CN108611383A - A method of improving Arteannuic acid fermentation yield - Google Patents
A method of improving Arteannuic acid fermentation yield Download PDFInfo
- Publication number
- CN108611383A CN108611383A CN201810449836.8A CN201810449836A CN108611383A CN 108611383 A CN108611383 A CN 108611383A CN 201810449836 A CN201810449836 A CN 201810449836A CN 108611383 A CN108611383 A CN 108611383A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- lactose
- solution
- acid
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 256
- 230000004151 fermentation Effects 0.000 title claims abstract description 256
- PLQMEXSCSAIXGB-SAXRGWBVSA-N (+)-artemisinic acid Chemical compound C1=C(C)CC[C@H]2[C@H](C)CC[C@@H](C(=C)C(O)=O)[C@H]21 PLQMEXSCSAIXGB-SAXRGWBVSA-N 0.000 title claims abstract description 144
- PLQMEXSCSAIXGB-UHFFFAOYSA-N artemisininic acid Natural products C1=C(C)CCC2C(C)CCC(C(=C)C(O)=O)C21 PLQMEXSCSAIXGB-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 40
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 83
- 239000008101 lactose Substances 0.000 claims abstract description 83
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 39
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 39
- 239000007788 liquid Substances 0.000 claims abstract description 36
- 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 abstract description 33
- 239000008103 glucose Substances 0.000 claims abstract description 33
- WTEVQBCEXWBHNA-UHFFFAOYSA-N Citral Natural products CC(C)=CCCC(C)=CC=O WTEVQBCEXWBHNA-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229940043350 citral Drugs 0.000 claims abstract description 23
- WTEVQBCEXWBHNA-JXMROGBWSA-N geranial Chemical compound CC(C)=CCC\C(C)=C\C=O WTEVQBCEXWBHNA-JXMROGBWSA-N 0.000 claims abstract description 23
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 claims abstract description 22
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 claims abstract description 22
- 230000007062 hydrolysis Effects 0.000 claims abstract description 19
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims description 109
- LZMOBPWDHUQTKL-RWMBFGLXSA-N artemisinic acid Natural products CC1=C[C@@H]2[C@@H](CCC[C@H]2C(=C)C(=O)O)CC1 LZMOBPWDHUQTKL-RWMBFGLXSA-N 0.000 claims description 61
- 239000002609 medium Substances 0.000 claims description 56
- 239000000413 hydrolysate Substances 0.000 claims description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 48
- 239000007787 solid Substances 0.000 claims description 32
- 229930182830 galactose Natural products 0.000 claims description 30
- 241000894006 Bacteria Species 0.000 claims description 27
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 16
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 16
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 claims description 16
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 14
- 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 14
- 230000003301 hydrolyzing effect Effects 0.000 claims description 14
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 11
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 11
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 11
- 239000005642 Oleic acid Substances 0.000 claims description 11
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 11
- 239000003242 anti bacterial agent Substances 0.000 claims description 11
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 11
- XUGNVMKQXJXZCD-UHFFFAOYSA-N isopropyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC(C)C XUGNVMKQXJXZCD-UHFFFAOYSA-N 0.000 claims description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052799 carbon Inorganic materials 0.000 claims description 10
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 10
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 10
- 229940073490 sodium glutamate Drugs 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 claims description 9
- 229940097277 hygromycin b Drugs 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 6
- 230000003115 biocidal effect Effects 0.000 claims description 5
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 4
- 239000013589 supplement Substances 0.000 claims 1
- 230000002068 genetic effect Effects 0.000 abstract description 4
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 65
- 239000007864 aqueous solution Substances 0.000 description 46
- 238000011218 seed culture Methods 0.000 description 29
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- 238000012258 culturing Methods 0.000 description 27
- 229910021645 metal ion Inorganic materials 0.000 description 23
- 229940088594 vitamin Drugs 0.000 description 22
- 235000013343 vitamin Nutrition 0.000 description 22
- 239000011782 vitamin Substances 0.000 description 22
- 229930003231 vitamin Natural products 0.000 description 22
- 150000003722 vitamin derivatives Chemical class 0.000 description 22
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 20
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 20
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 20
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 20
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 18
- 238000011081 inoculation Methods 0.000 description 18
- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 16
- 229960004191 artemisinin Drugs 0.000 description 16
- 229930101531 artemisinin Natural products 0.000 description 16
- 238000001514 detection method Methods 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 239000007836 KH2PO4 Substances 0.000 description 10
- 229910021529 ammonia Inorganic materials 0.000 description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 10
- 235000011130 ammonium sulphate Nutrition 0.000 description 10
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 10
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 10
- 230000001105 regulatory effect Effects 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 230000001502 supplementing effect Effects 0.000 description 10
- 229920001817 Agar Polymers 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- 229940088710 antibiotic agent Drugs 0.000 description 9
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- 238000007865 diluting Methods 0.000 description 9
- 238000001914 filtration Methods 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 9
- 239000008362 succinate buffer Substances 0.000 description 9
- 238000002604 ultrasonography Methods 0.000 description 9
- 230000004913 activation Effects 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000010353 genetic engineering Methods 0.000 description 5
- 238000009776 industrial production Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 4
- HIQIXEFWDLTDED-UHFFFAOYSA-N 4-hydroxy-1-piperidin-4-ylpyrrolidin-2-one Chemical compound O=C1CC(O)CN1C1CCNCC1 HIQIXEFWDLTDED-UHFFFAOYSA-N 0.000 description 4
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 4
- RADKZDMFGJYCBB-UHFFFAOYSA-N Pyridoxal Chemical compound CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 4
- NRAUADCLPJTGSF-ZPGVOIKOSA-N [(2r,3s,4r,5r,6r)-6-[[(3as,7r,7as)-7-hydroxy-4-oxo-1,3a,5,6,7,7a-hexahydroimidazo[4,5-c]pyridin-2-yl]amino]-5-[[(3s)-3,6-diaminohexanoyl]amino]-4-hydroxy-2-(hydroxymethyl)oxan-3-yl] carbamate Chemical compound NCCC[C@H](N)CC(=O)N[C@@H]1[C@@H](O)[C@H](OC(N)=O)[C@@H](CO)O[C@H]1\N=C/1N[C@H](C(=O)NC[C@H]2O)[C@@H]2N\1 NRAUADCLPJTGSF-ZPGVOIKOSA-N 0.000 description 4
- HMTAHNDPLDKYJT-CBBWQLFWSA-N amorpha-4,11-diene Chemical compound C1=C(C)CC[C@H]2[C@H](C)CC[C@@H](C(C)=C)[C@H]21 HMTAHNDPLDKYJT-CBBWQLFWSA-N 0.000 description 4
- HMTAHNDPLDKYJT-UHFFFAOYSA-N amorphadiene Natural products C1=C(C)CCC2C(C)CCC(C(C)=C)C21 HMTAHNDPLDKYJT-UHFFFAOYSA-N 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000000411 inducer Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- CZSSHKCZSDDOAH-UNQGMJICSA-N (+)-artemisinic alcohol Chemical compound C1=C(C)CC[C@H]2[C@H](C)CC[C@@H](C(=C)CO)[C@H]21 CZSSHKCZSDDOAH-UNQGMJICSA-N 0.000 description 2
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 2
- XJMMNTGIMDZPMU-UHFFFAOYSA-N 3-methylglutaric acid Chemical compound OC(=O)CC(C)CC(O)=O XJMMNTGIMDZPMU-UHFFFAOYSA-N 0.000 description 2
- 240000000011 Artemisia annua Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 2
- 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 2
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 2
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 2
- 229910018890 NaMoO4 Inorganic materials 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 108700005078 Synthetic Genes Proteins 0.000 description 2
- 229930003761 Vitamin B9 Natural products 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 229960004050 aminobenzoic acid Drugs 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 229960002079 calcium pantothenate Drugs 0.000 description 2
- 229960003677 chloroquine Drugs 0.000 description 2
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- JYGAZEJXUVDYHI-DGTMBMJNSA-N dihydroartemisinic acid Chemical compound C1CC(C)=C[C@@H]2[C@H]([C@@H](C)C(O)=O)CC[C@@H](C)[C@@H]21 JYGAZEJXUVDYHI-DGTMBMJNSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 201000004792 malaria Diseases 0.000 description 2
- 239000011565 manganese chloride Substances 0.000 description 2
- 229960003512 nicotinic acid Drugs 0.000 description 2
- 235000001968 nicotinic acid Nutrition 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229960003581 pyridoxal Drugs 0.000 description 2
- 235000008164 pyridoxal Nutrition 0.000 description 2
- 239000011674 pyridoxal Substances 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 239000011727 vitamin B9 Substances 0.000 description 2
- 235000019159 vitamin B9 Nutrition 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- 239000011686 zinc sulphate Substances 0.000 description 2
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- WCXDHFDTOYPNIE-RIYZIHGNSA-N (E)-acetamiprid Chemical compound N#C/N=C(\C)N(C)CC1=CC=C(Cl)N=C1 WCXDHFDTOYPNIE-RIYZIHGNSA-N 0.000 description 1
- 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
- 239000005875 Acetamiprid Substances 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 1
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 1
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 1
- 102100040069 Aldehyde dehydrogenase 1A1 Human genes 0.000 description 1
- 101710150756 Aldehyde dehydrogenase, mitochondrial Proteins 0.000 description 1
- 235000001405 Artemisia annua Nutrition 0.000 description 1
- 101100323110 Artemisia annua CYP71AV1 gene Proteins 0.000 description 1
- 101150081058 CPR gene Proteins 0.000 description 1
- 206010063094 Cerebral malaria Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 101150084072 ERG20 gene Proteins 0.000 description 1
- 108010022535 Farnesyl-Diphosphate Farnesyltransferase Proteins 0.000 description 1
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 1
- 101000780443 Homo sapiens Alcohol dehydrogenase 1A Proteins 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 101150053185 P450 gene Proteins 0.000 description 1
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 1
- 102100037997 Squalene synthase Human genes 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- HATRDXDCPOXQJX-UHFFFAOYSA-N Thapsigargin Natural products CCCCCCCC(=O)OC1C(OC(O)C(=C/C)C)C(=C2C3OC(=O)C(C)(O)C3(O)C(CC(C)(OC(=O)C)C12)OC(=O)CCC)C HATRDXDCPOXQJX-UHFFFAOYSA-N 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000078 anti-malarial effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000003430 antimalarial agent Substances 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- SVAPNGMAOHQQFJ-UNQGMJICSA-N artemisinic aldehyde Chemical compound C1=C(C)CC[C@H]2[C@H](C)CC[C@@H](C(=C)C=O)[C@H]21 SVAPNGMAOHQQFJ-UNQGMJICSA-N 0.000 description 1
- SVAPNGMAOHQQFJ-UHFFFAOYSA-N artemisinic aldehyde Natural products C1=C(C)CCC2C(C)CCC(C(=C)C=O)C21 SVAPNGMAOHQQFJ-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000008845 cholagoga Substances 0.000 description 1
- 229940124571 cholagogue Drugs 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 150000001993 dienes Chemical class 0.000 description 1
- JYGAZEJXUVDYHI-UHFFFAOYSA-N dihydroartemisininic acid Natural products C1CC(C)=CC2C(C(C)C(O)=O)CCC(C)C21 JYGAZEJXUVDYHI-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 125000000864 peroxy group Chemical group O(O*)* 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 229960005179 primaquine Drugs 0.000 description 1
- INDBQLZJXZLFIT-UHFFFAOYSA-N primaquine Chemical compound N1=CC=CC2=CC(OC)=CC(NC(C)CCCN)=C21 INDBQLZJXZLFIT-UHFFFAOYSA-N 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 201000004409 schistosomiasis Diseases 0.000 description 1
- 229930009674 sesquiterpene lactone Natural products 0.000 description 1
- 150000002107 sesquiterpene lactone derivatives Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
Classifications
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of methods improving Arteannuic acid fermentation yield, include so that the saccharomyces cerevisiae engineered yeast of Arteannuic acid can be produced as production bacterial strain, containing the lactose hydrolysis liquid that hydrolyzable is glucose and galactolipin, it ferments in the fermentation medium of the exogenous factors such as mevalonic acid and/or citral, the present invention improves the fermentation yield of Arteannuic acid, reduces the fermentation costs of Arteannuic acid, technical solution is simple and practicable, environmental-friendly, strain genetic stability is high, is easy to industrialized production and application.
Description
Technical Field
The invention relates to the technical field of industrial microbial fermentation, in particular to a method for improving the fermentation yield of artemisinic acid.
Background
Artemisinin is sesquiterpene lactone medicine with peroxy group extracted from stem and leaf of composite inflorescence plant Artemisia annua, and is discovered in 1971 by U-yo of Chinese pharmacy15H22O5. Artemisinin is the most effective antimalarial specific drug after acetamiprid, chloroquine and primaquine, particularly has the characteristics of quick response and low toxicity for cerebral malaria and chloroquine malaria, and has been called as the 'only effective malaria treatment drug in the world' by the world health organization. Artemisinin was widely used over the next 40 years, and statistics show that artemisinin and its derivatives are sold worldwide in the number of $ 15 billion each year. In recent years, artemisinin has shown attractive prospects in the treatment of other diseases such as schistosomiasis resistance, regulation or inhibition of the immune function of body fluid, improvement of the conversion rate of lymphocytes, cholagogue, phlegm elimination, cough relief, asthma relief and the like. Therefore, the artemisinin has very wide market prospect.
The artemisinin is mainly prepared by three production methods, one method is the traditional method for extracting the artemisinin from the sweet wormwood herb, and the method is greatly influenced by climate and regions and has unstable yield; secondly, the compound is obtained by chemical total synthesis, so that the method has great difficulty and high cost; the third method is to adopt genetic engineering technology to obtain arteannuic acid through microbial fermentation, and then chemically synthesize artemisinin, and the method has high yield, stable production, low production cost and green and environment-friendly process; the third method is to adopt genetic engineering technology to obtain arteannuic acid by microbial fermentation and then synthesize artemisinin, namely the arteannuic acid is obtained by a biosynthesis method, and then artemisinin is produced by chemical conversion. In 2013, the WHO also approved the artemisinin produced by the process to be applied to clinic.
The third method has great influence on the yield and cost of final products due to arteannuic acid, a key intermediate of artemisinin. The development of the microbial fermentation method of artemisinic acid starts from a supporting project of the 2004 Ganz Foundation, and the technologies adopted at home and abroad are yeast fermentation processes developed on the basis of the supporting project, but the actual industrial production has more problems, and the difficulty in realizing the industrial production is still high.
A model for the use of synthetic biological in pharmaceutical degradation (Chris J.Paddon and Jay D.Keasling, 2014, volume 12, p355) discloses that the requirement for fermentation to produce artemisinic acid is the presence of galactose, since the engineered Saccharomyces cerevisiae requires the initiation of a galactose-induced promoter to achieve the biosynthesis of artemisinic acid when constructing synthetic genes of artemisinic acid. In actual production, the addition amount of galactose is large, and the cost is very high (about 180-250 yuan/kg), so that the use of a large amount of galactose in the fermentation process directly causes the high fermentation cost of the arteannuic acid, and finally causes the cost of the biosynthetic artemisinin to still be unable to counterbalance the cost of the naturally extracted product, so that the large-scale industrial production of the semi-synthetic artemisinin is finally failed to be formed.
High-level semi-synthetic production of the potential anti-microbial arminins (CJ Paddon, PJ Westfall, DJ Pitera, et al. Nature,2013,496(7446):528-532) discloses a means of gene knockout and reconstruction, overcoming the defect that galactose must be used as an inducer for synthesizing a gene promoter in the conventional process operation, and obtaining a yeast engineering bacterium which can not need galactose to induce synthesis of artemisinic acid, however, the fermentation period of the strain is 144 hours and too long, and a large amount of isopropyl myristate (IPM) needs to be added in the fermentation process to prepare the artemisinic acid, but the large amount of isopropyl myristate (IPM) is not favorable for cost control, and the strain is poor in genetic stability, unstable in yield and unfavorable for industrial large-scale production. The Production of amorpha-4, 11-diene from amorphine yeast, and its conversion to dihydroartemisinic acid (PJ Westfall, DJ Pitera, JR Lenihan, et al. Procedents of the National Academy of sciences.2012,109(3): E111-E118) describes a method of genetically modifying Saccharomyces cerevisiae engineering bacteria to achieve high yield of amorpha-4, 11-diene, but conversion of amorpha-4, 11-diene to artemisinic acid is difficult to achieve in vivo, and conversion of amorpha-4, 11-diene to artemisinic acid needs to be achieved by chemical methods in vitro, and the conversion process is complex, low in yield, and introduces new impurities, and is high in cost, thus, and the industrial application thereof is really poor. Patent CN101338309A discloses a method for producing artemisinic acid by introducing CYP71AV1P450 gene and P450 reductase CPR gene into saccharomyces cerevisiae cells to obtain artemisinic acid genetic engineering bacteria, the method has less process optimization and yield description on artemisinic acid fermentation yield, and the patent does not describe the application aspect of whether the strains can be industrially produced.
Aiming at the defects of low yield, high cost, incapability of realizing industrial production, unstable strain performance and the like in the fermentation production of the artemisinic acid in the prior art, the artemisinic acid fermentation process which has high yield, low cost, no harm to human bodies, environmental friendliness, high strain genetic stability and capability of realizing industrial production is necessary to be found.
Disclosure of Invention
The invention provides a method for improving fermentation yield of artemisinic acid, which comprises the following steps: the method comprises the steps of taking saccharomyces cerevisiae engineering bacteria capable of producing artemisinic acid as production strains, and fermenting in a fermentation culture medium containing lactose hydrolysate and exogenous factors, wherein the lactose hydrolysate is a solution obtained by hydrolyzing lactose into glucose and galactose through enzyme, and the exogenous factors are one or two of mevalonic acid and citral.
In a preferred embodiment, the enzyme is β -galactosidase.
In a preferred embodiment, the mass ratio of β -galactosidase to lactose is 0.1-2: 100.
In a preferred embodiment, the mass-to-volume ratio of the amounts of glucose and galactose in the lactose hydrolysate to the fermentation medium is 15-30: 1 (unit is g/L).
In a preferred embodiment, the lactose is hydrolyzed by enzyme at a hydrolysis temperature of 50-55 deg.C for 3-5h with a hydrolysis rate of 90% -95% and a hydrolysis pH of 4-7, preferably 5.5.
In a preferred embodiment, the concentration of the exogenous factor in the fermentation medium is 0.1-0.5 g/L.
In a preferred embodiment, the fermentation medium further comprises: 8-15g/L of ammonium sulfate; 5-8g/L potassium dihydrogen phosphate; 3-6.2g/L magnesium sulfate heptahydrate; 8-12ml/L of vitamin solution; 6-10ml/L of metal ion solution; CuSO4·5H2O20-40 ug/L, water in balance, and pH of 5.0-5.5. Regulating the pH value by using an aqueous solution containing 13-14.5mol/L of ammonia;
the metal ion solution comprises the following components: ZnSO4·7H2O 5.75g/L；MnCl2·4H2O 0.32g/L；CoCl2·6H2O 0.47g/L；NaMoO4·2H2O 0.48g/L；CaCl2·2H2O 2.9g/L；FeSO4·7H2O2.8 g/L; 0.5MEDTA 80ml/L, the rest is water;
the vitamin solution comprises the following components: biotin 0.05 g/L; 1g/L of calcium pantothenate; 1g/L of nicotinic acid; inositol 25 g/L; vitamin B11 g/L; 1g/L pyridoxal; 0.2g/L of p-aminobenzoic acid, and the balance of water.
In a preferred embodiment, the fermentation time is between 84 and 120h and the fermentation temperature is between 22 and 35 ℃.
In a preferred embodiment, the fermentation further comprises feeding a carbon source, a nitrogen source and a water-insoluble oily liquid, wherein the water-insoluble oily liquid is one or two of isopropyl palmitate and oleic acid.
In a preferred embodiment, the carbon source is fed-batch for 10-18h from fermentation to fermentation end, and the carbon source is one or more of lactose hydrolysate, glycerol aqueous solution, sucrose aqueous solution and ethanol aqueous solution.
In a preferred embodiment, the concentration of the lactose hydrolysate is 400-700g/L, the concentration of the glycerol aqueous solution is 400-700g/L, the concentration of the sucrose aqueous solution is 400-700g/L, and the concentration of the ethanol aqueous solution is 80-99% (V/V).
In a preferred embodiment, the rate of feeding the carbon source is controlled such that the volume of carbon source fed per hour is 0.2% to 2% of the initial volume of the fermentation medium.
In a preferred embodiment, the time of feeding the nitrogen source is from 36 to 45 hours from the beginning of the fermentation to the end of the fermentation, and the fed nitrogen source is one or more of yeast powder soaking water solution, peptone water solution and sodium glutamate water solution.
In a preferred embodiment, the concentration of the yeast extract powder water solution is 200-500g/L, the concentration of the peptone water solution is 200-500g/L, and the concentration of the sodium glutamate water solution is 200-500 g/L.
In a preferred embodiment, the rate of feeding the nitrogen source is controlled such that the volume of the nitrogen source fed per hour is 0.05% to 0.5% of the initial volume of the fermentation medium.
In a preferred embodiment, after the time for feeding the water-insoluble oily liquid is 48-55h, the feeding mode is a one-time feeding mode.
In a preferred embodiment, the volume of the fed-in water-insoluble oily liquid is 5% to 40% of the initial volume of the fermentation medium.
In a preferred embodiment, the production strain may further be subjected to resistance selection on a plate solid medium containing an antibiotic, which is one or more of noursemycin, geneticin and hygromycin B.
In a preferred embodiment, the antibiotic is present in an amount of 50-300. mu.g/L on the plate solid medium.
In a preferred embodiment, the time for the plate culture is 30-50 h.
The fermentation process of the present invention has artemisinic acid yield as high as 42g/L, high artemisinic acid fermentation yield, and lactose hydrolysate capable of being hydrolyzed into glucose and galactose to replace galactose in available technology, so that one molecule of lactose may be hydrolyzed into one molecule of glucose and one molecule of galactose under the catalysis of enzyme, and 342.3g of lactose may be hydrolyzed into 180.16g of glucose and 180.16g of galactose at hydrolysis rate of 100%. Galactose is a promoter inducer of a synthetic gene necessary for fermentation of artemisinic acid, and the using amount is large, but the market price is about 6 times of lactose, so the fermentation cost is greatly reduced.
Compared with the production process of artemisinic acid in the prior art, the invention process of artemisinic acid mainly has the following beneficial effects:
(1) the arteannuic acid prepared by fermentation of the invention has the titer as high as 42g/L and high fermentation titer.
(2) According to the method, the lactose is hydrolyzed to obtain the hydrolysate containing high-concentration glucose and galactose, the glucose can be directly utilized, the galactose can be used as a promoter inducer of the transgene, the hydrolysate is used as a fermentation carbon source, a process of directly adding the galactose is abandoned, and the fermentation cost of the artemisinic acid is greatly reduced.
(3) According to the invention, exogenous factors such as mevalonic acid and/or citral are added into the fermentation medium, so that the fermentation titer of the artemisinic acid is further improved.
(4) According to the invention, non-water-soluble oily liquid such as isopropyl palmitate and/or oleic acid is added once in the fermentation process, the artemisinic acid is promoted to transfer from the fermentation culture medium to the oily liquid, the toxicity of artemisinic acid accumulation on fermentation thalli is reduced, the fermentation yield of the artemisinic acid is further improved, the isopropyl palmitate and the oleic acid are oily liquids without pungent odor and toxicity, the environment is friendly, the price is relatively low, and the industrial large-scale production is facilitated.
(5) The invention carries out resistance screening on the yeast genetic engineering bacteria capable of producing the artemisinic acid, and the genetic stability of the strains is higher.
Detailed Description
The following examples are intended to further illustrate the present invention, but it should be understood that the following examples are only illustrative of the present invention and are not to be construed as limiting the scope of the present invention, which is defined by the appended claims.
The strain used in the invention is any saccharomyces cerevisiae engineering bacteria capable of producing artemisinic acid, can be purchased commercially or constructed in laboratories, for example, saccharomyces cerevisiae purchased from China center for type culture Collection (Wuhan university) with the preservation number of CCTCC AY92011 is taken as a starting strain, gene engineering means is utilized to construct genes of a methylglutaric acid pathway (MVA), ADH1 (artemisinic alcohol dehydrogenase) and ALDH1 (artemisinic aldehyde dehydrogenase), overexpression of tHMGR and ERG20 genes increases the accumulation amount of farnesyl pyrophosphate FPP which is a common precursor of hemiterpenoid substances in yeast, the expression of squalene synthase is inhibited through overexpression of MVA pathway genes and ADS genes, so that the massive synthesis of artemisinic diene is realized, further, an exogenous gene CYP71AV1 (cytochrome P450 enzyme gene) is introduced so as to realize the synthesis of artemisinic acid, and the specific strain construction method can refer to Ro D K, Paradise E M, ouellet M, et al.production of the antigenic drug analogue in engineered layer [ J ] Nature,2006,440(7086) 940-: the detailed Methods described in Methods section 528-532; or Saccharomyces cerevisiae (Saccharomyces cerevisiae) disclosed in Chinese patent with publication No. CN101338309B, preferably Saccharomyces cerevisiae with biological collection number of CGMCC NO. 1861.
The strains used in the embodiment of the invention are saccharomyces cerevisiae engineering bacteria which can be fermented and produce artemisinic acid and are constructed by using genetic engineering means and by taking saccharomyces cerevisiae purchased from China center for type culture Collection (Wuhan university) with the preservation number of CCTCC AY92011 as an original strain, and the specific strain construction methods refer to the following documents Ro D K, Paraise E M, Ouelet M, et. Production of the anti-mental aromatic drug intensive amino acid expressed yeast [ J ]. Nature,2006, 7086, 940-: methods detailed in 528-532.
The various materials and reagents used in the examples of the present invention are those commonly used in the art and are commercially available in a conventional manner unless otherwise specified.
Noursemycin, available from Solarbio life science; geneticin, purchased from Inalco; hygromycin B, available from Solarbio life science.
The liquid phase detection method for detecting the artemisinin titer has the following conditions: a chromatographic column: diamonsil C18(250 mm. times.460 mm,5 μm), mobile phase: acetonitrile: 0.2% (mass to volume, g/L) phosphoric acid aqueous solution 65:35(v/v), gradient elution, retention time: 25min, flow rate: 1mL/min, sample size: 10 μ L, detection wavelength: 212nm, column temperature: (30. + -. 1) DEG C.
Taking Rg lactose dissolved in QL aqueous solution as an example, hydrolyzing with a certain amount of β -galactosidase, taking 5ml of lactose hydrolysate to fix the volume to a 500ml volumetric flask, namely diluting by 100 times, taking 1ml of lactose hydrolysate diluted by 100 times, detecting the glucose concentration by an enzyme method to obtain the glucose concentration, and calculating the hydrolysis rate Y after T (g/L) expression, wherein the specific calculation formula is as follows:
wherein,
t is glucose concentration detected by an enzyme method, g/L;
r is the amount of lactose, g;
q is the volume of water in which lactose is dissolved, L;
342.3 is the molar mass of lactose, g/mol;
180.16 is the molar mass of glucose, g/mol.
The seed culture medium used in the examples of the present invention was: glucose 19.5g/L, (NH)4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, 100ml/L succinate buffer (0.5M, pH 5.0), and the balance water, pH 5.0.
The specific formulas of the vitamin solution and the metal ion solution used in the seed culture medium, the flat solid culture medium and the fermentation culture medium in the embodiment of the invention are as follows:
the vitamin solution is as follows: biotin 0.05 g/L; 1g/L of calcium pantothenate; 1g/L of nicotinic acid; inositol 25 g/L; vitamin B11 g/L; 1g/L pyridoxal; 0.2g/L of p-aminobenzoic acid, and the balance of water.
The metal ion solution is: ZnSO4·7H2O 5.75g/L；MnCl2·4H2O 0.32g/L；CoCl2·6H2O0.47g/L；NaMoO4·2H2O 0.48g/L；CaCl2·2H2O 2.9g/L；FeSO4·7H2O2.8 g/L; 0.5M EDTA80ml/L, the remainder being water.
Comparative example 1:
(1) activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.5% (V/V), and culturing for 16h to obtain a strain liquid;
(2) and (3) screening the resistance of strains: the bacteria obtained in the step (1) are treatedDilution of seed liquor 10-7After doubling, sucking 100 mu L of the strain and coating the strain on a flat solid culture medium containing 200 mu g/L of hygromycin B, and culturing for 40h to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 24 hours to obtain a seed solution;
(4) fermentation culture: inoculating the seed solution obtained in the step (3) into 20L of fermentation medium without adding exogenous factors according to the inoculation amount of 10% (V/V);
the formula of the fermentation medium is as follows: 25g/L of glucose; 25g/L of galactose; ammonium sulfate is 10 g/L; 6g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 5 g/L; 10ml/L of vitamin solution; 8ml/L of metal ion solution; CuSO4·5H2O30 ug/L, and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 14mol/L ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
Continuously adding 700g/L of sucrose aqueous solution from 15h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 2% of the initial volume of 20L of the fermentation culture medium, continuously adding 300g/L of sodium glutamate aqueous solution from 40h of fermentation culture to the end of fermentation, the fed-batch volume per hour is 0.2% of the initial volume of 20L of the fermentation culture medium, and after 52h of fermentation culture, once supplementing isopropyl palmitate and oleic acid, wherein the supplemented volumes are respectively 15% of the initial volume of 20L of the fermentation culture medium; and after fermentation culture for 120h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 24.6 g/L.
Example 1
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.5 percent, and culturing for 16 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the strain and coating the strain on a flat solid culture medium containing 200 mu g/L of hygromycin B, and culturing for 40h to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 24 hours to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing mevalonic acid and citral according to the inoculation amount of 10% for culture, wherein the concentrations of the mevalonic acid and the citral in the culture medium are 0.1g/L respectively;
the formula of the fermentation medium is as follows: 0.1L/L lactose hydrolysate; ammonium sulfate is 10 g/L; 6g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 5 g/L; 10ml/L of vitamin solution; 8ml/L of metal ion solution; CuSO4·5H2O30 ug/L, mevalonic acid 0.1 g/L; 0.1g/L of citral; the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 14mol/L ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of lactose hydrolysate in fermentation medium comprises dissolving 1144g lactose in 2L water, adding β -galactosidase 12g, hydrolyzing at 53 deg.C and pH 4.0 for 4h with hydrolysis rate of 93%, mixing all lactose hydrolysate into fermentation medium with initial volume of 20L to make the mass volume ratio of glucose and galactose in lactose hydrolysate to fermentation medium 28:1(g/L)
Continuously adding 700g/L of sucrose aqueous solution from 15h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 2% of the initial volume of 20L of the fermentation culture medium, continuously adding 300g/L of sodium glutamate aqueous solution from 40h of fermentation culture to the end of fermentation, the fed-batch volume per hour is 0.2% of the initial volume of 20L of the fermentation culture medium, after 52h of fermentation culture, supplementing isopropyl palmitate and oleic acid once, and the supplemented volumes are 15% of the initial volume of 20L of the fermentation culture medium respectively; and after fermentation culture for 120h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 41.8 g/L.
The difference between comparative example 1 and example 1 is that ① example 1 added lactose hydrolysate, comparative example 1 added galactose, ② added exogenous factors mevalonic acid and citral in example 1, and no exogenous factors added in comparative example 1 compared with comparative example 1, galactose in comparative example 1 was replaced by lactose hydrolysate in the fermentation medium of example 1, and when lactose hydrolysate was used in the medium, a large amount of galactose alone was not added, so that the fermentation cost was significantly reduced, and further, the titer of comparative example 1 was 24.6g/L, the titer of example 1 was 41.8g/L, and the yield of artemisinic acid was significantly improved.
Example 2
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.1% (V/V), and culturing for 30h to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-5After doubling, sucking 100 mu L of the culture medium, coating the culture medium on a flat solid culture medium containing 300 mu g/L of nourseothricin, and culturing for 30h to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinic acid buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 16h to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step 3) into a 20L fermentation culture medium containing mevalonic acid according to the inoculation amount of 10% (V/V) for culture, wherein the concentration of mevalonic acid in the culture medium is 0.3 g/L;
the formula of the fermentation medium is as follows: 0.05L/L lactose hydrolysate; 8g/L of ammonium sulfate; 5g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 3 g/L; the vitamin solution is 8 ml/L; 6ml/L of metal ion solution; CuSO4·5H2O20 ug/L; 0.3g/L of mevalonic acid and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of lactose hydrolysate in fermentation medium comprises dissolving 633g lactose in 1L water, adding 0.633g β -galactosidase, hydrolyzing at 50 deg.C and pH of 7.0 for 5h to obtain hydrolysis rate of 90%, mixing all lactose hydrolysate with fermentation medium with initial volume of 20L to make the mass volume ratio of glucose and galactose in lactose hydrolysate to fermentation medium 15:1 (g/L).
Continuously adding 400g/L lactose hydrolysate from 10h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 1% of the initial volume of 20L of the fermentation culture medium, continuously adding 200g/L yeast extract powder aqueous solution from 36h of fermentation culture to the end of fermentation, the fed-batch volume per hour is 0.05% of the initial volume of 20L of the fermentation culture medium, and after 48h of fermentation culture, supplementing isopropyl palmitate once, wherein the supplemented volume is 5% of the initial volume of 20L of the fermentation culture medium; and after fermentation culture for 84h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
The preparation method of the fed-batch lactose hydrolysate comprises dissolving 8000g lactose in 20L water, adding 168.5g β -galactosidase, and hydrolyzing at 50 deg.C and pH of 5.5 for 5 hr to obtain 400g/L lactose hydrolysate.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 38.9 g/L.
Example 3
(1) Preparing strain liquid: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.3 percent, and culturing for 20 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-6After doubling, sucking 100 mu L of the culture medium, coating the culture medium on a flat solid culture medium containing 250 mu g/L of geneticin, and culturing for 35 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 20h to obtain a seed solution;
(4) fermentation culture: inoculating the seed solution obtained in the step 3) into a 20L fermentation culture medium containing citral according to the inoculation amount of 10% for culture, wherein the concentration of the citral in the culture medium is 0.4 g/L;
the formula of the fermentation medium is as follows: 0.075L/L of lactose hydrolysate; 15g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 6.2 g/L; the vitamin solution is 12 ml/L; 10ml/L of metal ion solution; CuSO4·5H2O40 ug/L; 0.4g/L of citral and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 14.5mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the steps of dissolving 800g of lactose in 1.5L of water, adding 10g of β -galactosidase, hydrolyzing for 3h under the conditions that the pH is 5.5 and the temperature is 55 ℃, wherein the hydrolysis rate is 95%, mixing all the lactose hydrolysate into the fermentation medium, and enabling the initial volume of the fermentation medium to be 20L and the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to be 20: 1(g/L) respectively.
Continuously feeding 450g/L of glycerol aqueous solution from 12h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 0.5 percent of the initial volume of 20L of the fermentation culture medium, continuously feeding 250g/L of peptone aqueous solution from 38h of fermentation culture to the end of fermentation, the fed-batch volume per hour is 0.1 percent of the initial volume of 20L of the fermentation culture medium, and replenishing oleic acid once after 50h of fermentation culture, wherein the replenished volume is 10 percent of the initial volume of 20L of the fermentation culture medium; and after fermentation culture for 96h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 39.7 g/L.
Example 4
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.8 percent, and culturing for 18 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media containing 150 mu g/L of nourseothricin and geneticin respectively, and culturing for 45 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 28h to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing mevalonic acid and citral according to the inoculation amount of 10% for culture, wherein the concentrations of the mevalonic acid and the citral in the culture medium are 0.2g/L respectively;
the formula of the fermentation medium is as follows: 0.1L/L lactose hydrolysate; 12g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; 6g/L magnesium sulfate heptahydrate; the vitamin solution is 11 ml/L; 8ml/L of metal ion solution; CuSO4·5H2O35 ug/L; 0.2g/L of mevalonic acid; 0.2g/L of citral and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13.5mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of lactose hydrolysate in the fermentation medium comprises the steps of dissolving 1212g of lactose in 2L of water, adding 24.2g of β -galactosidase, hydrolyzing for 3h under the conditions of pH 5.0 and 52 ℃, wherein the hydrolysis rate is 94%, mixing all lactose hydrolysate into the fermentation medium, and the initial volume of the fermentation medium is 20L, so that the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to the fermentation medium is 30: 1 (g/L).
Continuously adding 65% (V/V) ethanol aqueous solution from 16h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 1.2% of the initial volume of 20L of the fermentation culture medium, continuously adding 350g/L yeast extract powder aqueous solution and 350g/L peptone aqueous solution from 42h of fermentation culture to the end of fermentation, wherein the fed-batch volume of the nitrogen source per hour is 0.25% of the initial volume of 20L of the fermentation culture medium, supplementing isopropyl palmitate and oleic acid once after 55h of fermentation culture, and the supplementing volume is 20% of the initial volume of 20L of the fermentation culture medium; and after fermentation culture for 84h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of artemisinic acid of 40.6 g/L.
Example 5
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.4 percent, and culturing for 24 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-6After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media containing 50 mu g/L of nourseothricin and hygromycin B respectively, and culturing for 50h to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O 6.2g/LVitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 30h to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing mevalonic acid according to the inoculation amount of 10% for culture, wherein the concentration of mevalonic acid in the culture medium is 0.5 g/L;
the formula of the fermentation medium is as follows: 0.075L/L of lactose hydrolysate; 15g/L of ammonium sulfate; 7g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 5.2 g/L; the vitamin solution is 12 ml/L; 9ml/L of metal ion solution; CuSO4·5H2O40 ug/L; 0.5g/L of mevalonic acid and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13.8mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the steps of dissolving 800g of lactose in 1.5L of water, adding 10g of β -galactosidase, hydrolyzing for 3h under the conditions that the pH is 5.5 and the temperature is 55 ℃, wherein the hydrolysis rate is 95%, mixing all the lactose hydrolysate into the fermentation medium, and enabling the initial volume of the fermentation medium to be 20L and the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to be 20: 1(g/L) respectively.
Continuously adding 500g/L lactose hydrolysate and 500g/L glycerol aqueous solution from 18h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 0.8 percent of the initial volume of the fermentation culture medium of 20L, continuously adding 400g/L peptone aqueous solution and 400g/L sodium glutamate aqueous solution from 45h of fermentation culture to the end of fermentation, wherein the fed-batch volume per hour is 0.2 percent of the initial volume of the fermentation culture medium of 20L, and supplementing isopropyl palmitate once after 50h of fermentation culture, wherein the supplemented volume is 15 percent of the initial volume of the fermentation culture medium of 20L; and after fermentation culture for 96h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
The preparation method of the lactose hydrolysate comprises dissolving 10000g lactose in 20L water, adding 210g β -galactosidase, and hydrolyzing at 50 deg.C and pH of 5.5 for 5h to obtain 500g/L lactose hydrolysate.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 42.1 g/L.
Example 6
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.6 percent, and culturing for 16 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media containing 100 mu g/L of geneticin and hygromycin B respectively, and culturing for 35 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 24 hours to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing citral according to the inoculation amount of 10% to culture, wherein the concentration of the citral in the culture medium is 0.2 g/L;
the formula of the fermentation medium is as follows: lactose hydrolysisLiquid 0.075L/L; 15g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; 6g/L magnesium sulfate heptahydrate; the vitamin solution is 12 ml/L; 10ml/L of metal ion solution; CuSO4·5H2O40 ug/L; 0.2g/L of citral and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13.5mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the steps of dissolving 633g of lactose in 1.5L of water, adding 3.2g of β -galactosidase, hydrolyzing for 5 hours at 55 ℃ with the pH value of 5.5, wherein the hydrolysis rate is 90%, mixing all the lactose hydrolysate into the fermentation medium, and enabling the initial volume of the fermentation medium to be 20L and the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to the fermentation medium to be 15:1 (g/L).
Starting from 17h of fermentation culture until the end of fermentation, continuously adding 600g/L of glycerol aqueous solution and 600g/L of sucrose aqueous solution, wherein the fed-batch volume per hour is 0.7 percent of the initial volume of the fermentation culture medium of 20L respectively, starting from 40h of fermentation culture until the end of fermentation, continuously adding 450g/L of yeast extract powder aqueous solution, 450g/L of peptone aqueous solution and 450g/L of sodium glutamate aqueous solution, wherein the fed-batch volume per hour is 0.15 percent of the initial volume of the fermentation culture medium of 20L respectively, supplementing oleic acid once after 48h of fermentation culture, and the supplemented volume is 20 percent of the initial volume of the fermentation culture medium of 20L; and after fermentation culture for 120h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 38.5 g/L.
Example 7
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.7 percent, and culturing for 20 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-7After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media containing 80 mu g/L of geneticin and hygromycin B respectively, and culturing for 45 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 28h to obtain a seed solution;
(4) fermentation culture: inoculating the seed solution obtained in the step 3) into a 20L fermentation culture medium containing citral according to the inoculation amount of 10% for culture, wherein the concentration of the citral in the culture medium is 0.1 g/L;
the formula of the fermentation medium is as follows: 0.1L/L lactose hydrolysate; 13g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; magnesium sulfate heptahydrate 6.2 g/L; 10ml/L of vitamin solution; 10ml/L of metal ion solution; CuSO4·5H2O30 ug/L; 0.1g/L of citral and the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 14mol/L ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the steps of dissolving 1239g of lactose in 2L of water, adding 5.2g of β -galactosidase, hydrolyzing for 5 hours at 52 ℃ with the pH value of 5.5, wherein the hydrolysis rate is 92%, mixing all the lactose hydrolysate into the fermentation medium, and the initial volume of the fermentation medium is 20L, so that the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to the fermentation medium is 30: 1 (g/L).
Continuously adding 700g/L of sucrose aqueous solution and 70% (V/V) of ethanol aqueous solution from the beginning of fermentation culture for 13h until the end of fermentation, wherein the adding volume per hour is respectively 0.1% of the initial volume of the fermentation culture medium for 20L, continuously adding 500g/L of yeast extract powder aqueous solution and 500g/L of sodium glutamate aqueous solution from the beginning of fermentation culture for 44h until the end of fermentation, wherein the adding volume per hour is respectively 0.2% of the initial volume of the fermentation culture medium for 20L, and after the fermentation culture for 53h, supplementing isopropyl palmitate and oleic acid for one time, and the supplementing volume is respectively 18% of the initial volume of the fermentation culture medium for 20L; and after fermentation culture for 90 hours, ending fermentation to obtain fermentation liquor containing artemisinic acid.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 36.7 g/L.
Example 8
(1) Activation of strains: inoculating the strain of the saccharomyces cerevisiae engineering bacteria frozen at the temperature of minus 80 ℃ into a seed culture medium according to the inoculation amount of 0.2 percent, and culturing for 30 hours to obtain a strain liquid;
(2) and (3) screening the resistance of strains: diluting the strain liquid obtained in the step (1) by 10-5After doubling, sucking 100 mu L of the culture medium, coating the culture medium on flat solid culture media respectively containing 60 mu g/L of nourseothricin, geneticin and hygromycin B, and culturing for 48 hours to obtain a single colony of the saccharomyces cerevisiae engineering bacteria;
the formula of the flat solid culture medium is as follows: glucose 19.5g/L (NH4)2SO415g/L，KH2PO48g/L，MgSO4·7H2O6.2 g/L, vitamin solution 12ml/L, metal ion solution 10ml/L, CuSO4·5H2O40ug/L, succinate buffer 100ml/L (0.5M, pH 5.0), agar 20g/L, and water in balance.
(3) Seed culture: picking single colony from a flat solid culture medium containing antibiotics, inoculating the single colony into a seed culture medium, and culturing for 25h to obtain a seed solution;
(4) fermentation culture: inoculating the seed liquid obtained in the step (3) into a 20L fermentation culture medium containing mevalonic acid and citral according to the inoculation amount of 10% for culture, wherein the concentrations of the mevalonic acid and the citral in the culture medium are 0.25g/L respectively;
the formula of the fermentation medium is as follows: 0.1L/L lactose hydrolysate; 15g/L of ammonium sulfate; 8g/L potassium dihydrogen phosphate; 6g/L magnesium sulfate heptahydrate; the vitamin solution is 11 ml/L; 8ml/L of metal ion solution; CuSO4·5H2O38 ug/L; 0.25g/L of mevalonic acid; 0.25g/L of citral; the balance of water. In the fermentation process, the pH is regulated and controlled by an aqueous solution containing 13.5mol/L of ammonia, so that the pH is maintained between 5.0 and 5.5 in the fermentation process.
The preparation method of the lactose hydrolysate in the fermentation medium comprises the steps of dissolving 1156g of lactose in 2L of water, adding 9.8g of β -galactosidase, hydrolyzing for 5 hours at 50 ℃ with the pH value of 5.5, wherein the hydrolysis rate is 92%, mixing all the lactose hydrolysate into the fermentation medium, and enabling the initial volume of the fermentation medium to be 20L and the mass-to-volume ratio of glucose and galactose in the lactose hydrolysate to the fermentation medium to be 28:1 (g/L).
Continuously adding 550g/L lactose hydrolysate, 550g/L glycerol aqueous solution, 550g/L sucrose aqueous solution and 55% (V/V) ethanol aqueous solution from 15h of fermentation culture to the end of fermentation, wherein the fed-in volume per hour is 0.3 percent of the initial volume of the fermentation culture medium 20L, continuously adding 400g/L yeast extract powder aqueous solution, 400g/L peptone aqueous solution and 400g/L sodium glutamate aqueous solution from 39h of fermentation culture to the end of fermentation, wherein the fed-in volume per hour is 0.1 percent of the initial volume of the fermentation culture medium 20L, and supplementing isopropyl palmitate and oleic acid once after 51h of fermentation culture, wherein the supplemented volume is 5 percent of the initial volume of the fermentation culture medium 20L; and after fermentation culture for 120h, ending fermentation to obtain fermentation liquor containing artemisinic acid.
The preparation method of the lactose hydrolysate comprises dissolving 11000g of lactose in 20L of water, adding 232g of β -galactosidase, and hydrolyzing at 50 deg.C and pH of 5.5 for 5h to obtain 550g/L lactose hydrolysate.
(5) And (3) potency detection: extracting 0.5ml fermentation liquor containing artemisinic acid with 9.5ml methanol, oscillating for 30min with ultrasound, filtering, and detecting by HPLC to obtain the fermentation yield of the artemisinic acid of 39.4 g/L.
Claims (10)
1. The method for improving fermentation yield of artemisinic acid is characterized by comprising the steps of taking saccharomyces cerevisiae engineering bacteria capable of producing artemisinic acid as production strains, and fermenting the strains in a fermentation culture medium containing lactose hydrolysate and exogenous factors, wherein the lactose hydrolysate is a solution obtained by hydrolyzing lactose into glucose and galactose through enzyme, and the exogenous factors are one or two of mevalonic acid and citral.
2. The method according to claim 1, wherein the enzyme is β -galactosidase, preferably wherein the mass ratio of β -galactosidase to lactose is 0.1-2: 100.
3. The method according to claim 1 or 2, wherein the mass-to-volume ratio of the amounts of glucose and galactose in the lactose hydrolysate to the fermentation medium is 15-30: 1 (unit is g/L), more preferably, the hydrolysis temperature of the lactose by enzyme hydrolysis is 50-55 ℃, the hydrolysis time is 3-5h, the hydrolysis rate is 90% -95%, and the hydrolysis pH is 4-7, preferably 5.5.
4. The method according to any one of claims 1 to 3, wherein the concentration of the exogenous factor in the fermentation medium is 0.1 to 0.5 g/L.
5. The process according to any one of claims 1 to 4, wherein the fermentation time is 84 to 120 hours and the fermentation temperature is 22 to 35 ℃.
6. The method according to any one of claims 1 to 5, wherein the fermentation further comprises feeding a carbon source, a nitrogen source and a water-insoluble oily liquid, wherein the water-insoluble oily liquid is one or both of isopropyl palmitate and oleic acid.
7. The method according to any one of claims 1 to 6, wherein the time of feeding the carbon source is from fermentation 10 to 18h to fermentation completion, the carbon source is one or more of lactose hydrolysate, aqueous glycerol solution, aqueous sucrose solution and aqueous ethanol solution, preferably, the concentration of the lactose hydrolysate is 400-700g/L, the concentration of the aqueous glycerol solution is 400-700g/L, the concentration of the aqueous sucrose solution is 400-700g/L, and the concentration of the aqueous ethanol solution is 80-99% (V/V), more preferably, the speed of feeding the carbon source is controlled in the range of 0.2-2% of the initial volume of the fermentation medium per hour.
8. The method as claimed in any one of claims 1 to 7, wherein the time of feeding the nitrogen source is from 36 to 45 hours from the beginning of the fermentation to the end of the fermentation, the nitrogen source is one or more of a yeast extract water solution, a peptone water solution and a sodium glutamate water solution, preferably, the concentration of the yeast extract water solution is 200-500g/L, the concentration of the peptone water solution is 200-500g/L, the concentration of the sodium glutamate water solution is 200-500g/L, and more preferably, the speed of feeding the nitrogen source is controlled in the volume of feeding the nitrogen source per hour which is 0.05 to 0.5 percent of the initial volume of the fermentation medium.
9. The method according to any one of claims 1 to 8, wherein the water-insoluble oily liquid is fed in a way of one-time supplement after 48 to 55 hours, and preferably, the volume of the fed water-insoluble oily liquid is 5 to 40 percent of the initial volume of the fermentation medium.
10. The method according to any one of claims 1 to 9, wherein the production strain is further subjected to resistance selection on a plate solid medium containing an antibiotic, wherein the antibiotic is one or more of noursemycin, geneticin and hygromycin B, and preferably wherein the content of the antibiotic on the plate solid medium is 50 to 300 μ g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810449836.8A CN108611383B (en) | 2018-05-11 | 2018-05-11 | Method for improving fermentation yield of artemisinic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810449836.8A CN108611383B (en) | 2018-05-11 | 2018-05-11 | Method for improving fermentation yield of artemisinic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108611383A true CN108611383A (en) | 2018-10-02 |
| CN108611383B CN108611383B (en) | 2021-04-20 |
Family
ID=63663027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810449836.8A Active CN108611383B (en) | 2018-05-11 | 2018-05-11 | Method for improving fermentation yield of artemisinic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108611383B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257451A (en) * | 2019-06-17 | 2019-09-20 | 浙江海正药业股份有限公司 | A method of promoting sweet wormwood acid accumulation |
| CN113552274A (en) * | 2021-07-23 | 2021-10-26 | 重庆市中药研究院 | Method for establishing high-performance liquid phase fingerprint spectrum of artemisinin by-product and measuring content of artemisinin by-product |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009085068A1 (en) * | 2007-12-27 | 2009-07-09 | Amyris Biotechnologies, Inc. | Compositions comprising artemisinic acid and methods of their preparation |
| CN101338309B (en) * | 2006-11-15 | 2012-07-18 | 中国医学科学院药物研究所 | Process for producing amorpha-4,11-diene and artemisinic acid |
| CN105316372A (en) * | 2014-08-04 | 2016-02-10 | 重庆乾泰生物医药有限公司 | Method for producing artemisinic acid through fermentation |
| CN106367361A (en) * | 2016-10-08 | 2017-02-01 | 天津大学 | Saccharomyces cerevisiae engineering strain as well as construction method and application thereof |
-
2018
- 2018-05-11 CN CN201810449836.8A patent/CN108611383B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338309B (en) * | 2006-11-15 | 2012-07-18 | 中国医学科学院药物研究所 | Process for producing amorpha-4,11-diene and artemisinic acid |
| WO2009085068A1 (en) * | 2007-12-27 | 2009-07-09 | Amyris Biotechnologies, Inc. | Compositions comprising artemisinic acid and methods of their preparation |
| CN105316372A (en) * | 2014-08-04 | 2016-02-10 | 重庆乾泰生物医药有限公司 | Method for producing artemisinic acid through fermentation |
| CN106367361A (en) * | 2016-10-08 | 2017-02-01 | 天津大学 | Saccharomyces cerevisiae engineering strain as well as construction method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| LENIHAN 等: "Developing an industrial artemisinic acid fermentation process to support the cost-effective production of antimalarial artemisinin-based combination therapies", 《BIOTECHNOL. PROG.》 * |
| 刘硕谦 等: "青蒿素组合生物合成的研究进展", 《中草药》 * |
| 杨竞成: "酶法水解乳糖生成D-半乳糖的工艺条件优化", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 郝乾坤 主编: "《药用微生物技术》", 31 August 2015, 重庆大学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257451A (en) * | 2019-06-17 | 2019-09-20 | 浙江海正药业股份有限公司 | A method of promoting sweet wormwood acid accumulation |
| CN113552274A (en) * | 2021-07-23 | 2021-10-26 | 重庆市中药研究院 | Method for establishing high-performance liquid phase fingerprint spectrum of artemisinin by-product and measuring content of artemisinin by-product |
| CN113552274B (en) * | 2021-07-23 | 2022-11-29 | 重庆市中药研究院 | Method for establishing high-performance liquid phase fingerprint spectrum of artemisinin by-product and measuring content of artemisinin by-product |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108611383B (en) | 2021-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104342390B (en) | A kind of Sinorhizobium meliloti strain and combinations thereof and application | |
| CN103409485A (en) | Method for improving adenosine fermentation output through feeding organic nitrogen source | |
| CN107746871B (en) | Method for preparing rare ginsenoside by biotransformation of ginsenoside with schizophyllum commune | |
| CN108611383B (en) | Method for improving fermentation yield of artemisinic acid | |
| CN104561134B (en) | A kind of method of Production by Microorganism Fermentation 1,3- propylene glycol | |
| CN102936608B (en) | Method for producing avilamycin by fermenting | |
| CN104893985B (en) | A kind of microorganism Aspergillus aculeatus bacterial strain and its application | |
| CN107418994A (en) | Process for producing gibberellin GA1 and GA4 by fermentation of gibberellic fungi | |
| CN104328155A (en) | Method and application for producing pyrroloquinoline quinone by using gluconobacter oxydans | |
| CN101245362A (en) | Method for producing polypeptide antibiotic enramycin by fermentation method | |
| CN110257451B (en) | Method for promoting accumulation of arteannuic acid | |
| CN104531535B (en) | Production of pneumocandin B0Gene recombination strain, breeding method and application | |
| CN109182147A (en) | A kind of mould and its method for producing fumidil | |
| US20130330789A1 (en) | Method for producing coenzyme q10 by fermentation using stock culture from solid phase fermentation | |
| CN108823110B (en) | Strain for producing griseofulvin and application thereof | |
| CN114703073A (en) | Yarrowia lipolytica, application thereof and method for producing erythritol through fermentation | |
| CN109680032A (en) | It is a kind of to utilize Microbe synthesis 7, the method for the pregnant Gona-4-en-3-one of 21- dihydroxy -20- methyl | |
| CN108265096B (en) | Preparation of pneumocandin B by microbial fermentation0Method (2) | |
| CN114134056A (en) | Saccharomyces cerevisiae ZJS10041 and application thereof in fermentation production of S-adenosylmethionine | |
| CN104277989B (en) | One plant of Saccharomyces cerevisiae and its application in fermenting and producing DPN | |
| CN101671706A (en) | Carbohydrate supplementing method in fermentation process of mycophenolic acid | |
| CN113637607A (en) | Amycolatopsis and application thereof | |
| CN108588152A (en) | Poly sialic acid fermentation medium, the production method of poly sialic acid and poly sialic acid product | |
| CN105316372B (en) | A kind of method of fermenting and producing Arteannuic acid | |
| CN110527650A (en) | A kind of Selective medium and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |