg gctgtatcga gtcacatggt ccagcaaatc 660
aagcaaaata tcaatcaaaa ttttttaagt tcctcaaaag aaacggtggt ttctttggtc 720
gaagccttgg aacaggggaa atcagaaaag attatcgagc aagtagaagt agccagcaag 780
cttttagaag gcttgagtac agatatttac acgcctttgc ttagacagtt gaaagaagcc 840
agtcaagatt tgcaggccgt tgccaagagt agtggtgctg gtggtggtga ctgtggcatc 900
gccctgagtt ttgatgcgca atcaaccaaa accttaaaaa atcgttgggc cgatctgggg 960
attgagctct tatatcaaga aaggatagga catgacgaca aatcgtaa 1008
<210> 17
<211> 549
<212> DNA
<213> 人工序列
<400> 17
atgcaaacgg aacacgtcat tttattgaat gcacagggag ttcccacggg tacgctggaa 60
aagtatgccg cacacacggc agacacccgc ttacatctcg cgttctccag ttggctgttt 120
aatgccaaag gacaattatt agttacccgc cgcgcactga gcaaaaaagc atggcctggc 180
gtgtggacta actcggtttg tgggcaccca caactgggag aaagcaacga agacgcagtg 240
atccgccgtt gccgttatga gcttggcgtg gaaattacgc ctcctgaatc tatctatcct 300
gactttcgct accgcgccac cgatccgagt ggcattgtgg aaaatgaagt gtgtccggta 360
tttgccgcac gcaccactag tgcgttacag atcaatgatg atgaagtgat ggattatcaa 420
tggtgtgatt tagcagatgt attacacggt attgatgcca cgccgtgggc gttcagtccg 480
tggatggtga tgcaggcgac aaatcgcgaa gccagaaaac gattatctgc atttacccag 540
cttaaataa 549
<210> 18
<211> 1152
<212> DNA
<213> 人工序列
<400> 18
atgacaattg ggattgataa aattagtttt tttgtgcccc cttattatat tgatatgacg 60
gcactggctg aagccagaaa tgtagaccct ggaaaatttc atattggtat tgggcaagac 120
caaatggcgg tgaacccaat cagccaagat attgtgacat ttgcagccaa tgccgcagaa 180
gcgatcttga ccaaagaaga taaagaggcc attgatatgg tgattgtcgg gactgagtcc 240
agtatcgatg agtcaaaagc ggccgcagtt gtcttacatc gtttaatggg gattcaacct 300
ttcgctcgct ctttcgaaat caaggaagct tgttacggag caactgcagg cttacagtta 360
gctaagaatc acgtagcctt acatccagat aaaaaagtct tggtcgtagc agcagatatt 420
gcaaaatatg gcttaaattc tggcggtgag cctacacaag gagctggggc ggttgcaatg 480
ttagttgcta gtgaaccgcg cattttggct ttaaaagagg ataatgtgat gctgacgcaa 540
gatatctatg acttttggcg tccaacaggc catccatatc ctatggtcga tggtcctttg 600
tcaaacgaaa cctacatcca atcttttgcc caagtctggg atgaacataa aaaacgaacc 660
ggtcttgatt ttgcagatta tgatgcttta gcgttccata ttccttacac aaaaatgggc 720
aaaaaagcct tattagcaaa aatctccgac caaactgaag cagaacagga acgaatttta 780
gcccgttatg aagaaagcat catctatagt cgtcgcgtag gaaacttgta tacgggttca 840
ctttatctgg gactcatttc ccttttagaa aatgcaacga ctttaaccgc aggcaatcaa 900
attgggttat tcagttatgg ttctggtgct gtcgctgaat ttttcactgg tgaattagta 960
gctggttatc aaaatcattt acaaaaagaa actcatttag cactgctgga taatcggaca 1020
gaactttcta tcgctgaata tgaagccatg tttgcagaaa ctttagacac agacattgat 1080
caaacgttaa aagatgaatt aaaatatagt atttctgcta ttaataatac cgttcgttct 1140
tatcgaaact aa 1152
<210> 19
<211> 2412
<212> DNA
<213> 人工序列
<400> 19
atgaaaacag tagttattat tgatgcatta cgaacaccaa ttggaaaata taaaggcagc 60
ttaagtcaag taagtgccgt agacttagga acacatgtta caacacaact tttaaaaaga 120
cattccacta tttctgaaga aattgatcaa gtaatctttg gaaatgtttt acaagctgga 180
aatggccaaa atcccgcacg acaaatagca ataaacagcg gtttatctca tgaaattccc 240
gcaatgacag ttaatgaggt ctgcggatca ggaatgaagg ccgttatttt ggcgaaacaa 300
ttgattcaat taggagaagc ggaagtttta attgctggcg ggattgagaa tatgtcccaa 360
gcacctaaat tacaacgatt taattacgaa acagaaagct atgatgcgcc tttttctagt 420
atgatgtacg atgggttaac ggatgccttt agtggtcaag caatgggctt aactgctgaa 480
aatgtggccg aaaagtatca tgtaactaga gaagagcaag atcaattttc tgtacattca 540
caattaaaag cagctcaagc acaagcagaa gggatattcg ctgacgaaat agccccatta 600
gaagtatcag gaacgcttgt ggagaaagat gaagggattc gccctaattc gagcgttgag 660
aagctaggaa cgcttaaaac agtttttaaa gaagacggta ctgtaacagc agggaatgca 720
tcaaccatta atgatggggc ttctgctttg attattgctt cacaagaata tgccgaagca 780
cacggtcttc cttatttagc tattattcga gacagtgtgg aagtcggtat tgatccagcc 840
tatatgggaa tttcgccgat taaagccatt caaaaactgt tagcgcgcaa tcaacttact 900
acggaagaaa ttgatctgta tgaaatcaac gaagcatttg cagcaacttc aatcgtggtc 960
caaagagaac tggctttacc agaggaaaag gtcaacattt atggtggcgg tatttcatta 1020
ggtcatgcga ttggtgccac aggtgctcgt ttattaacga gtttaagtta tcaattaaat 1080
caaaaagaaa agaaatatgg agtggcttct ttatgtatcg gcggtggctt aggactcgct 1140
atgctactag agagacctca gcaaaaaaaa aacagccgat tttatcaaat gagtcctgag 1200
gaacgcctgg cttctcttct taatgaaggc cagatttctg ctgatacaaa aaaagaattt 1260
gaaaatacgg ctttatcttc gcagattgcc aatcatatga ttgaaaatca aatcagtgaa 1320
acagaagtgc cgatgggcgt tggcttacat ttaacagtgg acgaaactga ttatttggta 1380
ccaatggcga cagaagagcc ctcagtgatt gcggctttga gtaatggtgc aaaaatagca 1440
caaggattta aaacagtgaa tcaacaacgt ttaatgcgtg gacaaatcgt tttttacgat 1500
gttgcagacg ccgagtcatt gattgatgaa ctacaagtaa gagaaacgga aatttttcaa 1560
caagcagagt taagttatcc atctatcgtt aaacgcggcg gcggcttaag agatttgcaa 1620
tatcgtgctt ttgatgaatc atttgtatct gtcgactttt tagtagatgt taaggatgca 1680
atgggggcaa atatcgttaa cgctatgttg gaaggtgtgg ccgagttgtt ccgtgaatgg 1740
tttgcggagc aaaagatttt attcagtatt ttaagtaatt atgccacgga gtcggttgtt 1800
acgatgaaaa cggctattcc agtttcacgt ttaagtaagg ggagcaatgg ccgggaaatt 1860
gctgaaaaaa ttgttttagc ttcacgctat gcttcattag atccttatcg ggcagtcacg 1920
cataacaaag ggatcatgaa tggcattgaa gctgtcgttt tagctacagg aaatgataca 1980
cgcgctgtta gcgcttcttg tcatgctttt gcggtgaagg aaggtcgcta ccaaggtttg 2040
actagttgga cgctggatgg cgaacaacta attggtgaaa tttcagttcc gcttgcgtta 2100
gccacggttg gcggtgccac aaaagtctta cctaaatctc aagcagctgc tgatttgtta 2160
gcagtgacgg atgcaaaaga actaagtcga gtagtagcgg ctgttggttt ggcacaaaat 2220
ttagcggcgt tacgggcctt agtctctgaa ggaattcaaa aaggacacat ggctctacaa 2280
gcacgttctt tagcgatgac ggtcggagct actggtaaag aagttgaggc agtcgctcaa 2340
caattaaaac gtcaaaaaac gatgaaccaa gaccgagcct tggctatttt aaatgattta 2400
agaaaacaat aa 2412
<210> 20
<211> 879
<212> DNA
<213> 人工序列
<400> 20
atgacaaaaa aagttggtgt cggtcaggca catagtaaga taattttaat aggggaacat 60
gcggtcgttt acggttatcc tgccatttcc ctgcctcttt tggaggtgga ggtgacctgt 120
aaggtagttc ctgcagagag tccttggcgc ctttatgagg aggatacctt gtccatggcg 180
gtttatgcct cactggagta tttggatatc acagaagcct gcattcgttg tgagattgac 240
tcggctatcc ctgagaaacg ggggatgggt tcgtcagcgg ctatcagcat agcggccatt 300
cgtgcggtat ttgactacta tcaggctgat ctgcctcatg atgtactaga aatcttggtc 360
aatcgagctg aaatgattgc ccatatgaat cctagtggtt tggatgctaa gacctgtctc 420
agtgaccaac ctattcgctt tatcaagaac gtaggattta cagaacttga gatggattta 480
tccgcctatt tggtgattgc cgatacgggt gtttatggtc atactcgtga agccatccaa 540
gtggttcaaa ataagggcaa ggatgcccta ccgtttttgc atgccttggg agaattaacc 600
cagcaagcag aagttgcgat ttcacaaaaa gatgctgaag gactgggaca aatcctcagt 660
caagcgcatt tacatttaaa agaaattgga gtcagtagcc ctgaggcaga ctttttggtt 720
gaaacgactc ttagccatgg tgctctgggt gccaagatga gcggtggtgg gctaggaggt 780
tgtatcatag ccttggtaac caatttgaca cacgcacaag aactagcaga aagattagaa 840
gagaaaggag ctgttcagac atggatagag agcctgtaa 879
<210> 21
<211> 49
<212> DNA
<213> 人工序列
<400> 21
atttcacaca ggaaacagct ggttaaacca tgagcacact gagcgtcag 49
<210> 22
<211> 62
<212> DNA
<213> 人工序列
<400> 22
agttgctgcg gaaagtccat tttccgtaac ctccttggat ccttagacta tcatcggatg 60
ta 62
<210> 23
<211> 62
<212> DNA
<213> 人工序列
<400> 23
tacatccgat gatagtctaa ggatccaagg aggttacgga aaatggactt tccgcagcaa 60
ct 62
<210> 24
<211> 40
<212> DNA
<213> 人工序列
<400> 24
aactcacatt acaggtcgac ttatttatta cgctggatga 40
<210> 25
<211> 20
<212> DNA
<213> 人工序列
<400> 25
actggccgtc gttttacaac 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列
<400> 26
agctgtttcc tgtgtgaaat 20
<210> 27
<211> 40
<212> DNA
<213> 人工序列
<400> 27
tcatccagcg taataaataa gtcgacctgt aatgtgagtt 40
<210> 28
<211> 52
<212> DNA
<213> 人工序列
<400> 28
ataataacta ctgttttcat ctaaatgctc ctttatttaa gctgggtaaa tg 52
<210> 29
<211> 52
<212> DNA
<213> 人工序列
<400> 29
catttaccca gcttaaataa aggagcattt agatgaaaac agtagttatt at 52
<210> 30
<211> 40
<212> DNA
<213> 人工序列
<400> 30
gttgtaaaac gacggccagt ttagtttcga taagaacgaa 40
<210> 31
<211> 20
<212> DNA
<213> 人工序列
<400> 31
actggccgtc gttttacaac 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列
<400> 32
ttatttaagc tgggtaaatg 20
<210> 33
<211> 40
<212> DNA
<213> 人工序列
<400> 33
tttcacacag gaaacagacc atgacaaaaa aagttggtgt 40
<210> 34
<211> 40
<212> DNA
<213> 人工序列
<400> 34
atttgcgtag gaacgtactg ttacaggctc tctatccatg 40
<210> 35
<211> 40
<212> DNA
<213> 人工序列
<400> 35
catggataga gagcctgtaa cagtacgttc ctacgcaaat 40
<210> 36
<211> 40
<212> DNA
<213> 人工序列
<400> 36
aaaacagcca agcttgcatg ttatttaagc tgggtaaatg 40
<210> 37
<211> 20
<212> DNA
<213> 人工序列
<400> 37
catgcaagct tggctgtttt 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列
<400> 38
ggtctgtttc ctgtgtgaaa 20
<210> 39
<211> 38
<212> DNA
<213> 人工序列
<400> 39
tggcgtcgtc gtcgttggga ctatcatcgg atgtacaa 38
<210> 40
<211> 41
<212> DNA
<213> 人工序列
<400> 40
tgatgatgat cctcctcctc cgactatcat cggatgtaca a 41
<210> 41
<211> 40
<212> DNA
<213> 人工序列
<400> 41
ggaaagtcga ttcctcctcc gactatcatc ggatgtacaa 40
<210> 42
<211> 40
<212> DNA
<213> 人工序列
<400> 42
ggaaagtctc ctccgattcc gactatcatc ggatgtacaa 40
<210> 43
<211> 40
<212> DNA
<213> 人工序列
<400> 43
ggaaagtctc cgattcctcc gactatcatc ggatgtacaa 40
<210> 44
<211> 38
<212> DNA
<213> 人工序列
<400> 44
ccaacgacga cgacgccaga ctttccgcag caactcga 38
<210> 45
<211> 41
<212> DNA
<213> 人工序列
<400> 45
ggaggaggag gatcatcatc agactttccg cagcaactcg a 41
<210> 46
<211> 40
<212> DNA
<213> 人工序列
<400> 46
tgatagtcgg aggaggaatc gactttccgc agcaactcga 40
<210> 47
<211> 40
<212> DNA
<213> 人工序列
<400> 47
tgatagtcgg aagcggagga gactttccgc agcaactcga 40
<210> 48
<211> 40
<212> DNA
<213> 人工序列
<400> 48
tgatagtcgg aggaatcgga gactttccgc agcaactcga 40
<210> 49
<211> 2644
<212> DNA
<213> 人工序列
<400> 49
ggttaaacca tgagcacact gagcgtcagc accccgagct ttagcagcag ccctctgtcg 60
agcgtgaata agaacagcac caagcagcat gtcactcgta acagcgtgat ctttcacgac 120
tcgatttggg gggaccagtt cctggaatac aaagagaaat tcaacgttgc aaccgagaaa 180
cagcttatag aagagctgaa agaagaagtg cgtaacgaac tgatgattcg tgcatgtaat 240
gaagcgagcc ggtatatcaa actgatccag ctgatcgatg ttgttgaacg tctggggctg 300
gcctatcatt ttgaaaaaga gattgaggaa agcctccagc atatatatgt gacgtatggt 360
cataaatgga cgaattacaa caatattgag agcctgagtc tgtggttccg cctgcttcgt 420
caaaatggct ttaatgttag ctcggatata tttgaaaatc acattgatga gaaaggaaat 480
tttcaggaga gcctgtgcaa tgatccgcag gggatgctgg cgctgtatga agcggcatat 540
atgcgtgttg aaggagagat cattctggac aaagcactcg aatttaccaa gctgcatctg 600
gggatcatta gcaatgatcc tagctgtgat agcagcctac gtacggaaat caagcaggca 660
ctgaaacagc cactgcgccg gcggctgcca aggctggaag ccgttcgtta cattgccatt 720
tatcagcaga aggcgagcca tagcgaggtt ctgctgaagc tggccaaact ggacttcaac 780
gttctgcagg aaatgcacaa agacgaattg agccaaatat gcaaatggtg gaaagatctg 840
gatatacgta acaaactgcc ctatgttcgt gatcgtctga ttgaaggcta tttttggatt 900
ctgggtattt atttcgaacc gcaacactcc cgtacccgta tgttcctgat gaaaacctgt 960
atgtggctga tcgtgctgga cgatacgttt gataattacg gcacctatga agagttagag 1020
atctttaccc aagcagtcga acgttggagc attacctgtc tggatgaact gccagagtat 1080
atgaagctga tatatcacga gcaatttcgc gtgcatcagg aaatggagga aagcctggaa 1140
aaggagggta aggcctacca gattcattat atcaaagaaa tggccaaaga aggtactcgt 1200
tcgctgctgc tggaagcgaa atggctgaag gaaggctata tgcctaccct ggatgagtac 1260
ctgagcaaca gcctggtcac ctgcggctat gcactgatga ccgcacgcag ctacgttgcc 1320
cgtgacgacg gcattgttac cgaagatgca ttcaaatggg ttgcaacgca cccgccgatt 1380
gttaaagcag catgcaaaat tctgcgcctg atggacgaca ttgcaaccca taaagaggaa 1440
caggagcggg gacacattgc aagtagcatt gagtgttaca ggaaggaaac cggagctagc 1500
gaagaggagg cttgcatgga ctttctgaag caggttgaag atggttggaa agttattaat 1560
caagaaagcc tgatgccgac cgatgttccg ttccctctgc tgattccggc aattaacctg 1620
gcacgtgtga gcgacaccct gtacaaagac aacgatggtt ataatcatgc cgataaagag 1680
gttataggtt atattaaaag cctgtttgta catccgatga tagtctaaaa ggaggttacg 1740
gaaaatggac tttccgcagc aactcgaagc ctgcgttaag caggccaacc aggcgctgag 1800
ccgttttatc gccccactgc cctttcagaa cactcccgtg gtcgaaacca tgcagtatgg 1860
cgcattatta ggtggtaagc gcctgcgacc tttcctggtt tatgccaccg gtcatatgtt 1920
cggcgttagc acaaacacgc tggacgcacc cgctgccgcc gttgagtgta tccacgctta 1980
ctcattaatt catgatgatt taccggcaat ggatgatgac gatctgcgtc gcggtttgcc 2040
aacctgccat gtgaagtttg gcgaagcaaa cgcgattctc gctggcgacg ctttacaaac 2100
gctggcgttc tcgattttaa gcgatgccga tatgccggaa gtgtcggacc gcgacagaat 2160
ttcgatgatt tctgaactgg cgagcgccag tggtattgcc ggaatgtgcg gtggtcaggc 2220
attagattta gacgcggaag gcaaacacgt acctctggac gcgcttgagc gtattcatcg 2280
tcataaaacc ggcgcattga ttcgcgccgc cgttcgcctt ggtgcattaa gcgccggaga 2340
taaaggacgt cgtgctctgc cggtactcga caagtatgca gagagcatcg gccttgcctt 2400
ccaggttcag gatgacatcc tggatgtggt gggagatact gcaacgttgg gaaaacgcca 2460
gggtgccgac cagcaacttg gtaaaagtac ctaccctgca cttctgggtc ttgagcaagc 2520
ccggaagaaa gcccgggatc tgatcgacga tgcccgtcag tcgctgaaac aactggctga 2580
acagtcactc gatacctcgg cactggaagc gctagcggac tacatcatcc agcgtaataa 2640
ataa 2644
<210> 50
<211> 7168
<212> DNA
<213> 人工序列
<400> 50
taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 60
tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 120
ttacgaattc gagctcggta cccggggatc catggtgagc tgcagcgcgc cgggcaaaat 180
ttatctgttt ggcgaacatg cggtggtgta tggcgaaacc gcgattgcgt gcgcggtgga 240
actgcgcacc cgcgtgcgcg cggaactgaa cgatagcatt accattcaga gccagattgg 300
ccgcaccggc ctggattttg aaaaacatcc gtatgtgagc gcggtgattg aaaaaatgcg 360
caaaagcatt ccgattaacg gcgtgtttct gaccgtggat agcgatattc cggtgggcag 420
cggcctgggc agcagcgcgg cggtgaccat tgcgagcatt ggcgcgctga acgaactgtt 480
tggctttggc ctgagcctgc aggaaattgc gaaactgggc catgaaattg aaattaaagt 540
gcagggcgcg gcgagcccga ccgataccta tgtgagcacc tttggcggcg tggtgaccat 600
tccggaacgc cgcaaactga aaaccccgga ttgcggcatt gtgattggcg ataccggcgt 660
gtttagcagc accaaagaac tggtggcgaa cgtgcgccag ctgcgcgaaa gctatccgga 720
tctgattgaa ccgctgatga ccagcattgg caaaattagc cgcattggcg aacagctggt 780
gctgagcggc gattatgcga gcattggccg cctgatgaac gtgaaccagg gcctgctgga 840
tgcgctgggc gtgaacattc tggaactgag ccagctgatt tatagcgcgc gcgcggcggg 900
cgcgtttggc gcgaaaatta ccggcgcggg cggcggcggc tgcatggtgg cgctgaccgc 960
gccggaaaaa tgcaaccagg tggcggaagc ggtggcgggc gcgggcggca aagtgaccat 1020
taccaaaccg accgaacagg gcctgaaagt ggattaaatg gatagagagc ctgtaacagt 1080
acgttcctac gcaaatattg ctattatcaa atattgggga aagaaaaaag aaaaagagat 1140
ggtgcctgct actagcagta tttctctaac tttggaaaat atgtatacag agacgacctt 1200
gtcgccttta ccagccaatg taacagctga cgaattttac atcaatggtc agctacaaaa 1260
tgaggtcgag catgccaaga tgagtaagat tattgaccgt tatcgtccag ctggtgaggg 1320
ctttgtccgt atcgatactc aaaacaatat gcctactgca gcgggcctgt cctcaagttc 1380
tagtggtttg tccgccctgg tcaaggcttg taatgcttat ttcaagcttg gattggatag 1440
aagtcagttg gcacaggaag ccaaatttgc ctcaggctct tcttctcgga gtttttatgg 1500
accactagga gcctgggata aggatagtgg agaaatttac cctgtagaga cagacttgaa 1560
actagctatg attatgttgg tgctagagga caagaaaaaa ccaatctcta gccgtgacgg 1620
gatgaaactt tgtgtggaaa cctcgacgac ttttgacgac tgggttcgtc agtctgagaa 1680
ggactatcag gatatgctga tttatctcaa ggaaaatgat tttgccaaga ttggagaatt 1740
aacggagaaa aatgccctgg ctatgcatgc tacgacaaag actgctagtc cagccttttc 1800
ttatctgacg gatgcctctt atgaggctat ggactttgtt cgccagcttc gtgagaaagg 1860
agaggcctgc tactttacca tggatgctgg tcccaatgtt aaggtcttct gtcaggagaa 1920
agacttggag catttatcag aaattttcgg tcatcgttat cgcttgattg tgtcaaaaac 1980
aaaggatttg agtcaagatg attgctgtta aatgattgct gttaaaactt gcggaaaact 2040
ctattgggca ggtgaatatg ctattttaga gccagggcag ttagctttga taaaggatat 2100
tcccatctat atgagggctg agattgcttt ttctgacagc taccgtatct attcagatat 2160
gtttgatttc gcagtggact taaggcctaa tcctgactac agcttgattc aagaaacgat 2220
tgctttgatg ggagacttcc tcgctgttcg tggtcagaat ttaagacctt tttctctaga 2280
aatctgtggc aaaatggaac gagaagggaa aaagtttggt ctaggttcta gtggcagcgt 2340
cgttgtcttg gttgtcaagg ctttactggc tctgtatgat gtttctgttg atcaggagct 2400
cttgttcaag ctgactagcg ctgtcttgct caagcgagga gacaatggtt ccatgggcga 2460
ccttgcctgt attgtggcag aggatttggt tctctaccag tcatttgatc gccagaaggt 2520
ggctgcttgg ttagaagaag aaaacttggc gacagttctg gagcgtgatt ggggcttttc 2580
aatttcacaa gtgaaaccaa ctttagaatg tgatttctta gtgggatgga ccaaggaagt 2640
ggctgtatcg agtcacatgg tccagcaaat caagcaaaat atcaatcaaa attttttaag 2700
ttcctcaaaa gaaacggtgg tttctttggt cgaagccttg gaacagggga aatcagaaaa 2760
gattatcgag caagtagaag tagccagcaa gcttttagaa ggcttgagta cagatattta 2820
cacgcctttg cttagacagt tgaaagaagc cagtcaagat ttgcaggccg ttgccaagag 2880
tagtggtgct ggtggtggtg actgtggcat cgccctgagt tttgatgcgc aatcaaccaa 2940
aaccttaaaa aatcgttggg ccgatctggg gattgagctc ttatatcaag aaaggatagg 3000
acatgacgac aaatcgtaaa ggagagaaat tatgcaaacg gaacacgtca ttttattgaa 3060
tgcacaggga gttcccacgg gtacgctgga aaagtatgcc gcacacacgg cagacacccg 3120
cttacatctc gcgttctcca gttggctgtt taatgccaaa ggacaattat tagttacccg 3180
ccgcgcactg agcaaaaaag catggcctgg cgtgtggact aactcggttt gtgggcaccc 3240
acaactggga gaaagcaacg aagacgcagt gatccgccgt tgccgttatg agcttggcgt 3300
ggaaattacg cctcctgaat ctatctatcc tgactttcgc taccgcgcca ccgatccgag 3360
tggcattgtg gaaaatgaag tgtgtccggt atttgccgca cgcaccacta gtgcgttaca 3420
gatcaatgat gatgaagtga tggattatca atggtgtgat ttagcagatg tattacacgg 3480
tattgatgcc acgccgtggg cgttcagtcc gtggatggtg atgcaggcga caaatcgcga 3540
agccagaaaa cgattatctg catttaccca gcttaaataa aggagcattt agatgaaaac 3600
agtagttatt attgatgcat tacgaacacc aattggaaaa tataaaggca gcttaagtca 3660
agtaagtgcc gtagacttag gaacacatgt tacaacacaa cttttaaaaa gacattccac 3720
tatttctgaa gaaattgatc aagtaatctt tggaaatgtt ttacaagctg gaaatggcca 3780
aaatcccgca cgacaaatag caataaacag cggtttatct catgaaattc ccgcaatgac 3840
agttaatgag gtctgcggat caggaatgaa ggccgttatt ttggcgaaac aattgattca 3900
attaggagaa gcggaagttt taattgctgg cgggattgag aatatgtccc aagcacctaa 3960
attacaacga tttaattacg aaacagaaag ctatgatgcg cctttttcta gtatgatgta 4020
cgatgggtta acggatgcct ttagtggtca agcaatgggc ttaactgctg aaaatgtggc 4080
cgaaaagtat catgtaacta gagaagagca agatcaattt tctgtacatt cacaattaaa 4140
agcagctcaa gcacaagcag aagggatatt cgctgacgaa atagccccat tagaagtatc 4200
aggaacgctt gtggagaaag atgaagggat tcgccctaat tcgagcgttg agaagctagg 4260
aacgcttaaa acagttttta aagaagacgg tactgtaaca gcagggaatg catcaaccat 4320
taatgatggg gcttctgctt tgattattgc ttcacaagaa tatgccgaag cacacggtct 4380
tccttattta gctattattc gagacagtgt ggaagtcggt attgatccag cctatatggg 4440
aatttcgccg attaaagcca ttcaaaaact gttagcgcgc aatcaactta ctacggaaga 4500
aattgatctg tatgaaatca acgaagcatt tgcagcaact tcaatcgtgg tccaaagaga 4560
actggcttta ccagaggaaa aggtcaacat ttatggtggc ggtatttcat taggtcatgc 4620
gattggtgcc acaggtgctc gtttattaac gagtttaagt tatcaattaa atcaaaaaga 4680
aaagaaatat ggagtggctt ctttatgtat cggcggtggc ttaggactcg ctatgctact 4740
agagagacct cagcaaaaaa aaaacagccg attttatcaa atgagtcctg aggaacgcct 4800
ggcttctctt cttaatgaag gccagatttc tgctgataca aaaaaagaat ttgaaaatac 4860
ggctttatct tcgcagattg ccaatcatat gattgaaaat caaatcagtg aaacagaagt 4920
gccgatgggc gttggcttac atttaacagt ggacgaaact gattatttgg taccaatggc 4980
gacagaagag ccctcagtga ttgcggcttt gagtaatggt gcaaaaatag cacaaggatt 5040
taaaacagtg aatcaacaac gtttaatgcg tggacaaatc gttttttacg atgttgcaga 5100
cgccgagtca ttgattgatg aactacaagt aagagaaacg gaaatttttc aacaagcaga 5160
gttaagttat ccatctatcg ttaaacgcgg cggcggctta agagatttgc aatatcgtgc 5220
ttttgatgaa tcatttgtat ctgtcgactt tttagtagat gttaaggatg caatgggggc 5280
aaatatcgtt aacgctatgt tggaaggtgt ggccgagttg ttccgtgaat ggtttgcgga 5340
gcaaaagatt ttattcagta ttttaagtaa ttatgccacg gagtcggttg ttacgatgaa 5400
aacggctatt ccagtttcac gtttaagtaa ggggagcaat ggccgggaaa ttgctgaaaa 5460
aattgtttta gcttcacgct atgcttcatt agatccttat cgggcagtca cgcataacaa 5520
agggatcatg aatggcattg aagctgtcgt tttagctaca ggaaatgata cacgcgctgt 5580
tagcgcttct tgtcatgctt ttgcggtgaa ggaaggtcgc taccaaggtt tgactagttg 5640
gacgctggat ggcgaacaac taattggtga aatttcagtt ccgcttgcgt tagccacggt 5700
tggcggtgcc acaaaagtct tacctaaatc tcaagcagct gctgatttgt tagcagtgac 5760
ggatgcaaaa gaactaagtc gagtagtagc ggctgttggt ttggcacaaa atttagcggc 5820
gttacgggcc ttagtctctg aaggaattca aaaaggacac atggctctac aagcacgttc 5880
tttagcgatg acggtcggag ctactggtaa agaagttgag gcagtcgctc aacaattaaa 5940
acgtcaaaaa acgatgaacc aagaccgagc cttggctatt ttaaatgatt taagaaaaca 6000
ataaaggaga aaccttatga caattgggat tgataaaatt agtttttttg tgccccctta 6060
ttatattgat atgacggcac tggctgaagc cagaaatgta gaccctggaa aatttcatat 6120
tggtattggg caagaccaaa tggcggtgaa cccaatcagc caagatattg tgacatttgc 6180
agccaatgcc gcagaagcga tcttgaccaa agaagataaa gaggccattg atatggtgat 6240
tgtcgggact gagtccagta tcgatgagtc aaaagcggcc gcagttgtct tacatcgttt 6300
aatggggatt caacctttcg ctcgctcttt cgaaatcaag gaagcttgtt acggagcaac 6360
tgcaggctta cagttagcta agaatcacgt agccttacat ccagataaaa aagtcttggt 6420
cgtagcagca gatattgcaa aatatggctt aaattctggc ggtgagccta cacaaggagc 6480
tggggcggtt gcaatgttag ttgctagtga accgcgcatt ttggctttaa aagaggataa 6540
tgtgatgctg acgcaagata tctatgactt ttggcgtcca acaggccatc catatcctat 6600
ggtcgatggt cctttgtcaa acgaaaccta catccaatct tttgcccaag tctgggatga 6660
acataaaaaa cgaaccggtc ttgattttgc agattatgat gctttagcgt tccatattcc 6720
ttacacaaaa atgggcaaaa aagccttatt agcaaaaatc tccgaccaaa ctgaagcaga 6780
acaggaacga attttagccc gttatgaaga aagcatcatc tatagtcgtc gcgtaggaaa 6840
cttgtatacg ggttcacttt atctgggact catttccctt ttagaaaatg caacgacttt 6900
aaccgcaggc aatcaaattg ggttattcag ttatggttct ggtgctgtcg ctgaattttt 6960
cactggtgaa ttagtagctg gttatcaaaa tcatttacaa aaagaaactc atttagcact 7020
gctggataat cggacagaac tttctatcgc tgaatatgaa gccatgtttg cagaaacttt 7080
agacacagac attgatcaaa cgttaaaaga tgaattaaaa tatagtattt ctgctattaa 7140
taataccgtt cgttcttatc gaaactaa 7168
<210> 51
<211> 3402
<212> DNA
<213> 人工序列
<400> 51
atgacaaaaa aagttggtgt cggtcaggca catagtaaga taattttaat aggggaacat 60
gcggtcgttt acggttatcc tgccatttcc ctgcctcttt tggaggtgga ggtgacctgt 120
aaggtagttc ctgcagagag tccttggcgc ctttatgagg aggatacctt gtccatggcg 180
gtttatgcct cactggagta tttggatatc acagaagcct gcattcgttg tgagattgac 240
tcggctatcc ctgagaaacg ggggatgggt tcgtcagcgg ctatcagcat agcggccatt 300
cgtgcggtat ttgactacta tcaggctgat ctgcctcatg atgtactaga aatcttggtc 360
aatcgagctg aaatgattgc ccatatgaat cctagtggtt tggatgctaa gacctgtctc 420
agtgaccaac ctattcgctt tatcaagaac gtaggattta cagaacttga gatggattta 480
tccgcctatt tggtgattgc cgatacgggt gtttatggtc atactcgtga agccatccaa 540
gtggttcaaa ataagggcaa ggatgcccta ccgtttttgc atgccttggg agaattaacc 600
cagcaagcag aagttgcgat ttcacaaaaa gatgctgaag gactgggaca aatcctcagt 660
caagcgcatt tacatttaaa agaaattgga gtcagtagcc ctgaggcaga ctttttggtt 720
gaaacgactc ttagccatgg tgctctgggt gccaagatga gcggtggtgg gctaggaggt 780
tgtatcatag ccttggtaac caatttgaca cacgcacaag aactagcaga aagattagaa 840
gagaaaggag ctgttcagac atggatagag agcctgtaaa tggatagaga gcctgtaaca 900
gtacgttcct acgcaaatat tgctattatc aaatattggg gaaagaaaaa agaaaaagag 960
atggtgcctg ctactagcag tatttctcta actttggaaa atatgtatac agagacgacc 1020
ttgtcgcctt taccagccaa tgtaacagct gacgaatttt acatcaatgg tcagctacaa 1080
aatgaggtcg agcatgccaa gatgagtaag attattgacc gttatcgtcc agctggtgag 1140
ggctttgtcc gtatcgatac tcaaaacaat atgcctactg cagcgggcct gtcctcaagt 1200
tctagtggtt tgtccgccct ggtcaaggct tgtaatgctt atttcaagct tggattggat 1260
agaagtcagt tggcacagga agccaaattt gcctcaggct cttcttctcg gagtttttat 1320
ggaccactag gagcctggga taaggatagt ggagaaattt accctgtaga gacagacttg 1380
aaactagcta tgattatgtt ggtgctagag gacaagaaaa aaccaatctc tagccgtgac 1440
gggatgaaac tttgtgtgga aacctcgacg acttttgacg actgggttcg tcagtctgag 1500
aaggactatc aggatatgct gatttatctc aaggaaaatg attttgccaa gattggagaa 1560
ttaacggaga aaaatgccct ggctatgcat gctacgacaa agactgctag tccagccttt 1620
tcttatctga cggatgcctc ttatgaggct atggactttg ttcgccagct tcgtgagaaa 1680
ggagaggcct gctactttac catggatgct ggtcccaatg ttaaggtctt ctgtcaggag 1740
aaagacttgg agcatttatc agaaattttc ggtcatcgtt atcgcttgat tgtgtcaaaa 1800
acaaaggatt tgagtcaaga tgattgctgt taaatgattg ctgttaaaac ttgcggaaaa 1860
ctctattggg caggtgaata tgctatttta gagccagggc agttagcttt gataaaggat 1920
attcccatct atatgagggc tgagattgct ttttctgaca gctaccgtat ctattcagat 1980
atgtttgatt tcgcagtgga cttaaggcct aatcctgact acagcttgat tcaagaaacg 2040
attgctttga tgggagactt cctcgctgtt cgtggtcaga atttaagacc tttttctcta 2100
gaaatctgtg gcaaaatgga acgagaaggg aaaaagtttg gtctaggttc tagtggcagc 2160
gtcgttgtct tggttgtcaa ggctttactg gctctgtatg atgtttctgt tgatcaggag 2220
ctcttgttca agctgactag cgctgtcttg ctcaagcgag gagacaatgg ttccatgggc 2280
gaccttgcct gtattgtggc agaggatttg gttctctacc agtcatttga tcgccagaag 2340
gtggctgctt ggttagaaga agaaaacttg gcgacagttc tggagcgtga ttggggcttt 2400
tcaatttcac aagtgaaacc aactttagaa tgtgatttct tagtgggatg gaccaaggaa 2460
gtggctgtat cgagtcacat ggtccagcaa atcaagcaaa atatcaatca aaatttttta 2520
agttcctcaa aagaaacggt ggtttctttg gtcgaagcct tggaacaggg gaaatcagaa 2580
aagattatcg agcaagtaga agtagccagc aagcttttag aaggcttgag tacagatatt 2640
tacacgcctt tgcttagaca gttgaaagaa gccagtcaag atttgcaggc cgttgccaag 2700
agtagtggtg ctggtggtgg tgactgtggc atcgccctga gttttgatgc gcaatcaacc 2760
aaaaccttaa aaaatcgttg ggccgatctg gggattgagc tcttatatca agaaaggata 2820
ggacatgacg acaaatcgta aaggagagaa attatgcaaa cggaacacgt cattttattg 2880
aatgcacagg gagttcccac gggtacgctg gaaaagtatg ccgcacacac ggcagacacc 2940
cgcttacatc tcgcgttctc cagttggctg tttaatgcca aaggacaatt attagttacc 3000
cgccgcgcac tgagcaaaaa agcatggcct ggcgtgtgga ctaactcggt ttgtgggcac 3060
ccacaactgg gagaaagcaa cgaagacgca gtgatccgcc gttgccgtta tgagcttggc 3120
gtggaaatta cgcctcctga atctatctat cctgactttc gctaccgcgc caccgatccg 3180
agtggcattg tggaaaatga agtgtgtccg gtatttgccg cacgcaccac tagtgcgtta 3240
cagatcaatg atgatgaagt gatggattat caatggtgtg atttagcaga tgtattacac 3300
ggtattgatg ccacgccgtg ggcgttcagt ccgtggatgg tgatgcaggc gacaaatcgc 3360
gaagccagaa aacgattatc tgcatttacc cagcttaaat aa 3402
<210> 52
<211> 2640
<212> DNA
<213> 人工序列
<400> 52
ggttaaacca tgagcacact gagcgtcagc accccgagct ttagcagcag ccctctgtcg 60
agcgtgaata agaacagcac caagcagcat gtcactcgta acagcgtgat ctttcacgac 120
tcgatttggg gggaccagtt cctggaatac aaagagaaat tcaacgttgc aaccgagaaa 180
cagcttatag aagagctgaa agaagaagtg cgtaacgaac tgatgattcg tgcatgtaat 240
gaagcgagcc ggtatatcaa actgatccag ctgatcgatg ttgttgaacg tctggggctg 300
gcctatcatt ttgaaaaaga gattgaggaa agcctccagc atatatatgt gacgtatggt 360
cataaatgga cgaattacaa caatattgag agcctgagtc tgtggttccg cctgcttcgt 420
caaaatggct ttaatgttag ctcggatata tttgaaaatc acattgatga gaaaggaaat 480
tttcaggaga gcctgtgcaa tgatccgcag gggatgctgg cgctgtatga agcggcatat 540
atgcgtgttg aaggagagat cattctggac aaagcactcg aatttaccaa gctgcatctg 600
gggatcatta gcaatgatcc tagctgtgat agcagcctac gtacggaaat caagcaggca 660
ctgaaacagc cactgcgccg gcggctgcca aggctggaag ccgttcgtta cattgccatt 720
tatcagcaga aggcgagcca tagcgaggtt ctgctgaagc tggccaaact ggacttcaac 780
gttctgcagg aaatgcacaa agacgaattg agccaaatat gcaaatggtg gaaagatctg 840
gatatacgta acaaactgcc ctatgttcgt gatcgtctga ttgaaggcta tttttggatt 900
ctgggtattt atttcgaacc gcaacactcc cgtacccgta tgttcctgat gaaaacctgt 960
atgtggctga tcgtgctgga cgatacgttt gataattacg gcacctatga agagttagag 1020
atctttaccc aagcagtcga acgttggagc attacctgtc tggatgaact gccagagtat 1080
atgaagctga tatatcacga gcaatttcgc gtgcatcagg aaatggagga aagcctggaa 1140
aaggagggta aggcctacca gattcattat atcaaagaaa tggccaaaga aggtactcgt 1200
tcgctgctgc tggaagcgaa atggctgaag gaaggctata tgcctaccct ggatgagtac 1260
ctgagcaaca gcctggtcac ctgcggctat gcactgatga ccgcacgcag ctacgttgcc 1320
cgtgacgacg gcattgttac cgaagatgca ttcaaatggg ttgcaacgca cccgccgatt 1380
gttaaagcag catgcaaaat tctgcgcctg atggacgaca ttgcaaccca taaagaggaa 1440
caggagcggg gacacattgc aagtagcatt gagtgttaca ggaaggaaac cggagctagc 1500
gaagaggagg cttgcatgga ctttctgaag caggttgaag atggttggaa agttattaat 1560
caagaaagcc tgatgccgac cgatgttccg ttccctctgc tgattccggc aattaacctg 1620
gcacgtgtga gcgacaccct gtacaaagac aacgatggtt ataatcatgc cgataaagag 1680
gttataggtt atattaaaag cctgtttgta catccgatga tagtcccaac gacgacgacg 1740
ccagactttc cgcagcaact cgaagcctgc gttaagcagg ccaaccaggc gctgagccgt 1800
tttatcgccc cactgccctt tcagaacact cccgtggtcg aaaccatgca gtatggcgca 1860
ttattaggtg gtaagcgcct gcgacctttc ctggtttatg ccaccggtca tatgttcggc 1920
gttagcacaa acacgctgga cgcacccgct gccgccgttg agtgtatcca cgcttactca 1980
ttaattcatg atgatttacc ggcaatggat gatgacgatc tgcgtcgcgg tttgccaacc 2040
tgccatgtga agtttggcga agcaaacgcg attctcgctg gcgacgcttt acaaacgctg 2100
gcgttctcga ttttaagcga tgccgatatg ccggaagtgt cggaccgcga cagaatttcg 2160
atgatttctg aactggcgag cgccagtggt attgccggaa tgtgcggtgg tcaggcatta 2220
gatttagacg cggaaggcaa acacgtacct ctggacgcgc ttgagcgtat tcatcgtcat 2280
aaaaccggcg cattgattcg cgccgccgtt cgccttggtg cattaagcgc cggagataaa 2340
ggacgtcgtg ctctgccggt actcgacaag tatgcagaga gcatcggcct tgccttccag 2400
gttcaggatg acatcctgga tgtggtggga gatactgcaa cgttgggaaa acgccagggt 2460
gccgaccagc aacttggtaa aagtacctac cctgcacttc tgggtcttga gcaagcccgg 2520
aagaaagccc gggatctgat cgacgatgcc cgtcagtcgc tgaaacaact ggctgaacag 2580
tcactcgata cctcggcact ggaagcgcta gcggactaca tcatccagcg taataaataa 2640
<210> 53
<211> 2643
<212> DNA
<213> 人工序列
<400> 53
ggttaaacca tgagcacact gagcgtcagc accccgagct ttagcagcag ccctctgtcg 60
agcgtgaata agaacagcac caagcagcat gtcactcgta acagcgtgat ctttcacgac 120
tcgatttggg gggaccagtt cctggaatac aaagagaaat tcaacgttgc aaccgagaaa 180
cagcttatag aagagctgaa agaagaagtg cgtaacgaac tgatgattcg tgcatgtaat 240
gaagcgagcc ggtatatcaa actgatccag ctgatcgatg ttgttgaacg tctggggctg 300
gcctatcatt ttgaaaaaga gattgaggaa agcctccagc atatatatgt gacgtatggt 360
cataaatgga cgaattacaa caatattgag agcctgagtc tgtggttccg cctgcttcgt 420
caaaatggct ttaatgttag ctcggatata tttgaaaatc acattgatga gaaaggaaat 480
tttcaggaga gcctgtgcaa tgatccgcag gggatgctgg cgctgtatga agcggcatat 540
atgcgtgttg aaggagagat cattctggac aaagcactcg aatttaccaa gctgcatctg 600
gggatcatta gcaatgatcc tagctgtgat agcagcctac gtacggaaat caagcaggca 660
ctgaaacagc cactgcgccg gcggctgcca aggctggaag ccgttcgtta cattgccatt 720
tatcagcaga aggcgagcca tagcgaggtt ctgctgaagc tggccaaact ggacttcaac 780
gttctgcagg aaatgcacaa agacgaattg agccaaatat gcaaatggtg gaaagatctg 840
gatatacgta acaaactgcc ctatgttcgt gatcgtctga ttgaaggcta tttttggatt 900
ctgggtattt atttcgaacc gcaacactcc cgtacccgta tgttcctgat gaaaacctgt 960
atgtggctga tcgtgctgga cgatacgttt gataattacg gcacctatga agagttagag 1020
atctttaccc aagcagtcga acgttggagc attacctgtc tggatgaact gccagagtat 1080
atgaagctga tatatcacga gcaatttcgc gtgcatcagg aaatggagga aagcctggaa 1140
aaggagggta aggcctacca gattcattat atcaaagaaa tggccaaaga aggtactcgt 1200
tcgctgctgc tggaagcgaa atggctgaag gaaggctata tgcctaccct ggatgagtac 1260
ctgagcaaca gcctggtcac ctgcggctat gcactgatga ccgcacgcag ctacgttgcc 1320
cgtgacgacg gcattgttac cgaagatgca ttcaaatggg ttgcaacgca cccgccgatt 1380
gttaaagcag catgcaaaat tctgcgcctg atggacgaca ttgcaaccca taaagaggaa 1440
caggagcggg gacacattgc aagtagcatt gagtgttaca ggaaggaaac cggagctagc 1500
gaagaggagg cttgcatgga ctttctgaag caggttgaag atggttggaa agttattaat 1560
caagaaagcc tgatgccgac cgatgttccg ttccctctgc tgattccggc aattaacctg 1620
gcacgtgtga gcgacaccct gtacaaagac aacgatggtt ataatcatgc cgataaagag 1680
gttataggtt atattaaaag cctgtttgta catccgatga tagtcggagg aggaggatca 1740
tcatcagact ttccgcagca actcgaagcc tgcgttaagc aggccaacca ggcgctgagc 1800
cgttttatcg ccccactgcc ctttcagaac actcccgtgg tcgaaaccat gcagtatggc 1860
gcattattag gtggtaagcg cctgcgacct ttcctggttt atgccaccgg tcatatgttc 1920
ggcgttagca caaacacgct ggacgcaccc gctgccgccg ttgagtgtat ccacgcttac 1980
tcattaattc atgatgattt accggcaatg gatgatgacg atctgcgtcg cggtttgcca 2040
acctgccatg tgaagtttgg cgaagcaaac gcgattctcg ctggcgacgc tttacaaacg 2100
ctggcgttct cgattttaag cgatgccgat atgccggaag tgtcggaccg cgacagaatt 2160
tcgatgattt ctgaactggc gagcgccagt ggtattgccg gaatgtgcgg tggtcaggca 2220
ttagatttag acgcggaagg caaacacgta cctctggacg cgcttgagcg tattcatcgt 2280
cataaaaccg gcgcattgat tcgcgccgcc gttcgccttg gtgcattaag cgccggagat 2340
aaaggacgtc gtgctctgcc ggtactcgac aagtatgcag agagcatcgg ccttgccttc 2400
caggttcagg atgacatcct ggatgtggtg ggagatactg caacgttggg aaaacgccag 2460
ggtgccgacc agcaacttgg taaaagtacc taccctgcac ttctgggtct tgagcaagcc 2520
cggaagaaag cccgggatct gatcgacgat gcccgtcagt cgctgaaaca actggctgaa 2580
cagtcactcg atacctcggc actggaagcg ctagcggact acatcatcca gcgtaataaa 2640
taa 2643
<210> 54
<211> 2634
<212> DNA
<213> 人工序列
<400> 54
ggttaaacca tgagcacact gagcgtcagc accccgagct ttagcagcag ccctctgtcg 60
agcgtgaata agaacagcac caagcagcat gtcactcgta acagcgtgat ctttcacgac 120
tcgatttggg gggaccagtt cctggaatac aaagagaaat tcaacgttgc aaccgagaaa 180
cagcttatag aagagctgaa agaagaagtg cgtaacgaac tgatgattcg tgcatgtaat 240
gaagcgagcc ggtatatcaa actgatccag ctgatcgatg ttgttgaacg tctggggctg 300
gcctatcatt ttgaaaaaga gattgaggaa agcctccagc atatatatgt gacgtatggt 360
cataaatgga cgaattacaa caatattgag agcctgagtc tgtggttccg cctgcttcgt 420
caaaatggct ttaatgttag ctcggatata tttgaaaatc acattgatga gaaaggaaat 480
tttcaggaga gcctgtgcaa tgatccgcag gggatgctgg cgctgtatga agcggcatat 540
atgcgtgttg aaggagagat cattctggac aaagcactcg aatttaccaa gctgcatctg 600
gggatcatta gcaatgatcc tagctgtgat agcagcctac gtacggaaat caagcaggca 660
ctgaaacagc cactgcgccg gcggctgcca aggctggaag ccgttcgtta cattgccatt 720
tatcagcaga aggcgagcca tagcgaggtt ctgctgaagc tggccaaact ggacttcaac 780
gttctgcagg aaatgcacaa agacgaattg agccaaatat gcaaatggtg gaaagatctg 840
gatatacgta acaaactgcc ctatgttcgt gatcgtctga ttgaaggcta tttttggatt 900
ctgggtattt atttcgaacc gcaacactcc cgtacccgta tgttcctgat gaaaacctgt 960
atgtggctga tcgtgctgga cgatacgttt gataattacg gcacctatga agagttagag 1020
atctttaccc aagcagtcga acgttggagc attacctgtc tggatgaact gccagagtat 1080
atgaagctga tatatcacga gcaatttcgc gtgcatcagg aaatggagga aagcctggaa 1140
aaggagggta aggcctacca gattcattat atcaaagaaa tggccaaaga aggtactcgt 1200
tcgctgctgc tggaagcgaa atggctgaag gaaggctata tgcctaccct ggatgagtac 1260
ctgagcaaca gcctggtcac ctgcggctat gcactgatga ccgcacgcag ctacgttgcc 1320
cgtgacgacg gcattgttac cgaagatgca ttcaaatggg ttgcaacgca cccgccgatt 1380
gttaaagcag catgcaaaat tctgcgcctg atggacgaca ttgcaaccca taaagaggaa 1440
caggagcggg gacacattgc aagtagcatt gagtgttaca ggaaggaaac cggagctagc 1500
gaagaggagg cttgcatgga ctttctgaag caggttgaag atggttggaa agttattaat 1560
caagaaagcc tgatgccgac cgatgttccg ttccctctgc tgattccggc aattaacctg 1620
gcacgtgtga gcgacaccct gtacaaagac aacgatggtt ataatcatgc cgataaagag 1680
gttataggtt atattaaaag cctgtttgta catccgatga tagtcggagg aggaatcgac 1740
tttccgcagc aactcgaagc ctgcgttaag caggccaacc aggcgctgag ccgttttatc 1800
gccccactgc cctttcagaa cactcccgtg gtcgaaacca tgcagtatgg cgcattatta 1860
ggtggtaagc gcctgcgacc tttcctggtt tatgccaccg gtcatatgtt cggcgttagc 1920
acaaacacgc tggacgcacc cgctgccgcc gttgagtgta tccacgctta ctcattaatt 1980
catgatgatt taccggcaat ggatgatgac gatctgcgtc gcggtttgcc aacctgccat 2040
gtgaagtttg gcgaagcaaa cgcgattctc gctggcgacg ctttacaaac gctggcgttc 2100
tcgattttaa gcgatgccga tatgccggaa gtgtcggacc gcgacagaat ttcgatgatt 2160
tctgaactgg cgagcgccag tggtattgcc ggaatgtgcg gtggtcaggc attagattta 2220
gacgcggaag gcaaacacgt acctctggac gcgcttgagc gtattcatcg tcataaaacc 2280
ggcgcattga ttcgcgccgc cgttcgcctt ggtgcattaa gcgccggaga taaaggacgt 2340
cgtgctctgc cggtactcga caagtatgca gagagcatcg gccttgcctt ccaggttcag 2400
gatgacatcc tggatgtggt gggagatact gcaacgttgg gaaaacgcca gggtgccgac 2460
cagcaacttg gtaaaagtac ctaccctgca cttctgggtc ttgagcaagc ccggaagaaa 2520
gcccgggatc tgatcgacga tgcccgtcag tcgctgaaac aactggctga acagtcactc 2580
gatacctcgg cactggaagc gctagcggac tacatcatcc agcgtaataa ataa 2634
<210> 55
<211> 2634
<212> DNA
<213> 人工序列
<400> 55
ggttaaacca tgagcacact gagcgtcagc accccgagct ttagcagcag ccctctgtcg 60
agcgtgaata agaacagcac caagcagcat gtcactcgta acagcgtgat ctttcacgac 120
tcgatttggg gggaccagtt cctggaatac aaagagaaat tcaacgttgc aaccgagaaa 180
cagcttatag aagagctgaa agaagaagtg cgtaacgaac tgatgattcg tgcatgtaat 240
gaagcgagcc ggtatatcaa actgatccag ctgatcgatg ttgttgaacg tctggggctg 300
gcctatcatt ttgaaaaaga gattgaggaa agcctccagc atatatatgt gacgtatggt 360
cataaatgga cgaattacaa caatattgag agcctgagtc tgtggttccg cctgcttcgt 420
caaaatggct ttaatgttag ctcggatata tttgaaaatc acattgatga gaaaggaaat 480
tttcaggaga gcctgtgcaa tgatccgcag gggatgctgg cgctgtatga agcggcatat 540
atgcgtgttg aaggagagat cattctggac aaagcactcg aatttaccaa gctgcatctg 600
gggatcatta gcaatgatcc tagctgtgat agcagcctac gtacggaaat caagcaggca 660
ctgaaacagc cactgcgccg gcggctgcca aggctggaag ccgttcgtta cattgccatt 720
tatcagcaga aggcgagcca tagcgaggtt ctgctgaagc tggccaaact ggacttcaac 780
gttctgcagg aaatgcacaa agacgaattg agccaaatat gcaaatggtg gaaagatctg 840
gatatacgta acaaactgcc ctatgttcgt gatcgtctga ttgaaggcta tttttggatt 900
ctgggtattt atttcgaacc gcaacactcc cgtacccgta tgttcctgat gaaaacctgt 960
atgtggctga tcgtgctgga cgatacgttt gataattacg gcacctatga agagttagag 1020
atctttaccc aagcagtcga acgttggagc attacctgtc tggatgaact gccagagtat 1080
atgaagctga tatatcacga gcaatttcgc gtgcatcagg aaatggagga aagcctggaa 1140
aaggagggta aggcctacca gattcattat atcaaagaaa tggccaaaga aggtactcgt 1200
tcgctgctgc tggaagcgaa atggctgaag gaaggctata tgcctaccct ggatgagtac 1260
ctgagcaaca gcctggtcac ctgcggctat gcactgatga ccgcacgcag ctacgttgcc 1320
cgtgacgacg gcattgttac cgaagatgca ttcaaatggg ttgcaacgca cccgccgatt 1380
gttaaagcag catgcaaaat tctgcgcctg atggacgaca ttgcaaccca taaagaggaa 1440
caggagcggg gacacattgc aagtagcatt gagtgttaca ggaaggaaac cggagctagc 1500
gaagaggagg cttgcatgga ctttctgaag caggttgaag atggttggaa agttattaat 1560
caagaaagcc tgatgccgac cgatgttccg ttccctctgc tgattccggc aattaacctg 1620
gcacgtgtga gcgacaccct gtacaaagac aacgatggtt ataatcatgc cgataaagag 1680
gttataggtt atattaaaag cctgtttgta catccgatga tagtcggaag cggaggagac 1740
tttccgcagc aactcgaagc ctgcgttaag caggccaacc aggcgctgag ccgttttatc 1800
gccccactgc cctttcagaa cactcccgtg gtcgaaacca tgcagtatgg cgcattatta 1860
ggtggtaagc gcctgcgacc tttcctggtt tatgccaccg gtcatatgtt cggcgttagc 1920
acaaacacgc tggacgcacc cgctgccgcc gttgagtgta tccacgctta ctcattaatt 1980
catgatgatt taccggcaat ggatgatgac gatctgcgtc gcggtttgcc aacctgccat 2040
gtgaagtttg gcgaagcaaa cgcgattctc gctggcgacg ctttacaaac gctggcgttc 2100
tcgattttaa gcgatgccga tatgccggaa gtgtcggacc gcgacagaat ttcgatgatt 2160
tctgaactgg cgagcgccag tggtattgcc ggaatgtgcg gtggtcaggc attagattta 2220
gacgcggaag gcaaacacgt acctctggac gcgcttgagc gtattcatcg tcataaaacc 2280
ggcgcattga ttcgcgccgc cgttcgcctt ggtgcattaa gcgccggaga taaaggacgt 2340
cgtgctctgc cggtactcga caagtatgca gagagcatcg gccttgcctt ccaggttcag 2400
gatgacatcc tggatgtggt gggagatact gcaacgttgg gaaaacgcca gggtgccgac 2460
cagcaacttg gtaaaagtac ctaccctgca cttctgggtc ttgagcaagc ccggaagaaa 2520
gcccgggatc tgatcgacga tgcccgtcag tcgctgaaac aactggctga acagtcactc 2580
gatacctcgg cactggaagc gctagcggac tacatcatcc agcgtaataa ataa 2634
<210> 56
<211> 2634
<212> DNA
<213> 人工序列
<400> 56
ggttaaacca tgagcacact gagcgtcagc accccgagct ttagcagcag ccctctgtcg 60
agcgtgaata agaacagcac caagcagcat gtcactcgta acagcgtgat ctttcacgac 120
tcgatttggg gggaccagtt cctggaatac aaagagaaat tcaacgttgc aaccgagaaa 180
cagcttatag aagagctgaa agaagaagtg cgtaacgaac tgatgattcg tgcatgtaat 240
gaagcgagcc ggtatatcaa actgatccag ctgatcgatg ttgttgaacg tctggggctg 300
gcctatcatt ttgaaaaaga gattgaggaa agcctccagc atatatatgt gacgtatggt 360
cataaatgga cgaattacaa caatattgag agcctgagtc tgtggttccg cctgcttcgt 420
caaaatggct ttaatgttag ctcggatata tttgaaaatc acattgatga gaaaggaaat 480
tttcaggaga gcctgtgcaa tgatccgcag gggatgctgg cgctgtatga agcggcatat 540
atgcgtgttg aaggagagat cattctggac aaagcactcg aatttaccaa gctgcatctg 600
gggatcatta gcaatgatcc tagctgtgat agcagcctac gtacggaaat caagcaggca 660
ctgaaacagc cactgcgccg gcggctgcca aggctggaag ccgttcgtta cattgccatt 720
tatcagcaga aggcgagcca tagcgaggtt ctgctgaagc tggccaaact ggacttcaac 780
gttctgcagg aaatgcacaa agacgaattg agccaaatat gcaaatggtg gaaagatctg 840
gatatacgta acaaactgcc ctatgttcgt gatcgtctga ttgaaggcta tttttggatt 900
ctgggtattt atttcgaacc gcaacactcc cgtacccgta tgttcctgat gaaaacctgt 960
atgtggctga tcgtgctgga cgatacgttt gataattacg gcacctatga agagttagag 1020
atctttaccc aagcagtcga acgttggagc attacctgtc tggatgaact gccagagtat 1080
atgaagctga tatatcacga gcaatttcgc gtgcatcagg aaatggagga aagcctggaa 1140
aaggagggta aggcctacca gattcattat atcaaagaaa tggccaaaga aggtactcgt 1200
tcgctgctgc tggaagcgaa atggctgaag gaaggctata tgcctaccct ggatgagtac 1260
ctgagcaaca gcctggtcac ctgcggctat gcactgatga ccgcacgcag ctacgttgcc 1320
cgtgacgacg gcattgttac cgaagatgca ttcaaatggg ttgcaacgca cccgccgatt 1380
gttaaagcag catgcaaaat tctgcgcctg atggacgaca ttgcaaccca taaagaggaa 1440
caggagcggg gacacattgc aagtagcatt gagtgttaca ggaaggaaac cggagctagc 1500
gaagaggagg cttgcatgga ctttctgaag caggttgaag atggttggaa agttattaat 1560
caagaaagcc tgatgccgac cgatgttccg ttccctctgc tgattccggc aattaacctg 1620
gcacgtgtga gcgacaccct gtacaaagac aacgatggtt ataatcatgc cgataaagag 1680
gttataggtt atattaaaag cctgtttgta catccgatga tagtcggagg aatcggagac 1740
tttccgcagc aactcgaagc ctgcgttaag caggccaacc aggcgctgag ccgttttatc 1800
gccccactgc cctttcagaa cactcccgtg gtcgaaacca tgcagtatgg cgcattatta 1860
ggtggtaagc gcctgcgacc tttcctggtt tatgccaccg gtcatatgtt cggcgttagc 1920
acaaacacgc tggacgcacc cgctgccgcc gttgagtgta tccacgctta ctcattaatt 1980
catgatgatt taccggcaat ggatgatgac gatctgcgtc gcggtttgcc aacctgccat 2040
gtgaagtttg gcgaagcaaa cgcgattctc gctggcgacg ctttacaaac gctggcgttc 2100
tcgattttaa gcgatgccga tatgccggaa gtgtcggacc gcgacagaat ttcgatgatt 2160
tctgaactgg cgagcgccag tggtattgcc ggaatgtgcg gtggtcaggc attagattta 2220
gacgcggaag gcaaacacgt acctctggac gcgcttgagc gtattcatcg tcataaaacc 2280
ggcgcattga ttcgcgccgc cgttcgcctt ggtgcattaa gcgccggaga taaaggacgt 2340
cgtgctctgc cggtactcga caagtatgca gagagcatcg gccttgcctt ccaggttcag 2400
gatgacatcc tggatgtggt gggagatact gcaacgttgg gaaaacgcca gggtgccgac 2460
cagcaacttg gtaaaagtac ctaccctgca cttctgggtc ttgagcaagc ccggaagaaa 2520
gcccgggatc tgatcgacga tgcccgtcag tcgctgaaac aactggctga acagtcactc 2580
gatacctcgg cactggaagc gctagcggac tacatcatcc agcgtaataa ataa 2634
Claims (19)
1.一种产(-)-α-红没药醇的重组基因工程菌，其特征在于：它是包含(-)-α-红没药醇合成酶MrBBS基因、法尼基二磷酸合酶ispA基因、MVA途径基因的重组大肠杆菌；所述(-)-α-红没药醇合成酶MrBBS基因与法尼基二磷酸合酶ispA基因之间通过SEQ ID NO.7、SEQ IDNO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列连接。
2.根据权利要求1所述的重组基因工程菌，其特征在于：所述(-)-α-红没药醇合成酶MrBBS基因中的终止密码子TAA被SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列代替，并与无起始密码子ATG的法尼基二磷酸合酶ispA基因连接。
3.根据权利要求1所述的重组基因工程菌，其特征在于：所述(-)-α-红没药醇合成酶MrBBS基因5’端带有SEQ ID NO.1所示核苷酸序列。
4.根据权利要求1所述的重组基因工程菌，其特征在于：所述(-)-α-红没药醇合成酶MrBBS基因来自春黄菊花；所述法尼基二磷酸合酶ispA基因来自大肠杆菌。
5.根据权利要求4所述的重组基因工程菌，其特征在于：所述(-)-α-红没药醇合成酶MrBBS基因的核苷酸序列如SEQ ID NO.12所示；所述法尼基二磷酸合酶ispA基因的核苷酸序列如SEQ ID NO.13所示。
6.根据权利要求5所述的重组基因工程菌，其特征在于：所述(-)-α-红没药醇合成酶MrBBS基因与法尼基二磷酸合酶ispA基因连接后的核苷酸序列如SEQ ID NO.52、SEQ IDNO.53、SEQ ID NO.54、SEQ ID NO.55或SEQ ID NO.56所示。
7.根据权利要求1所述的重组基因工程菌，其特征在于：所述MVA途径基因包括甲羟戊酸激酶mvaKmm基因、甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因、异戊烯基二磷酸δ-异构酶idi基因、3-羟基-3-甲基戊二酰CoA合酶mvaS基因、乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因和/或甲羟戊酸激酶mvaK1基因。
8.根据权利要求7所述的重组基因工程菌，其特征在于：所述甲羟戊酸激酶mvaKmm基因来自甲烷八叠球古菌Methanosarcina mazei；
所述甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因和甲羟戊酸激酶mvaK1基因来自肺炎链球菌Streptococcus pneumoniae；
所述异戊烯二磷酸δ异构酶idi基因来自大肠杆菌Escherichia coli；
所述3-羟基-3-甲基戊二酰CoA合酶mvaS基因和乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因来自粪肠球菌Enterococcus faecalis。
9.根据权利要求8所述的重组基因工程菌，其特征在于：所述甲羟戊酸激酶mvaKmm基因的核苷酸序列如SEQ ID NO.14所示，甲羟戊酸5-焦磷酸脱羧酶mvaD基因的核苷酸序列如SEQ ID NO.15所示，磷酸甲羟戊酸激酶mvaK2基因的核苷酸序列如SEQ ID NO.16所示，异戊烯二磷酸δ异构酶idi基因的核苷酸序列如SEQ ID NO.17所示，3-羟基-3-甲基戊二酰CoA合酶mvaS基因的核苷酸序列如SEQ ID NO.18所示，乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因的核苷酸序列如SEQ ID NO.19所示，甲羟戊酸激酶mvaK1基因的核苷酸序列如SEQ ID NO.20所示。
10.根据权利要求9所述的重组基因工程菌：其特征在于：所述异戊烯基二磷酸δ-异构酶idi基因5’端带有核苷酸序列SEQ ID NO.3，乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因5’端带有核苷酸序列SEQ ID NO.4，3-羟基-3-甲基戊二酰CoA合酶mvaS基因5’端带有核苷酸序列SEQ ID NO.5，甲羟戊酸激酶mvaKmm基因5’端带有核苷酸序列SEQID NO.6。
11.根据权利要求7所述的重组基因工程菌：其特征在于：所述MVA途径基因与(-)-α-红没药醇合成酶MrBBS基因和法尼基二磷酸合酶ispA基因连接在两个质粒上，其中一个质粒连接的核苷酸序列包括SEQ ID NO.52、SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55或SEQID NO.56，以及SEQ ID NO.50，另一个质粒连接的核苷酸序列包括SEQ ID NO.51；
所述质粒优选为质粒pSTV28和质粒pTrc99A。
12.根据权利要求1～11任一所述的重组基因工程菌，其特征在于：所述重组大肠杆菌为重组大肠杆菌E.coliDH5α或E.coli W3110。
13.一种权利要求1～12任一项所述重组基因工程菌的制备方法，其特征在于：它包括如下步骤：
1)取(-)-α-红没药醇合成酶MrBBS基因和法尼基二磷酸合酶ispA基因融合，融合产物与线性化表达载体连接，再导入大肠杆菌，提取重组表达载体；
所述(-)-α-红没药醇合成酶MrBBS基因中的终止密码子TAA被如SEQ ID NO.7、SEQ IDNO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列代替；
所述法尼基二磷酸合酶ispA基因中的起始密码子ATG被如SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的核苷酸序列代替；
2)取MVA途径基因融合，融合产物与酶切后的步骤1)所得重组表达载体连接，再导入大肠杆菌，提取重组表达载体；
3)取甲羟戊酸激酶mvaK1基因，与包含甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因和异戊烯基二磷酸δ-异构酶idi基因的基因片段融合，融合产物与线性化表达载体连接，再导入大肠杆菌，提取重组表达载体；
4)将步骤2)所得重组表达载体和步骤3)所得重组表达载体，导入大肠杆菌中，即得重组基因工程菌。
14.根据权利要求13所述的制备方法：其特征在于：步骤2)所述取MVA途径基因融合是取包含甲羟戊酸激酶mvaKmm基因、甲羟戊酸5-焦磷酸脱羧酶mvaD基因、磷酸甲羟戊酸激酶mvaK2基因和异戊烯基二磷酸δ-异构酶idi基因的基因片段，与包含3-羟基-3-甲基戊二酰CoA合酶mvaS基因和乙酰乙酰CoA硫解酶/3-羟基-3-甲基戊二酰CoA还原酶mvaE基因的基因片段融合。
15.根据权利要求14所述的制备方法：其特征在于：步骤1)所述表达载体为质粒pSTV28，和/或，步骤3)所述表达载体为质粒pTrc99A。
16.权利要求1-12任一所述的重组基因工程菌在制备(-)-α-红没药醇及其制剂中的用途。
17.一种产(-)-α-红没药醇的方法，其特征在于：它包括如下步骤：
取权利要求1～12任一所述的重组基因工程菌，接种于种子培养基，培养8～10h，取种子液，接种于发酵培养基，加正十二烷，发酵培养30～60h；
所述种子培养基的配方为：胰蛋白胨5～15g/L、酵母粉2～8g/L、氯化钠5～15g/L、氨苄青霉素终浓度50～150mg/L和氯霉素终浓度30～40mg/L；
所述发酵培养基的配方为：葡萄糖或甘油5～15g/L、磷酸二氢钾2～3g/L、磷酸氢二钾2.5～3.0g/L、酵母粉20～28g/L、酵母蛋白胨10～20g/L、IPTG 0.1～0.2mM、氨苄青霉素终浓度50～150mg/L和氯霉素终浓度为30～38mg/L。
18.根据权利要求17所述的方法：其特征在于：所述种子液、发酵培养基、正十二烷的体积比为2：25：5；所述培养为振荡培养，温度30℃，转速200rpm；所述发酵培养到3h添加培养容器容量4～8×10-4的0.25M IPTG。
19.根据权利要求18所述的方法：其特征在于：所述种子培养基的配方为：胰蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L、氨苄青霉素终浓度100mg/L和氯霉素终浓度34mg/L；
所述发酵培养基的配方为葡萄糖或甘油10g/L、磷酸二氢钾2.2g/L、磷酸氢二钾2.9g/L、酵母粉24g/L、酵母蛋白胨12g/L、IPTG 0.1mM、氨苄青霉素终浓度100mg/L和氯霉素终浓度为34mg/L。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210535851.0A CN115074302A (zh) | 2022-05-17 | 2022-05-17 | 一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210535851.0A CN115074302A (zh) | 2022-05-17 | 2022-05-17 | 一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115074302A true CN115074302A (zh) | 2022-09-20 |
Family
ID=83247792
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210535851.0A Pending CN115074302A (zh) | 2022-05-17 | 2022-05-17 | 一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115074302A (zh) |
-
2022
- 2022-05-17 CN CN202210535851.0A patent/CN115074302A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101511639B1 (ko) | 재조합 미생물 및 이의 사용 방법 | |
| KR101420889B1 (ko) | 생물 유기 화합물을 제조하기 위한 장치 | |
| CN113234652B (zh) | 高效合成麦角硫因的工程菌的构建方法与应用 | |
| KR20170121147A (ko) | 발효 경로를 통해 플럭스 증가를 나타내는 재조합 미생물 | |
| CN107771214A (zh) | 用于具有增加的2,4‑二羟基丁酸外排物的优化的2,4‑二羟基丁酸产生的修饰的微生物 | |
| CN111434773A (zh) | 一种高产檀香油的重组酵母菌及其构建方法与应用 | |
| CN113122490B (zh) | 双基因缺陷型工程菌及其在提高n-乙酰氨基葡萄糖产量的应用 | |
| CN114181877B (zh) | 一种合成香兰素的基因工程菌及其应用 | |
| CN115873836A (zh) | 一种橙花叔醇合成酶及应用 | |
| CA2935979C (en) | Recombinant microorganism having enhanced d(-) 2,3-butanediol producing ability and method for producing d(-) 2,3-butanediol using the same | |
| KR20200010285A (ko) | 증가된 nadph를 유도하는 생합성 경로의 게놈 공학 | |
| CN111690585B (zh) | rcsB基因缺失的重组粘质沙雷氏菌及其应用 | |
| EP1354954A1 (en) | Process for producing prenyl alcohol | |
| CN113583925B (zh) | 一种代谢工程大肠杆菌发酵制备广藿香醇的方法 | |
| CN115667518A (zh) | 重组的微生物和方法 | |
| CN114540261A (zh) | 一种产氨基己二酸的基因工程菌 | |
| CN107223152B (zh) | 具有改变的一氧化碳脱氢酶(codh)活性的遗传工程细菌 | |
| CN115074302A (zh) | 一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途 | |
| CN112708569A (zh) | 发酵生产硫酸软骨素的酵母工程菌及其应用 | |
| CN115094015A (zh) | 一种产(-)-α-红没药醇的重组基因工程菌及其制备方法和用途 | |
| CN108913732B (zh) | 一种莫纳可林j异源生产的方法及应用 | |
| KR20210151928A (ko) | 호열성 단백질을 이용한 재조합 시험관내 전사 및 해독을 위한 시스템, 방법 및 조성물 | |
| CN110982723A (zh) | 一种重组酿酒酵母及其在生产α-红没药醇中的应用 | |
| CN110982773A (zh) | 重组枯草芽孢杆菌及其在生产2,3-丁二醇中的应用 | |
| CN114058602B (zh) | 新疆紫草咖啡酸及迷迭香酸糖基转移酶及编码基因与应用 |
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 |