CN116590161B - Recombinant amycolatopsis for producing vanillin, construction method and application thereof - Google Patents
Recombinant amycolatopsis for producing vanillin, construction method and application thereof Download PDFInfo
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- CN116590161B CN116590161B CN202310519438.XA CN202310519438A CN116590161B CN 116590161 B CN116590161 B CN 116590161B CN 202310519438 A CN202310519438 A CN 202310519438A CN 116590161 B CN116590161 B CN 116590161B
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- amycolatopsis
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- vanillin
- coniferyl
- pkc1139
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- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 title claims abstract description 58
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 235000012141 vanillin Nutrition 0.000 title claims abstract description 57
- 241000187643 Amycolatopsis Species 0.000 title claims abstract description 42
- 238000010276 construction Methods 0.000 title claims description 15
- RRAFCDWBNXTKKO-UHFFFAOYSA-N eugenol Chemical compound COC1=CC(CC=C)=CC=C1O RRAFCDWBNXTKKO-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000000855 fermentation Methods 0.000 claims abstract description 31
- 230000004151 fermentation Effects 0.000 claims abstract description 31
- NPBVQXIMTZKSBA-UHFFFAOYSA-N Chavibetol Natural products COC1=CC=C(CC=C)C=C1O NPBVQXIMTZKSBA-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000005770 Eugenol Substances 0.000 claims abstract description 24
- UVMRYBDEERADNV-UHFFFAOYSA-N Pseudoeugenol Natural products COC1=CC(C(C)=C)=CC=C1O UVMRYBDEERADNV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229960002217 eugenol Drugs 0.000 claims abstract description 24
- 108010024679 Coniferyl-alcohol dehydrogenase Proteins 0.000 claims abstract description 19
- 108010000798 Coniferyl-aldehyde dehydrogenase Proteins 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 108010005214 Vanillyl-alcohol oxidase Proteins 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 239000013612 plasmid Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 16
- 241001522168 Amycolatopsis sp. Species 0.000 claims description 14
- 238000005520 cutting process Methods 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 238000012546 transfer Methods 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 10
- 241000219194 Arabidopsis Species 0.000 claims description 9
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims description 8
- 241001474374 Blennius Species 0.000 claims description 8
- 241000223218 Fusarium Species 0.000 claims description 8
- 241000219823 Medicago Species 0.000 claims description 8
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 claims description 8
- 244000290333 Vanilla fragrans Species 0.000 claims description 8
- 235000009499 Vanilla fragrans Nutrition 0.000 claims description 8
- 235000012036 Vanilla tahitensis Nutrition 0.000 claims description 8
- 230000021615 conjugation Effects 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
- 230000002194 synthesizing effect Effects 0.000 claims description 8
- 108010025188 Alcohol oxidase Proteins 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 230000001131 transforming effect Effects 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 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 5
- 239000008103 glucose Substances 0.000 claims description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- 238000001976 enzyme digestion Methods 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 239000008272 agar Substances 0.000 claims description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 claims description 2
- 229960000210 nalidixic acid Drugs 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 238000011218 seed culture Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims 4
- UGXQOOQUZRUVSS-ZZXKWVIFSA-N [5-[3,5-dihydroxy-2-(1,3,4-trihydroxy-5-oxopentan-2-yl)oxyoxan-4-yl]oxy-3,4-dihydroxyoxolan-2-yl]methyl (e)-3-(4-hydroxyphenyl)prop-2-enoate Chemical compound OC1C(OC(CO)C(O)C(O)C=O)OCC(O)C1OC1C(O)C(O)C(COC(=O)\C=C\C=2C=CC(O)=CC=2)O1 UGXQOOQUZRUVSS-ZZXKWVIFSA-N 0.000 claims 1
- 229920000617 arabinoxylan Polymers 0.000 claims 1
- 239000011248 coating agent Substances 0.000 claims 1
- 238000000576 coating method Methods 0.000 claims 1
- 238000004321 preservation Methods 0.000 claims 1
- 108091008146 restriction endonucleases Proteins 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 13
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 abstract description 8
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 abstract description 8
- 229940114124 ferulic acid Drugs 0.000 abstract description 8
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 abstract description 8
- 235000001785 ferulic acid Nutrition 0.000 abstract description 8
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 238000009630 liquid culture Methods 0.000 abstract description 2
- 239000002243 precursor Substances 0.000 abstract description 2
- JMFRWRFFLBVWSI-NSCUHMNNSA-N coniferol Chemical compound COC1=CC(\C=C\CO)=CC=C1O JMFRWRFFLBVWSI-NSCUHMNNSA-N 0.000 description 22
- 239000000243 solution Substances 0.000 description 15
- 241000894006 Bacteria Species 0.000 description 14
- 229940119526 coniferyl alcohol Drugs 0.000 description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 241000219195 Arabidopsis thaliana Species 0.000 description 6
- 101150104988 calA gene Proteins 0.000 description 6
- 101150044474 calB gene Proteins 0.000 description 6
- DKZBBWMURDFHNE-UHFFFAOYSA-N trans-coniferylaldehyde Natural products COC1=CC(C=CC=O)=CC=C1O DKZBBWMURDFHNE-UHFFFAOYSA-N 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- DKZBBWMURDFHNE-NSCUHMNNSA-N coniferyl aldehyde Chemical compound COC1=CC(\C=C\C=O)=CC=C1O DKZBBWMURDFHNE-NSCUHMNNSA-N 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000026165 Alternaria arborescens Species 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241000970829 Mesorhizobium Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- 244000046052 Phaseolus vulgaris Species 0.000 description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 3
- 241000208422 Rhododendron Species 0.000 description 3
- 244000299461 Theobroma cacao Species 0.000 description 3
- 241000607598 Vibrio Species 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 235000009566 rice Nutrition 0.000 description 3
- 241001196073 Alsophila spinulosa Species 0.000 description 2
- 241000187654 Nocardia Species 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 235000009470 Theobroma cacao Nutrition 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003851 biochemical process Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 101150044508 key gene Proteins 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000002304 perfume Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- YQUVCSBJEUQKSH-UHFFFAOYSA-N protochatechuic acid Natural products OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- WKOLLVMJNQIZCI-UHFFFAOYSA-N vanillic acid Chemical compound COC1=CC(C(O)=O)=CC=C1O WKOLLVMJNQIZCI-UHFFFAOYSA-N 0.000 description 2
- TUUBOHWZSQXCSW-UHFFFAOYSA-N vanillic acid Natural products COC1=CC(O)=CC(C(O)=O)=C1 TUUBOHWZSQXCSW-UHFFFAOYSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 101100355949 Caenorhabditis elegans spr-1 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000723185 Cyathea Species 0.000 description 1
- 240000006497 Dianthus caryophyllus Species 0.000 description 1
- 235000009355 Dianthus caryophyllus Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- BJIOGJUNALELMI-ONEGZZNKSA-N Isoeugenol Natural products COC1=CC(\C=C\C)=CC=C1O BJIOGJUNALELMI-ONEGZZNKSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000520900 Pseudomonas resinovorans Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 1
- 241000082085 Verticillium <Phyllachorales> Species 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 235000001046 cacaotero Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 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 description 1
- BJIOGJUNALELMI-ARJAWSKDSA-N cis-isoeugenol Chemical compound COC1=CC(\C=C/C)=CC=C1O BJIOGJUNALELMI-ARJAWSKDSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- AILKHAQXUAOOFU-UHFFFAOYSA-N hexanenitrile Chemical compound CCCCCC#N AILKHAQXUAOOFU-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 235000019640 taste Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- BJIOGJUNALELMI-UHFFFAOYSA-N trans-isoeugenol Natural products COC1=CC(C=CC)=CC=C1O BJIOGJUNALELMI-UHFFFAOYSA-N 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- 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)
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- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
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- 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)
- C12Y101/01194—Coniferyl-alcohol dehydrogenase (1.1.1.194)
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- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03038—Vanillyl-alcohol oxidase (1.1.3.38)
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- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01068—Coniferyl-aldehyde dehydrogenase (1.2.1.68)
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Abstract
The invention provides a recombinant amycolatopsis engineering strain for high-yield vanillin by taking eugenol as a substrate, wherein the recombinant amycolatopsis contains key genes capable of accumulating ferulic acid which is a precursor substance synthesized by vanillin, and proteins coded by the key genes are vanillyl alcohol oxidase and coniferyl alcohol dehydrogenase coniferyl aldehyde dehydrogenase. The recombinant amycolatopsis can ferment with eugenol as a substrate to produce vanillin, the concentration of vanillin in a seed liquid culture obtained after the recombinant amycolatopsis is fermented and cultured can reach 9.34g/L, the conversion rate reaches 84.68%, the concentration of vanillin can be 20.2g/L in a 5L fermentation tank, and the yield of vanillin is obviously higher than that of vanillin of other types.
Description
Technical Field
The invention belongs to the technical field of genetic engineering. In particular to a recombinant amycolatopsis for producing vanillin, and also relates to a construction method and application of the strain.
Background
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most important aromatic flavor compounds in the food and perfume manufacturing industries. As with many other low molecular weight phenolic compounds, vanillin has antioxidant and antibacterial properties and thus has potential as a food preservative. It is active against both gram-positive and gram-negative food spoilage bacteria and has been shown to be effective against both pure fruit bacteria and yeasts and molds in laboratory growth media. Vanillin is considered one of the most important tastes in the world. It is widely used in the food, beverage, perfume and pharmaceutical industries.
At present, the production modes of vanillin in the market mainly comprise plant extraction, chemical synthesis and microbial transformation. Because of the shortcomings of natural extraction and chemical synthesis, attention is paid to the production of vanillin by microbial transformation, wherein eugenol, isoeugenol and ferulic acid are the main substrates for the production of vanillin by the method. Eugenol is widely used as an inexpensive raw material for medicines, foods, fragrances, antioxidants, etc., and is expected to be a main component of an eco-friendly chemical synthesis substance. Tadasa for the first time reported that corynebacteria are capable of converting eugenol into vanillin, and the corynebacteria can obtain ferulic acid and vanillin by using eugenol as a unique carbon source and energy source. Ashengroph et al isolated a Pseudomonas resina (Pseudomonas resinovorans SPR 1) strain capable of converting eugenol to vanillin and related phenolic aromatic products. After 30h and 60h of bioconversion 0.24g/L vanillin (conversion 10%) and 1.1g/L vanillic acid (conversion 44%) were produced, respectively, before further optimizing the conditions. Researchers also establish a two-step bioconversion process from eugenol to vanillin, in which, in the first step, recombinant E.coli XL1-Blue (pSKvaomPcalAmcalB) is used to convert eugenol into ferulic acid with a maximum concentration of up to 14.7g/L; in the second step, ferulic acid was converted to vanillin with E.coli (pSKechE/Hfcs) at a yield of 0.3g/L.
According to the method, the yield of vanillin is low, and according to the current clear synthetic route of vanillin, amycolatopsis capable of metabolizing toxic substances and low in vanillin degradation rate is used as a research platform, and different sources of genes are excavated for synthesizing key proteins of vanillin, so that amycolatopsis capable of producing vanillin can be provided through genetic modification.
Disclosure of Invention
The invention aims to provide a recombinant amycolatopsis engineering strain for producing vanillin by taking eugenol as a substrate through genetic modification, and also provides a construction method and application of the engineering strain.
In order to achieve the above object, the present invention provides a recombinant amycolatopsis engineering strain for producing vanillin by using eugenol as a substrate, wherein the recombinant amycolatopsis contains a recombinant plasmid, the recombinant plasmid contains a key gene capable of accumulating ferulic acid (Ferulic acid) which is a precursor substance for synthesizing vanillin, and the protein encoded by the key gene is vanillyl alcohol oxidase and coniferyl aldehyde dehydrogenase.
In the present invention, preferably, the vanillyl alcohol oxidase is derived from azalea, alternaria arborescens, fusarium verticillatum, alsophila spinulosa or cocoa beans; the coniferyl alcohol dehydrogenase is selected from the group consisting of Lenocardia, alfalfa, du Genshi, carnation and mesorhizobium; the coniferyl aldehyde dehydrogenase is derived from Arabidopsis thaliana, vibrio cyclophilus, rice, brazilian bacillus or seaweed.
As a particularly preferred embodiment, the vanillyl alcohol oxidase is derived from Fusarium verticillium, and the nucleotide sequence of the vanillyl alcohol oxidase is shown as SEQ ID NO. 1; the coniferyl alcohol dehydrogenase is from alfalfa, and the nucleotide sequence of the coniferyl alcohol dehydrogenase is shown as SEQ ID NO. 2; the coniferyl aldehyde dehydrogenase is derived from seaweed, and the nucleotide sequence of the coniferyl aldehyde dehydrogenase is shown as SEQ ID NO. 3.
Based on the above, the invention also provides a construction method of the recombinant amycolatopsis, which comprises the following steps:
(1) Construction of recombinant engineering strains containing Vanilla alcohol oxidase Gene
Obtaining and synthesizing vanillyl alcohol oxidase (vaoA) gene sequences from azalea (DrvaoA), alternaria arborescens (AavaoA), fusarium verticillatum (FvvaoA), alsophila spinulosa (XcvaoA) and cacao bean (LtvaoA), respectively taking BamHI/NsiI as enzyme cutting site double enzyme cutting carrier pKC1139 and connecting to obtain recombinant plasmids pKC1139-DrvaoA, pKC 1139-AovaoA, pKC1139-FvvaoA, pKC1139-XcvaoA and pKC1139-LtvaoA, transforming the above plasmids into Amycolatopsis (Amycolatopsis sp.) by a method of conjugation transfer, and verifying the correctness of the recombinant engineering strains selected by an arabidopsis resistance screen by a PCR method;
construction of recombinant engineering Strain containing Vanilla alcohol oxidase and coniferyl alcohol dehydrogenase genes
Obtaining and synthesizing coniferyl alcohol dehydrogenase (calA) gene sequences from nocardia (NgcalA), alfalfa (MscalA), du Genshi bacteria (DvcalA), caryophyllus (TccalA) and mesorhizobium (MlcalA), respectively taking XbaI/SpeI as a double-enzyme-cutting carrier pKC1139-FvvaoA of enzyme cutting sites and connecting to obtain recombinant plasmids pKC1139-FvvaoANgcalA, pKC1139-FvvaoAMscalA, pKC1139-FvvaoADvcalA, pKC1139-FvvaoATccalA and pKC1139-FvvaoAMlcalA, transforming the plasmids into Amycolatopsis (Amycolatopsis sp.) by a joint transfer method, and verifying the correctness of the recombinant engineering strains selected by an arabiamycin resistance screen by a PCR mode;
(3) Construction of recombinant engineering strains containing Vanilla alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase genes
Obtaining and synthesizing coniferyl aldehyde dehydrogenase (calB) gene sequences from arabidopsis thaliana (AtcalB), vibrio cyclophilis (VccalB), rice (ObcalB), brazilian bacillus (PbcalB) or seaweed (MncalB), respectively taking XbaI/SpeI as enzyme cutting site double enzyme cutting vectors pKC1139-FvvaoAMscalA and connecting to obtain recombinant plasmids pKC1139-FvvaoAMscalAAtcalB、pKC1139-FvvaoAMscalAVccalB、pKC1139-FvvaoAMscalAObcalB、pKC1139-FvvaoAMscalAPbcalB、pKC1139-FvvaoAMscalAMncalB,, transforming the plasmids into Amycolatopsis (Amycolatopsis sp.) by a method of conjugation transfer, and verifying the correctness of the recombinant engineering strains by a PCR method through a recombinant engineering strain selected by using the resistance of arabidopsis.
The invention provides and verifies the application of the gene recombinant amycolatopsis in the production of vanillin by taking eugenol as a substrate.
In the present invention, when recombinant Amycolatopsis is constructed, the starting strain when the recombinant plasmid is transformed into Amycolatopsis (Amycolatopsis sp.) is Amycolatopsis (Amycolatopsis sp.) HM-141, which has been deposited in the China general microbiological culture Collection center, china center for culture Collection, having the accession number CGMCC No. 22871, at the X.3835, west 3, national academy of sciences of microorganisms in the Korean area North Star, beijing at the month 9 of 2021. The amycolatopsis HM-141 is also described in the Chinese patent application CN 2021109397312.
Amycolatopsis sp.HM-141 can produce vanillin by using ferulic acid as a substrate, the molar conversion rate is as high as 87%, the impurity content in the product is low, the detection amount of vanillin is 0, and the detection amount of vanillic acid is 0.25g/L.
The invention verifies that recombinant plasmids containing vanillin synthesis genes vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase with different sources are respectively constructed, the recombinant plasmids are transformed into different amycolatopsis by a joint transfer mode, and recombinant bacteria for improving the vanillin yield are screened out through the resistance of arabidopsis. The recombinant amycolatopsis contains vanillyl alcohol oxidase (vaoA) derived from fusarium verticillatum (FvvaoA), coniferyl alcohol dehydrogenase (calA) derived from alfalfa (MscalA) and coniferyl aldehyde dehydrogenase (calB) derived from seaweed (MncalB), and can ferment with eugenol as a substrate to produce vanillin, the concentration of vanillin in a seed liquid culture obtained after fermentation culture of the recombinant amycolatopsis can reach 9.34g/L, the conversion rate can reach 84.68%, and the concentration of vanillin can be 20.2g/L after fermentation in a 5L fermentation tank, which is remarkably higher than the yields of vanillin of other types.
Drawings
FIG. 1 is a vanillin biosynthetic pathway;
FIG. 2 is a comparison of coniferyl alcohol yield and mass conversion rate for each strain of example 1;
FIG. 3 is a comparison of coniferyl aldehyde yield and mass conversion rate for each strain of example 2;
FIG. 4 is a comparison of vanillin yield and mass conversion of each strain of example 3;
FIG. 5 shows the concentration trend of eugenol and vanillin in the 5L fermentor of the optimal strain of example 4.
Detailed Description
The following examples serve to illustrate the technical solution of the invention without limiting it.
In the invention, unless otherwise specified, "%" for explaining the concentration is mass percent ": "all are mass ratios.
The invention relates to the following media:
The formula of the LB culture medium is as follows: 10g/L peptone, 5g/L yeast extract and 10g/L sodium chloride.
The formula of the GYM medium is as follows: glucose 4g/L, yeast extract 4g/L, malt extract 10g/L.
The formula of the GYM solid culture medium is as follows: glucose 4g/L, yeast extract 4g/L, malt extract 10g/L, calcium carbonate 2g/L, and agar powder 20g/L.
The formula of the M1 culture medium is as follows: 25g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of monopotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, and regulating the pH to 7.2.
The bond transfer experiments used in the following examples included the following steps:
(1) Amycolatopsis sp (or other engineered strain) was activated on GYM solid medium, cultured at 30℃for 3-4d to develop colonies, and then inoculated into 50mL of GYM liquid medium, cultured at 30℃for 2d at 200 rpm.
(2) The constructed plasmid was heat-shocked into E.coli ET12567 (pUZ 8002) strain, spread on LB solid plate containing 25. Mu.g/mL chloramphenicol, 25. Mu.g/mL kanamycin and 50. Mu.g/mL arabinomycin resistance, cultured at 37℃for 12 hours until single colony developed, inoculated single colony was cultured overnight at 37℃in 4mL LB liquid medium containing resistance at 200rpm, and then inoculated in 20mL LB at 37℃at 200rpm for 4-5 hours to OD600 of 0.4-0.6 according to 1% inoculum size.
(3) 2ML of Amycolatopsis sp.bacteria solution and 1mL of E.coli ET12567 (pUZ 8002) bacteria solution carrying target plasmid are respectively taken, 1mL and 5000g of the bacteria solution are centrifuged for 1min, the bacteria solution is washed twice by antibiotic-free LB, 100 mu L of antibiotic-free LB culture medium is added, the Amycolatopsis and the E.coli ET12567 (pUZ 8002) are mixed according to the volume ratio of 7:1, and 30 mu L of the mixed bacteria solution is taken and placed on the antibiotic-free GYM solid culture medium for 30 ℃ for 14h for forward culture.
(4) The resulting plaque was scraped off, spread on a GYM solid medium containing 50. Mu.g/mL of arabinomycin and 25. Mu.g/mL of nalidixic acid solution, cultured at 30℃for 4d until single colonies were grown, and then PCR verification was performed on the single colonies.
The method for analyzing coniferyl alcohol content in the strain by HPLC used in the following examples includes:
(1) Preparation of coniferyl alcohol standard curve: 10mg of coniferyl alcohol is accurately weighed by a ten-thousandth balance, dissolved and diluted to 1g/L mother liquor by 10mL of pure methanol, and then diluted to 120 mug/mL, 100 mug/mL, 80 mug/mL, 60 mug/mL, 40 mug/mL and 20 mug/mL coniferyl alcohol solution by sterilized water in a gradient manner. All samples were filtered through a 0.22 μm aqueous pinhole filter head prior to HPLC injection.
(2) Preparation of the test sample: 1mL of the fermentation broth was centrifuged at 12000rpm for 5min in a 1.5mL centrifuge tube, 10. Mu.L of the supernatant was taken and added with 990. Mu.L of sterilized water to dilute to 100 times, and the mixture was filtered by a 0.22 μm aqueous pinhole filter.
(3) HPLC detection conditions: mobile phase: 0.5% phosphoric acid aqueous solution (A), capronitrile (B); volume ratio: 90% A/10% B; flow rate: 1mL/min; detection wavelength: 280nm; column temperature: 30 ℃; chromatographic column: diamonsil SB-C18 column, 4.6 mm X250 mm.
The present invention relates to the following genes:
TABLE 1 Vanillin oxidase genes and sources thereof
TABLE 2 coniferyl alcohol dehydrogenase genes and sources thereof
TABLE 3 coniferyl aldehyde dehydrogenase genes and sources thereof
The invention relates to the following primers:
The colony PCR verification reaction system related to the invention is as follows:
The PCR procedure was as follows:
EXAMPLE 1 construction of recombinant engineering bacteria containing Vanilla alcohol oxidase Gene (vaoA)
Vanilla oxidase (vaoA) gene sequences derived from azalea (DrvaoA), alternaria arborescens (AavaoA), fusarium verticillatum (FvvaoA), cyathea (XcvaoA, MU375925.1 308097-310471 (-)) and cocoa beans (Ltvaoa, KZ107832.1 1234575-1236406 (+)) were obtained from NCBI and sent to the synthesis. The recombinant plasmids pKC1139-DrvaoA, pKC 1139-AovaoA, pKC1139-FvvaoA, pKC1139-XcvaoA and pKC1139-LtvaoA are obtained by respectively using BamHI/NsiI as enzyme cutting site double enzyme cutting carrier and gene vaoA. The above recombinant plasmids were transformed into Amycolatopsis (Amycolatopsis sp.) by conjugation transfer, and the recombinant engineering strains screened for resistance to arabidopsis were obtained as recombinant strains 1,2, 3, 4, and 5, and were confirmed to be correct by colony PCR.
Detecting the content of coniferyl alcohol in the fermentation liquor to screen the recombinant strain with highest coniferyl alcohol yield.
The fermentation conditions were as follows:
mu.L of the strain was activated on GYM plate solid (plus Arabidopsis thaliana resistance) at a constant temperature of 30℃for 60 hours, then the cells were scraped off and diluted with 200. Mu.L of sterilized water, and 5% inoculated in 50ml of M1 seed solution (plus Arabian resistance) and subjected to shaking fermentation at a constant temperature of 30℃for 72 hours at 200 rpm. After adjusting the pH of the seed solution to 8.0 with NaOH aqueous solution, adding 1% eugenol (addition concentration: 10.57 g/L) for fermentation for 48 hours, centrifuging 1ml fermentation liquor, sucking the supernatant to dilute to 100 times, and filtering for HPLC analysis.
As shown in FIG. 2, the coniferyl alcohol concentration of each recombinant strain is compared, and the highest coniferyl alcohol concentration of the recombinant strain 3 is found, and the molar conversion rate reaches 88.78 percent, which is 9.88 g/L.
EXAMPLE 2 construction of recombinant engineering bacteria containing vanillyl alcohol oxidase Gene (vaoA) and coniferyl alcohol dehydrogenase Gene (calA)
The coniferyl alcohol dehydrogenase (calA) gene sequences derived from nocardia (NgcalA), alfalfa (MscalA), du Genshi (DvcalA), caryophyllus (TccalA) and mesorhizobium (MlcalA) were obtained from NCBI and synthesized by the biochemical process. The recombinant plasmids pKC1139-FvvaoANgcalA, pKC1139-FvvaoAMscalA, pKC1139-FvvaoADvcalA, pKC1139-FvvaoATccalA and pKC1139-FvvaoAMlcalA are obtained by double-enzyme cutting of the vector pKC1139-FvvaoA with the highest yield of coniferyl alcohol with XbaI/SpeI as enzyme cutting sites and connecting the vector pKC1139-FvvaoA with the gene calA. The above recombinant plasmids were transformed into Amycolatopsis (Amycolatopsis sp.) by conjugation transfer, and the recombinant engineering strains screened for resistance to arabidopsis were obtained as recombinant strains 6, 7, 8, 9, and 10, and were confirmed to be correct by colony PCR.
Detecting the content of coniferyl aldehyde in the fermentation liquor to screen the recombinant strain with highest coniferyl aldehyde yield.
The fermentation conditions were as follows:
mu.L of the strain was activated on GYM plate solid (plus Arabidopsis thaliana resistance) at a constant temperature of 30℃for 60 hours, then the cells were hung and diluted with 200. Mu.L of sterilized water, and 5% inoculated in 50ml of M1 seed solution (plus Arabian resistance) and subjected to shaking fermentation at a constant temperature of 30℃for 72 hours at 200 rpm. After adjusting the pH of the seed solution to 8.0 with NaOH aqueous solution, adding 1% eugenol (addition concentration: 10.57%) for further fermentation for 48 hours, centrifuging 1ml of fermentation liquor, sucking the supernatant to dilute to 100 times, and filtering for HPLC analysis.
As shown in FIG. 3, the concentration of coniferyl aldehyde of each recombinant strain is compared, and the highest coniferyl aldehyde concentration of the recombinant strain 7 is found to reach 9.53g/L, and the molar conversion rate reaches 86.12%.
EXAMPLE 3 construction of recombinant engineering Strain containing coniferyl aldehyde dehydrogenase (calB) Gene
Coniferyl aldehyde dehydrogenase (calB) derived from Arabidopsis thaliana (AtcalB), vibrio cyclophilia (VccalB), rice (ObcalB, NC_023163.2 17132108-17139602 (+)), brazilian bacillus (PbcalB) and seaweed (MncalB) was obtained from NCBI and sent to the biochemical process. The recombinant plasmid pKC1139-FvvaoAMscalAAtcalB、pKC1139-FvvaoAMscalAVccalB、pKC1139-FvvaoAMscalAObcalB、pKC1139-FvvaoAMscalAPbcalB、pKC1139-FvvaoAMscalAMncalB. obtained by double enzyme digestion of the vector pKC1139-FvvaoAMscalA with the highest yield of coniferyl alcohol with XbaI/SpeI as enzyme digestion sites is connected with the gene calB, the recombinant plasmid is transformed into Amycolatopsis (Amycolatopsis sp.) by a conjugation transfer method, and the recombinant engineering strain screened by the resistance of the arabidopsis is obtained, so that the following recombinant bacteria 11, 12, 13, 14 and 15 are verified to be correct by adopting a colony PCR mode.
Detecting the content of vanillin in the fermentation broth to screen recombinant strains with highest vanillin yield.
The fermentation conditions were as follows:
mu.L of the strain was activated on GYM plate solid (plus Arabidopsis thaliana resistance) at a constant temperature of 30℃for 60 hours, then the cells were hung and diluted with 200. Mu.L of sterilized water, and 5% inoculated in 50ml of M1 seed solution (plus Arabian resistance) and subjected to shaking fermentation at a constant temperature of 30℃for 72 hours at 200 rpm. After adjusting the pH of the seed solution to 8.0 with NaOH aqueous solution, adding 1% eugenol (adding concentration: 10.57 g/L) for fermentation for 48 hours, centrifuging 1ml fermentation liquor, sucking the supernatant to dilute to 100 times, and filtering for HPLC analysis.
As shown in FIG. 4, the concentration of vanillin in each recombinant strain was compared, and it was found that the concentration of vanillin in recombinant strain 15 was the highest, reaching 9.34g/L, and the molar conversion rate reached 84.68%.
EXAMPLE 4 fermentation in a 5L fermentor of the strain producing the highest concentration of vanillin
Inoculating 200 μl of recombinant strain 15 into 50mL of seed culture medium M1, and culturing at 30deg.C and 200rpm for 48 hr; the seed solution was inoculated into a 5L fermenter containing 4L of fermentation medium M1, which had been adjusted to pH 8.0 and to which had been added arabinomycin resistance, at 30℃and a stirring speed of 800rpm and a aeration ratio of 1vvm, and after fermentation for 24 hours, 21.34g/L (total 80ml, eugenol concentration calculated as 4L broth volume) of the substrate eugenol was added, and fermentation was continued for 72 hours. The concentration of vanillin in the fermentation broth was determined by HPLC method to be 20.2g/L. The results are shown in FIG. 5.
The results show that the recombinant engineering bacteria obtained by comparing and selecting recombinant plasmids of vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase with different sources finally confirm that the recombinant engineering bacteria containing the vanillyl alcohol oxidase (vaoA) from fusarium verticillatum (FvvaoA), the coniferyl alcohol dehydrogenase (calA) from alfalfa (MscalA) and the coniferyl aldehyde dehydrogenase (calB) from seaweed (MncalB) can ferment with eugenol as a substrate to produce vanillin, the concentration of vanillin in a culture obtained after fermentation culture of the recombinant bacteria can reach 9.34g/L, the conversion rate reaches 84.68%, and the concentration of vanillin can be realized in a 5L fermentation tank by fermentation to be 20.2g/L, which is obviously higher than that of vanillin of the same type.
Claims (7)
1. Recombinant amycolatopsis for producing vanillin by taking eugenol as a substrate is characterized in that the recombinant amycolatopsis expresses vanillyl alcohol oxidase gene FvvaoA from fusarium verticillatum, coniferyl alcohol dehydrogenase gene MscalA from alfalfa and coniferyl aldehyde dehydrogenase gene MncalB from seaweed; the Amycolatopsis is Amycolatopsis (Amycolatopsis sp.) HM-141, and the strain is preserved in China general microbiological culture Collection center (CGMCC) of China national institute of sciences of microbiology, china No. 3, which is the national academy of sciences of China, at North Star, in the Korean area of Beijing, 7, 9, and has a preservation number of CGMCC No.22871.
2. The recombinant amycolatopsis according to claim 1, characterized in that the nucleotide sequence of vanillyl alcohol oxidase is shown in SEQ ID No. 1; the nucleotide sequence of the coniferyl alcohol dehydrogenase is shown as SEQ ID NO. 2; the nucleotide sequence of the coniferyl aldehyde dehydrogenase is shown as SEQ ID NO. 3.
3. The use of the recombinant amycolatopsis of claim 1 or 2 for producing vanillin using eugenol as a substrate.
4. The use according to claim 3, characterized in that the recombinant amycolatopsis produces vanillin by fermentation in seed medium M1 with eugenol as substrate.
5. The use according to claim 4, characterized in that the recombinant amycolatopsis is inoculated in 50mL of seed culture M1 and cultivated at 30 ℃ at 200rpm for 72h;
adjusting the pH of the M1 seed solution to 8.0, adding a substrate eugenol of 10.67g/L, continuing fermenting for 48 hours, and measuring the concentration of vanillin in the fermentation liquor by HPLC after the fermentation is finished;
Wherein, the formula of the M1 culture medium is as follows: 25g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of monopotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, and regulating the pH to 7.2.
6. The method for constructing recombinant amycolatopsis according to claim 1, which comprises the following steps:
(1) Construction of recombinant engineering strains containing Vanilla alcohol oxidase Gene
Obtaining and synthesizing a vanillyl alcohol oxidase (FvvaoA) gene sequence from fusarium verticillatum, respectively taking BamHI/NsiI as a double-enzyme-cutting carrier pKC1139 of enzyme cutting sites and connecting to obtain recombinant plasmids pKC1139-FvvaoA, transforming the plasmids into Amycolatopsis (Amycolatopsis sp.) by a method of conjugation transfer, and screening recombinant engineering strains by using resistance of arabidopsis, wherein the obtained strains are verified to be correct by a PCR mode;
(2) Construction of recombinant engineering Strain containing Vanilla alcohol oxidase and coniferyl alcohol dehydrogenase genes
Obtaining and synthesizing a coniferyl alcohol dehydrogenase (MscalA) gene sequence derived from alfalfa; respectively taking XbaI/SpeI as a double-restriction enzyme digestion vector pKC1139-FvvaoA of restriction enzyme digestion sites and connecting to obtain recombinant plasmids pKC1139-FvvaoAMscalA, transforming the plasmids into Amycolatopsis (Amycolatopsis sp.) by a conjugation transfer method, and screening recombinant engineering strains by using the resistance of arabidopsis, wherein the obtained strains are verified to be correct by a PCR mode;
(3) Construction of recombinant engineering strains containing Vanilla alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase genes
Obtaining and synthesizing coniferyl aldehyde dehydrogenase (MncalB) gene sequences from seaweed, respectively taking XbaI/SpeI as enzyme cutting site double enzyme cutting vectors pKC1139-FvvaoAMscalA and connecting to obtain recombinant plasmids pKC1139-FvvaoAMscalAMncalB, transforming the plasmids into Amycolatopsis (Amycolatopsis sp.) by a conjugation transfer method, and screening recombinant engineering strains by using the resistance of arabidopsis, wherein the obtained strains are verified to be correct by a PCR mode.
7. The construction method according to claim 6, wherein the arabinoxylan resistance screening is carried out by coating recombinant amycolatopsis in GYM solid culture medium containing 50 μg/mL arabinomycin and 25 μg/mL nalidixic acid solution, culturing at 30deg.C for 4d until single colony grows, and the single colony is positive mutant;
The formula of the GYM solid culture medium is as follows: glucose 4g/L, yeast extract 4g/L, malt extract 10g/L, calcium carbonate 2g/L, and agar powder 20g/L.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820375A (en) * | 2013-12-13 | 2014-05-28 | 安徽师范大学 | Engineering strain for biologically producing ferulic acid and establishing method of engineering strain |
| CN104928224A (en) * | 2015-05-08 | 2015-09-23 | 安徽师范大学 | Ferulic acid producing engineering strain, construction method and biotransformation method |
| CN113755422A (en) * | 2021-10-09 | 2021-12-07 | 陕西海斯夫生物工程有限公司 | Recombinant amycolatopsis capable of producing vanillin at high yield, and construction method and application thereof |
| CN114703113A (en) * | 2022-03-29 | 2022-07-05 | 陕西海斯夫生物工程有限公司 | Recombinant amycolatopsis, construction method and application thereof |
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| JP6596009B2 (en) * | 2013-11-04 | 2019-10-23 | ビージーエヌ テック エルエルシー | Process for producing vanillin via microbial fermentation of ferulic acid from eugenol using plant dehydrogenase |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820375A (en) * | 2013-12-13 | 2014-05-28 | 安徽师范大学 | Engineering strain for biologically producing ferulic acid and establishing method of engineering strain |
| CN104928224A (en) * | 2015-05-08 | 2015-09-23 | 安徽师范大学 | Ferulic acid producing engineering strain, construction method and biotransformation method |
| CN113755422A (en) * | 2021-10-09 | 2021-12-07 | 陕西海斯夫生物工程有限公司 | Recombinant amycolatopsis capable of producing vanillin at high yield, and construction method and application thereof |
| CN114703113A (en) * | 2022-03-29 | 2022-07-05 | 陕西海斯夫生物工程有限公司 | Recombinant amycolatopsis, construction method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli;Jorg Overhage et al.;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;第69卷(第11期);6569-6576 * |
| 香兰素生物合成的研究进展;党玥等;《微生物学通报》;第47卷(第11期);3678-3688 * |
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