ES2330602A1 - Method for the production of phytoene and/or phytofluene or mixtures of carotenoids having a high content of same - Google Patents
Method for the production of phytoene and/or phytofluene or mixtures of carotenoids having a high content of same Download PDFInfo
- Publication number
- ES2330602A1 ES2330602A1 ES200800817A ES200800817A ES2330602A1 ES 2330602 A1 ES2330602 A1 ES 2330602A1 ES 200800817 A ES200800817 A ES 200800817A ES 200800817 A ES200800817 A ES 200800817A ES 2330602 A1 ES2330602 A1 ES 2330602A1
- Authority
- ES
- Spain
- Prior art keywords
- phytofluene
- paracoccus
- phytoene
- carotenoids
- production
- 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
- YVLPJIGOMTXXLP-UHFFFAOYSA-N 15-cis-phytoene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C YVLPJIGOMTXXLP-UHFFFAOYSA-N 0.000 title claims abstract description 106
- OVSVTCFNLSGAMM-KGBODLQUSA-N cis-phytofluene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/C=C/C=C(C)/CCC=C(/C)CCC=C(C)C)C)C)C)C OVSVTCFNLSGAMM-KGBODLQUSA-N 0.000 title claims abstract description 83
- 235000002677 phytofluene Nutrition 0.000 title claims abstract description 83
- OVSVTCFNLSGAMM-UZFNGAIXSA-N phytofluene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=C\C=C(/C)\C=C\C=C(C)CCC=C(C)CCC=C(C)C OVSVTCFNLSGAMM-UZFNGAIXSA-N 0.000 title claims abstract description 83
- ZYSFBWMZMDHGOJ-SGKBLAECSA-N phytofluene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)C=CC=C(/C)CCC=C(C)C)C)C)C)C ZYSFBWMZMDHGOJ-SGKBLAECSA-N 0.000 title claims abstract description 83
- ZIUDAKDLOLDEGU-UHFFFAOYSA-N trans-Phytofluen Natural products CC(C)=CCCC(C)CCCC(C)CC=CC(C)=CC=CC=C(C)C=CCC(C)CCCC(C)CCC=C(C)C ZIUDAKDLOLDEGU-UHFFFAOYSA-N 0.000 title claims abstract description 83
- YVLPJIGOMTXXLP-UUKUAVTLSA-N 15,15'-cis-Phytoene Natural products C(=C\C=C/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C YVLPJIGOMTXXLP-UUKUAVTLSA-N 0.000 title claims abstract description 53
- YVLPJIGOMTXXLP-BAHRDPFUSA-N 15Z-phytoene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)CCC=C(/C)CCC=C(C)C)C)C)C)C YVLPJIGOMTXXLP-BAHRDPFUSA-N 0.000 title claims abstract description 53
- 235000011765 phytoene Nutrition 0.000 title claims abstract description 53
- 235000021466 carotenoid Nutrition 0.000 title claims abstract description 52
- 150000001747 carotenoids Chemical class 0.000 title claims abstract description 52
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 40
- 239000000203 mixture Substances 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims description 30
- 241000589598 Paracoccus sp. Species 0.000 claims abstract description 63
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims description 40
- 230000004151 fermentation Effects 0.000 claims description 40
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 30
- 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 13
- 239000008103 glucose Substances 0.000 claims description 13
- 238000010352 biotechnological method Methods 0.000 abstract description 3
- 239000002054 inoculum Substances 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- 235000008210 xanthophylls Nutrition 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 description 8
- 102100025327 Dynamin-like 120 kDa protein, mitochondrial Human genes 0.000 description 7
- 101100283604 Caenorhabditis elegans pigk-1 gene Proteins 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 description 6
- JEVVKJMRZMXFBT-XWDZUXABSA-N Lycophyll Natural products OC/C(=C/CC/C(=C\C=C\C(=C/C=C/C(=C\C=C\C=C(/C=C/C=C(\C=C\C=C(/CC/C=C(/CO)\C)\C)/C)\C)/C)\C)/C)/C JEVVKJMRZMXFBT-XWDZUXABSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 240000008042 Zea mays Species 0.000 description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 235000010633 broth Nutrition 0.000 description 6
- 235000005822 corn Nutrition 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- KBPHJBAIARWVSC-RGZFRNHPSA-N lutein Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\[C@H]1C(C)=C[C@H](O)CC1(C)C KBPHJBAIARWVSC-RGZFRNHPSA-N 0.000 description 6
- 229960005375 lutein Drugs 0.000 description 6
- 235000012661 lycopene Nutrition 0.000 description 6
- 239000001751 lycopene Substances 0.000 description 6
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 description 6
- 229960004999 lycopene Drugs 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- ZCIHMQAPACOQHT-ZGMPDRQDSA-N trans-isorenieratene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/c1c(C)ccc(C)c1C)C=CC=C(/C)C=Cc2c(C)ccc(C)c2C ZCIHMQAPACOQHT-ZGMPDRQDSA-N 0.000 description 6
- FJHBOVDFOQMZRV-XQIHNALSSA-N xanthophyll Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C=C(C)C(O)CC2(C)C FJHBOVDFOQMZRV-XQIHNALSSA-N 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 150000003735 xanthophylls Chemical class 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000003471 mutagenic agent Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000012064 sodium phosphate buffer Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- OINNEUNVOZHBOX-QIRCYJPOSA-K 2-trans,6-trans,10-trans-geranylgeranyl diphosphate(3-) Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\COP([O-])(=O)OP([O-])([O-])=O OINNEUNVOZHBOX-QIRCYJPOSA-K 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 3
- 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 3
- OINNEUNVOZHBOX-XBQSVVNOSA-N Geranylgeranyl diphosphate Natural products [P@](=O)(OP(=O)(O)O)(OC/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)O OINNEUNVOZHBOX-XBQSVVNOSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 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 description 3
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 235000013734 beta-carotene Nutrition 0.000 description 3
- 239000011648 beta-carotene Substances 0.000 description 3
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 description 3
- 229960002747 betacarotene Drugs 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 230000003505 mutagenic effect Effects 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 239000000377 silicon dioxide Substances 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 3
- JKQXZKUSFCKOGQ-JLGXGRJMSA-N (3R,3'R)-beta,beta-carotene-3,3'-diol Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C[C@@H](O)CC1(C)C JKQXZKUSFCKOGQ-JLGXGRJMSA-N 0.000 description 2
- ATCICVFRSJQYDV-UHFFFAOYSA-N (6E,8E,10E,12E,14E,16E,18E,20E,22E,26E)-2,6,10,14,19,23,27,31-octamethyldotriaconta-2,6,8,10,12,14,16,18,20,22,26,30-dodecaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC=C(C)CCC=C(C)C ATCICVFRSJQYDV-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 description 2
- HRQKOYFGHJYEFS-UHFFFAOYSA-N Beta psi-carotene Chemical compound CC(C)=CCCC(C)=CC=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C HRQKOYFGHJYEFS-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 2
- 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 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- JKQXZKUSFCKOGQ-LQFQNGICSA-N Z-zeaxanthin Natural products C([C@H](O)CC=1C)C(C)(C)C=1C=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC1=C(C)C[C@@H](O)CC1(C)C JKQXZKUSFCKOGQ-LQFQNGICSA-N 0.000 description 2
- QOPRSMDTRDMBNK-RNUUUQFGSA-N Zeaxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCC(O)C1(C)C)C=CC=C(/C)C=CC2=C(C)CC(O)CC2(C)C QOPRSMDTRDMBNK-RNUUUQFGSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- JKQXZKUSFCKOGQ-LOFNIBRQSA-N all-trans-Zeaxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2=C(C)CC(O)CC2(C)C JKQXZKUSFCKOGQ-LOFNIBRQSA-N 0.000 description 2
- ANVAOWXLWRTKGA-XHGAXZNDSA-N all-trans-alpha-carotene Chemical compound CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1C(C)=CCCC1(C)C ANVAOWXLWRTKGA-XHGAXZNDSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000002518 antifoaming agent Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 235000013793 astaxanthin Nutrition 0.000 description 2
- 239000001168 astaxanthin Substances 0.000 description 2
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 description 2
- 229940022405 astaxanthin Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000011663 gamma-carotene Substances 0.000 description 2
- 235000000633 gamma-carotene Nutrition 0.000 description 2
- HRQKOYFGHJYEFS-RZWPOVEWSA-N gamma-carotene Natural products C(=C\C=C\C(=C/C=C/C=C(\C=C\C=C(/C=C/C=1C(C)(C)CCCC=1C)\C)/C)\C)(\C=C\C=C(/CC/C=C(\C)/C)\C)/C HRQKOYFGHJYEFS-RZWPOVEWSA-N 0.000 description 2
- GVVPGTZRZFNKDS-JXMROGBWSA-N geranyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O GVVPGTZRZFNKDS-JXMROGBWSA-N 0.000 description 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
- 230000000813 microbial effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- CBIDRCWHNCKSTO-UHFFFAOYSA-N prenyl diphosphate Chemical compound CC(C)=CCO[P@](O)(=O)OP(O)(O)=O CBIDRCWHNCKSTO-UHFFFAOYSA-N 0.000 description 2
- 238000011137 process chromatography Methods 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 235000010930 zeaxanthin Nutrition 0.000 description 2
- 239000001775 zeaxanthin Substances 0.000 description 2
- 229940043269 zeaxanthin Drugs 0.000 description 2
- BIWLELKAFXRPDE-UHFFFAOYSA-N zeta-Carotene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)CCC=C(C)CCC=C(C)C BIWLELKAFXRPDE-UHFFFAOYSA-N 0.000 description 2
- 101710165761 (2E,6E)-farnesyl diphosphate synthase Proteins 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-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
- YECXHLPYMXGEBI-DOYZGLONSA-N Adonixanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)C(=O)C(O)CC1(C)C)C=CC=C(/C)C=CC2=C(C)CC(O)CC2(C)C YECXHLPYMXGEBI-DOYZGLONSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000004212 Cryptoxanthin Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101710156207 Farnesyl diphosphate synthase Proteins 0.000 description 1
- 102100035111 Farnesyl pyrophosphate synthase Human genes 0.000 description 1
- 101710125754 Farnesyl pyrophosphate synthase Proteins 0.000 description 1
- 101710089428 Farnesyl pyrophosphate synthase erg20 Proteins 0.000 description 1
- 108010007508 Farnesyltranstransferase Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- GVVPGTZRZFNKDS-YFHOEESVSA-N Geranyl diphosphate Natural products CC(C)=CCC\C(C)=C/COP(O)(=O)OP(O)(O)=O GVVPGTZRZFNKDS-YFHOEESVSA-N 0.000 description 1
- YTZIWAULTIDEEY-UHFFFAOYSA-N Isomeres zeta-Carotin Natural products CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)C=CC=C(C)C=CC=C(C)CCC=C(C)C YTZIWAULTIDEEY-UHFFFAOYSA-N 0.000 description 1
- 108010065958 Isopentenyl-diphosphate Delta-isomerase Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- ATCICVFRSJQYDV-DDRHJXQASA-N Neurosporene Natural products C(=C\C=C\C(=C/C=C/C=C(\C=C\C=C(/CC/C=C(\CC/C=C(\C)/C)/C)\C)/C)\C)(\C=C\C=C(/CC/C=C(\C)/C)\C)/C ATCICVFRSJQYDV-DDRHJXQASA-N 0.000 description 1
- 241000235401 Phycomyces blakesleeanus Species 0.000 description 1
- 101710150389 Probable farnesyl diphosphate synthase Proteins 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- YECXHLPYMXGEBI-ZNQVSPAOSA-N adonixanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)C[C@@H](O)CC1(C)C YECXHLPYMXGEBI-ZNQVSPAOSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 239000011795 alpha-carotene Substances 0.000 description 1
- 235000003903 alpha-carotene Nutrition 0.000 description 1
- ANVAOWXLWRTKGA-HLLMEWEMSA-N alpha-carotene Natural products C(=C\C=C\C=C(/C=C/C=C(\C=C\C=1C(C)(C)CCCC=1C)/C)\C)(\C=C\C=C(/C=C/[C@H]1C(C)=CCCC1(C)C)\C)/C ANVAOWXLWRTKGA-HLLMEWEMSA-N 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940072107 ascorbate Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 238000011138 biotechnological process Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 235000012682 canthaxanthin Nutrition 0.000 description 1
- FDSDTBUPSURDBL-DKLMTRRASA-N canthaxanthin Chemical compound CC=1C(=O)CCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)C(=O)CCC1(C)C FDSDTBUPSURDBL-DKLMTRRASA-N 0.000 description 1
- -1 capsantin Chemical compound 0.000 description 1
- 150000001746 carotenes Chemical class 0.000 description 1
- 235000005473 carotenes Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 101150081158 crtB gene Proteins 0.000 description 1
- 101150000046 crtE gene Proteins 0.000 description 1
- 101150011633 crtI gene Proteins 0.000 description 1
- 101150085103 crtY gene Proteins 0.000 description 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- BIWLELKAFXRPDE-PCYOLSTGSA-N di-cis-zeta-carotene Natural products CC(C)=CCCC(C)=CCCC(C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(\C)CCC=C(C)CCC=C(C)C BIWLELKAFXRPDE-PCYOLSTGSA-N 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 235000006486 human diet Nutrition 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 235000012680 lutein Nutrition 0.000 description 1
- 239000001656 lutein Substances 0.000 description 1
- ORAKUVXRZWMARG-WZLJTJAWSA-N lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C ORAKUVXRZWMARG-WZLJTJAWSA-N 0.000 description 1
- 108060004506 lycopene beta-cyclase Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 235000008665 neurosporene Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 108010001545 phytoene dehydrogenase Proteins 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- BIWLELKAFXRPDE-XXKNMTJFSA-N zeta-Carotene Natural products C(=C\C=C\C=C(/C=C/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)\C)(\C=C\C=C(/CC/C=C(\CC/C=C(\C)/C)/C)\C)/C BIWLELKAFXRPDE-XXKNMTJFSA-N 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 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
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Método de producción de fitoeno y/o fitoflueno, o mezclas de carotenoides con alto contenido en los mismos.Production method of phytoene and / or phytofluene, or mixtures of carotenoids with high content therein.
La presente invención describe un método biotecnológico que permite obtener de forma simple, efectiva y a gran escala, preparaciones estabilizadas de fitoeno y/o fitoflueno, o mezclas de carotenoides con alto contenido en los mismos, mediante la utilización de cepas mutadas de Paracoccus sp. superproductoras de fitoeno y/o fitoflueno. Por lo tanto, la presente invención es susceptible de ser aplicada en diversos campos de las ciencias de la vida, como por ejemplo en el campo alimentario, el farmacéutico o el cosmético.The present invention describes a biotechnological method that makes it possible to obtain, in a simple, effective and large-scale manner, stabilized preparations of phytoene and / or phytofluene, or mixtures of carotenoids with high content thereof, by using mutated strains of Paracoccus sp . Phytoenne and / or phytofluene superproducers. Therefore, the present invention is capable of being applied in various fields of life sciences, such as in the food, pharmaceutical or cosmetic fields.
Los carotenoides son pigmentos de naturaleza isoprenoide sintetizados por ciertas bacterias, hongos y organismos fotosintéticos. Pueden clasificarse en dos tipos:Carotenoids are pigments of nature Isoprenoid synthesized by certain bacteria, fungi and organisms photosynthetic They can be classified into two types:
- (i) (i)
- Carotenos: son hidrocarburos puros entre los que se encuentran compuestos como \beta-caroteno, \alpha-caroteno, \gamma-caroteno, licopeno, fitoeno o fitoflueno; yCarotenes: they are pure hydrocarbons among which find compounds such as β-carotene, α-carotene, γ-carotene, lycopene, phytoen or phytofluene; Y
- (ii) (ii)
- Xantofilas: son moléculas que contienen oxígeno en diferentes formas (grupos hidroxi, epoxi, etc.), entre las que se encuentran compuestos como astaxantina, zeaxantina, capsantina, cantaxantina, luteína, etc.Xanthophylls: these are molecules that contain oxygen in different forms (hydroxy, epoxy groups, etc.), among which find compounds such as astaxanthin, zeaxanthin, capsantin, cantaxanthin, lutein, etc.
Todos estos compuestos juegan un papel importante en la dieta humana como antioxidantes y como precursores de la vitamina A. Por lo tanto, debido a sus características, los carotenoides son eficaces contra el cáncer y otras enfermedades. Debido a sus efectos beneficiosos para la salud y a sus atractivos colores, los carotenoides poseen una gran importancia comercial como colorantes y aditivos alimentarios [Ninet L. y Renaut J. (1979) En: Peppler HJ., Perlman D. (eds). Microbial Technology, 2^{nd}. Edn, vol. 1 Academic Press, NY, pp. 529-544].All these compounds play a role important in the human diet as antioxidants and as precursors of vitamin A. Therefore, due to its characteristics, Carotenoids are effective against cancer and other diseases. Due to its beneficial effects on health and its attractiveness colors, carotenoids have great commercial importance as dyes and food additives [Ninet L. and Renaut J. (1979) In: Peppler HJ., Perlman D. (eds). Microbial Technology, 2 nd. Edn, vol. 1 Academic Press, NY, pp. 529-544].
Diversas investigaciones apoyan la teoría de que el daño oxidativo sobre el ADN, proteínas y lípidos está directamente relacionado con la esperanza de vida, y que los antioxidantes naturales tales como ascorbato, tocoferol y carotenoides son compuestos importantes en la prevención de estas oxidaciones [Stadtman ER (1992) Science 257: 1220-1222; Sohal et al. (1993) Proceedings of the National Academic of Sciences USA 90: 7255-7256]. Particularmente, en el caso del fitoeno han sido descritos efectos beneficiosos para la salud al tratarse de un compuesto con actividad antitumoral [Mathews-Roth MM (1982) Oncology 39: 33-37].Several investigations support the theory that oxidative damage on DNA, proteins and lipids is directly related to life expectancy, and that natural antioxidants such as ascorbate, tocopherol and carotenoids are important compounds in the prevention of these oxidations [Stadtman ER (1992) Science 257: 1220-1222; Sohal et al . (1993) Proceedings of the National Academic of Sciences USA 90: 7255-7256]. Particularly, in the case of phytoene, beneficial health effects have been described as it is a compound with antitumor activity [Mathews-Roth MM (1982) Oncology 39: 33-37].
La producción de carotenoides por biosíntesis microbiana es un ejemplo clásico de competencia entre los procesos químicos y los biológicos. Los procesos biotecnológicos muestran, entre otras, la ventaja de permitir obtener de forma simple los carotenoides de estructura más compleja, así como los isómeros conformacionales que sólo existen de forma natural.The production of carotenoids by biosynthesis Microbial is a classic example of competition between processes Chemical and biological. Biotechnological processes show, among others, the advantage of allowing to obtain in a simple way the carotenoids of more complex structure, as well as isomers conformational that only exist naturally.
A pesar de la diversidad estructural y funcional de los carotenoides y xantofilas, su ruta biosintética es similar en los diferentes organismos, siendo el fitoeno la molécula precursora en todos los casos [Arrach N. et al. (2001) Proceedings of the National Academic of Sciences USA 98: 1687-1692; Velayos A. et al. (2000) European Journal of Biochemistry 267: 5509-5519; Lee & Schmidt-Dannert (2002) Appl. Microbiol. Biotechnol. 60: 1-11; Sandmann G (2003) Chem. Biol. 10: 478-479; Umeno et al. (2005) Microbiol. Mol. Biol. Rev. 69: 51-78].Despite the structural and functional diversity of carotenoids and xanthophylls, their biosynthetic pathway is similar in different organisms, with phytoene being the precursor molecule in all cases [Arrach N. et al . (2001) Proceedings of the National Academic of Sciences USA 98: 1687-1692; Velayos A. et al . (2000) European Journal of Biochemistry 267: 5509-5519; Lee & Schmidt-Dannert (2002) Appl. Microbiol Biotechnol 60: 1-11; Sandmann G (2003) Chem. Biol. 10: 478-479; Umeno et al . (2005) Microbiol. Mol. Biol. Rev. 69: 51-78].
La biosíntesis (Esquema 1) se inicia con la isomerización de isopentenil pirofosfato (IPP, C_{5}) a dimetilalil pirofosfato (DMAPP, C_{5}), catalizada por la enzima IPP isomerasa. Seguidamente una molécula de DMAPP se condensa con una molécula de IPP para generar geranil pirofosfato (GPP, C_{10}), el cual se condensa nuevamente con una molécula de IPP para generar farnesil pirofosfato (FPP, C_{15}); ambas reacciones están catalizadas por la enzima FPP sintasa. Una nueva condensación de una molécula de FPP con una molécula de IPP da lugar a geranilgeranil pirofosfato (GGPP, C_{20}); esta reacción está catalizada por la enzima GGPP sintasa (codificada por el gen crtE). La condensación de dos moléculas de GGPP catalizada por la enzima fitoeno sintasa (codificada por el gen crtB) genera fitoeno (C_{40}), molécula precursora de la ruta biosintética de carotenoides. La enzima fitoeno desaturasa (codificada por el gen crtI) introduce cuatro dobles enlaces en la molécula de fitoeno para sintetizar consecutivamente fitoflueno, \zeta-caroteno, neurosporeno y licopeno (C_{40}). Finalmente, la enzima licopeno ciclasa (codificada por el gen crtY) se encarga de convertir los extremos acíclicos de la molécula de licopeno en anillos \beta para formar secuencialmente \gamma-caroteno y \beta-caroteno (C_{40}).Biosynthesis (Scheme 1) begins with the isomerization of isopentenyl pyrophosphate (IPP, C 5) to dimethylalkyl pyrophosphate (DMAPP, C 5), catalyzed by the enzyme IPP isomerase. Next, a DMAPP molecule is condensed with an IPP molecule to generate geranyl pyrophosphate (GPP, C 10), which is condensed again with an IPP molecule to generate farnesyl pyrophosphate (FPP, C 15); both reactions are catalyzed by the enzyme FPP synthase. A new condensation of an FPP molecule with an IPP molecule results in geranylgeranyl pyrophosphate (GGPP, C20); This reaction is catalyzed by the enzyme GGPP synthase (encoded by the crtE gene). The condensation of two GGPP molecules catalyzed by the enzyme phytoen synthase (encoded by the crtB gene) generates phytoen (C 40), a precursor molecule of the biosynthetic pathway of carotenoids. The enzyme phytoene desaturase (encoded by the crtI gene) introduces four double bonds in the phytoen molecule to synthesize phytofluene, zeta-carotene, neurosporene and lycopene (C 40) consecutively. Finally, the lycopene cyclase enzyme (encoded by the crtY gene) is responsible for converting the acyclic ends of the lycopene molecule into β rings to sequentially form γ-carotene and β-carotene (C 40).
En el caso de Phycomyces blakesleeanus, el color amarillo de su micelio puede ser modificado mediante mutación dando lugar a cepas con micelio de color rojo, blanco o varias gradaciones de amarillo. Los mutantes rojos acumulan licopeno, mientras que los blancos carecen de producción de carotenoides o acumulan fitoeno [Metha B.J. y Cerdá-Olmedo E. (1995) Applied Microbiology and Biotechnology 42: 836-838].In the case of Phycomyces blakesleeanus, the yellow color of my mycelium can be modified by mutation, resulting in strains with red, white or several yellow gradations. Red mutants accumulate lycopene, while whites lack carotenoid production or accumulate phytoeno [Metha BJ and Cerdá-Olmedo E. (1995) Applied Microbiology and Biotechnology 42: 836-838].
La utilización de Paracoccus sp. para la producción de astaxantina, adonixantina, equinenona, 3-hidroxiequinenona, zeaxantina, \beta-caroteno, licopeno, o \beta-criptoxantina está recogida en las patentes EP 635576, EP 1138208, EP 1229126, EP 1676925, o US 5935808.The use of Paracoccus sp. For the production of astaxanthin, adonixanthin, equinenone, 3-hydroxyequinenone, zeaxanthin,? -carotene, lycopene, or? -cryptoxanthin is contained in EP 635576, EP 1138208, EP 1229126, EP 1676925, or US 5935808.
La presente invención describe un método biotecnológico, en adelante método de la invención, que permite obtener de forma simple, efectiva y a gran escala preparaciones estabilizadas de fitoeno y/o fitoflueno, o mezclas de carotenoides con alto contenido en los mismos, mediante la utilización de nuevas cepas mutadas de Paracoccus sp. (FA1 y FA3) superproductoras de fitoeno y/o fitoflueno.The present invention describes a biotechnological method, hereinafter the method of the invention , which allows to obtain, in a simple, effective and large-scale way, stabilized preparations of phytoene and / or phytofluene, or mixtures of carotenoids with high content thereof, by means of the use of new mutated strains of Paracoccus sp. (FA1 and FA3) superproducers of phytoene and / or phytofluene.
Así, el problema técnico resuelto por la presente invención es la obtención de forma simple, efectiva y a gran escala de preparaciones estabilizadas de fitoeno y fitoflueno, o mezclas de carotenoides con alto contenido en fitoeno y fitoflueno, las cuales puedan aplicarse directamente en los campos alimentario, farmacéutico y cosmético.Thus, the technical problem solved by the The present invention is to obtain in a simple, effective and large scale stabilized preparations of phytoene and phytofluene, or mixtures of carotenoids with a high phytoen content and phytofluene, which can be applied directly in the fields Food, pharmaceutical and cosmetic.
Tal y como se cita en el estado de la técnica, existen varios documentos de patente que describen el uso de Paracoccus sp. para la producción de diversos carotenoides distintos del fitoeno y del fitoflueno. Sin embargo, ninguno de ellos se refiere a la utilización de las cepas de Paracoccus sp. mutadas (FA1 y FA3) conseguidas en la presente invención para la producción de fitoeno o fitoflueno. A la luz de los documentos citados en el estado de la técnica, se considera que la presente invención presenta: (i) novedad, ya que ninguno de los documentos del estado de la técnica divulga el uso de las cepas de Paracoccus sp. mutadas (FA1 y FA3) para la producción simple, efectiva y a gran escala de preparaciones estabilizadas de fitoeno y/o fitoflueno; y (ii) actividad inventiva, ya que un experto medio en la materia no podría combinar de forma obvia los documentos expuestos en el estado de la técnica y resolver el problema técnico arriba planteado.As cited in the prior art, there are several patent documents that describe the use of Paracoccus sp. for the production of various carotenoids other than phytoene and phytofluene. However, none of them refers to the use of the strains of Paracoccus sp. mutates (FA1 and FA3) achieved in the present invention for the production of phytoene or phytofluene. In the light of the documents cited in the prior art, the present invention is considered to present: (i) novelty, since none of the documents of the prior art discloses the use of the Paracoccus sp . mutated (FA1 and FA3) for simple, effective and large-scale production of stabilized preparations of phytoene and / or phytofluene; and (ii) inventive activity, since an average expert in the field could not obviously combine the documents set forth in the prior art and solve the technical problem raised above.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Figura 1Figure one
A: Izquierda: Cromatograma HPLC que muestra un pico mayoritario con RT 9,46 correspondiente a fitoeno. Derecha: Espectro de absorción obtenido por diodoarray del pico con RT 9,46. El máximo de absorción obtenido (286 nm) es característico del fitoeno (ver Tabla 1).A: Left: HPLC chromatogram showing a Major peak with RT 9.46 corresponding to phytoen. Right: Absorption spectrum obtained by diodearray of the peak with RT 9.46. The maximum absorption obtained (286 nm) is characteristic of phytoeno (see Table 1).
B: Izquierda: Cromatograma HPLC que muestra un pico mayoritario con RT 8,23 correspondiente a fitoflueno. Derecha: Espectro de absorción obtenido por diodoarray del pico con RT 8,23. El máximo de absorción obtenido (348 nm) es característico del fitoflueno (ver Tabla 1).B: Left: HPLC chromatogram showing a Major peak with RT 8.23 corresponding to phytofluene. Right: Absorption spectrum obtained by diodearray of the peak with RT 8.23. The maximum absorption obtained (348 nm) is characteristic of phytofluene (see Table 1).
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Figura 2Figure 2
A: Izquierda: Cromatograma HPLC que muestra un pico mayoritario con RT 9,52 correspondiente a fitoeno. Derecha: Espectro de absorción obtenido por diodoarray del pico con RT 9,52. El máximo de absorción obtenido (286 nm) es característico del fitoeno (ver Tabla 1).A: Left: HPLC chromatogram showing a Major peak with RT 9.52 corresponding to phytoen. Right: Absorption spectrum obtained by diodearray of the peak with RT 9.52. The maximum absorption obtained (286 nm) is characteristic of phytoeno (see Table 1).
B: Izquierda: Cromatograma HPLC que muestra un pico mayoritario con RT 8,29 correspondiente a fitoflueno. Derecha: Espectro de absorción obtenido por diodoarray del pico con RT 8,29. El máximo de absorción obtenido (348 nm) es característico del fitoflueno (ver Tabla 1).B: Left: HPLC chromatogram showing a Major peak with RT 8.29 corresponding to phytofluene. Right: Absorption spectrum obtained by diodearray of the peak with RT 8.29. The maximum absorption obtained (348 nm) is characteristic of phytofluene (see Table 1).
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Figura 3Figure 3
A: Producción de fitoeno y fitoflueno mediante fermentación en matraz de la cepa Paracoccus sp. FA1. Ordenadas: fitoeno (\blacklozenge) y fitoflueno (\Box) (mg/L). Abscisas: tiempo de fermentación (h). Los datos se representan en la Tabla 5.A: Production of phytoene and phytofluene by flask fermentation of the strain Paracoccus sp . FA1. Ordered: phytoene (\ blacklozenge) and phytofluene (\ Box) (mg / L). Abscissa: fermentation time (h). The data is represented in Table 5.
B: Producción de fitoeno y fitoflueno mediante fermentación en matraz de la cepa Paracoccus sp. FA3. Ordenadas: fitoeno (\blacklozenge) y fitoflueno (\Box) (mg/L). Abscisas: tiempo de fermentación (h). Los datos se representan en la Tabla 6.B: Production of phytoeno and phytofluene by flask fermentation of the strain Paracoccus sp . FA3. Ordered: phytoene (\ blacklozenge) and phytofluene (\ Box) (mg / L). Abscissa: fermentation time (h). The data is represented in Table 6.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Figura 4Figure 4
A: Producción de fitoeno y fitoflueno mediante fermentación en tanque fermentador de la cepa Paracoccus sp. FA1. Ordenadas: fitoeno (\blacklozenge) y fitoflueno (\Box) (mg/L). Abscisas: tiempo de fermentación (h). Los datos se representan en la Tabla 7.A: Production of phytoeno and phytofluene by fermentation in a fermenting tank of the Paracoccus sp. FA1. Ordered: phytoene (\ blacklozenge) and phytofluene (\ Box) (mg / L). Abscissa: fermentation time (h). The data is represented in Table 7.
\newpage\ newpage
B: Producción de fitoeno y fitoflueno mediante fermentación en tanque fermentador de la cepa Paracoccus sp. FA3. Ordenadas: fitoeno (\blacklozenge) y fitoflueno (\Box) (mg/L). Abscisas: tiempo de fermentación (h). Los datos se representan en la Tabla 8.B: Production of phytoeno and phytofluene by fermentation in a fermenting tank of the Paracoccus sp. FA3. Ordered: phytoene (\ blacklozenge) and phytofluene (\ Box) (mg / L). Abscissa: fermentation time (h). The data is represented in Table 8.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Tal y como se ha comentado anteriormente, el método de la invención permite obtener de forma simple, efectiva y a gran escala preparaciones estabilizadas de fitoeno y/o fitoflueno, o mezclas de carotenoides con alto contenido en los mismos, mediante la utilización de cepas mutadas de Paracoccus sp. superproductoras de fitoeno y/o fitoflueno (FA1 y FA3).As mentioned above, the method of the invention makes it possible to obtain, in a simple, effective and large-scale manner, stabilized preparations of phytoene and / or phytofluene, or mixtures of carotenoids with high content thereof, by using mutated strains of Paracoccus sp. superproducers of phytoen and / or phytofluene (FA1 and FA3).
El método de la invención comprende:The method of the invention comprises:
- (i) (i)
- obtención y selección de mutantes superproductores de fitoeno y fitoflueno a partir de una cepa de Paracoccus sp. productora de xantofilas;Obtaining and selecting mutants superproducers of phytoene and phytofluene from a strain of Paracoccus sp. xanthophyll producer;
- (ii) (ii)
- desarrollo de condiciones mejoradas de fermentación para conseguir una máxima producción de fitoeno y/o fitoflueno, ydevelopment of improved fermentation conditions to achieve maximum production of phytoeno and / or phytofluene, Y
- (iii) (iii)
- extracción y purificación del fitoeno y del fitoflueno producido.extraction and purification of phytoen and phytofluene produced.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
En primer lugar y con la finalidad de obtener cepas de Paracoccus sp. superproductoras de fitoeno y fitoflueno, se desarrolló un procedimiento mutagénico de Paracoccus sp. utilizando los agentes mutagénicos etilmetanosulfonato (EMS) y N-metil-N'-nitro-N-nitrosoguanidina (NTG). Las suspensiones de células a mutar se obtuvieron a partir de cultivo en medio líquido, lavando con solución salina para eliminar restos del medio. El procedimiento de mutación con EMS consistió en la incubación de aproximadamente 10^{11} células/mL en una solución de EMS al 3% en tampón fosfato sódico 0,1 M pH 7,0 a temperatura ambiente durante 30 a 180 minutos, consiguiendo tasas de mortalidad de entre el 90 y el 99,9%. Las células mutadas se lavaron tres veces con solución salina. El procedimiento de mutación con NTG consistió en la incubación de aproximadamente 10^{11} células/mL en una solución que contenía 250 \mug/mL de NTG y tampón fosfato sódico 0,1 M pH 7,0 a temperatura ambiente durante 90 minutos, consiguiendo tasas de mortalidad de alrededor del 99%. Las células mutadas se lavaron tres veces con solución salina. Con las células mutadas se sembraron placas Petri con medio sólido y se incubaron a 28ºC durante 4 días para obtener colonias aisladas.In the first place and with the purpose of obtaining strains of Paracoccus sp. Superproducers of phytoene and phytofluene, a mutagenic procedure of Paracoccus sp . using the mutagenic agents ethylmethanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The suspensions of cells to be mutated were obtained from culture in liquid medium, washing with saline solution to remove remains of the medium. The mutation procedure with EMS consisted of the incubation of approximately 10 11 cells / mL in a solution of 3% EMS in 0.1 M sodium phosphate buffer pH 7.0 at room temperature for 30 to 180 minutes, achieving Mortality rates between 90 and 99.9%. The mutated cells were washed three times with saline. The NTG mutation procedure consisted of the incubation of approximately 10 11 cells / mL in a solution containing 250 µg / mL of NTG and 0.1 M sodium phosphate buffer pH 7.0 at room temperature for 90 minutes , obtaining mortality rates of around 99%. The mutated cells were washed three times with saline. With the mutated cells, Petri dishes were seeded with solid medium and incubated at 28 ° C for 4 days to obtain isolated colonies.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
La estrategia utilizada para la selección de cepas superproductoras de fitoeno y fitoflueno a partir de una cepa de Paracoccus sp. productora de xantofilas se basó en el color de la colonia. Para ello, las células mutadas de Paracoccus sp. se sembraron en placas Petri y, una vez crecidas a 28ºC, se seleccionaron aquellas colonias que poseían color crema (la cepa parental posee color naranja). Tras analizar alrededor de 100.000 colonias se aislaron 2 colonias con color crema denominadas FA1 y FA3 productoras de fitoeno y fitoflueno.The strategy used for the selection of superproductive strains of phytoene and phytofluene from a strain of Paracoccus sp. Xanthophyll producer was based on the color of the colony. To do this, the mutated cells of Paracoccus sp. they were seeded in Petri dishes and, once grown at 28 ° C, those colonies that had a cream color were selected (the parental strain has an orange color). After analyzing about 100,000 colonies, 2 cream-colored colonies called FA1 and FA3 producing phytoene and phytophenol were isolated.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Las cepas Paracoccus sp. FA1 y Paracoccus sp. FA3 se fermentaron en matraz con la finalidad de determinar el nivel de producción de fitoeno y fitoflueno en medio líquido. El análisis de fitoeno y fitoflueno se determinó mediante cromatografia líquida HPLC en fase reversa, demostrándose que con ambas cepas es posible producir fitoeno y fitoflueno junto con una mezcla de carotenoides en la que el fitoeno es el carotenoide mayoritario. Asimismo, las cepas Paracoccus sp. FA1 y Paracoccus sp. FA3 se fermentaron en fermentadores pre-industriales con la finalidad de determinar el nivel de producción de fitoeno y fitoflueno. En este caso se desarrollaron dos condiciones de fermentación diferentes: una basada en la utilización de glucosa como fuente de carbono y otra basada en la utilización de glicerol como fuente de carbono. En ambos casos se demuestra que las cepas FA1 y FA3 producen fitoeno y fitoflueno junto con una mezcla de carotenoides en la que el fitoeno es el carotenoide mayoritario.The Paracoccus sp . FA1 and Paracoccus sp. FA3 were fermented in a flask in order to determine the production level of phytoen and phytofluene in liquid medium. The analysis of phytoene and phytofluene was determined by reverse phase HPLC liquid chromatography, demonstrating that with both strains it is possible to produce phytoene and phytofluene together with a mixture of carotenoids in which phytoen is the majority carotenoid. Also, the strains Paracoccus sp . FA1 and Paracoccus sp. FA3 were fermented in pre-industrial fermenters in order to determine the production level of phytoene and phytofluene. In this case, two different fermentation conditions developed: one based on the use of glucose as a carbon source and another based on the use of glycerol as a carbon source. In both cases, it is shown that the FA1 and FA3 strains produce phytoene and phyto-glune together with a mixture of carotenoids in which phytoene is the majority carotenoid.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
La extracción y purificación de fitoeno y fitoflueno se realizó mediante la centrifugación del caldo de fermentación para obtener una biomasa húmeda, la cual se extrajo con alcohol isopropílico. El extracto obtenido se concentró y se purificó mediante dos separaciones cromatográficas sucesivas en gel de sílice.The extraction and purification of phytoen and Phytofluene was performed by centrifuging the broth of fermentation to obtain a wet biomass, which was extracted with isopropyl alcohol. The extract obtained was concentrated and purified by two successive gel chromatographic separations of silica
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Tal y como se ha expuesto anteriormente, en la presente invención se han obtenido y seleccionado los mutantes FA1 y FA3 de la cepa de Paracoccus sp., útiles para la obtención de preparaciones estabilizadas de fitoeno y/o fitoflueno de forma simple, efectiva y a gran escala. Los datos relativos al depósito de material biológico son los siguientes:As described above, the mutants FA1 and FA3 of the strain of Paracoccus sp ., Useful for obtaining stabilized preparations of phytoene and / or phyto-fluphane in a simple, effective and large manner have been obtained and selected in the present invention. scale. The data related to the deposit of biological material are the following:
Autoridad Internacional de Depósito: Colección Española de Cultivos Tipo (CECT). International Deposit Authority: Spanish Type Crops Collection (CECT).
Dirección de la Autoridad Internacional de Depósito: Universidad de Valencia; Edificio de investigación; Campus de Burjassot; 46100 Burjassot (Valencia). Management of the International Deposit Authority: University of Valencia; Research building; Burjassot Campus; 46100 Burjassot (Valencia).
Número y fecha de depósito de la cepa Paracoccus sp. FA1: CECT 7383; depositada en la fecha 4 de Marzo de 2008. Number and date of deposit of the strain Paracoccus sp. FA1: CECT 7383; deposited on March 4, 2008.
Número y fecha de depósito de la cepa Paracoccus sp. FA3: CECT 7384; depositada en la fecha 4 de Marzo de 2008. Number and date of deposit of the strain Paracoccus sp. FA3: CECT 7384; deposited on March 4, 2008.
A continuación se disponen los Ejemplos de realización, los cuales tienen el objetivo de ilustrar la presente invención sin limitar la misma.The Examples of embodiment, which are intended to illustrate the present invention without limiting it.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
En primer lugar se desarrolló un procedimiento mutagénico de una cepa de Paracoccus sp. productora de xantofilas, para lo cual se analizaron: (i) diferentes tipos de agentes mutagénicos, (ii) concentración del mutágeno, (iii) concentración de células, (iv) pH de incubación y (v) tiempo de tratamiento. De esta forma se seleccionaron como agentes mutagénicos etilmetanosulfonato (EMS) y N-metil-N'-nitro-N-nitrosoguanidina (NTG).First, a mutagenic procedure of a strain of Paracoccus sp . Producer of xanthophylls, for which we analyzed: (i) different types of mutagenic agents, (ii) concentration of the mutagen, (iii) concentration of cells, (iv) incubation pH and (v) treatment time. Thus, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) were selected as mutagenic agents.
Las suspensiones de células a mutar se obtuvieron a partir de cultivos en un medio líquido, descrito en EP 1229126, cuya composición es la siguiente: corn steep liquor 30,0 g/L, sacarosa 30,0 g/L, KH_{2}PO_{4} 0,54 g/L, K_{2}HPO_{4} 2,78 g/L, MgSO_{4}\cdot7H_{2}O 12,0 g/L, CaCl_{2}\cdot2H_{2}O 0,1 g/L, y FeSO_{4}\cdot7H_{2}O 0,3 g/L, ajustado a un pH final de 7,2 con NaOH. Una vez crecidas las células se lavaron dos veces en solución salina (NaCl 9 g/L) y se ajustó la concentración de células en la suspensión a aproximadamente 10^{11} células/mL.The suspensions of cells to be mutated are obtained from cultures in a liquid medium, described in EP 1229126, whose composition is as follows: corn steep liquor 30.0 g / L, sucrose 30.0 g / L, KH 2 PO 4 0.54 g / L, K 2 HPO 4 2.78 g / L, MgSO 4 • 7 H 2 O 12.0 g / L, CaCl 2 • 2H 2 O 0.1 g / L, and FeSO 4 • 7 H 2 O 0.3 g / L, adjusted to a final pH of 7.2 with NaOH. Once grown the cells were washed twice in saline (9 g / L NaCl) and were adjusted the concentration of cells in the suspension to approximately 10 11 cells / mL.
El procedimiento de mutación con EMS consistió en la incubación de aproximadamente 10^{11} células/mL en una solución de EMS al 3% en tampón fosfato sódico 0,1 M pH 7,0 a temperatura ambiente durante 30 a 180 minutos, consiguiendo tasas de mortalidad de alrededor del 90-99,9%. Las células mutadas se lavaron tres veces con solución salina, centrifugando a 3.000 r.p.m. y 15ºC durante 5 minutos.The mutation procedure with EMS consisted of in the incubation of approximately 10 11 cells / mL in a 3% EMS solution in 0.1 M sodium phosphate buffer pH 7.0 a room temperature for 30 to 180 minutes, getting rates of mortality of around 90-99.9%. The cells mutates were washed three times with saline, centrifuging at 3,000 r.p.m. and 15 ° C for 5 minutes.
El procedimiento de mutación con NTG consistió en la incubación de aproximadamente 10^{11} células/mL en una solución que contenía 250 \mug/mL de NTG y tampón fosfato sódico 0,1 M pH 7,0 a temperatura ambiente durante 90 minutos, consiguiendo tasas de mortalidad de alrededor del 99%. Las células mutadas se lavaron tres veces con solución salina, centrifugando a 3.000 r.p.m. y 15ºC durante 5 minutos.The NTG mutation procedure consisted of in the incubation of approximately 10 11 cells / mL in a solution containing 250 µg / mL of NTG and sodium phosphate buffer 0.1 M pH 7.0 at room temperature for 90 minutes, getting mortality rates of around 99%. The cells mutates were washed three times with saline, centrifuging at 3,000 r.p.m. and 15 ° C for 5 minutes.
Con las células mutadas se sembraron placas Petri que contenían un medio sólido con la siguiente composición por litro: corn steep liquor 30,0 g/L, sacarosa 30,0 g/L, KH_{2}PO_{4} 0,54 g/L, K_{2}HPO_{4} 2,78 g/L, MgSO_{4}\cdot7H_{2}O 12,0 g/L, CaCl_{2}\cdot2H_{2}O 0,1 g/L, FeSO_{4}\cdot7H_{2}O 0,3 g/L, y agar 15 g/L, ajustado a un pH final de 7,2 con NaOH. Las placas sembradas se incubaron a 28ºC durante 4 días para obtener colonias aisladas.Plates were seeded with the mutated cells Petri containing a solid medium with the following composition per liter: corn steep liquor 30.0 g / L, sucrose 30.0 g / L, KH 2 PO 4 0.54 g / L, K 2 HPO 4 2.78 g / L, MgSO 4 • 7H 2 O 12.0 g / L, CaCl 2 • 2 O 0.1 g / L, FeSO4 \ 7H2O 0.3 g / L, and agar 15 g / L, adjusted to a final pH of 7.2 with NaOH. The seeded plates were incubated at 28 ° C for 4 days to obtain isolated colonies.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
La estrategia de selección de cepas de Paracoccus sp. productoras de fitoeno y fitoflueno a partir de una cepa de Paracoccus sp. productora de xantofilas se basó en el color de la colonia. La selección de los citados mutantes se realizó a partir de células mutadas con EMS y NTG tal y como se describe en el Ejemplo 1. Las células mutadas se sembraron en el medio sólido descrito en el Ejemplo 1 y se incubaron a 28ºC durante 5 días. Transcurrido este tiempo se seleccionaron aquellas colonias que poseían color crema en lugar del color naranja típico de la cepa parental Paracoccus sp. productora de xantofilas. De esta forma, a partir de aproximadamente 100.000 colonias analizadas se aislaron 12 colonias con color crema, las cuales podrían ser productoras de fitoeno y fitoflueno. Con la finalidad de determinar los carotenoides producidos por estos mutantes se realizó una extracción de la biomasa obtenida directamente del medio sólido con una mezcla de tetrahidrofurano:metanol:hexano (20:1:40) (v/v) y el extracto se analizó mediante cromatografia líquida HPLC con los siguientes métodos: (I) Para el análisis de \beta-caroteno, licopeno, fitoeno y fitoflueno se empleó una columna Hypersil ODS 5 \mum (100 x 4,6 mm) con una fase móvil de metanol:acetonitrilo:cloroformo (47:47:6) (v/v) a un flujo de 1 mL/min. (II) Para el análisis de xantofilas se empleó una columna nucleosil 100 NH2 5 \mum (250 x 4,6 mm) con una fase móvil de hexano:acetato de etilo (1:1) (v/v) a un flujo de 1 mL/min. Se utilizó un detector de diodoarray para adaptarse a las longitudes de onda de máxima absorción de los distintos carotenoides, las cuales se muestran en la Tabla 1.The strain selection strategy of Paracoccus sp . producers of phytoeno and phytofluene from a strain of Paracoccus sp . Xanthophyll producer was based on the color of the colony. The selection of said mutants was made from cells mutated with EMS and NTG as described in Example 1. The mutated cells were seeded in the solid medium described in Example 1 and incubated at 28 ° C for 5 days. After this time, those colonies that had cream color were selected instead of the typical orange color of the parental strain Paracoccus sp . Xanthophyll producer. In this way, from approximately 100,000 colonies analyzed, 12 cream-colored colonies were isolated, which could be producers of phytoene and phytofluene. In order to determine the carotenoids produced by these mutants, an extraction of the biomass obtained directly from the solid medium was performed with a mixture of tetrahydrofuran: methanol: hexane (20: 1: 40) (v / v) and the extract was analyzed by HPLC liquid chromatography with the following methods: (I) For the analysis of β-carotene, lycopene, phytoene and phytofluene, a Hypersil ODS 5 µm column (100 x 4.6 mm) was used with a mobile phase of methanol: acetonitrile : chloroform (47: 47: 6) (v / v) at a flow of 1 mL / min. (II) For the analysis of xanthophylls a 100 NH2 5 µm (250 x 4.6 mm) nucleosyl column with a mobile phase of hexane: ethyl acetate (1: 1) (v / v) was used at a flow of 1 mL / min A diodearray detector was used to adapt to the maximum absorption wavelengths of the different carotenoids, which are shown in Table 1.
De los 12 mutantes inicialmente aislados se seleccionaron 2, denominados FA1 y FA3, los cuales acumulaban fitoeno y fitoflueno, siendo fitoeno el carotenoide principal. Las Figuras 1 y 2 muestran los cromatogramas HPLC de los carotenoides acumulados por ambas cepas. El nivel de producción de fitoeno y fitoflueno de las cepas FA1 y FA3 se analizó posteriormente en medio líquido tal y como se describe en los Ejemplos 3, 4 y 5.Of the 12 initially isolated mutants, they selected 2, called FA1 and FA3, which accumulated phytoeno and phytofluene, phytoeno being the main carotenoid. The Figures 1 and 2 show the HPLC chromatograms of the carotenoids accumulated by both strains. The phytoen production level and Phytofluene from strains FA1 and FA3 was subsequently analyzed in liquid medium as described in Examples 3, 4 and 5.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Las cepas Paracoccus sp. FA1 y Paracoccus sp. FA3, seleccionadas tal y como se describe en los Ejemplos 1 y 2, se fermentaron en matraz con la finalidad de determinar el nivel de producción de fitoeno y fitoflueno en medio líquido. Para ello, se preparó un medio de inóculo, descrito en EP 1229126, con la siguiente composición por litro: corn steep liquor 30,0 g/L, sacarosa 30,0 g/L, KH_{2}PO_{4} 0,54 g/L, K_{2}HPO_{4} 2,78 g/L, MgSO_{4}\cdot7H_{2}O 12,0 g/L, CaCl_{2}\cdot2H_{2}O 0,1 g/L, y FeSO_{4}\cdot7H_{2}O 0,3 g/L, ajustado a un pH final de 7,2 con NaOH. Cada matraz de inóculo de 250 mL con 50 mL de medio se sembró con 100 \mul del cultivo congelado y se incubó a 28ºC, 250 r.p.m. y 5 cm de excentricidad durante 48 horas.The Paracoccus sp . FA1 and Paracoccus sp . FA3, selected as described in Examples 1 and 2, were fermented in a flask in order to determine the production level of phytoene and phytofluene in liquid medium. For this, an inoculum medium, described in EP 1229126, was prepared with the following composition per liter: corn steep liquor 30.0 g / L, sucrose 30.0 g / L, KH 2 PO 4 0, 54 g / L, K 2 HPO 4 2.78 g / L, MgSO 4 • 12.0 g / L, CaCl 2 • 2H 2 O 0.1 g / L, and FeSO 4 • 7 H 2 O 0.3 g / L, adjusted to a final pH of 7.2 with NaOH. Each 250 mL inoculum flask with 50 mL of medium was seeded with 100 µl of the frozen culture and incubated at 28 ° C, 250 rpm and 5 cm eccentricity for 48 hours.
El medio de fermentación, descrito en EP 1229126, posee la siguiente composición por litro: corn steep liquor 30,0 g/L, glucosa 30,0 g/L, KH_{2}PO_{4} 1,5 g/L, Na_{2}HPO_{4}\cdot12H_{2}O 3,8 g/L, MgSO_{4}\cdot7H_{2}O 3,0 g/L, CaCl_{2}\cdot2H_{2}O 0,2 g/L, y FeSO_{4}\cdot7H_{2}O 1,0 g/L, ajustado a un pH final de 7,2 con NaOH. El medio se repartió en matraces Erlenmeyer de 250 mL a razón de 20 mL por matraz. Los matraces con el medio de fermentación se sembraron con el inóculo anteriormente descrito y se incubaron a 28ºC y 250 r.p.m. Una vez concluida la fermentación (5-6 días), se preparó una mezcla de caldo de fermentación y alcohol isopropíllico (1/1). La concentración y pureza de fitoeno y fitoflueno se determinó mediante el uso de cromatografia líquida HPLC en fase reversa.The fermentation medium, described in EP 1229126, has the following composition per liter: corn steep liquor 30.0 g / L, glucose 30.0 g / L, KH 2 PO 4 1.5 g / L, Na 2 HPO 4 • 12H 2 O 3.8 g / L, MgSO 4 • 7H 2 O 3.0 g / L, CaCl 2 • 2 O 0.2 g / L, and FeSO4 \ 7H2 O 1.0 g / L, adjusted to a final pH of 7.2 with NaOH. The medium was distributed in 250 Erlenmeyer flasks mL at a rate of 20 mL per flask. The flasks with the medium of fermentation were seeded with the inoculum described above and were incubated at 28 ° C and 250 r.p.m. Once the fermentation is finished (5-6 days), a broth mixture of fermentation and isopropyl alcohol (1/1). Concentration and Purity of phytoene and phytofluene was determined by the use of HPLC liquid chromatography in reverse phase.
Los niveles de producción de fitoeno, fitoflueno y otros carotenoides obtenidos con las cepas FA1 y FA3 se muestran en las Tablas 2, 5 y 6. En la Figura 3 se muestra la evolución de la producción de fitoeno y fitoflueno a lo largo de la fermentación en matraz.Production levels of phytoene, phytofluene and other carotenoids obtained with FA1 and FA3 strains are shown in Tables 2, 5 and 6. Figure 3 shows the evolution of the production of phytoen and phytofluene throughout the fermentation in flask
Este Ejemplo demuestra con claridad que con ambas cepas y el procedimiento descrito es posible producir fitoeno y fitoflueno junto con una mezcla de carotenoides en la que fitoeno es el carotenoide mayoritario.This Example clearly demonstrates that with both strains and the described procedure it is possible to produce phytoen and phytofluene together with a mixture of carotenoids in which phytoene It is the majority carotenoid.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Las cepas Paracoccus sp. FA1 y Paracoccus sp. FA3, seleccionadas tal y como se describe en los Ejemplos 1, 2, y 3, se cultivaron en fermentadores pre-industriales con la finalidad de determinar el nivel de producción de fitoeno y fitoflueno. Para ello, se preparó un medio de pre-inóculo con la composición descrita en el Ejemplo 3 para el medio de inóculo. Los pre-inóculos se sembraron en matraces de 500 mL con 100 mL de medio y se incubaron a 28ºC y 250 r.p.m. con 5 cm de excentricidad durante 48 horas. Posteriormente se sembraron inóculos en matraces de 500 mL con 100 mL de medio de inóculo (Ejemplo 3) con el cultivo de pre-inóculo, y se incubaron a 28ºC y 250 r.p.m. con 5 cm de excentricidad durante 30 horas.The Paracoccus sp . FA1 and Paracoccus sp . FA3, selected as described in Examples 1, 2, and 3, were grown in pre-industrial fermenters for the purpose of determining the production level of phytoen and phytofluene. For this, a pre-inoculum medium was prepared with the composition described in Example 3 for the inoculum medium. The pre-inoculums were seeded in 500 mL flasks with 100 mL of medium and incubated at 28 ° C and 250 rpm with 5 cm eccentricity for 48 hours. Inoculants were subsequently seeded in 500 mL flasks with 100 mL of inoculum medium (Example 3) with the pre-inoculum culture, and incubated at 28 ° C and 250 rpm with 5 cm eccentricity for 30 hours.
Con el cultivo de inóculo se sembraron fermentadores de 30 L con 16 litros de un medio de fermentación con la siguiente composición por litro: corn steep liquor 30,0 g/L, glucosa 30,0 g/L, KH_{2}PO_{4} 1,5 g/L, Na_{2}HPO_{4}\cdot12H_{2}O 3,8 g/L, MgSO_{4}\cdot7H_{2}O 3,0 g/L, CaCl_{2}\cdot2H_{2}O 0,2 g/L, FeSO_{4}\cdot7H_{2}O 1,0 g/L, y antiespumante PPG 1 mL/L. El pH se ajustó a 7,2 con amoniaco. La fermentación se incubó durante 96-144 horas a una temperatura de 28ºC con agitación variable entre 150 y 500 r.p.m. y una aireación de 0,25-1 v/v/m.With the inoculum culture they were sown 30 L fermenters with 16 liters of a fermentation medium with The following composition per liter: corn steep liquor 30.0 g / L, glucose 30.0 g / L, KH 2 PO 4 1.5 g / L, Na 2 HPO 4 • 12H 2 O 3.8 g / L, MgSO 4 • 7H 2 O 3.0 g / L, CaCl 2 • 2 O 0.2 g / L, FeSO 4 • 7 H 2 O 1.0 g / L, and PPG antifoam 1 mL / L. The pH was adjusted to 7.2 with ammonia. The fermentation was incubated. for 96-144 hours at a temperature of 28 ° C with variable agitation between 150 and 500 r.p.m. and an aeration of 0.25-1 v / v / m.
Desde las 20 horas hasta las 90 horas se mantuvo una adición de glucosa al 70% para mantener un rango de 10 a 30 g/L de glucosa en el caldo de fermentación.From 20 hours to 90 hours it remained a 70% glucose addition to maintain a range of 10 to 30 g / L of glucose in the fermentation broth.
La valoración de la concentración y pureza de fitoeno y fitoflueno al final de la fermentación se realizó tal y como se describe en el Ejemplo 2. Los niveles de producción de fitoeno, fitoflueno y otros carotenoides se muestran en la Tabla 3.The assessment of the concentration and purity of phytoene and phytofluene at the end of the fermentation was performed as and as described in Example 2. Production levels of phytoeno, phytofluene and other carotenoids are shown in the Table 3.
Este Ejemplo demuestra con claridad que con ambas cepas y el procedimiento descrito es posible producir fitoeno y fitoflueno junto con una mezcla de carotenoides en la que fitoeno es el carotenoide mayoritario.This Example clearly demonstrates that with both strains and the described procedure it is possible to produce phytoen and phytofluene together with a mixture of carotenoids in which phytoene It is the majority carotenoid.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Las cepas Paracoccus sp. FA1 y Paracoccus sp. FA3, seleccionadas tal y como se describe en los Ejemplos 1, 2, y 3, se cultivaron en fermentadores pre-industriales con la finalidad de determinar el nivel de producción de fitoeno y fitoflueno. Para ello, se preparó un medio de pre-inóculo con la composición descrita en el Ejemplo 3 para el medio de inóculo. Los pre-inóculos se sembraron en matraces de 500 mL con 100 mL de medio y se incubaron a 28ºC y 250 r.p.m. con 5 cm de excentricidad durante 48 horas. Posteriormente se sembraron inóculos en matraces de 500 mL con 100 mL de medio de inóculo (Ejemplo 3) con el cultivo de pre-inóculo, y se incubaron a 28ºC y 250 r.p.m. con 5 cm de excentricidad durante 30 horas.The Paracoccus sp . FA1 and Paracoccus sp . FA3, selected as described in Examples 1, 2, and 3, were grown in pre-industrial fermenters for the purpose of determining the production level of phytoen and phytofluene. For this, a pre-inoculum medium was prepared with the composition described in Example 3 for the inoculum medium. The pre-inoculums were seeded in 500 mL flasks with 100 mL of medium and incubated at 28 ° C and 250 rpm with 5 cm eccentricity for 48 hours. Inoculants were subsequently seeded in 500 mL flasks with 100 mL of inoculum medium (Example 3) with the pre-inoculum culture, and incubated at 28 ° C and 250 rpm with 5 cm eccentricity for 30 hours.
Con el cultivo de inóculo se sembraron fermentadores de 30 L con 16 litros de un medio de fermentación con la siguiente composición por litro: corn steep liquor 30 g/L, glicerol 30 g/L, KH_{2}PO_{4} 1,5 g/L, Na_{2}HPO_{4}\cdot12H_{2}O 3,8 g/L, MgSO_{4}\cdot7H_{2}O 3,0 g/L, CaCl_{2}\cdot2H_{2}O 0,2 g/L, FeSO_{4}\cdot7H_{2}O 1,0 g/L, y antiespumante PPG 1 mL/L. El pH se ajustó a 7,2 con amoniaco. La fermentación se incubó durante 96-144 horas a una temperatura de 28ºC con agitación variable entre 150 y 500 r.p.m. y una aireación de 0,25-1 v/v/m.With the inoculum culture they were sown 30 L fermenters with 16 liters of a fermentation medium with The following composition per liter: corn steep liquor 30 g / L, glycerol 30 g / L, KH 2 PO 4 1.5 g / L, Na 2 HPO 4 • 12H 2 O 3.8 g / L, MgSO 4 • 7H 2 O 3.0 g / L, CaCl 2 • 2 O 0.2 g / L, FeSO 4 • 7 H 2 O 1.0 g / L, and PPG antifoam 1 mL / L. The pH was adjusted to 7.2 with ammonia. The fermentation was incubated. for 96-144 hours at a temperature of 28 ° C with variable agitation between 150 and 500 r.p.m. and an aeration of 0.25-1 v / v / m.
Desde las 20 horas hasta las 90 horas se mantuvo una adición de glicerol al 66% para mantener un rango de 10 a 30 g/L de glicerol en el caldo de fermentación.From 20 hours to 90 hours it remained an addition of 66% glycerol to maintain a range of 10 to 30 g / L glycerol in the fermentation broth.
La valoración de la concentración y pureza de fitoeno y fitoflueno al final de la fermentación se realizó tal y como se describe en el Ejemplo 2. Los niveles de producción de fitoeno, fitoflueno y otros carotenoides obtenidos se muestran en la Tabla 4.The assessment of the concentration and purity of phytoene and phytofluene at the end of the fermentation was performed as and as described in Example 2. Production levels of phytoeno, phytofluene and other carotenoids obtained are shown in the Table 4
Este Ejemplo demuestra con claridad que con ambas cepas y el procedimiento descrito es posible producir fitoeno y fitoflueno junto con una mezcla de carotenoides en la que fitoeno es el carotenoide mayoritario.This Example clearly demonstrates that with both strains and the described procedure it is possible to produce phytoen and phytofluene together with a mixture of carotenoids in which phytoene It is the majority carotenoid.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
El caldo de fermentación obtenido tal y como se describe en los Ejemplos 3, 4 y 5 se centrifugó, recuperando la biomasa húmeda, la cual se extrajo con al menos 2 volúmenes por peso de alcohol isopropílico, preferentemente 6 volúmenes por peso. La mezcla se agitó durante 1 hora a temperatura ambiente y seguidamente se filtró a través de un embudo Büchner. El líquido obtenido se concentró bajo vacío a sequedad, obteniendo un extracto que se analizó espectrofotométricamente a 286 nm [Britton G., Liaaen-Jensen S. and Pfander H. (1995) Carotenoids. Volume 1B: Spectroscopy, pag. 60, Birkhauser, Basel] mostrando la presencia de 15-25% fitoeno y 0.5-1% fitoflueno.The fermentation broth obtained as it is described in Examples 3, 4 and 5 was centrifuged, recovering the wet biomass, which was extracted with at least 2 volumes per weight of isopropyl alcohol, preferably 6 volumes by weight. The mixture was stirred for 1 hour at room temperature and It was then filtered through a Büchner funnel. The liquid obtained was concentrated under vacuum to dryness, obtaining an extract which was analyzed spectrophotometrically at 286 nm [Britton G., Liaaen-Jensen S. and Pfander H. (1995) Carotenoids. Volume 1B: Spectroscopy, p. 60, Birkhauser, Basel] showing the presence of 15-25% phytoeno and 0.5-1% phytofluene.
Seguidamente se realizó la primera purificación cromatográfica en gel de sílice utilizando al menos 5 gramos por cada gramo de extracto a purificar, preferentemente 10 gramos por cada gramo. La preparación de la columna se realizó mezclando gel de sílice y diclorometano. El extracto concentrado, obtenido tal y como se describe anteriormente, se disolvió en diclorometano y se cargó en la columna. Las fracciones eluidas tras el proceso cromatográfico se analizaron espectrofotométricamente y aquellas que presentaban el espectro UV/visible coincidente con la presencia de fitoeno y fitoflueno se evaporaron a sequedad. El producto pre-purificado obtenido se analizó espectrofotométricamente mostrando la presencia de 70-80% fitoeno y 1.0-2.0% fitoflueno. El citado producto pre-purificado debe ser almacenado a -20ºC en atmósfera inerte para evitar degradación química.Then the first purification was performed chromatographic silica gel using at least 5 grams per each gram of extract to be purified, preferably 10 grams per each gram The column preparation was done by mixing gel silica and dichloromethane. The concentrated extract, obtained as and as described above, it was dissolved in dichloromethane and loaded in the column. Fractions eluted after the process chromatography were analyzed spectrophotometrically and those that presented the UV / visible spectrum coinciding with the presence of phytoene and phytofluene evaporated to dryness. The product pre-purified obtained was analyzed spectrophotometrically showing the presence of 70-80% phytoeno and 1.0-2.0% phytofluene The aforementioned pre-purified product must be stored at -20ºC in an inert atmosphere to avoid degradation chemistry.
Por último se realizó la segunda purificación cromatográfica en gel de sílice utilizando al menos 10 gramos de gel de sílice por cada gramo de producto pre-purificado, preferentemente 20 gramos por cada gramo. La preparación de la columna se realizó mezclando gel de sílice y ciclohexano o n-hexano. El producto pre-purificado, obtenido tal y como se describe anteriormente, se disolvió en ciclohexano o n-hexano y se cargó en la columna. Al final de la cromatografía es necesario incrementar la polaridad adicionando etil acetato, eluyendo con mezclas de polaridad creciente ciclohexano o n-hexano:etil acetato 98:2, 97:3, ó 96:4.Finally the second purification was performed chromatographic silica gel using at least 10 grams of silica gel per gram of product pre-purified, preferably 20 grams per gram. The column preparation was done by mixing gel silica and cyclohexane or n-hexane. The product pre-purified, obtained as described previously, it was dissolved in cyclohexane or n-hexane and loaded in the column. At the end of chromatography it is necessary increase polarity by adding ethyl acetate, eluting with mixtures of increasing polarity cyclohexane or n-hexane: ethyl acetate 98: 2, 97: 3, or 96: 4.
Las fracciones eluidas tras el proceso cromatográfico se analizaron espectrofotométricamente y aquellas que presentaban el espectro UV/visible coincidente con la presencia de fitoeno y fitoflueno se evaporaron a sequedad. El producto obtenido se analizó espectrofotométricamente mostrando la presencia de 80-90% fitoeno y 0.5-1.5% fitoflueno.Fractions eluted after the process chromatography were analyzed spectrophotometrically and those that presented the UV / visible spectrum coinciding with the presence of phytoene and phytofluene evaporated to dryness. The product obtained was analyzed spectrophotometrically showing the presence 80-90% phytoeno and 0.5-1.5% phytofluene
\newpage\ newpage
Esquema 1Scheme one
Ruta biosintética de carotenoides y xantofilasBiosynthetic Route of carotenoids and xanthophylls
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Claims (16)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES200800817A ES2330602B1 (en) | 2008-03-19 | 2008-03-19 | METHOD OF PRODUCTION OF PHYTOENE AND / OR PHYTOFLUENE, OR CAROTENOID MIXTURES WITH HIGH CONTENT IN THE SAME |
PCT/ES2009/070025 WO2009115629A1 (en) | 2008-03-19 | 2009-02-13 | Method for the production of phytoene and/or phytofluene or mixtures of carotenoids having a high content of same |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES200800817A ES2330602B1 (en) | 2008-03-19 | 2008-03-19 | METHOD OF PRODUCTION OF PHYTOENE AND / OR PHYTOFLUENE, OR CAROTENOID MIXTURES WITH HIGH CONTENT IN THE SAME |
Publications (2)
Publication Number | Publication Date |
---|---|
ES2330602A1 true ES2330602A1 (en) | 2009-12-11 |
ES2330602B1 ES2330602B1 (en) | 2010-09-30 |
Family
ID=41090523
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ES200800817A Active ES2330602B1 (en) | 2008-03-19 | 2008-03-19 | METHOD OF PRODUCTION OF PHYTOENE AND / OR PHYTOFLUENE, OR CAROTENOID MIXTURES WITH HIGH CONTENT IN THE SAME |
Country Status (2)
Country | Link |
---|---|
ES (1) | ES2330602B1 (en) |
WO (1) | WO2009115629A1 (en) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0635576A1 (en) * | 1993-07-22 | 1995-01-25 | NIPPON OIL Co. Ltd. | Bacteria belonging to new genus and process for production of carotenoids using same |
WO1999006586A1 (en) * | 1997-07-29 | 1999-02-11 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Novel carotenoid-producing bacterial species and process for production of carotenoids using same |
EP1229126A1 (en) * | 2000-06-12 | 2002-08-07 | Nippon Mitsubishi Oil Corporation | Process for producing carotenoid pigments |
WO2002099095A2 (en) * | 2001-06-06 | 2002-12-12 | Roche Vitamins Ag | Improved isoprenoid production |
JP2003180387A (en) * | 2001-12-19 | 2003-07-02 | Nikken Sohonsha Corp | Method for producing carotenoid |
US20060053513A1 (en) * | 2003-01-09 | 2006-03-09 | Sabine Steiger | Method for producing ketocarotenoids by cultivating genetically modified organisms |
EP1676925A1 (en) * | 2003-09-17 | 2006-07-05 | Nippon Oil Corporation | Process for producing carotenoid compound |
-
2008
- 2008-03-19 ES ES200800817A patent/ES2330602B1/en active Active
-
2009
- 2009-02-13 WO PCT/ES2009/070025 patent/WO2009115629A1/en active Application Filing
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0635576A1 (en) * | 1993-07-22 | 1995-01-25 | NIPPON OIL Co. Ltd. | Bacteria belonging to new genus and process for production of carotenoids using same |
WO1999006586A1 (en) * | 1997-07-29 | 1999-02-11 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Novel carotenoid-producing bacterial species and process for production of carotenoids using same |
EP1229126A1 (en) * | 2000-06-12 | 2002-08-07 | Nippon Mitsubishi Oil Corporation | Process for producing carotenoid pigments |
WO2002099095A2 (en) * | 2001-06-06 | 2002-12-12 | Roche Vitamins Ag | Improved isoprenoid production |
JP2003180387A (en) * | 2001-12-19 | 2003-07-02 | Nikken Sohonsha Corp | Method for producing carotenoid |
US20060053513A1 (en) * | 2003-01-09 | 2006-03-09 | Sabine Steiger | Method for producing ketocarotenoids by cultivating genetically modified organisms |
EP1676925A1 (en) * | 2003-09-17 | 2006-07-05 | Nippon Oil Corporation | Process for producing carotenoid compound |
Non-Patent Citations (1)
Title |
---|
BASE DE DATOS WPI [online], Thomson Corp., Philadelphia, USA, [recuperado el 12.05.2009]. Recuperado de WPI en EPOQUENET, (EPO), DW 200372, Nº DE ACCESO 2003-759458 & JP 2003180387 A (NIKKEN SOHONSHA KK) 02.07.2003, (resumen) * |
Also Published As
Publication number | Publication date |
---|---|
ES2330602B1 (en) | 2010-09-30 |
WO2009115629A1 (en) | 2009-09-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Schmidt-Dannert | Engineering novel carotenoids in microorganisms | |
CN103857786B (en) | The method of retinoid is manufactured from microorganism | |
KR0178871B1 (en) | Keto group introducing enzyme dna coding for the same and process for producing ketocarotenoid | |
US9963731B2 (en) | Method for producing carotenoids each having 50 carbon atoms | |
NO313204B1 (en) | Bacteria belonging to a new genus and process for the preparation of carotenoids using them | |
Ye et al. | Defining the biosynthesis of ketocarotenoids in Chromochloris zofingiensis | |
Albrecht et al. | Synthesis of atypical cyclic and acyclic hydroxy carotenoids in Escherichia coli transformants | |
US7745170B2 (en) | Process for producing carotenoid compound | |
Misawa et al. | Carotenoids: Carotenoid and apocarotenoid analysis—Use of E. coli to produce carotenoid standards | |
EP1367131B1 (en) | Method of producing beta-carotene by means of mixed culture fermentation using (+) and (-) strains of blakeslea trispora | |
JPH0779796A (en) | Production of carotenoid pigment | |
ES2330602B1 (en) | METHOD OF PRODUCTION OF PHYTOENE AND / OR PHYTOFLUENE, OR CAROTENOID MIXTURES WITH HIGH CONTENT IN THE SAME | |
US20220340949A1 (en) | Methods of Isoprenoid Synthesis Using a Genetically Engineered Hydrocarbonoclastic Organism in a Biofilm Bioreactor | |
Matselyukh et al. | Isolation of Streptomyces globisporus and Blakeslea trispora mutants with increased carotenoid content | |
JP3429563B2 (en) | New microorganism | |
Shindo et al. | 4-Ketoantheraxanthin, a novel carotenoid produced by the combination of the bacterial enzyme β-carotene ketolase CrtW and endogenous carotenoid biosynthetic enzymes in higher plants | |
Lee et al. | Microbial Production of Zeaxanthin | |
Rezanka et al. | Yeast need not be used only in beer production. I. The carotenoid astaxanthin produced by yeast. | |
KR20240121366A (en) | Method for production of carotenoid derived from microorganisms | |
Sandmann | Production of carotenoids by gene combination in Escherichia coli | |
Chen et al. | De Novo Biosynthesis of Glycosylated Carotenoids in Escherichia Coli | |
Mochimaru¹ et al. | CHAPTER TWENTY THREE | |
Meléndez Granados | Carotenoids in bacteria: general scheme on the diversity of the biosynthetic pathways | |
Roth | Comparison of the methylerythritol phosphate pathway and the isopentenol utilization pathway for astaxanthin production in Escherichia coli | |
Breierová et al. | 25 Biotechnological Production and Properties of Carotenoid Pigments |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EC2A | Search report published |
Date of ref document: 20091211 Kind code of ref document: A1 |
|
FG2A | Definitive protection |
Ref document number: 2330602B1 Country of ref document: ES |
|
PC2A | Transfer of patent |
Owner name: DSM IP ASSETS B.V. Effective date: 20130430 |