WO2023214072A2 - Nouveau procédé - Google Patents

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Publication number
WO2023214072A2
WO2023214072A2 PCT/EP2023/062052 EP2023062052W WO2023214072A2 WO 2023214072 A2 WO2023214072 A2 WO 2023214072A2 EP 2023062052 W EP2023062052 W EP 2023062052W WO 2023214072 A2 WO2023214072 A2 WO 2023214072A2
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solvent
process according
vitamin
fermentation
isopar
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WO2023214072A3 (fr
Inventor
Valmik Kanubhai VYAS
Ethan LAM
Peter Louis HOUSTON
Reed Chadbourne DOTEN
Nathan Charles TICHOVOLSKY
Michael Benjamin Johnson
James Dittmar
Qiang Yan
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DSM IP Assets BV
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DSM IP Assets BV
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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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • 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
    • C12P33/00—Preparation of steroids
    • 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
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/04—Solvent extraction of solutions which are liquid
    • B01D11/0492—Applications, solvents used

Definitions

  • the present invention is related to fermentative production of isoprenoids, lipids, and sterols, particularly various forms of vitamin E and vitamin D3, as well as various polyunsaturated fatty acids (PUFAs), including derivatives or intermediates, comprising cultivation of a suitable host cell, such as fungal host cell, particularly oleaginous host cell, in an improved two-phase culture system.
  • a suitable host cell such as fungal host cell, particularly oleaginous host cell
  • the so-called two- phase cultivation systems have been developed, wherein the fermentation products are collected outside the cell in the so-called second phase comprising lipophilic solvents such as e.g. Drakeol®, silicone oil or n-dodecane (see W02020/141168 or Jang et al., Microbial Cell Factories 10:59, 2011).
  • lipophilic solvents such as e.g. Drakeol®, silicone oil or n-dodecane (see W02020/141168 or Jang et al., Microbial Cell Factories 10:59, 2011).
  • the yield as well as impurity profile with the known solvents is not satisfactory and/or the fermentation products are stored in lipid bodies that are not accessible by the second phase solvents.
  • the present invention is directed to a two-phase culture system including an in vitro extraction system for fermentative production of isoprenoids, lipids, and sterols, particularly various forms of vitamin E and vitamin D3, as well as various polyunsaturated fatty acids (PUFAs), including derivatives thereof, wherein a suitable host cell, particularly fungal host cell, such as e.g. an oleaginous yeast, e.g. Yarrowia or Saccharomyces, is grown on a suitable carbon source and in the presence of a lipophilic solvent as defined herein.
  • a suitable host cell particularly fungal host cell, such as e.g. an oleaginous yeast, e.g. Yarrowia or Saccharomyces, is grown on a suitable carbon source and in the presence of a lipophilic solvent as defined herein.
  • the present invention allows for increased productivity of intracellular lipids, wherein the second phase allows for in-situ extraction of certain lipids as defined herein, particularly PUFAs, more particularly omega-3 fatty acids, which thereby allows for more PUFAs to be made intracellularly.
  • PUFAs lipids as defined herein, particularly PUFAs, more particularly omega-3 fatty acids
  • lipophilic solvents i.e. second phase solvents to be used for the present invention
  • second phase solvents might be selected from lipophilic solvents comprising mixtures of n-alkanes, isoalkanes, hydrocarbons.
  • the solvents might be natural or synthetic ones.
  • commercially available useful solvents might be selected from Exxon Mobil, as e.g. commercially available under the tradenames Exxsol D60, D80, D95 or DT10. It is understood that useful solvents include the above listed commercially available solvents as well as the respective solvents with the same or equivalent properties but known/available from other suppliers.
  • a solvent has equivalent or identical properties as Exxsol D60, D80, D95, D110.
  • Exxsol Ds are narrow boiling distillation cuts from cracked hydrocarbons that have been reduced by catalytic hydrogenation to remove aromatics and unsaturation.
  • a suitable sterol that is produced in the two-phase culture system according to the present invention might be selected from vitamin D3, including but not limited to intermediates and derivatives thereof, e.g. 7-DHC, 25- hydroxycholecalciferol (HyD), 25-7-DHC (HyDHC), cholesta-5,7,24(25)-trienol, Zymosterol, lanosterol, lathosterol, cholesta-5,8,24(25)-trienol, i.e. fermentation products, that can pass through the cell wall of the host cell to be collected outside the cell in the second phase solvent as defined herein.
  • intermediates and derivatives thereof e.g. 7-DHC, 25- hydroxycholecalciferol (HyD), 25-7-DHC (HyDHC), cholesta-5,7,24(25)-trienol, Zymosterol, lanosterol, lathosterol, cholesta-5,8,24(25)
  • Using the two-phase culture system comprising a lipophilic solvent as defined herein results in a mix of sterols comprising 7-DHC, zymosterol, trienols and/or hydroxylated forms such as e.g. HyDHC, wherein the percentage of HyDHC based on total sterols in the mix is in the range of at least about 65%, such as e.g. 70, 75, 80, 85, 90, 95 or more HyDHC based on total sterols in the mix.
  • Suitable (n-3 and n-6) PUFAs include but are not limited to docosahexaenoic acid (DHA, 22:6 omega-3), suitably from algae or fungi, such as the (dinoflagellate) Crypthecodinium or the (fungus) Thraustochytrium; y-linolenic acid (GLA, 18:3 omega-6); alpha-linolenic acid (ALA, 18:3 omega-3); conjugated linoleic acid (octadecadienoic acid, CLA); dihomo-gamma-linolenic acid (DGLA, 20:3 omega-6); arachidonic acid (ARA, 20:4 omega-6); and eicosapentaenoic acid (EPA, 20:5 omega-3).
  • DHA docosahexaenoic acid
  • fungi such as the (dinoflagellate) Crypthecodinium or the (fungus) Thraustochytrium
  • Preferred PUFAs include arachidonic acid (ARA), docosohexaenoic acid (DHA), eicosapentaenoic acid (EPA) and/or gamma-linolenic acid (GLA).
  • ARA arachidonic acid
  • DHA docosohexaenoic acid
  • EPA eicosapentaenoic acid
  • GLA gamma-linolenic acid
  • ARA is preferred.
  • the PUFA may be produced by the cells pasteurised in the process of the invention, such as a microbial cell.
  • This may be a bacteria, algae, fungus or yeast cell.
  • Fungi are preferred, preferably selected from Mucorales, such as e.g., Mortierella, Phycomyces, Blakeslea, Aspergillus, Thraustochytrium, Pythium or Entomophthora.
  • the preferred source of ARA is from Mortierella alpina, Blakeslea trispora, Aspergillus terreus or Pythium insidiosum.
  • Algae can be dinoflagellate and/or include Porphyridium, Nitszchia, or Crypthecodinium (e.g.
  • the PUFAs are in the form of triglycerides, such as e.g. at least about 50%, 60, 70% of the PUFA is in triglyceride form.
  • the percentage of triglycerides may be higher, such as at least about 85%, preferably at least about 90%, 93%, 95% of the oil.
  • at least about 40% such as at least about 50% to 60% of the PUFA is present at the a-position of the glycerol (present in the triglyceride backbone), also known at the 1 or 3 position. It is preferred that at least about 20%, such as at least about 30 to 40% 40% of the PUFA is at the b(2) position.
  • the present invention is related to a process using a two-phase cultivation system for fermentative production of isoprenoids, lipids, and sterols, particularly various forms of vitamin E and vitamin D3, as well as various polyunsaturated fatty acids (PUFAs), including derivatives or intermediates thereof, wherein a lipophilic solvent is used as a second phase and as defined herein, wherein a suitable host cell, particularly fungal host cell, such as e.g. an oleaginous yeast, e.g. Yarrowia or Saccharomyces, is grown on a suitable carbon source and in the presence of said lipophilic solvent that is not disappearing during the fermentation.
  • a suitable host cell particularly fungal host cell, such as e.g. an oleaginous yeast, e.g. Yarrowia or Saccharomyces
  • the two-phase culture system as defined herein including the use of the solvents as defined herein results in a disappearance of solvents of about 20% or less, i.e., wherein at least about 80%, such as e.g. 85, 90, 92, 95, 97, 98, 99, 100% of solvent present at the start of the fermentation is still present at the end of the fermentation process.
  • Particularly useful solvents that are still present at the end of the fermentation might be selected from solvents identical or equivalent to isopars, more particularly Isopar M.
  • the present invention is related to a process for reducing or abolishing the consumption of the second phase solvent, i.e., consumption of the second phase solvent by the host cell, as compared to consumption using n- dodecane as second phase.
  • the consumption of the second phase might be reduced by at least about 50%, such as e.g. 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 100% compared to consumption using n-dodecane as second phase solvent via a process using a lipophilic solvent as described herein.
  • the lipophilic solvent is selected from solvents known under the tradename Isopar M, Isopar N, Isopar H, Isopar K, Isopar L, Exxsol D110 or solvents with equivalent or identical properties but from other suppliers.
  • a stable solvent means that the evaporation and/or consumption is reduced by at least about 50% compared to known solvents such as e.g. n-dodecane, silicone oil or hexadecane.
  • solvents such as e.g. n-dodecane, silicone oil or hexadecane.
  • non-stable solvents are n-dodecane, silicone oil or hexadecane.
  • isoparaffins according to the present invention by the host organism, such as e.g. Saccharomyces or Yarrowia, is detection of oxygen consumption (measured by a dissolved oxygen probe) in the presence of the second phase in the absence of another carbon source, wherein consumption of oxygen above the background being the same as with no solvent correlates with consumption of the solvent.
  • the host organism such as e.g. Saccharomyces or Yarrowia
  • measurement/ mass spec analysis of off-gas or condensation in a cold trap from fermentation can be used to measure evaporation.
  • the present invention features a fermentative process for production of isoprenoids, lipids and sterols, particularly various forms of vitamin E and vitamin D3, as well as various PUFAs as defined herein using a two-phase culture system, wherein the formation of by-products could be reduced, particularly reduced by at least about 10% in comparison to the use of a non-stable solvent, such as e.g. in the range of about 15, 20, 25, 30, 35, 40, 45, 50% or more.
  • a non-stable solvent such as e.g. in the range of about 15, 20, 25, 30, 35, 40, 45, 50% or more.
  • the terms "by-products", “side-products” or “undesired fermentation products” in connection with fermentative production of target fermentation products as defined herein, i.e. isoprenoids lipids, and sterols, according to the definition given in the present application, according to the present invention are used interchangeably herein and refer to undesired coproduction of competing products, i.e. impurities, which have to be separated from the desired fermentation products and/or limit the yield and/or productivity of the desired fermentation products. Furthermore, it relates to undesired conversion processes, i.e., wherein the conversion of an intermediate into the desired fermentation product is competing with undesired conversion of the intermediate into a side-product or undesired by-product.
  • the terms "isoprenoids or sterols according to the present invention” and (desired) fermentation or target fermentation products are used interchangeably herein.
  • undesired side-products include but are not limited to formation of saturated fatty acids, diacylglyceride and monoglyceride fatty acids as well as free fatty acids (FFAs).
  • FFAs free fatty acids
  • Yarrowia or Saccharomyces or host cells selected from Mortierella, Phycomyces, Blakeslea, Aspergillus, Thraustochytrium, Pythium or Entomophthora, Porphyridium, Nitszchia, or Crypthecodinium, particularly Mortierella alpina, Blakeslea trispora, Aspergillus terreus or Pythium insidiosum, Crypthecodinium cohnii said host cell being grown on a suitable carbon source.
  • a list of suitable host cells for production of lipids can be found in US10385289, including particularly cells from Stramenopiles, Hamatores, Proteromonads, Opalines, Develpayella, Diplophrys, Labrinthulids, Thraustochytrids, Biosecids, Oomycetes, Hypochytridiomycetes, Commotion, Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms, Xanthophytes, Phaeophytes, Eustigmatophytes, Raphidophytes, Synurids, Axodines (including Rhizochromulinaales, Pedinellales, Dictyochales), Chrysomeridales, Sarcinochrysidales, Hydrurales, Hibberdiales, and Chromulinales.
  • Axodines including Rhizochromulinaales, Pe
  • Labyrinthulaceae were previously considered to be members of the order Thraustochytriales, but in more recent revisions of the taxonomic classification of such organisms, the family Labyrinthulaceae is now considered to be a member of the order Labyrinthulales. Both Labyrinthulales and Thraustochytriales are considered to be members of the phylum Labyrinthulomycota. Taxonomic theorists now generally place both of these groups of microorganisms with the algae or algae-like protists of the Stramenopile lineage.
  • the current taxonomic placement of the thraustochytrids and labyrinthu lids can be summarized as follows: Realm: Stramenopila (Chromista); Phylum: Labyrinthulomycota (Heterokonta); Class: Labyrinthulomycetes (Labyrinthulae); Order: Labyrinthulales, Family: Labyrinthulaceae; Order: Thraustochytriales, Family: Thraustochytriaceae.
  • strains of microbial cells described as thraustochytrids include the following organisms: Order: Thraustochytriales; Family: Thraustochytriaceae; Genera: Thraustochytrium (Species: sp., arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum, and striatum), Ulkenia (Species: sp., amoeboidea, kerguelensis, minuta, profunda, radiata, sailens, sarkariana, schizochytrops, visurgensis, yorkensis, and sp.
  • Schizochytrium (Species: sp., aggregatum, limnaceum, mangrovei, minutum, and octosporum), Japonochytrium (Species: sp., marinum), Aplanochytrium (Species: sp., haliotidis, kerguelensis, profunda, and stocchinoi), Althornia (Species: sp., crouchii), or Elina (Species: sp., marisalba, and sinorifica).
  • species described within Ulkenia will be considered to be members of the genus Thraustochytrium.
  • Microbial cells suitable for use with the present invention include, but are not limited to, Labyrinthulids selected from: Order: Labyrinthulales, Labyrinthulaceae, Genera: Labyrinthula (Species: sp., algeriensis, coenocystis, chattonii, macrocystis, macrocystis atlantica, macrocystis macrocystis, marina, minuta, roscoffensis, valkanovii, vitellina, vitellina pacifica, vitellina vitellina, and zopfii), Labyrinthuloides (Species: sp., haliotidis, and yorkensis), Labyrinthomyxa (Species: sp., marina), Diplophrys (Species: sp., archeri), Pyrrhosorus (Species: sp., marinus), Sorodiploph
  • Host cells of the phylum Labyrinthulomycota include, but are not limited to, deposited strains PTA-10212, PTA-10213, PTA-10214, PTA-10215, PTA-9695, PTA- 9696, PTA-9697, PTA-9698, PTA-10208, PTA-10209, PTA-10210, PTA-10211, the microorganism deposited as SAM2179 (named "Ulkenia SAM2179” by the depositor), any Thraustochytrium species (including former Ulkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U. profunda, U. radiata, U.
  • Ulkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U. profunda, U. radiata, U.
  • Thraustochytriales include but are not limited to Thraustochytrium sp. (23B) (ATCC 20891); Thraustochytrium striatum (Schneider) (ATCC 24473); Thraustochytrium aureum (Goldstein) (ATCC 34304); Thraustochytrium roseum (Goldstein) (ATCC 28210); Japonochytrium sp.
  • Schizochytrium include, but are not limited to Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium sp. (S31) (ATCC 20888), Schizochytrium sp. (58) (ATCC 20889), Schizochytrium sp.
  • the host cell is a Schizochytrium or a Thraustochytrium.
  • Schizochytrium can replicate both by successive bipartition and by forming sporangia, which ultimately release zoospores.
  • Suitable carbon sources to be used for the present invention might be selected from linear alkanes, free fatty acids, including triglycerides, particularly vegetable oil, such as e.g., selected from the group consisting of oil originated from corn, soy, olive, sunflower, canola, cottonseed, rapeseed, sesame, safflower, grapeseed or mixtures thereof, including the respective free fatty acids, such as e.g., oleic acid, palmitic acid or linoleic acid.
  • Suitable carbon sources might furthermore be selected from ethanol, glycerol or glucose and mixtures of one or more of the above-listed carbon sources.
  • vitamin D3 as defined herein is produced under suitable culture conditions in a two-phase culture system as described herein, preferably using lipophilic solvents selected from IsoparTM fluids, particular selected from Isopar M, Isopar K, Isopar L, Isopar H or solvents with equivalent or identical properties but from other suppliers.
  • the present invention is directed to a process for the production of isoprenoids, lipids, and sterols, particularly various forms of vitamin E and vitamin D3, as well as various PUFAs, including derivatives or intermediates, i.e.
  • a two-phase culture system in the presence of a lipophilic solvent as defined herein, wherein a respective host cell, particularly fungal host cell such as Saccharomyces or Yarrowia, is cultivated under suitable culture conditions, wherein the lipophilic solvent, preferably selected from solvents that are commercially available as IsoparTM fluids, particularly selected from Isopar M, Isopar N, Isopar K, Isopar L, Isopar H or solvents with equivalent or identical properties but from other suppliers, is not consumed or evaporated during the fermentation process.
  • a lipophilic solvent preferably selected from solvents that are commercially available as IsoparTM fluids, particularly selected from Isopar M, Isopar N, Isopar K, Isopar L, Isopar H or solvents with equivalent or identical properties but from other suppliers, is not consumed or evaporated during the fermentation process.
  • the present invention is directed to a process for the production of sterols, in particular production of vitamin D3, i.e. a two-phase culture system in the presence of a lipophilic solvent as defined herein, wherein a respective host cell, particularly fungal host cell such as Saccharomyces or Yarrowia, is cultivated under suitable culture conditions, wherein the percentage of 7-DHC could be increased by at least about 5%, such as e.g. about 8, 10, 12, 14, 15, 18, 20% or more as compared to a process without the addition of said lipophilic solvent as defined herein.
  • the present invention is directed to a process for the production of sterols, in particular production of vitamin D3, i.e. a two-phase culture system in the presence of a lipophilic solvent as defined herein, wherein a respective host cell, particularly fungal host cell such as Saccharomyces or Yarrowia, is cultivated under suitable culture conditions, wherein the percentage of HyDHC could be increased by at least about 15%, such as e.g. about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150% or more as compared to a process without the addition of said lipophilic solvent as defined herein.
  • the present invention is directed to a process for the production of sterols, in particular production of vitamin D3, i.e. a two-phase culture system in the presence of a lipophilic solvent as defined herein, wherein a respective host cell, particularly fungal host cell such as Saccharomyces or Yarrowia, is cultivated under suitable culture conditions, wherein the percentage of zymosterol could be reduced by at least about 5%, such as e.g. about 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 5, 60, 65, 70% or more as compared to a process without the addition of said lipophilic solvent as defined herein.
  • organisms such as e.g.
  • microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code).
  • the term "specific activity” or "activity” with regards to enzymes means its catalytic activity, i.e., its ability to catalyze formation of a product from a given substrate.
  • the specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature.
  • specific activity is expressed in pmol substrate consumed or product formed per min per mg of protein.
  • An enzyme is active, if it performs its catalytic activity in vivo, i.e.
  • Conversion according to the present invention is defined as specific enzymatic activity, i.e. catalytic activity of enzymes described herein, including but not limited to the enzymatic activity of hydroxylases, such as e.g. cholesterol C25- hydroxylase involved in conversion of 7-DHC into HyDHC.
  • hydroxylases such as e.g. cholesterol C25- hydroxylase involved in conversion of 7-DHC into HyDHC.
  • triglycerides and “triglyceride oils” are used interchangeably herein.
  • Fermentative production of isoprenoids or sterols as backbone in a two- phase culture system comprising in situ extraction of said isoprenoids or sterols, wherein a suitable host cell is cultivated in the presence of a carbon source and a lipophilic solvent, wherein the lipophilic solvent is different from the carbon source and with a minimal loss of solvent during the fermentation process, and wherein the isoprenoid is selected from vitamin E and the sterol is selected from vitamin D3 including derivatives and intermediates of vitamin E or vitamin D3.
  • Embodiment (1) or (2) wherein the lipophilic solvent present at the end of the fermentation is in the range of at least 80% of the solvent present at the start of the fermentation process.
  • the carbon source is selected from linear alkanes, free fatty acids, ethanol, glucose, including triglycerides, particularly vegetable oil, such as e.g. selected from the group consisting of oil originated from corn, soy, olive, sunflower, canola, cottonseed, rapeseed, sesame, safflower, grapeseed or mixtures thereof, including the respective free fatty acids, such as e.g. o
  • Embodiment (1), (2), (3), (4), (5) or (6) wherein 20% or less of the solvent is lost during the fermentation, preferably wherein the evaporation of the solvent is reduced by at least about 50%.
  • Embodiment (1), (2), (3), (4), (5), (6) or (7) wherein the consumption of the solvent by the host cell is reduced by at least about 50%.
  • 5% such as e.g. about 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 5, 60, 65, 70% or more as compared to a process without the addition of said lipophilic solvent as defined herein.
  • fungal host cells particularly oleaginous host cells, such as e.g. yeast, preferably selected from Yarrowia or Saccharomyces.
  • a HyDHC producing Saccharomyces cerevisiae strain was constructed as described in W02011067144, wherein the cholesterol C25- hydroxylase from S. scrofa has been integrated (see SEQ ID NO: 27 in W02011067144).
  • a HyDHC producing Yarrowia lipolytica was constructed accordingly (Table 1), i.e. wherein the Erg5 and Erg6 genes are knocked out, a heterologous cholesterol C25- hydroxylase is expressed and a heterologous sterol A24- reductase such as selected from Danio rerio, rat or human.
  • Table 1 list of analytes using C18 UPLC chromatography. "RT” means retention time. For more details, see text.
  • Fermentation conditions (Saccharomyces): Fed-batch fermentations using Isopar M as a second phase were performed in a stirred tank in a bench top reactor with 0.5L initial batch volume.
  • the batch medium carbon source composition and feed media are glucose and ethanol. Feeds were added using two different feed profiles between hours 2-60 for glucose and hours 5-120 for ethanol.
  • Fermentation conditions (Yarrowia): Fed-batch fermentations were similar to the previously described conditions except using Isopar M as a second phase in a stirred tank bench top reactor with 0.6L initial batch volume (see WO2016172282).
  • the batch medium carbon source and feed medium are glucose. Feeding was initiated after the initial batch carbon had been consumed, with feed added in a controlled manner to maintain a dissolved oxygen level (DO) setpoint.
  • DO dissolved oxygen level
  • glucose was added (5% w/w final concentration) along with 60mL Isopar M as the second phase if used.
  • the fermentation was inoculated with 60 ml of an overnight shake flask culture (YPD medium grown with 250RPM agitation at 30°C). Fermentation parameters were agitation at 1300RPM, airflow at 0.7LPM, pH controlled at 5.5 control with NH 4 0H, and the temperature set to 30°C.
  • feed 65% glucose was added to maintain the DO setpoint at 60%.
  • the DO setpoint was ramped down to 20% in a linear fashion over the following 24 hours by increased feed rate. The DO was then maintained at 20% via feed addition for the remainder of the fermentation.
  • Example 2 Vitamin D3 production using a two-phase culture system in Saccharomyces and Yarrowia
  • Table 6 effect of Isopar M (10%) on formation of HyDHC and 7-DHC, wherein the increase in % is shown as compared to the numbers without the use of Isopar M in the second phase (i.e. without the use of a two-phase culture system). For more details, see text.
  • Example 3 PUFA production using a two-phase culture system in Yarrowia
  • Table 7 effect of Isopar M (10%) on formation of PUFAs, wherein the increase in % is shown as compared to the numbers without the use of Isopar M in the second phase (i.e. without the use of a two-phase culture system: "control"), which is set as lOO%For more details, see text.

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Abstract

La présente invention concerne la production par fermentation d'isoprénoïdes, de lipides et de stérols, en particulier diverses formes de vitamine E et de vitamine D3, ainsi que divers acides gras polyinsaturés (PUFAs), y compris des dérivés ou des intermédiaires, comprenant la culture d'une cellule hôte appropriée, telle qu'une cellule hôte fongique, en particulier une cellule hôte oléagineuse, dans un système de culture à deux phases amélioré.
PCT/EP2023/062052 2022-05-05 2023-05-05 Nouveau procédé Ceased WO2023214072A2 (fr)

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WO2019057998A1 (fr) * 2017-09-25 2019-03-28 Dsm Ip Assets B.V. Production de rétinol
US12049658B2 (en) * 2017-09-25 2024-07-30 Dsm Ip Assets B.V. Production of retinyl esters
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WO2025099223A1 (fr) * 2023-11-09 2025-05-15 Dsm Ip Assets B.V. Nouveau procédé

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