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WO2009115629A1 - Procédé de production de phytoène et/ou de phytofluène, ou de mélanges de caroténoïdes à haute teneur en ceux-ci - Google Patents

Procédé de production de phytoène et/ou de phytofluène, ou de mélanges de caroténoïdes à haute teneur en ceux-ci Download PDF

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Publication number
WO2009115629A1
WO2009115629A1 PCT/ES2009/070025 ES2009070025W WO2009115629A1 WO 2009115629 A1 WO2009115629 A1 WO 2009115629A1 ES 2009070025 W ES2009070025 W ES 2009070025W WO 2009115629 A1 WO2009115629 A1 WO 2009115629A1
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Prior art keywords
phytofluene
paracoccus
phytoene
fal
carotenoids
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Spanish (es)
Inventor
Juan Luis De La Fuente Moreno
Marta RODRIGUEZ SÁIZ
Javier Costa Pérez
Antonio Estrella De Castro
José Luis BARREDO FUENTE
Juan Francisco López Ortiz
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Vitatene SA
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Vitatene SA
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    • 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

Definitions

  • the present invention describes a logical biotech 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 super producers Therefore, the present invention is capable of being applied in various fields of life sciences, such as in the food, pharmaceutical or cosmetic fields.
  • Carotenoids are pigments of an isoprenoid nature synthesized by certain bacteria, fungi and photosynthetic organisms. They can be classified into two types:
  • Carotenes they are pure hydrocarbons, among which are compounds such as ⁇ -carotene, ⁇ -carotene, ⁇ -carotene, lycopene, phytoene or phytofluene; Y
  • Xanto rows they are molecules that contain oxygen in different forms (hydroxy, epoxy groups, etc.), among which are compounds such as astaxanthin, zeaxanthin, capsantin, canthaxanthin, lutein, etc.
  • Biosynthesis begins with the isomerization of isopentenyl pyrophosphate (IPP, C 5 ) to dimethylalkyl pyrophosphate (DMAPP, C 5 ), catalyzed by the enzyme IPP isomerase.
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylalkyl pyrophosphate
  • a DMAPP molecule is condensed with an IPP molecule to generate geranyl pyrophosphate (GPP, Cio), which is condensed again with an IPP molecule to generate farnesyl pyrophosphate (FPP, Ci 5 ); both reactions are catalyzed by the enzyme FPP synthase.
  • GPP geranyl pyrophosphate
  • FPP farnesyl pyrophosphate
  • 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).
  • GGPP synthase encoded by the crtE gene
  • the condensation of two GGPP molecules catalyzed by the enzyme phytoeno synthase generates phytoen (C 40 ), a precursor molecule of the biosynthetic pathway of carotenoids.
  • the enzyme phytoene desaturase (encoded by the crtl gene) introduces four double bonds in the phytoen molecule to synthesize phytofluene, ⁇ -carotene, neurosporene and lycopene (C 40 ) consecutively.
  • the lycopene cyclase enzyme (encoded by the crtY gene) is responsible for converting the acyclic ends of the lycopene molecule into ⁇ rings to sequentially form ⁇ -carotene and ⁇ -carotene (C 40 ).
  • the present invention describes a logical biotech method, hereinafter the method of the invention, which makes it possible to obtain, in a simple, effective and large-scale manner, stabilized preparations of phytoene and / or phytophane, or mixtures of carotenoids with high content thereof, by using of new mutated strains of Paracoccus sp. (FAl and FA3) superproducers of phytoen and / or phytofluene.
  • the technical problem solved by the present invention is to obtain, in a simple, effective and large-scale manner, stabilized preparations of phytoene and phytofluene, or mixtures of carotenoids with a high phytoene and phytofluene content, which can be applied directly in the food fields , pharmaceutical and cosmetic.
  • the method of the invention allows simple, effective and large-scale stabilized preparations of phytoene and / or phytofluene, or mixtures of carotenoids with high content thereof, to be obtained by using mutated strains of Paracoccus sp. superproducers of phytoen and / or phytofluene (FAl and FA3).
  • the method of the invention comprises:
  • the mutated cells were washed three times with saline.
  • the method of mutation with NTG consisted of incubating about November 10 cells / mL in a solution containing 250 g / mL of NTG and 0.1 M sodium phosphate buffer pH 7.0 at room temperature environment for 90 minutes, achieving mortality rates of around 99%.
  • the mutated cells were washed three times with saline.
  • the mutated cells were seeded Petri dishes with solid medium and incubated at 28 0 C for 4 days to obtain isolated colonies.
  • 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. were sown in Petri dishes and once grown at 28 0 C, those colonies that possessed cream were selected (the parent strain has orange). After analyzing about 100,000 colonies, 2 cream-colored colonies called FAl and FA3 producing phytoene and phytophenol were isolated.
  • the Paracoccus sp. FAl 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.
  • the strains Paracoccus sp. FAl and Paracoccus sp. FA3 were fermented in pre-industrial fermenters in order to determine the production level of phytoene and phytofluene.
  • the extraction and purification of phytoene and phytofluene was performed by centrifuging the fermentation broth to obtain a wet biomass, which was extracted with isopropyl alcohol.
  • the extract obtained was concentrated and purified by two successive chromatographic separations on silica gel.
  • the FAl and FA3 mutants of the Paracoccus sp. Strain, 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:
  • Example 1 Obtaining mutants of Paracoccus sp.
  • a mutagenic procedure of a strain of Paracoccus sp. Xanthophyll producer 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.
  • EMS ethyl methanesulfonate
  • NTG N-methyl-N'-nitro-N-nitrosoguanidine
  • the suspensions of cells to be mutated were 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 VH 2 O 12.0 g / L, CaCl 2 -2H 2 O 0.1 g / L, and FeSO 4 -7H 2 O 0.3 g / L, adjusted to a final pH of 7.2 with NaOH.
  • saline solution NaCl 9 g / L
  • the EMS mutation procedure consisted of the incubation of approximately 10 11 cells / mL in a 3% EMS solution in 0.1 M sodium phosphate buffer pH 7.0 at room temperature for 30 to 180 minutes, achieving mortality rates of about 90-99.9%.
  • the mutated cells were washed three times with saline, and centrifuged at 3000 rpm and 15 0 C for 5 minutes.
  • the NTG mutation procedure consisted of the incubation of approximately 10 11 cells / mL in a solution containing 250 ⁇ g / mL of NTG and 0.1 M sodium phosphate buffer pH 7.0 at room temperature for 90 minutes, achieving rates of mortality of about 99%.
  • the mutated cells were washed three times with saline, and centrifuged at 3000 rpm and 15 0 C for 5 minutes.
  • Petri dishes containing a solid medium with the following composition per liter were seeded: 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 -2H 2 O 0.1 g / L, FeSO 4 -7H 2 O 0.3 g / L, and 15 g / L agar, adjusted to a final pH of 7.2 with NaOH.
  • the seeded plates were incubated at 28 0 C for 4 days to obtain isolated colonies.
  • Example 2 Selection of mutants of Paracoccus sp. producers of phytoeno and ⁇ toflueno from a strain of Paracoccus sp. xanthophyll producer
  • 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 mutants was performed cited from mutated with EMS and NTG cells as described in Example 1.
  • the mutated cells were plated on solid medium described in Example 1 and incubated at 28 0 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.
  • 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 VH 2 O 12.0 g / L, CaCl 2 -2H 2 O 0.1 g / L, and FeSO 4 VH 2 O 0.3 g / L, adjusted to a final pH of 7.2 with NaOH.
  • Each flask inoculum of 250 mL with 50 mL of medium was seeded with 100 .mu.l of frozen culture and incubated at 28 0 C, 250 rpm and 5 cm eccentricity for 48 hours.
  • 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 VH 2 O 3.0 g / L, CaCl 2 -2H 2 O 0.2 g / L, and FeSO 4 VH 2 O 1.0 g / L, adjusted at a final pH of 7.2 with NaOH.
  • the medium was distributed in 250 mL Erlenmeyer flasks at a rate of 20 mL per flask.
  • Flasks with fermentation medium were seeded with inoculum described above and incubated at 28 0 C and 250 rpm After completion of the fermentation (5-6 days), a mixture of fermentation broth and isoprop ⁇ llico alcohol (1 / was prepared one). The concentration and purity of phytoene and phytofluene was determined by the use of reverse phase HPLC liquid chromatography.
  • Example 4 Procedure for the production of phytoeno and phytofluene by fermentation in a fermentor tank of the Paracoccus sp. FAl and Paracoccus sp. FA3 using a specific glucose addition program
  • the Paracoccus sp. FAl 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.
  • 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 0 C and 250 rpm with 5 cm eccentricity for 48 hours.
  • inocula were seeded in 500 mL flasks with 100 mL of inoculation medium (Example 3) with the pre-inoculum culture and incubated at 28 0 C and 250 rpm with 5 cm eccentricity for 30 hours.
  • Example 5 Procedure for the production of phytoene and phytofluene by fermentation in fermentation tank of the Paracoccus sp. FAl and Paracoccus sp. FA3 using a specific glycerol addition program
  • the Paracoccus sp. FAl 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.
  • 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 medium and incubated at 28 0 C and 250 rpm with 5 cm eccentricity for 48 hours.
  • inocula were seeded in 500 mL flasks with 100 mL of inoculation medium (Example 3) with the pre-inoculum culture and incubated at 28 0 C and 250 rpm with 5 cm eccentricity for 30 hours.
  • Example 2 The titration of the concentration and purity of phytoene and phytofluene at the end of the fermentation was performed as described in Example 2.
  • the production levels of phytoene, phytofluene and other carotenoids obtained are shown in Table 4. This Example clearly demonstrates that with both strains and the described procedure it is possible to produce phytoene and phytofluene together with a mixture of carotenoids in which phytoene is the majority carotenoid.
  • Example 6 Procedure for recovery and purification of phytoen and phytofluene from the fermentation broths of Paracoccus sp. FAl and Paracoccus sp. FA3
  • the fermentation broth obtained as described in Examples 3, 4 and 5 was centrifuged, recovering the wet biomass, which was extracted with at least 2 volumes by weight of isopropyl alcohol, preferably 6 volumes by weight.
  • the mixture was stirred for 1 hour at room temperature and then filtered through a Büchner funnel.
  • the liquid obtained was concentrated under vacuum to dryness, obtaining an extract that was analyzed spectrophotometrically at 286 nm [Britton G., Liaaen-Jensen S. and Pfander H. (1995) Carotenoids. Volume IB: Spectroscopy, p. 60, Birkhauser, Basel] showing the presence of 15-25% phytoene and 0.5-1% phytofluene.
  • the first chromatographic purification was carried out on silica gel using at least 5 grams per gram of extract to be purified, preferably 10 grams per gram.
  • the column preparation was done by mixing silica gel and dichloromethane.
  • the concentrated extract, obtained as described above, was dissolved in dichloromethane and loaded on the column.
  • the fractions eluted after the graphical chromate process were analyzed spectrophotometrically and those with the UV / visible spectrum coinciding with the presence of phytoene and phytofluene evaporated to dryness.
  • the pre-purified product obtained was analyzed spectrophotometrically showing the presence of 70-80% phytoene and 1.0-2.0% phytofluene.
  • Said pre-purified product must be stored at -2O 0 C in an inert atmosphere to avoid chemical degradation.
  • the second chromatographic purification was performed on silica gel using at least 10 grams of silica gel per gram of pre-purified product, preferably 20 grams per gram.
  • the column preparation was performed mixing silica gel and hexane or n-hexane cycle.
  • the pre-purified product, obtained as described above, was dissolved in cyclohexane or n-hexane and loaded onto the column.
  • it is necessary to increase the polarity by adding ethyl acetate, eluting with mixtures of increasing polarity cyclohexane or n-hexane: ethyl acetate 98: 2, 97: 3, or 96: 4.
  • the fractions eluted after the chromatographic process 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 of 80-90% phytoene and 0.5-1.5% phytofluene.
  • Table 3 Production of phytoene, phytofluene and other carotenoids of the FAl and FA3 strains in a 30 L fermenter with added glucose at 96 hours of fermentation. The production values of each carotenoid are expressed as a percentage of the total carotenoids produced.
  • Table 5 Values (mg / L) of phytoene and phytofluene production obtained by the FAl strain in flask. These values are represented in Figure 3A.
  • Table 7 Values (mg / L) of phytoene and phytofluene production obtained by the FAl strain in a fermentation tank with glucose. These values are represented in Figure 4A.
  • Table 8 Values (mg / L) of phytoene and phytofluene production obtained by the FA3 strain in glucose fermentor tank. These values are represented in Figure 4B.

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Abstract

L'invention concerne un procédé de production de phytoène et/ou de phytofluène, ou de mélanges de caroténoïdes à haute teneur en ceux-ci. La présente invention concerne un procédé biotechnologique permettant d'obtenir, de manière simple et efficace et en grande quantité, des préparations stabilisées de phytoène et/ou de phytofluène, ou des mélanges de caroténoïdes à haute teneur en ceux-ci, par utilisation de nouvelles souches mutées de Paracoccus sp. (FA1 et FA3) hyperproductrices de phytoène et/ou de phytofluène.
PCT/ES2009/070025 2008-03-19 2009-02-13 Procédé de production de phytoène et/ou de phytofluène, ou de mélanges de caroténoïdes à haute teneur en ceux-ci Ceased WO2009115629A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200800817 2008-03-19
ES200800817A ES2330602B1 (es) 2008-03-19 2008-03-19 Metodo de produccion de fitoeno y/o fitoflueno, o mezclas de carotenoides con alto contenido en los mismos.

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WO2009115629A1 true WO2009115629A1 (fr) 2009-09-24

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0635576A1 (fr) * 1993-07-22 1995-01-25 NIPPON OIL Co. Ltd. Bactéries de genre nouveau et procédé de production de caroténoides par utilisation de ces bactéries
WO1999006586A1 (fr) * 1997-07-29 1999-02-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Nouvelles especes bacteriennes produisant des carotenoides et procede de production de carotenoides au moyen de ces nouvelles especes
EP1229126A1 (fr) * 2000-06-12 2002-08-07 Nippon Mitsubishi Oil Corporation Procede pour produire des pigments carotenoides
WO2002099095A2 (fr) * 2001-06-06 2002-12-12 Roche Vitamins Ag Production amelioree d'isoprenoides
JP2003180387A (ja) * 2001-12-19 2003-07-02 Nikken Sohonsha Corp カロチノイドの製造方法
US20060053513A1 (en) * 2003-01-09 2006-03-09 Sabine Steiger Method for producing ketocarotenoids by cultivating genetically modified organisms
EP1676925A1 (fr) * 2003-09-17 2006-07-05 Nippon Oil Corporation Procede de production d'un compose carotenoide

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0635576A1 (fr) * 1993-07-22 1995-01-25 NIPPON OIL Co. Ltd. Bactéries de genre nouveau et procédé de production de caroténoides par utilisation de ces bactéries
WO1999006586A1 (fr) * 1997-07-29 1999-02-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Nouvelles especes bacteriennes produisant des carotenoides et procede de production de carotenoides au moyen de ces nouvelles especes
EP1229126A1 (fr) * 2000-06-12 2002-08-07 Nippon Mitsubishi Oil Corporation Procede pour produire des pigments carotenoides
WO2002099095A2 (fr) * 2001-06-06 2002-12-12 Roche Vitamins Ag Production amelioree d'isoprenoides
JP2003180387A (ja) * 2001-12-19 2003-07-02 Nikken Sohonsha Corp カロチノイドの製造方法
US20060053513A1 (en) * 2003-01-09 2006-03-09 Sabine Steiger Method for producing ketocarotenoids by cultivating genetically modified organisms
EP1676925A1 (fr) * 2003-09-17 2006-07-05 Nippon Oil Corporation Procede de production d'un compose carotenoide

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ES2330602A1 (es) 2009-12-11

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