EP3687486A1 - Biosynthèse de rétinoïdes - Google Patents
Biosynthèse de rétinoïdesInfo
- Publication number
- EP3687486A1 EP3687486A1 EP18770063.8A EP18770063A EP3687486A1 EP 3687486 A1 EP3687486 A1 EP 3687486A1 EP 18770063 A EP18770063 A EP 18770063A EP 3687486 A1 EP3687486 A1 EP 3687486A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- host cell
- trans
- retinol
- carotenoid
- retinyl acetate
- 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.)
- Pending
Links
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Classifications
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- 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
- 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
- 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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/07—Retinol compounds, e.g. vitamin A
Definitions
- the present invention is related to a novel enzymatic process for production of retinoids via a multi-step process, which process includes the use of
- heterologous enzymes having activity in a carotene-producing host cell, particularly wherein such process results in high percentage of retinoids, in trans-isoform.
- Retinoids including vitamin A, are one of very important and indispensable nutrient factors for human beings which have to be supplied via nutrition.
- Retinoids promote well-being of humans, inter alia in respect of vision, the immune system and growth.
- the biological systems that produce retinoids are industrially intractable and/or produce the compounds at such low levels that commercial scale isolation is not practicable. There are several reasons for this, including instability of the retinoids in such biological systems or the relatively high production of by-products.
- carotenoid-producing host cell particularly fungal host cell, comprising and expressing genes involved in the conversion of beta-carotene to retinyl acetate, with a total conversion of at least about 10% towards generation of retinol and with a percentage of trans-retinyl acetate of at least 65%.
- the present invention is directed to a host cell, particularly a carotenoid-producing host cell, such as a fungal host cell, comprising (1 ) a stereoselective/trans-selective beta-carotene oxidase (BCO) catalyzing the conversion of beta-carotene to a retinal mix with a percentage of at least 65% present as trans-retinal, and (2) acetyl transferases (ATFs) catalyzing the conversion of retinol to a retinyl acetate mix with a total conversion of at least 10% of retinol acetylated into retinyl esters, particularly retinyl acetate and wherein the ATFs have a preference for acetylation of trans-retinol.
- at least 80% of the retinyl esters are in the form of retinyl acetate, preferably as trans-retinyl acetate.
- a carotenoid-producing host cell, particularly fungal host cell, according to the present invention is optionally furthermore comprising (3) (preferably
- RDH heterologous retinol dehydrogenase
- a carotenoid-producing host cell, particularly fungal host cell, according to the present invention is optionally furthermore comprising (4) a modification in the endogenous acyltransferase activity, i.e. activity towards acylating retinol into long chain retinyl esters, said modification leading to reduction or abolishment of said endogenous acyltransferase activity.
- yeast host cell includes particularly yeast as host cell, such as e.g. Yarrowia or Saccharomyces.
- the terms "stereoselective”, “selective”, “trans-selective” or “trans-isomer selective” enzyme with regards to BCO are used interchangeably herein. They refer to enzymes, i.e. BCOs as disclosed herein, with increased catalytic activity towards trans-isomers, i.e. increased activity towards catalysis of beta-carotene into trans-retinal.
- An enzyme according to the present invention is trans-specific, if the percentage of trans-isoforms, such as e.g.
- trans-retinal is in the range of at least about 65% based on the total amounts of retinoids produced by such an enzyme or such carotene-producing host cell, particularly fungal host cell, comprising/expressing such enzyme.
- beta-carotene oxidizing enzyme As used herein, the terms "beta-carotene oxidizing enzyme”, “beta-carotene oxygenase”, “enzyme having beta-carotene oxidizing activity” or “BCO” are used interchangeably herein and refer to enzymes which are capable of catalyzing the conversion of beta-carotene into retinal in a trans-isomer selective way, leading to a retinal mix with at least about 65%, such as e.g. 68, 70, 75, 80, 85, 90, 95, 98% or up to 100%, of retinal in trans-isoform, based on the total amount of retinoids including retinal produced by said host cell.
- Trans-selective BCOs as defined herein might be obtained from any source, such as e.g. plant, animal, bacteria, fungi, algae.
- Particular useful stereoselective BCOs are obtained from fungi, in particular Dikarya, including but not limited to fungi selected from Ascomycota or Basidiomycota, preferably obtained from Fusarium or Ustilago, more preferably isolated from F. fujikuroi or U. maydis, such as e.g. FfCarX (polypeptide sequence derived from AJ854252), UmCCOI (polypeptide sequence derived from EAK81726).
- particularly useful stereoselective BCOs are obtained from insects, in particular Diptera, preferably obtained from Drosophila, more preferably from D. melanogaster, such as e.g. DmNinaB or DmBCO (polypeptide sequence derived from NP_650307.2).
- stereoselective BCOs are obtained from plants, in particular Angiosperms, preferably obtained from Crocus, more preferably from C. sativus, such as e.g. CsZCO (polypeptide sequence derived from
- stereoselective BCOs are obtained from eukaryotes, in particular pesces, preferably obtained from Danio or lctalurus, more preferably from D. rerio or I. punctatus, such as e.g. DrBCOI , IpBCO (polypeptide sequence derived from XP_017333634).
- the present invention is directed to a carotenoid -producing host cell, particularly a fungal host cell, used for biosynthesis of retinoids including vitamin A, said host cell comprising a polypeptide with at least 60%, such as e.g. 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% or up to 100% identity to a polypeptide known from the database selected from the group consisting of SEQ ID NOs:1 , 3, 5, 7 or polynucleotides encoding such sequences, or a polypeptide with at least 50%, such as e.g.
- the host cell furthermore comprises (2) acetyl transferases (ATFs) catalyzing the conversion of retinol to a retinyl acetate mix with a total conversion of at least 10% of retinol acetylated into retinyl esters, particularly retinyl acetate and wherein the ATFs have a preference for acetylation of trans-retinol.
- ATFs acetyl transferases
- acetyl transferase As used herein, the terms “acetyl transferase”, “retinol acetylating enzyme”, “enzyme having retinol acetylating activity” or “ATF” are used interchangeably herein and refer to enzymes [EC 2.3.1 .84] which are capable of catalyzing the conversion of retinol into retinyl acetate with an amount of at least 80%, about 87, 90, 92, 95, 97, 99 or up to 100% of produced retinyl acetate in the trans- isoform.
- Said ATFs are capable of converting retinol, preferably trans-retinol, into retinyl ester, particularly retinyl acetate, with a total conversion of at least about 10%, preferably 12, 15, 20, 30, 40, 50, 80, 90 or even 100% (based on the total amount of retinoids within the retinoid mix produced by said host cell) towards generation of retinyl esters, e.g. retinyl acetate.
- a preferred isoform is ATF1 .
- ATFs as defined herein might be obtained from any source, such as e.g. plants, animals, including humans, algae, fungi, including yeast, or bacteria.
- Particular useful ATFs, preferably ATF1 enzymes are obtained from yeast, in particular Saccharomyces or Lachancea, preferably obtained from Saccharomyces bayanus, such as e.g. SbATFI (polypeptide sequence derived from AHX23958.1 ),
- Lachancea mirantina (LmATFI ; SEQ ID NO: 33), or Lachancea fermentati such as Lf ATF1 (polypeptide sequence derived from SCW02964.1 ) or Lff ATF1 polypeptide sequence derived from LT598487).
- Lf ATF1 polypeptide sequence derived from SCW02964.1
- Lff ATF1 polypeptide sequence derived from LT598487 Lff ATF1 polypeptide sequence derived from LT598487.
- particularly useful ATF1 enzymes are obtained from plants, including but not limited to plants selected from Petunia, Euonymus, Malus, or Fragaria, preferably obtained from P.
- hybrida such as PhATF (polypeptide sequence derived from ABG75942.1 ), E. alatus, such as EaCAcT (polypeptide sequence derived from ADF57327.1 ), M. domestica (polypeptide sequence derived from AY517491 ) or F. ananassa
- ATF1 enzymes are obtained from Escherichia, preferably E. coli, such as e.g. EcCAT (polypeptide sequence derived from EDS05563.1 ).
- the present invention is directed to a carotenoid-producing host cell, particularly a fungal host cell, used for biosynthesis of retinoids including vitamin A, said host cell comprising:
- a polypeptide with at least 60% such as e.g. 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% or up to 100% identity to a polypeptide selected from the group consisting of SEQ ID NOs:1 , 3, 5 and 7 or a polypeptide with at least 50%, such as e.g. 55, 60, 65, 70, 75, 80, 85, 90, 93, 95, 97, 98, 99% or up to 100% identity to a polypeptide selected from SEQ ID NO:9, 11 , 13, 15 or 17; and
- polypeptide with at least 60% such as e.g. 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to a polypeptide selected from the group consisting of SEQ ID NO:21 , 23, 25, 27, 29, 31 , 33, 36, or 38 as encoded by a polynucleotide including a nucleotide sequence according to SEQ ID NO: 22, 24, 26, 28, 30, 32, 34, 35, 37 or 39.
- the carotenoid-producing host cell comprising (1 ) stereoselective BCOs as defined herein and (2) trans-acting ATFs, preferably ATF1 enzymes, used in a process for production of retinyl acetate with a percentage of at least 80% present as trans-isoforms in the retinyl acetate mix, said host cell further comprising selective retinol dehydrogenases (RDHs) catalyzing the reduction of retinal into retinol with a total conversion of at least 90% towards production of retinol.
- RDHs selective retinol dehydrogenases
- enzyme having retinal reducing activity or “RDH” are used interchangeably herein and refer to enzymes [EC 1.1.1.105] which nearly exclusively (90% or more) are capable of catalyzing the conversion of retinal into retinol, i.e. which are capable of catalyzing the conversion of retinal to retinol with a total conversion of at least about 90%, preferably 92, 95, 97, 98, 99 or even 100% towards retinol formation.
- any retinal reducing enzyme which results in an increase of at least about 18%, such as e.g. at least about 20, 30, 40, 50, 60, 70, 80, 90, 100% towards formation of retinol can be used in a process as defined herein, such increase being calculated on the retinol formation using endogenous RDHs present in suitable carotenoid-producing host cells, particularly fungal host cells, such as e.g. strains of Yarrowia or
- RDHs with activity towards retinol formation i.e. retinal reduction reaction
- any source such as e.g. plants, animals, including humans, algae, fungi, including yeast, or bacteria.
- Particular useful RDHs are obtained from fungi, in particular Dikarya, including but not limited to fungi selected from Ascomycota, preferably obtained from Fusarium, more preferably isolated from F. fujikuroi, such as e.g. FfRDH12 (SEQ ID NO: 19).
- the present invention is directed to a carotenoid- producing host cell, particularly a fungal host cell, used for biosynthesis of retinoids including vitamin A, said host cell comprising:
- a polypeptide with at least 60% such as e.g. 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% or up to 100% identity to a polypeptide selected from the group consisting of SEQ ID NOs: 1 , 3, 5 and 7 or a polypeptide with at least 50%, such as e.g. 55, 60, 65, 70, 75, 80, 85, 90, 93, 95, 97, 98, 99% or up to 100% identity to a polypeptide selected from SEQ ID NO: 9, 11 , 13, 15 or 17;
- polypeptide with at least 60%, such as e.g. 65, 70, 75, 80, 85, 90, 92, 95,
- polypeptide with at least 60% such as e.g. 65, 70, 75, 80, 85, 90, 95, 97,
- the carotenoid-producing host cell according to the present invention is used in a process for production of retinyl acetate with a percentage of at least 65% present as trans-isoforms, said host cell comprising (1 ) stereoselective or trans-selective BCOs as defined herein, (2) ATFs as defined herein, such as ATF1 , with preference for acetylation of trans-retinol, (3) RDHs with about 90% or more activity towards formation of retinol via reduction of retinal, optionally further comprising modifications in the endogenous acyltransferase activity, such as reduced or abolished
- acyltransferase As used herein, the terms “acyltransferase”, “retinol acylating enzyme”, “enzyme having retinol acylating activity” are used interchangeable herein and refer to enzymes which are capable of catalyzing the conversion of retinol into long chain retinyl esters. Suitable acylating enzymes might be selected from acyl-CoA:diacylglycerol acyltransferase family members [EC 2.3.1], including but not limited to DGATs [EC 2.3.1.20] such as e.g. DGAT1 or DGAT2, ARATs, mdy.
- the present invention is directed to a carotenoid- producing host cell, particularly a fungal host cell, used for biosynthesis of retinoids including vitamin A, said host cell comprising:
- a polypeptide with at least 60% such as e.g. 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% or up to 100% identity to a polypeptide selected from the group consisting of SEQ ID NOs: 1 , 3, 5 and 7 or a polypeptide with at least 50%, such as e.g. 55, 60, 65, 70, 75, 80, 85, 90, 93, 95, 97, 98, 99% or up to 100% identity to a polypeptide selected from SEQ ID NO:9, 11 , 13, 15 or 17;
- polypeptide with at least 60%, such as e.g. 65, 70, 75, 80, 85, 90, 92, 95,
- polypeptide with at least 60% such as e.g. 65, 70, 75, 80, 85, 90, 95, 97,
- Modification with regards to acylation activity in a process for production of retinoids using the carotenoid-producing host cell, particularly fungal host cell, as defined herein, means a reduction or abolishment of the endogenous gene(s) encoding acyltransferase activity, such that the activity of endogenous acyltransferases is reduced or abolished, preferably abolished, said host cell being capable or used for production of a retinyl acetate mix comprising at least about 65% in trans-isoform compared to a host cell expressing the respective endogenous acyltransferases prior to the modification of the host cell, i.e.
- the percentage of trans-isoforms can be increased to about 65% or more, preferably such as 68, 70, 75, 80, 85, 90, 95, 98 or up to 100% based on the total amount of retinyl esters.
- Reduction or abolishment of endogenous gene/protein activity might be achieved by, e.g. introducing mutation(s) into the endogenous gene(s) coding for enzymes having said activity, such as acyltransferase activity.
- the skilled person knows how to genetically manipulate a host cell as defined herein resulting in reduction or abolishment of such activity, e.g. acyltransf erase activity.
- These genetic manipulations include, but are not limited to, e.g. gene replacement, gene amplification, gene disruption, transfection, transformation using plasmids, viruses, or other vectors.
- 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 how to introduce mutations.
- Modifications in order to have the host cell as defined herein produce less or no copies of genes and/or proteins may include the use of weak promoters, or the mutation (e.g. insertion, deletion or point mutation) of (parts or) the respective enzymes (as described herein), in particular its regulatory elements.
- An example of such a genetic manipulation may for instance affect the interaction with DNA that is mediated by the N -terminal region of enzymes as defined herein or interaction with other effector
- modifications leading to reduced or abolished specific enzyme activity may be carried out in functional, such as functional for the catalytic activity, parts of the proteins.
- reduction or abolishment of enzyme specific activity might be achieved by contacting said enzymes with specific inhibitors or other substances that specifically interact with them.
- the respective enzymes such as e.g. the acylating enzymes as defined herein, may be expressed and tested for activity in the presence of compounds suspected to inhibit their activity.
- Modifications in order to have the host cell as defined herein produce more copies of genes and/or proteins may include the use of strong promoters, suitable
- transcriptional- and/or translational enhancers or the introduction of one or more gene copies into the carotenoid-producing host cell, particularly fungal host cell, leading to increased accumulation of the respective enzymes in a given time.
- the skilled person knows which techniques to use in dependence of the host cell.
- the increase or reduction of gene expression can be measured by various methods, such as e.g. Northern, Southern or Western blot technology as known in the art.
- conversion", “oxidation”, “reduction”, “acylation”, “acetylation” in connection with enzymatic catalysis of enzymes as defined herein are art- recognized and refer to actions of the enzymes towards formation /production of retinoids, in particular retinyl acetate.
- the enzymes used in a process for production of retinoids are expressed as heterologous enzymes. They might be integrated on suitable expression vectors or might be integrated in the chromosomal DNA.
- Such carotenoid-producing host cell particularly fungal host cell, comprising a heterologous polynucleotide either on an expression vector or integrated into the chromosomal DNA of the host cell encoding enzymes involved in retinoid production, in particular production of retinyl acetate as described herein, is called a recombinant host cell.
- the present invention is related to a carotenoid- producing host cell, particularly fungal host cell, carrying one or more (genetic) modifications as defined herein, to be used in a process for production of retinoids, in particular retinyl acetate with at least about 65-90% of the retinyl acetate in trans-isoform and wherein the percentage of acetylated retinol forms, i.e. retinyl esters, such as retinyl acetate, is about at least 10% based on the total amount of retinoids produced by said host cell.
- extracellular retinoids produced with a carotenoid-producing host cell as defined herein can be increased, in particular using a carotenoid-producing host cell which is selected from fungi including yeast, such as e.g. Yarrowia or
- a process as described herein leads to at least 80% of retinoids exported outside of the cell, such as e.g. 85, 90, 92, 95, 98, 99 or up to 100% of the retinoids, in particular retinyl acetate with preferably a percentage of about at least 80% in trans-isoform. This is in particular useful with regards to further isolation and purification steps.
- Suitable carotenoid-producing host cells used for the process as described herein might be selected from any (micro)organisms, which is suitable for
- carotenoid/ retinoid production and which allows expression of the nucleic acids encoding one of the enzymes as disclosed herein, including functional
- carotenoid/retinoid-producing host (micro)organisms are bacteria, algae, fungi, including yeasts, plant or animal cells.
- Preferred bacteria are those of the genera Escherichia, such as, for example, Escherichia coli, Streptomyces, Pantoea (Erwinia), Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, Mixococcus, Brevibacterium, Bradyrhizobium, Gordonia, Dietzia, Muricauda, Sphingomonas, Synochocystis, Paracoccus, such as, for example, Paracoccus zeaxanthinifaciens.
- Preferred eukaryotic such as, for example, Escherichia coli, Streptomyces, Pantoea (Erwinia), Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Coryn
- microorganisms in particular fungi including yeast, are selected from
- Saccharomyces such as Saccharomyces cerevisiae, Aspergillus, such as
- Pichia such as Pichia pastoris
- Hansenula such as Hansenula polymorpha
- Phycomyces such as Phycomyces blakesleanus, Mucor
- Rhodotorula Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, such as e.g. Blakeslea trispora, or Yarrowia, such as Yarrowia lipolytica.
- a fungal host cell such as e.g. Yarrowia or
- Saccharomyces or expression in Escherichia, more preferably expression in Yarrowia lipolytica or Saccharomyces cerevisiae.
- 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 polynucleotides as defined herein such as e.g. the polynucleotides encoding BCOs, RDHs, ATFs as defined herein, might be optimized for expression in the respective host cell.
- the skilled person knows how to generate such modified polynucleotides. It is understood that the polynucleotides as defined herein also encompass such host-optimized nucleic acid molecules as long as they still express the polypeptide with the respective activities as defined herein.
- the present invention is directed to a carotenoid- producing host cell, particularly fungal host cell, comprising polynucleotides encoding BCOs, ATFs, and/or RDHs as defined herein which are optimized for expression in said host cell, with no impact on growth of expression pattern of the host cell or the enzymes.
- a carotenoid-producing host cell is selected from Yarrowia, such as Yarrowia lipolytica, comprising optimized polynucleotide sequences selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 , 24, 26, 28, 30, 32 , 35 , 37 and 39 or sequences with at least 60%, such as e.g. 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity thereto.
- the present invention is directed to a process for production of a retinyl ester mix comprising retinyl acetate, preferably with a percentage of at least 65% a trans-retinyl acetate, via enzymatic activity of (1 ) stereospecific BCO as defined herein, comprising contacting beta-carotene with said BCO leading to a retinal mix with a percentage of at least 65%, such as e.g. at least 65-90%, of trans- retinal, and (2) one of the Atf1 enzymes as defined herein, comprising
- the invention is directed to a process for production of vitamin A, said process comprising (a) introducing a nucleic acid molecule encoding (1 ) one of the stereoselective BCO enzymes as defined herein and (2) one of the Atf1 enzymes as defined herein into a suitable carotenoid-producing host cell, particularly fungal host cell, as defined herein, (b) enzymatic conversion of beta-carotene into retinal, with at least about 65% of trans-retinal, enzymatic conversion, i.e. acetylation, of retinol, preferably with a percentage of at least 65-90% of trans-retinol, via action of said
- Atf1 into a mix of trans- and cis-retinyl acetate, and (3) conversion of said retinyl acetate into vitamin A under suitable conditions known to the skilled person.
- sequence identity in order to determine the percentage of sequence homology or 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 using the Needleman and Wunsch algorithm for the alignment of two sequences
- 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 using the 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.
- enzymes originated from plants as defined herein the skilled person knows plant-derived enzymes might contain a chloroplast targeting signal which is to be cleaved via specific enzymes, such as e.g.
- chloroplast processing enzymes CPEs
- the enzymes as defined herein also encompasses enzymes carrying amino acid substitution(s) which do not alter enzyme activity, i.e. which show the same properties with respect to the wild-type enzyme and catalyze the conversion of beta-carotene to retinal, retinal to retinol, retinol to retinyl acetate, in particular with a total conversion of at least about 65%, such as e.g. at least about 65-90%, towards production of trans-isoform of retinyl acetate.
- Such mutations are also called "silent mutations", which do not alter the (enzymatic) activity of the enzymes as described herein.
- a nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence provided by the present invention, for example a fragment which may be used as a probe or primer or a fragment encoding a portion of an enzyme as defined herein.
- the nucleotide sequence determined from the cloning of the genes encoding the BCOs, ATFs and/or RDHs as defined herein allows for the generation of probes and primers designed for use in identifying and/or cloning other homologues from other species.
- the probe/primer typically comprises substantially purified oligonucleotides which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, more preferably about 22 or 25, even more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequences described herein.
- Highly stringent conditions include, for example, 2 h to 4 days incubation at 42 °C using a digoxigenin (DIG)-labeled DNA probe (prepared by using a DIG labeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solution such as DigEasyHyb solution (Roche Diagnostics GmbH) with or without 100 Mg/ml salmon sperm DNA, or a solution comprising 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate, 0.1% N- lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics GmbH), followed by washing the filters twice for 5 to 15 minutes in 2x SSC and 0.1% SDS at room temperature and then washing twice for 15-30 minutes in 0.5x SSC and 0.1% SDS or 0.1x SSC and 0.1% SDS at 65-68°C.
- DIG digoxigenin
- the carotenoid-producing host cell particularly fungal host cell, as defined herein, which is able to express beta-carotene producing genes, the beta- carotene oxidases as described herein, the retinol acetylating enzymes as defined herein, the retinal reducing enzymes as defined herein, and/or optionally further genes required for biosynthesis of vitamin A, may be cultured in an aqueous medium supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person for the different host cells. Optionally, such cultivation is in the presence of proteins and/or co- factors involved in transfer of electrons, as defined herein.
- the carotenoid-producing host cell particularly fungal host cell, as defined herein, which is able to express beta-carotene producing genes, the beta- carotene oxidases as described herein, the retinol acetylating enzymes as defined herein, the retinal reducing enzymes as defined herein, and/or optionally
- cultivation /growth of the host cell may be conducted in batch, fed-batch, semi- continuous or continuous mode.
- production of retinoids such as e.g. vitamin A and precursors such as retinal
- retinol can vary, as it is known to the skilled person.
- Cultivation and isolation of beta-carotene and retinoid-producing host cells selected from Yarrowia is described in e.g. WO2008042338.
- methods are described in e.g. Jang et al, Microbial Cell Factories, 10:95 (2011 ).
- Specific methods for production of beta-carotene and retinoids in yeast host cells such as e.g. Saccharomyces cerevisiae, are disclosed in e.g. WO201 096992.
- the present invention is directed to a process for production of retinoids, in particular retinyl acetate with at least about 65% present in trans-isoform and a percentage of at least about 10% in acetylated form, i.e. as retinyl acetate based on the total amount of retinoids produced by the respecitve host cell, in a carotenoid-producing host cell under conditions as described herein.
- the produced retinoids, in particular retinyl acetate might be isolated and optionally further purified from the medium and/or host cell.
- 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 ⁇ 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.
- a suitable substrate within the host cell as defined herein or within a system in the presence of a suitable substrate.
- the skilled person knows how to measure enzyme activity, in particular activity of BCOs, RDHs or ATFs as defined herein.
- Analytical methods to evaluate the capability of a suitable enzyme as defined herein for retinoid production are known in the art, such as e.g. described in Example 4 of WO2014096992.
- titers of products retinoids and carotenoids and the like can be measured by HPLC.
- a carotenoid-producing host cell is a host cell, wherein the respective polypeptides are expressed and active in vivo leading to production of carotenoids, e.g. beta-carotene.
- carotenoids e.g. beta-carotene.
- the genes and methods to generate carotenoid-producing host cells are known in the art, see e.g. WO2006102342. Depending on the carotenoid to be produced, different genes might be involved.
- a retinoid-producing host cell particularly fungal host cell, is a host cell wherein, the respective polypeptides are expressed and active in vivo, leading to production of retinoids, e.g.
- the beta-carotene is converted into retinal via action of beta-carotene oxidizing enzymes, the retinal is further converted into retinol via action of RDHs as defined herein, and the retinol, preferably trans-retinol, is converted into retinol acetate via action of acetyl- transferase enzymes, such as e.g. ATF1.
- the retinol acetate might be the retinoid of choice which is isolated from the host cell.
- 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.
- Long chain retinyl esters as used herein define hydrocarbon esters that consists of at least about 8, such as e.g. 9, 10, 12, 13, 15 or 20 carbon atoms and up to about 26, such as e.g. 25, 22, 21 or less carbon atoms, with preferably up to about 6 unsaturated bonds, such as e.g.
- Long chain retinyl esters include but are not limited to linoleic acid, oleic acid or palmitic acid. Biosynthesis of retinoids is described in e.g. WO2008042338.
- 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
- 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. WO2006102342.
- Vitamin A as used herein may be any chemical form of vitamin A found in aqueous solutions, such as for instance undissociated, in its free acid form or dissociated as an anion.
- the term as used herein includes all precursors or intermediates in the biotechnological vitamin A pathway. It also includes vitamin A acetate.
- the present invention features the present embodiments:
- percentage of trans-retinal in the mix is at least about 65%, preferably 68, 70, 75, 80, 85, 90, 95, 98% or up to 100% produced by said host cell;
- an acetyl transferase [EC 2.3.1.84], preferably an enzyme with acetyl transferase 1 (Atf 1 ) activity, said enzyme catalyzing the conversion of retinol, preferably trans-retinol, to a retinyl acetate mix, with a percentage of at least 10% of acetylated retinol, i.e. retinyl acetate, based on the total amount of retinoids produced by said host cell.
- ATF acetyl transferase
- a carotenoid-producing host cell particularly fungal host cell, as above and defined herein, wherein the acetyl transferase [EC 2.3.1.84], preferably an enzyme with acetyl transferase 1 activity, catalyzes the conversion of retinol to a retinyl acetate mix, wherein the mix comprises at least about 65%, preferably 68, 70, 75, 80, 85, 90, 95, 98% or up to 100% retinyl acetate, such as e.g. at least 65-90% retinyl acetate, in trans-isoform.
- the carotenoid-producing host cell particularly fungal host cell, as above and defined herein, further comprising a (preferably heterologous) retinol
- RDH dehydrogenase
- fungi in particular Dikarya, including but not limited to fungi selected from Ascomycota, more preferably obtained from Fusarium, even more preferably isolated from F. fujikuroi, such as a polypeptide with at least about 60% identity to FfRDH12 (SEQ ID NO: 19).
- the carotenoid-producing host cell particularly fungal host cell, as above and defined herein, furthermore comprising a modification in the endogenous acyltransferase activity, wherein the endogenous acyltransferase activity, preferably [EC 2.3.1 ] activity, more preferably acyltransferase [EC 2.3.1.20] activity, has been reduced or abolished.
- the carotenoid-producing host cell particularly fungal host cell, as above and defined herein, wherein the BCO is selected from fungi, plants or animal, preferably selected from Fusarium, Ustilago, Crocus, Drosophila, Danio, lctalurus, Esox, Latimeria, more preferably selected from Fusarium fujikuroi, Ustilago maydis, Crocus sativus, Drosophila melanogaster, Danio rerio, lctalurus punctatus, Esox lucius, Latimeria chalumnae even more preferably selected from a polypeptide with at least about 60% identity to a polypeptide according to SEQ ID NOs:1 , 3, 5 or 7 or a polypeptide with at least about 50% identity to a polypeptide sequence according to SEQ ID NOs:9, 11 , 13, 15 or 17.
- the carotenoid-producing host cell particularly fungal host cell, as above and defined herein, wherein the acetyl transferase, preferably Atf1 , is selected from plants, animals, including humans, algae, fungi, including yeast or bacteria, preferably selected from Saccharomyces, Fragaria, Escherichia, Euonymus, Malus, Petunia or Lachancea, more preferably selected from Saccharomyces bayanus, Fragaria ananassa, Escherichia coli, Euonymus alatus, Malus domestica, Petunia hybrida, Lachancea mirantina or Lachancea fermentati, even more preferably selected from a polypeptide with at least about 60% identity to a polypeptide according to SEQ ID NOs:21 , 23, 25, 27, 29, 31 , 33, 36, or 38.
- the carotenoid-producing host cell particularly fungal host cell, as above and defined herein, producing a retinyl acetate mix comprising at least about 65%, preferably 68, 70, 75, 80, 85, 90, 95, 98% or up to 100% trans-retinyl acetate isoform, such as at least 65-90% trans-retinyl acetate isoform.
- plants, fungi, algae or microorganisms preferably selected from Escherichia, Streptomyces, Pantoea, Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, Mixococcus, Brevibacterium, Bradyrhizobium, Gordonia, Dietzia, Muricauda, Sphingomonas
- the carotenoid-producing host cell particularly fungal host cell, as above and defined herein used in a process for conversion of beta-carotene into vitamin A.
- a process for production of trans-retinyl acetate comprising cultivation of the carotenoid-producing host cell, particularly fungal host cell, as above and defined herein in an aqueous medium under suitable culture conditions and isolating and optionally further purifying said trans-retinyl acetate from the medium and/or host cell.
- Example 1 General Methods, strains, and plasmids All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998).
- Shake plate assay Typically, 800 ⁇ of 0.075% Yeast extract, 0.25% peptone (0.25X YP) is inoculated with 10 ⁇ of freshly grown Yarrowia and overlaid with 800 ⁇ of mineral oil (Drakeol 5, Penreco Personal Care Products, Karns City, PA, USA) carbon source 5% corn oil in mineral oil and/or 5% in glucose in aqueous phase. Transformants were grown in 24 well plates (Microplate Devices 24 Deep Well Plates Whatman 7701 -5102), covered with mat seal (Analytical Sales and Services Inc.
- mineral oil Drakeol 5, Penreco Personal Care Products, Karns City, PA, USA
- MB5082 'URA3' marker could be reused due to gratuitous repeated flanking sequences that enable selection of circular excisants of the URA3 cassette on FOA.
- the NatR and HygR markers can be removed by transient expression of Cre recombinase that results in excisants due to the flanking Lox sites.
- Plasmid list Plasmid, strains, nucleotide and amino acid sequences to be used are listed in Table 1 , 2 and the sequence listing. Nucleotide sequence ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 35, 37, and 39 are codon optimized for expression in Yarrowia.
- Table 1 list of plasmids used for construction of the strains carrying the heterologous BCO, RDH and ATF1 -genes.
- the sequence ID NOs refer to the inserts. For more details, see text.
- Table 2 list of Yarrowia strains used for production of retinoids carrying the heterologous BCO, RDH and ATF1 -genes. For more details, see text.
- Normal phase retinol method A Waters 1525 binary pump attached to a Waters 717 auto sampler were used to inject samples. A Phenomenex Luna 3 ⁇ Silica (2), 150 x 4.6 mm with a security silica guard column kit was used to resolve retinoids.
- the mobile phase consists of either, 1000 mL hexane, 30 mL isopropanol, and 0.1 mL acetic acid for astaxanthin related compounds, or 1000 mL hexane, 60 mL isopropanol, and 0.1 mL acetic acid for zeaxanthin related compounds. The flow rate for each is 0.6 mL per minute. Column temperature is ambient. The injection volume is 20 ⁇ . The detector is a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 3.
- Table 3 list of analytes using normal phase retinol method. The addition of all added intermediates gives the amount of total retinoids. For more details, see text.
- Sample preparation Samples were prepared by various methods depending on the conditions. For whole broth or washed broth samples the broth was placed in a Precellys® tube weighed and mobile phase was added, the samples were processed in a Precellys® homogenizer (Bertin Corp, Rockville, MD, USA) on the highest setting 3X according to the manufactures directions. In the washed broth the samples were spun in a 1.7 ml tube in a microfuge at lOOOOrpm for 1 minute, the broth decanted, 1 ml water added mixed pelleted and decanted and brought up to the original volume the mixture was pelleted again and brought up in appropriate amount of mobile phase and processed by Precellys® bead beating.
- the sample was spun at 4000RPM for 10 minutes and the oil was decanted off the top by positive displacement pipet (Eppendorf, Hauppauge, NY, USA) and diluted into mobile phase mixed by vortexing and measured for retinoid concentration by HPLC analysis.
- positive displacement pipet Eppendorf, Hauppauge, NY, USA
- Fermentation conditions Fermentations were identical to the previously described conditions using preferably a silicone oil or a mineral oil overlay and stirred tank that was preferably glucose or corn oil fed in a bench top reactor with 0.5L to 5L total volume (see WO2016172282). Generally, the same results were observed with a fed batch stirred tank reactor with an increased
- a beta carotene strain ML17544 was transformed with purified linear DNA fragment by Hindi I and Xbal mediated restriction endonucleotide cleavage and gel purification of beta carotene oxidase (BCO) containing codon optimized fragments linked to a URA3 nutritional marker.
- BCO beta carotene oxidase
- Transforming DNA were derived from MB6702 Drosophila NinaB BCO gene, MB6703 Crocus BCO gene, MB8456 Fusarium BCO gene, and MB8457
- Ustilago BCO gene and MB6098 Dario BCO gene whereby the codon-optimized sequences (SEQ ID NOs:2, 4, 6, 8, 10, 12) had been used.
- the genes were then grown screening 6-8 isolates in a shake plate analysis, and isolates that performed well were run in a fed batch stirred tank reaction for 8-10 days.
- Detection of cis-and trans-retinal was made by HPLC using standard parameters as described in WO2014096992, but calibrated with purified standards for the retinoid analytes.
- the amount of trans-retinal in the retinal mix could be increased to 90% (using the Crocus BCO), 95% (using the Fusarium BCO), 98% (using the Ustilago BCO) and 98% (using Dario BCO), respectively.
- a comparison with the BCO from Drosophila melanogaster SEQ ID NO:7) resulted in 61% of trans-retinal based on the total amount of retinal (see Table 4).
- the beta carotene strain ML17767 was transformed with purified HinDIII/Xbal fragments derived from plasmids containing retinol dehydrogenase (RDH) gene fragments linker to a URA3 promoter. Six to eight isolates were screened for a decrease in the
- retinol retinal ratio in a shake plate assay and successful isolates were run in a fed batch stirred tank reactor for eight days which showed an order of magnitude increase in the productivity of the process which indicates a utility in large scale production.
- Example 4 Conversion of retinol to retinyl acetate in Yarrowia lipolytica
- the trans retinol producing strain ML17968 was transformed with purified Pvull gene fragments containing acetyltransferase gene fragments linked to a Hygromycin resistance marker (HygR) for selection rich media (YPD) containing 100ug/ml hygromycin.
- HygR Hygromycin resistance marker
- YPD selection rich media
- Isolates were screened for acylation in shake plate assays and successful isolates were screened in fed batch stirred tank reactor which showed an order of magnitude increased productivity indicating utility in the production of retinoids. The data from the analysis are shown in Table 5).
- heterologous ATF1 the trans retinol producing strain ML17968 was transformed with purified Pvull gene fragments containing acetyltransferase gene fragments linked to a Hygromycin resistance marker (HygR) for selection rich media (YPD) containing 100ug/ml hygromycin. Prior to plating the cultures were outgrown in YPD for four hours to synthesize the antibiotic resistance genes.
- HygR Hygromycin resistance marker
- YPD selection rich media
- Isolates were screened for acylation in shake plate assays, specifically using 10% glucose as a carbon source in 0.25X YP with silicone oil as an overlay and successful isolates were further screened in fed batch stirred tank reactor with glucose feed and silicone oil overlay, which showed an order of magnitude increased productivity indicating utility in the production of retinoids.
- the data from the analysis are shown in Table 5.
- a beta carotene strain is transformed with heterologous genes encoding for enzymes such as geranylgeranyl synthase, phytoene synthase, lycopene synthase, lycopene cyclase constructed that is producing beta carotene according to standard methods as known in the art (such as e.g. as described in US20160130628 or WO2009126890).
- beta carotene oxidase genes retinal can be produced.
- retinol dehydrogenase then retinol can be produced.
- the retinol can be acetylated by transformation with genes encoding alcohol acetyl transferases.
- the endogenous retinol acylating genes can be deleted.
- the enzymes can be selected to produce and acylate the trans form of retinol to yield all trans retinyl acetate, and long chain esters of trans retinol,
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