WO2023006179A1 - Method to produce retinyl acetate - Google Patents
Method to produce retinyl acetate Download PDFInfo
- Publication number
- WO2023006179A1 WO2023006179A1 PCT/EP2021/070909 EP2021070909W WO2023006179A1 WO 2023006179 A1 WO2023006179 A1 WO 2023006179A1 EP 2021070909 W EP2021070909 W EP 2021070909W WO 2023006179 A1 WO2023006179 A1 WO 2023006179A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fermentation
- host cell
- ethanol
- retinyl acetate
- process according
- Prior art date
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/67—Vitamins
- A61K8/671—Vitamin A; Derivatives thereof, e.g. ester of vitamin A acid, ester of retinol, retinol, retinal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- 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
- C12P7/62—Carboxylic acid esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01084—Alcohol O-acetyltransferase (2.3.1.84)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/86—Products or compounds obtained by genetic engineering
Definitions
- the present invention is related to a novel process for production of retinyl acetate in a host cell, particularly oleaginous yeast such as e.g. Yarrowia, wherein the product purity could be increased with reduction of unwanted side- products.
- the novel process comprises fermentation in the presence of ethanol, such as e.g. in a fed-batch fermentation process. Such process is especially useful in a biotechnological process for production of vitamin A.
- the biological systems that produce retinoids are industrially intractable and/or produce the compounds at such low levels that commercial scale isolation is not practical.
- the most limiting factors include instability of intermediates in such biological systems and/or the relatively high production of by-products, such as e.g. fatty acid retinyl esters (FAREs), particularly using oleaginous host cells grown on vegetable oils or glucose as carbon source.
- FAREs fatty acid retinyl esters
- the present invention is directed to a process for production of retinyl acetate in a fungal host cell, preferably oleaginous yeast cell such as e.g. Yarrowia, comprising cultivation of the host cell in the presence of ethanol added during the fermentation, particularly in a fed-batch fermentation process, wherein the formation of by-products including FARES is reduced or abolished, preferably reduced by about 50-100% based on total retinoids, compared to fermentation without ethanol, particularly compared to a (fermentation) process in the presence of triglycerides, particularly vegetable oil, as defined herein.
- a fungal host cell preferably oleaginous yeast cell such as e.g. Yarrowia
- by-products and “side-products” in connection with fermentative production of retinoids that are used interchangeably herein means retinoids generated during the fermentation process, with the exception of retinyl acetate.
- the formation of by-products which is to be reduced or abolished during the fermentation includes particularly the formation of FARE, optionally furthermore presence of retinol or retinal present in the cultivation medium and which might be purified from the retinyl acetate.
- a fermentation “in the "presence of ethanol” or “ethanol added during the fermentation” particularly refers to a fed-batch fermentation process, wherein the batch phase comprises a concentration of about 5% ethanol (v/v) or less, such as e.g. an amount of about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1% added to the cultivation medium during the batch phase of the fermentation.
- Ethanol feed is controlled via measurement of the pH and/or DO as known in the art, typically with feeding of about 100% ethanol and with DO setpoint of about 40 to 20%
- either the batch phase or the feed further comprises glucose, such as e.g.
- a fermentation "in the "presence of triglycerides, particularly vegetable oil” or “triglycerides, particularly vegetable oil, added during the fermentation” particularly refers to a fed-batch fermentation process, wherein the batch phase comprises a concentration of about 10% triglycerides(v/v) or less, particularly vegetable oil, such as e.g. an amount of about 10, 8, 6, 5, 4, 3, 2, 1% added to the cultivation medium during the batch phase of the fermentation.
- the oil to be used is particularly vegetable oil, such as e.g.
- 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.
- the present invention is related to a process as described herein for reducing FARE-production in retinyl acetate production process, wherein the percentage of by-products including FARES could be reduced by about 100%, i.e. more or less abolished, said process comprising the fermentation in the presence of ethanol as defined herein.
- the formation of FARES could be reduced by at least about 50%, such as e.g. about 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%, i.e. abolishment of FARE formation.
- the present invention is directed to a retinyl acetate production process as defined herein, wherein the production of FARE formed during the fermentation is less than about 25%, such as e.g. less than about 20, 15, 10, 8, 5, 3, 2, 1% based on total retinoids.
- the present invention is directed to a process for production of retinyl acetate in a fungal host cell, preferably oleaginous yeast cell such as e.g. Yarrowia, said process comprising fermentation in the presence of ethanol as defined herein, wherein the percentage of retinyl acetate formation could be increased by at least about 25%, such as e.g. by about 30, 35, 40, 45, 50, 55, 60,
- the present invention is related to a process as described herein for production of retinyl acetate in a fungal host cell as defined herein, wherein the percentage of retinyl acetate formed during the fermentation is in the range of at least about 50-80%, such as e.g. a percentage of at least about 50, 55, 60, 65, 70, 75, 80, 85, 87, 90, 95, 98% or up to 100% based on total retinoids, said process comprising the fermentation in the presence of ethanol as defined herein.
- the present invention is directed to a process for increasing total retinoids, with an increase in retinoids in the range of at least about 30%, such as e.g. an increase of about 30, 35, 40, 45, 50, 60, 70, 80% or more, said process comprising the fermentation in the presence of ethanol as defined herein, as compared to the respective process in the absence of ethanol, particularly compared to a fermentation process in the presence of triglycerides as defined herein.
- the present invention is directed to a process as defined herein, wherein a retinyl acetate producing host cell, preferably oleaginous yeast cell such as e.g. Yarrowia, is cultivated under suitable culture conditions comprising fermentation in the presence of ethanol as defined herein, wherein the percentage of FARES formed during the fermentation is in the range of less than 25%, preferably less than about 10%, and the percentage of retinyl acetate is the range of at least about 50-80%, preferably in the range of at least about 70, 80, 85, 87, 90, 95, 98% based on total retinoids.
- a retinyl acetate producing host cell preferably oleaginous yeast cell such as e.g. Yarrowia
- the percentage of FARES formed during the fermentation is in the range of less than 25%, preferably less than about 10%
- the percentage of retinyl acetate is the range of at least about 50-80%, preferably in the range of at least about 70, 80
- the percentage of retinyl acetate might be increased by about 100% or more and the percentage of FARES might be reduced to a range of about 10 to 1%, or even to less than about 1%, or even abolished, based on total retinoids.
- the present invention is directed to the use of a retinyl acetate producing host cell, particularly fungal host cell, preferably oleaginous yeast cell such as e.g. such as e.g. Rhodosporidium, Lipomyces or Yarrowia, preferably Yarrowia, more preferably Yarrowia lipolytica, in a process as defined herein, e.g. in a fermentation process comprising addition of ethanol during the fermentation as defined herein, wherein the process is particularly a fed-batch fermentation, wherein the batch phase comprises a concentration of about 5% ethanol (v/v) or less, particularly 2 to 1% (v/v) ethanol in the batch phase.
- oleaginous yeast cell such as e.g. such as e.g. Rhodosporidium, Lipomyces or Yarrowia, preferably Yarrowia, more preferably Yarrowia lipolytica
- suitable host cells are expressing genes coding for heterologous enzymes EC class [EC 2.3.1.84] catalyzing the enzymatic conversion of retinol into retinyl acetate.
- EC class [EC 2.3.1.84] catalyzing the enzymatic conversion of retinol into retinyl acetate.
- Suitable strains expressing such ATFs are described in e.g. W02019058001 or W02020141168.
- the host cell to be used in the present invention is expressing a heterologous ATF, particularly fungal ATF, comprising a highly conserved partial amino acid sequence of at least 7 amino acid residues selected from [NDEHCS]- H-x(3)-D-[GA] (motifs are in Prosite syntax, as defined in https://prosite.expasy.org/scanprosite/scanprosite_doc.html), wherein "x" denotes an arbitrary amino acid and with the central histidine being part of the enzyme's binding pocket, preferably wherein the 7 amino acid motif is selected from [NDE]-H-x(3)-D-[GA], more preferably selected from [ND]-H-x(3)-D-[GA], most preferably selected from N-H-x(3)-D-[GA] corresponding to position N218 to G224 in the polypeptide according to SEQ ID N0:18.
- motifs are in Prosite syntax, as defined in https://prosite.expasy.org/scanprosite/scanprosite_doc
- Such enzymes might be particularly selected from L. mirantina, L. fermentati, S. bayanus, or W. anomalus, such as e.g. LmATFI according to SEQ ID N0:18, SbATFI, LffATFI,
- Wa1ATF1 or Wa3ATF1 as disclosed in W02019058001 more preferably said ATFs comprising one or more amino acid substitution(s) in a sequence with at least about 20%, such as e.g. 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:18, wherein the one or more amino acid substitution(s) are located at position(s) corresponding to amino acid residue(s) selected from the group consisting of position 68, 69, 72, 73, 171, 174, 176, 178, 291, 292, 294, 301, 307, 308, 311, 312, 320, 322, 334, 362, 405, 407, 409, 480, 483, 484, 490, 492, 520, 521, 522, 524, 525, 526 and combinations thereof and as particularly exemplified in Table 4 of W02020141168
- the host cell to be used for the process according to the present invention comprises an amino acid substitution at a position corresponding to residue 69 in the polypeptide according to SEQ ID NO:18 leading to asparagine, serine or alanine at said residue, such as e.g. via substitution of histidine by asparagine (H69N), serine (H69S) or alanine (H69A), with preference for H69A.
- Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus, preferably from L.
- mirantina optionally being combined with amino acid substitution at a position corresponding to residue 407 in the polypeptide according to SEQ ID NO:18 leading to isoleucine at said residue, such as e.g. via substitution of valine by isoleucine (V407I), optionally being combined with an amino acid substitution at a position corresponding to residue 409 in the polypeptide according to SEQ ID NO:18 leading to alanine at said residue, such as e.g.
- the ATF to be used for the process according to the present invention is a modified ATF comprising amino acid substitutions S480Q_G409A_V407I_H69A_I484L and is obtainable from Lachancea mirantina.
- the term "host cell” includes retinyl-acetate producing cells, i.e. capable of synthesizing retinol and expressing ATF as defined herein resulting in retinyl acetate with a percentage as defined herein based on total retinoids produced by said host cell.
- retinyl-acetate producing cells i.e. capable of synthesizing retinol and expressing ATF as defined herein resulting in retinyl acetate with a percentage as defined herein based on total retinoids produced by said host cell.
- such host cell is furthermore capable of producing carotenoids.
- a "fungal host cell” particularly includes yeast cells, i.e. retinyl acetate-producing yeast cells, including but not limited to Yarrowia, Rhodosporidium, or Lipomyces.
- the host cell such as e.g. Yarrowia, capable of producing retinyl acetate from conversion of retinol
- the host cell is expressing further enzymes used for biosynthesis of beta-carotene and/or additionally used for catalyzing conversion of beta-carotene into retinal and/or retinal into retinol.
- the skilled person knows which genes to be used/expressed for either biosynthesis of beta- carotene and/or bio-conversion of beta-carotene into retinol.
- Such host cell further being capable of expressing ATF genes as defined herein and/or further genes required for biosynthesis of vitamin A, is cultured in an aqueous medium comprising addition of ethanol during the fermentation, optionally supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person to enable production of retinyl acetate.
- the fermentation is performed in fed-batch, wherein the batch phase comprises a concentration of about 5% or less ethanol, particularly 2 to 1% ethanol, and with feed of 100% ethanol as particularly exemplified herein.
- fermentations are run in fed-batch stirred tank reactors. Fermentations can be run for 5 to 14 days, such as e.g. for around 118 h.
- Fermentation products including retinyl acetate may be harvested from the cultivation at a suitable moment, e.g. when the tank fills due to addition of the feed.
- retinoids such as e.g. vitamin A, precursors and/or derivatives thereof such as retinal, retinol, retinyl acetate, particularly retinyl acetate
- the retinoids including but not limited to retinol, retinyl acetate, vitamin A might be used as ingredients/formulations in the food, feed, pharma or cosmetic industry.
- Cultivation and isolation of beta-carotene and retinoid- producing host cells selected from Saccharomyces is described in e.g. W02008042338.
- the host cell to be used for the process according to the present invention might comprise further modifications, such as modification in endogenous enzyme activities leading to conversion of retinol into FARES.
- modifications include deletion of endogenous lipase activities, i.e. activities in enzymes involved in pre-digestion of triglyceride oils such as e.g. vegetable oil into glycerol and fatty acids that are normally expressed in oleaginous host cells.
- Suitable enzymes to be modified in a host cell used in the process as defined herein might be selected from endogenous enzymes belonging to EC class 3.1.1.
- an enzyme having activity corresponding to the respective LIP activity in Yarrowia includes not only the genes originating from Yarrowia, e.g. Yarrowia lipolytica, such as e.g. Yarrowia LIP2, LIP3, LIP4, LIP8, TGL-1, LIP16, LIP17, LIP18 or combinations thereof according to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15 but also includes enzymes having equivalent enzymatic activity but are originated from another source organism, particularly retinyl acetate-producing oleaginous host cell.
- Yarrowia lipolytica such as e.g. Yarrowia LIP2, LIP3, LIP4, LIP8, TGL-1, LIP16, LIP17, LIP18 or combinations thereof according to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15
- enzymes having equivalent enzymatic activity but are originated from another source organism, particularly retinyl acetate-producing oleaginous host cell.
- the host cell comprises deletion of endogenous lipase activities corresponding to Yarrowia lipolytica lipase activities 2, 3, 4, 8, or combinations thereof, in particular wherein the host cell is Yarrowia lipolytica comprising deletion of endogenous lipase activities such as lip8 or combinations of lip8, such as combination of lip8 with lip2, combination of lip8 with lip2 and lip3, or combination of lip8 with lip2, lip3, and lip4 activities.
- mutagenesis may be performed in different ways, such as for instance by random or side- directed mutagenesis, physical damage caused by agents such as for instance radiation, chemical treatment, or insertion of a genetic element.
- agents such as for instance radiation, chemical treatment, or insertion of a genetic element.
- the skilled person knows howto introduce mutations.
- sequence identity in order to determine the percentage of sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids.
- sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.
- the percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined usingthe Needleman and Wunsch algorithm for the alignment of two sequences (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm.
- the Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE.
- the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/).
- EMBOSS European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/).
- EBLOSUM62 is used for the substitution matrix.
- EDNAFULL is used for nucleotide sequence.
- the optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
- the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
- the identity as defined herein can be obtained from NEEDLE by usingthe NOBRIEF option and is labeled in the output of the program as "longest identity”. If both amino acid sequences which are compared do not differ in any of their amino acids, they are identical or have 100% identity.
- the enzymes as defined herein to be expressed in a suitable host cell to be used in the present invention also encompass enzymes carrying (further) amino acid substitution(s) which do not alter enzyme activity, i.e. which show the same properties with respect to the enzymes defined herein. Such mutations are also called “silent mutations", which do not alter the (enzymatic) activity of the enzymes according to the present invention.
- 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).
- strain Lachancea mirantina is a synonym of strain Zygosaccharomyces sp. IFO 11066, originated from Japan.
- the present invention is directed to a process for production of retinyl acetate, wherein the retinyl acetate is generated via acetylation of retinol (particularly at least 65% as trans-retinol) as disclosed herein by the action of modified/non- modified ATF as described herein, wherein the acetylating enzymes are heterologous expressed in a suitable host cell under suitable conditions as described herein and wherein the host cell is cultivated in a medium comprising an effective amount of ethanol added during the fermentation.
- the produced retinyl acetate might be isolated and optionally further purified from the medium and/or host cell.
- Said acetylated retinoids defined herein can be used as building blocks in a multi-step process leading to vitamin A. Vitamin A might be isolated and optionally further purified from the medium and/or host cell as known in the art.
- 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.
- Suitable host cells comprising specific enzymes involved in biosynthesis of beta-carotene and that are expressed and active in vivo leading to production of carotenoids, e.g. beta-carotene
- both genes and methods to generate carotenoid-producing host cells are known in the art, see e.g. W02006102342.
- different genes might be involved.
- a "retinol-producing host cell” is a host cell, wherein the respective polypeptides are expressed and active in vivo, leading to production of retinoids, e.g. vitamin A and its precursors including retinol, via enzymatic activity of the ATFs as described herein.
- retinoids e.g. vitamin A and its precursors including retinol
- the genes of the vitamin A pathway and methods to generate retinoid-producing host cells are known in the art.
- the term retinoid includes retinol, which is used as a substrate for the modified acetylating enzymes as defined herein.
- a "retinyl acetate-producing host cell” is the respective host cell capable of acetylation of retinol into retinyl acetate.
- Retinoids as used herein include beta-carotene cleavage products also known as apocarotenoids, including but not limited to retinal, retinolic acid, retinol, retinoic methoxide, retinyl acetate, retinyl esters, 4-keto-retinoids, 3 hydroxy- retinoids or combinations thereof. Biosynthesis of retinoids is described in e.g. W02008042338.
- Retinal as used herein is known under lUPAC name (2E,4E,6E,8E)-3,7-Dimethyl- 9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenal. It is herein interchangeably referred to as retinaldehyde or vitamin A aldehyde and includes both cis- and trans-isoforms, such as e.g. 11-cis retinal, 13-cis retinal, trans- retinal and all-trans retinal.
- carotenoids as used herein is well known in the art. It includes long, 40 carbon conjugated isoprenoid polyenes that are formed in nature by the ligation of two 20 carbon geranylgeranyl pyrophosphate molecules. These include but are not limited to phytoene, lycopene, and carotene, such as e.g. beta-carotene, which can be oxidized on the 4-keto position or 3-hydroxy position to yield canthaxanthin, zeaxanthin, or astaxanthin. Biosynthesis of carotenoids is described in e.g. W02006102342.
- Vitamin A as used herein may be any chemical form of vitamin A found in solutions, in solids and formulations, and includes retinol, retinyl acetate and retinyl esters. It also includes retinoic acid, such as for instance undissociated, in its free acid form or dissociated as an anion.
- a recombinant fungal host cell particularly a yeast cell such as e.g. a Socchoromyces cerevisioe cell, more particularly an oleaginous yeast cell such as e.g. a recombinant Yorrowio cell (suitably modified to be able to produce steviol glycosides), in the presence of ethanol.
- a yeast cell such as e.g. a Socchoromyces cerevisioe cell
- an oleaginous yeast cell such as e.g. a recombinant Yorrowio cell (suitably modified to be able to produce steviol glycosides)
- the production of said products was increased when ethanol was provided as a carbon source, particularly in a fed-batch fermentation process, when compared to the respective process in the absence of ethanol. More particularly, the production of said products was increased with the use of pure ethanol feeds or mixed ethanol feeds (e.g. ethanol/glucose mixture) following the batch phase
- the present disclosure is directed to a process for the production of steviol glycosides in a recombinant fungal host cell, preferably a yeast cell such as e.g. a Socchoromyces cerevisioe cell, more preferably an oleaginous yeast cell such as e.g. a recombinant Yorrowio cell, more preferably Yorrowio lipolytico (suitably modified to be able to produce steviol glycosides), said process comprising fermentation in the presence of ethanol as defined herein.
- the present disclosure is also directed to the use of a host cell that produces steviol glycosides, preferably a yeast cell such as e.g.
- a Socchoromyces cerevisioe cell more preferably an oleaginous yeast cell such as e.g. a recombinant Yorrowio cell, more preferably Yorrowio lipolytico (suitably modified to be able to produce steviol glycosides), in a fermentation process comprising addition of ethanol duringthe fermentation as defined herein.
- an oleaginous yeast cell such as e.g. a recombinant Yorrowio cell, more preferably Yorrowio lipolytico (suitably modified to be able to produce steviol glycosides)
- Suitable fungal host cells for the production of steviol glycosides are known in the art and are described in e.g. WO2011153378, WO2013022989, WO2014122227, WO2013110673, and W02015007748.
- the produced steviol glycosides might be isolated and optionally further purified from the medium and/or host cell according to a method known to the skilled person in the art.
- Episomal hygromycin resistance marker plasmids were cured by passage on non-selective media, with identification of Hyg-sensitive colonies by replica plating colonies from non-selective media to hygromycin containing media (100 pg/mL).
- Plasmids MB9523 containing expression systems for DrBCO, LmATF-S480Q_G409A_V407l_H69A_l484L, and FfRDH (SEQ ID NO:17) was synthesized at Genscript (Piscataway, NJ, USA). Plasmid MB9523 contains the 'URA3' for marker selection in Yarrowia lipolytica transformations. For gene insertion by random nonhomologous end joining of the gene and marker, Sfil- digested MB9523 plasmid fragment of interest was purified by gel electrophoresis and Qiagen gel purification column. Clones were verified by sequencing. Typically, genes are synthesized by a synthetic biology at GenScript (Piscataway, NJ).
- Plasmid list Plasmid, strains, nucleotide and amino acid sequences that were used are listed in Table 1, 2 and the sequence listing. In general, all non- modified sequences referred to herein are the same as the accession sequence in the database for reference strain CLIB122 (Dujon B, et al, Nature. 2004 Jul 1;430(6995):35-44).
- Table 1 list of plasmids used for construction of the strains for overexpression or deletion of the respective genes indicated as "insert”.
- LmATFl-mut refers to Lachancea mirantina (LmATFI; SEQ ID NO:13 in W02019058001) carrying aa substitutions S480Q_G409A_V407I_H69A_I484L.
- DrBCO refers to BCO originated from Danio rerio (see SEQ ID NO:16 in WQ2020141168);
- FfRDH refers to RDH originated from Fusarium fujikuroi (see SEQ ID NO:22 in W02020141168). For more explanation, see text.
- Table 2 list of Yarrowia lipolytica strains used. Construction of ML17544 is described in Table 2 of W02020141168. For more details, see text.
- the fermentations were run in 3.0L flood volume in glass New Brunswick or Eppendorf fermentation systems.
- the fermentor was batched with the following components: 2228mL of deionized water, MgS0 4* 7H 2 0 is 1.96g/kg and NaCl is 0.20g/kg, 10.46mL, 1.04g CaCl 2 -2H 2 0, 26.18g (NH 4 )2S0 4 , 27.10g KH 2 P0 4 , 19.62g Tastone yeast extract (Marcor, Leominster, MA) , 26mL DF204 antifoam, 0.654pL, thiamine HCl at 4 mg/ml, trace elements stock solution 3.27ml/L containing: 200g/kg citric acid, 27.3 g/ kg FeS0 4* 7H 2 0, 19.6 g/ kg Na 2 Mo0 4* 2H 2 0,
- Retinoid quantification Analysis of retinoids were carried out with a C4 reverse phase retinoid method (see below) and C18 as described elsewhere
- Table 4A list of analytes using C4-reverse phase method. The addition of all added intermediates gives the total amount retinoids. "RT” means retention time. For more details, see text.
- Table 4B UPLC Method Gradient with solvent A: acetonitrile; solvent B: water; solvent C: water/acetonitrile/methanesulfonic acid 1000:25:1. For more details, see text.
- Method Calibration Method is calibrated using high purity retinyl acetate received from DSM Nutritional Products, Kaiseraugst, CH. Retinols and retinal are quantitated against retinyl acetate. Dilutions were prepared as follows. 40 mg of retinyl acetate was weighed into a 100 mL volumetric flask, and dissolved in ethanol, yielding a 400 pg/mL solution. This solution was sonicated as required to ensure dissolution.
- 5mL of this 400 pg/mL solution was diluted into 50 mL (1/10 dilution, final concentration 40pg/mL), 5mL into 100mL (1/20 dilution, final concentration 20pg/mL), 5mL of 40pg/mL into 50mL (1/10 dilution, final concentration 4pg/mL), 5mL of 20 pg/mL into 50mL (1/10 dilution, 2pg/mL), using 50/50 methanol/ methyl tert-butyl ether(MTBE) as the dilutent. All dilutions were done in volumetric flasks.
- Example 2 Impact of carbon source on retinoid production in fed-batch fermentations using Yarrowia
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