Novel acetyl transferase

Abstract

The present invention relates to the production of retinol acetate produced via the enzymatic conversion of retinol, said method comprising the use of a modified enzyme having improved activity.

Description

[0001] The present invention relates to the production of retinyl acetate via the enzymatic conversion of retinol, the method comprising the use of modified enzymes with improved activity.

[0002] Retinyl acetate is an important intermediate or precursor in the production of retinoids, particularly vitamin A. Retinoids, including vitamin A, are essential nutrients for humans and animals that must be provided through the diet. Retinoids promote health, particularly in the areas of vision, the immune system, and growth.

[0003] Current chemical production methods for retinoids, particularly vitamin A and its precursors, have some undesirable characteristics, such as high energy consumption, complex purification steps and / or undesirable by-products. Therefore, over the past few decades, other methods for producing retinoids, particularly vitamin A and its precursors, including microbial conversion steps, have been investigated, which would be more economical and ecologically sound.

[0004] Typically, biological systems that produce retinoids are industrially intractable and / or produce the compounds at such low levels that their isolation on an industrial scale is not economically viable. This is due to several reasons, including the instability of retinoids in such biological systems or the relatively high production of by-products.

[0005] The acetylation of carotenoids (such as astaxanthin or zeaxanthin) by the action of Atf1 from Pasteurella has been previously reported (WO2014096992), where for example the acetylation of zeaxanthin is in the range of up to 90%. However, these acetyltransferases generally have different substrate specificities for different alcohol substrates, which is determined by the local structural environment of the alcohol functional group on the molecule to be acetylated. For example, the hydroxyl group to be acetylated in carotenoids (such as zeaxanthin) is located on the β-ionone ring structure, while the hydroxyl group to be acetylated in retinol is not located on the ionone ring structure, but at the other end on the CH2 carbon at the end of the polyene chain of the molecule. Due to this different local molecular context of the acetylated hydroxyl group, it is difficult to predict the acetylation of retinol from the acetylation data of carotenoids.

[0006] For the acetylation of retinoids, it was found that the enzyme (i.e., acetyltransferase) derived from Lachancea, particularly Lachanceae mirantina (i.e., LmATF1) is particularly useful for the acetylation of retinol to retinol acetate. Using wild-type LmATF in retinol-producing Yarrowia lipolytica strains, up to 40% by weight of retinol acetate (based on total retinoids) can be obtained. By substituting certain amino acids, increases of more than 80% by weight have been achieved (see WO2020141168).

[0007] However, in order to use this enzymatic process on an industrial scale, the production efficiency of retinyl acetate must be further improved.

[0008] Surprisingly, we can now identify amino acid positions in the fungal acetyltransferases, particularly ATF enzymes, derived from Lachancea mirantina disclosed in WO2019058001 that are crucial for the formation of acetylated retinoids, in particular the conversion of retinol into retinol acetate. Compared to the corresponding wild-type enzyme, modification of certain amino acids results in increased formation of retinol acetate, for example, by at least about 10-20% when compared to the acetylation of retinol using the corresponding unmodified enzyme (e.g., wt-ATF1 from Lachancea mirantina disclosed in WO2019058001). In particular, the percentage of retinol acetate can be even further increased compared to the best enzymes known to date (as disclosed in WO2020141168).

[0009] In particular, the present invention relates to modified enzymes and methods for producing the modified enzymes, which participate in the acetylation of retinol to retinyl acetate in a suitable retinol-producing host cell, wherein the percentage of retinyl acetate based on total retinoids is at least about 81%, in particular, in the presence of a residue corresponding to SEQ ID NO: 1 ( Figure 1 ) or SEQ ID No: 3( Figure 2 ) a fungal enzyme comprising one or more modifications, such as amino acid substitutions, in a sequence having at least about 20% (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 at a position corresponding to an amino acid residue selected from the group consisting of position 68, 451, 452, 473, 483, 512, and combinations thereof in a polypeptide according to SEQ ID No: 1, in particular comprising at least one amino acid substitution at a position corresponding to A451, T473 and / or L483 in a polypeptide according to SEQ ID No: 1, wherein upon introduction of said amino acid substitutions, the percentage of retinyl acetate is increased by at least 10-20% by weight compared to a method using the same conditions but using the corresponding or corresponding wild-type enzyme, including an ATF enzyme according to SEQ ID NO: 1.

[0010] This modified enzyme is used in a method for producing retinoids, wherein the modified enzyme is expressed (particularly heterologously expressed) in a suitable host cell, particularly a fungal host cell capable of producing retinol, resulting in an increase of at least about 10% in retinyl acetate based on the total retinoids present / produced in the modified host cell compared to a method using the same conditions but using the ATF enzyme according to SEQ ID NO: 1.

[0011] The terms "acetyltransferase," "retinol acetylase," "enzyme having retinol acetylation activity," "ATF," or "ATF1" are used interchangeably herein and refer to enzymes of EC class [EC 2.3.1.84] capable of catalyzing the conversion of retinol to retinol acetate, particularly wherein the acetylated form comprises about 30 to 90 weight percent of the total retinoid, including naturally occurring enzymes and enzymes produced synthetically with the aid of artificial intelligence. Such enzymes as used herein are referred to as "unmodified" ATF. Examples of such unmodified enzymes are shown in SEQ ID NO: 1 or 3, for example, isolated from or derived from Figure 1 or the enzyme (LmATF1) of Lachanceamirantina shown in 2 .

[0012] A "modified" ATF as defined herein, in particular a "modified" ATF based on an "unmodified" ATF, e.g. an enzyme having at least about 20% identity to SEQ ID NO: 1 or SEQ ID NO: 3, shows an increase, e.g., in particular an increase of at least about 10%, in the formation of retinyl acetate from retinol, based on total retinoids and compared to the use of the enzyme according to SEQ ID NO: 1.

[0013] Suitable unmodified enzymes include enzymes having at least about 20% (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: 1 or 3 (including enzymes isolated / derived from Lachancea mirantina), is an enzyme obtainable from a fungal enzyme comprising a highly conserved partial amino acid sequence of at least 7 amino acid residues selected from NHx(3)-D-[GA] (motif in Prosite syntax, as defined in https: / / prosite.expasy.org / scanprosite / scanprosite_doc.html), wherein "x" represents any amino acid and the central histidine is part of the binding pocket of the enzyme, preferably wherein the 7 amino acid motif is selected from NHCSSDG, NHCLCDG or NHILKDG, more preferably selected from NHCSSDG corresponding to positions N218 to G224 in the polypeptide according to SEQ ID NO: 1.

[0014] The modified ATF as defined herein is capable of converting retinol into retinol acetate, in particular with a conversion rate increased by at least about 10% compared to the conversion of retinol into retinol acetate using the unmodified enzyme according to SEQ ID NO: 1, and can be obtained, for example, by expressing the modified ATF under suitable culture conditions, including but not limited to cultivation on glucose, galactose, xylose with or without combination with ethanol.

[0015] The enzymes as defined herein are used to convert retinol to retinol acetate, wherein the substrate (i.e., retinol) can be cis-, trans-, or a mixture of cis- / trans-retinol in any possible ratio. Preferably, the retinol mixture used as the substrate has a high percentage of trans-retinol, for example, at least about 65 to 98% by weight of the trans isomer based on the total retinol in the host cell. Acetylation of the retinol mixture of at least about 65-98% by weight of trans-retinol will result in retinol acetate having approximately the same ratio of trans- to cis-retinol acetate based on the total retinol acetate produced by the host cell.

[0016] In a specific embodiment, the present invention relates to the conversion of retinol to retinol acetate using a suitable host cell as defined herein, said host cell comprising and expressing a modified enzyme as defined herein, wherein the retinol is a mixture of trans- and cis-retinol, and wherein the percentage of trans-retinol based on total retinol is in the range of at least about 65 to 98 weight %.

[0017] The terms "conversion," "enzymatic conversion," "acetylation," or "enzymatic acetylation" in connection with the enzymatic catalysis of retinol are used interchangeably herein and refer to the action of a modified or unmodified ATF in catalyzing the conversion of retinol to retinol acetate, resulting in a percentage of retinol acetate based on total retinoids present / produced by a suitable host cell when expressing the ATF, wherein an increase of at least 10 weight percent retinol acetate based on total retinoids can be achieved using a modified ATF as defined herein.

[0018] Suitable host cells according to the present invention include fungal host cells and cells from Escherichia coli. As used herein, the term "fungal host cell" particularly includes yeast cells, wherein the cell is a retinol-producing host cell, particularly a retinol acetate-producing host cell, such as a retinol acetate-producing fungal host cell, including but not limited to a host cell of the genus Yarrowia or Saccharomyces, such as a host cell of Yarrowia lipolytica or Saccharomyces cerevisiae.

[0019] The modified ATF enzyme can be used in an isolated form (e.g., in a cell-free system) or can be expressed in a suitable host cell, for example, in a host cell producing retinol, particularly in a fungal host cell as defined herein. The enzyme can be expressed as an endogenous enzyme or a heterologous enzyme. Preferably, the modified enzyme as described herein is introduced as a heterologous enzyme and expressed in a suitable host cell, for example, in a host cell producing retinol, particularly in a fungal host cell as defined herein.

[0020] In one embodiment, the modified ATF enzyme as defined herein for producing retinyl acetate as defined herein comprises an amino acid substitution at a position corresponding to residue 68 in the polypeptide according to SEQ ID NO: 1 or 3, resulting in a leucine at said residue, for example, by replacing glutamine with leucine (Q68L). The modified enzyme may be derived from Lachancea, such as L. mirantina, L. Fermentati, preferably from L. mirantina. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase of at least about 10%, e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or more, based on total retinoids from retinol acetylation, compared to a corresponding process using an enzyme according to SEQ ID NO: 1. Furthermore, the mutations may be combined with additional mutations as defined herein, e.g. in particular with one or more amino acid substitutions at positions corresponding to residues 451 and / or 452 and / or 473 and / or 483 and / or 512 in the polypeptide according to SEQ ID NO: 1 or 3.

[0021] In one embodiment, the modified ATF enzyme as defined herein for producing retinyl acetate as defined herein comprises an amino acid substitution at a position corresponding to residue 451 in a polypeptide according to SEQ ID NO: 1 or 3, resulting in a leucine or methionine at said residue, e.g., via substitution of alanine with leucine (A451L) or substitution of alanine with methionine (A451M). The modified enzyme may be derived from Lachancea, e.g., L. mirantina, L. Fermentati, preferably from L. mirantina. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase of at least about 20%, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, 200%, 220%, 250%, 280%, 300%, 350%, 400%, 450%, 500% or more, based on total retinoids from retinol acetylation, compared to a corresponding process using an enzyme according to SEQ ID NO: 1. Furthermore, the mutations may be combined with additional mutations as defined herein, e.g. in particular with one or more amino acid substitutions at positions corresponding to residues 68 and / or 452 and / or 473 and / or 483 and / or 512 in the polypeptide according to SEQ ID NO: 1 or 3.

[0022] In one embodiment, the modified ATF enzyme as defined herein for producing retinyl acetate as defined herein comprises an amino acid substitution at a position corresponding to residue 452 in a polypeptide according to SEQ ID NO: 1 or 3, resulting in a phenylalanine at said residue, e.g., via substitution of leucine with phenylalanine (L452F). The modified enzyme may be derived from Lachancea, e.g., L. mirantina, L. Fermentati, preferably from L. mirantina. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase of at least about 20%, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, 200%, 220%, 250%, 280%, 300%, 350%, 400%, 450%, 500% or more, based on total retinoids from retinol acetylation, compared to a corresponding process using an enzyme according to SEQ ID NO: 1. Furthermore, the mutations may be combined with additional mutations as defined herein, e.g. in particular with one or more amino acid substitutions at positions corresponding to residues 68 and / or 451 and / or 473 and / or 483 and / or 512 in the polypeptide according to SEQ ID NO: 1 or 3.

[0023] In one embodiment, the modified ATF enzyme as defined herein for producing retinyl acetate as defined herein comprises an amino acid substitution at a position corresponding to residue 473 in a polypeptide according to SEQ ID NO: 1 or 3, resulting in a leucine or alanine at said residue, e.g., via substitution of threonine with leucine (T473L) or substitution of threonine with alanine (T473A). The modified enzyme may be derived from Lachancea, e.g., L. mirantina, L. Fermentati, preferably from L. mirantina. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase of at least about 20%, e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, 200%, 220%, 250%, 280%, 300%, 350%, 400%, 450%, 500% or more, based on total retinoids from retinol acetylation, compared to a corresponding process using an enzyme according to SEQ ID NO: 1. Furthermore, the mutations may be combined with additional mutations as defined herein, e.g. in particular with one or more amino acid substitutions at positions corresponding to residues 68 and / or 451 and / or 452 and / or 483 and / or 512 in the polypeptide according to SEQ ID NO: 1 or 3.

[0024] In one embodiment, the modified ATF enzyme as defined herein for producing retinyl acetate as defined herein comprises an amino acid substitution at a position corresponding to residue 512 in the polypeptide according to SEQ ID NO: 1 or 3, resulting in a phenylalanine at said residue, for example, by replacing asparagine with phenylalanine (N512F). The modified enzyme may be derived from Lachancea, such as L. mirantina, L. Fermentati, preferably from L. mirantina. The use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase of at least about 20%, for example 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150% or more of retinyl acetate based on total retinoids from retinol acetylation compared to a corresponding process using an enzyme according to SEQ ID NO: 1. In addition, the mutations can be combined with additional mutations as defined herein, for example, in particular with one or more amino acid substitutions at positions corresponding to residues 68 and / or 451 and / or 452 and / or 473 and / or 483 in a polypeptide according to SEQ ID NO: 1 or 3.

[0025] Host cells as described herein are capable of converting retinol to retinol acetate with an increase in conversion rate of at least about 10-20% or more compared to conversion via an enzyme according to SEQ ID NO: 1, particularly an increase in the production of retinol acetate in the range of 10% to 50% or more (based on the total amount of retinoids produced by the host cell), such as can be obtained by expressing a modified ATF under suitable culture conditions, including but not limited to culture on xylose, glucose, galactose, and a suitable host cell in the presence or absence of ethanol, such as fungal host cells (including Yarrowia or Saccharomyces) or other microbial host cells (e.g., E. coli). Suitable conditions can be culture in a fed-batch fermentation for, for example, 80, 90, 100, 110, 120, 130 hours.

[0026] The modified host cell as defined herein comprises one or more copies of a modified ATF as defined herein, preferably wherein the ATF is heterologously expressed in said modified host cell. In order for the host cell as defined herein to produce more copies of a gene and / or protein, e.g. more copies of a modified ATF as defined herein having selectivity for forming retinyl acetate, modifications (including with the use of a modified ATF according to SEQ ID NO: 1 ( Figure 1 ) compared to a method for producing LmATF1 based on the total amount of retinoid produced by the host cell, the conversion rate towards the production of retinyl acetate is increased by at least about 10-20%, such as can be obtained by expressing the modified ATF under appropriate culture conditions, including but not limited to culture on xylose, glucose, galactose in the presence or absence of ethanol.) This can include the use of a strong promoter, suitable transcription and / or translation enhancers, or the introduction of one or more gene copies into a retinol-producing host cell, particularly a fungal host cell, resulting in increased accumulation of the corresponding enzyme within a given time. The skilled person will know which technique to use depending on the host cell. The increase or decrease in gene expression can be measured by various methods, such as Northern, Southern or Western blotting techniques known in the art.

[0027] The generation of mutations in nucleic acids or amino acids (i.e., mutagenesis) can be performed in different ways, for example by random or lateral mutagenesis, physical damage caused by an agent such as radiation, chemical treatment or insertion of a genetic element. The skilled person knows how to introduce mutations.

[0028] Therefore, the present invention relates to production retinol host cells, particularly fungal host cells as described herein, which comprise expression vectors or polynucleotides encoding modified ATF as described herein, which have been integrated into the chromosomal DNA of the host cell. This type of production retinol host cells, particularly fungal host cells, which are included in the expression vector or are integrated into the chromosomal DNA of the modified ATF encoding as described herein, are referred to as recombinant or modified host cells. The host cell producing retinol, particularly fungal host cells, can contain one or more copies of the gene encoding the modified ATF as defined herein, which comprises a sudden change as defined herein, resulting in the overexpression of this type of gene encoding the modified ATF, particularly Atf1 as defined herein. The increase in gene expression can be measured by various methods, such as Northern, Southern or Western blotting techniques known in the art.

[0029] The present invention particularly relates to the use of this novel modified ATF enzyme in a method for producing retinyl acetate, particularly under conditions where the amount of other retinyl esters, particularly long-chain retinyl esters, is reduced. The skilled person is aware of how to create such conditions (see, for example, WO2021136689 or WO2022090548). Retinyl acetate can be further converted to vitamin A by the action of (known) suitable chemical or biotechnological mechanisms.

[0030] The terms "sequence identity" and "% identity" are used interchangeably herein. For the purposes of the present invention, it is defined herein that in order to determine the sequence identity percentage of two amino acid sequences or two nucleic acid sequences, sequences are aligned for optimal comparison purposes. In order to optimize the comparison between the two sequences, a gap can be introduced in any of the two sequences being compared. This comparison can be carried out over the full length of the compared sequence. Alternatively, the comparison can 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 on the reported comparison region. The sequence identity percentage between two amino acid sequences or between two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for comparing the two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be compared by an algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS software package (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 / ) is used. For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5. The skilled person will understand that all of these different parameters will produce slightly different results, but the overall percent identity of the two sequences will not change significantly when different algorithms are used.

[0031] After alignment by the program NEEDLE as described above, the percentage of sequence identity between the query sequence and the sequence of the invention is calculated as follows: the number of corresponding positions in the alignment showing the same amino acid or the same nucleotide in the two sequences divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is labeled "longest identity" in the program's output. Two amino acid sequences are identical or have 100% identity if they do not differ in any of their amino acids.

[0032] Modified ATF enzymes as defined herein also include enzymes carrying additional amino acid substitutions that do not change the enzymatic activity, i.e., enzymes that demonstrate the same properties as the enzymes defined herein and catalyze the conversion of retinol described herein into retinol acetate. Such mutations are also referred to as "silent mutations," which do not change the (enzymatic) activity of the enzyme according to the present invention.

[0033] The expression of enzyme / polynucleotide encoding one of the modified enzymes as defined herein can be achieved in any host system, including (microorganism) organisms, which are suitable for retinoid (including retinol) production and allow expression of nucleic acids encoding one of the enzymes as described herein, including functional equivalents or derivatives as described herein. Examples of suitable host (microorganism) organisms for producing retinol are bacteria, algae, fungi (including yeast), plants or animal cells. Preferred bacteria are those of the genus Escherichia, such as Escherichia coli, Streptomyces, Pantoea (Erwinia), Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, Mixococcus (Mixococcus), Brevibacterium, Bradyrhizobium, Gordonia, Dill, Murine Tau, Sphingomonas, Synechocystis (Synochocystis), Paracoccus, such as Paracoccus zeaxanthinifaciens. Preferred eukaryotic microorganisms, in particular fungi including yeast, are selected from the group consisting of: Saccharomyces, such as Saccharomyces cerevisiae; Aspergillus, such as Aspergillus niger; Pichia, such as Pichia pastoris; Hansenula, such as Hansenula polymorpha; Kluyveromyces, such as Kluyveromyces lactis; Phycomyces, such as Phycomyces blakesleanus; Mucor; Rhodotorula; Sporoblosarcoma; Phaffia; Phaffia; Blakesleatrispora; or Yarrowia, such as Yarrowia lipolytica. Particularly preferred is expression in a fungal host cell (e.g., Yarrowia or Saccharomyces), or expression in Escherichia, more preferably in Yarrowia lipolytica or Saccharomyces cerevisiae.

[0034] Depending on the host cell, the polynucleotides for retinol acetylation as defined herein may be optimized for expression in the corresponding host cell. A skilled artisan will appreciate how to generate such further modified polynucleotides. It will be understood that the polynucleotides as defined herein also encompass such host-optimized nucleic acid molecules, as long as they still express a polypeptide having the corresponding activity as defined herein.

[0035] Thus, in one embodiment, the present invention relates to a retinol-producing host cell, in particular a fungal host cell, comprising a polynucleotide encoding a modified ATF enzyme as defined herein, which is optimized for expression in said host cell and which is used for the production of retinol acetate. In particular, the retinol-producing host cell, in particular a fungal host cell, is selected from a yeast, such as a Yarrowia or Saccharomyces, such as Saccharomyces cerevisiae or Yarrowia lipolytica, wherein the polynucleotide encoding the modified ATF enzyme as defined herein is selected from a polynucleotide expressing a modified polypeptide comprising one or more amino acid substitutions in a sequence having at least 20% (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: 1 or 3, for example introducing one or more amino acid substitutions at a position as defined herein corresponding to a residue selected from positions 68, 451, 452, 473, 483, 512 and combinations thereof, in particular corresponding to a residue in a sequence according to SEQ ID NO: 1 ( Figure 1 ) in a polypeptide of the present invention, wherein after introduction of said amino acid substitution, the percentage of retinol acetate is increased by at least 10-20% by weight compared to a method using the same conditions but the corresponding or corresponding wild-type enzyme (including the ATF enzyme according to SEQ ID NO: 1), and preferably comprises a highly conserved partial amino acid sequence, i.e. a common active site or a "Prosite-motif" of at least 7 amino acid residues selected from NHx(3)-DG corresponding to positions N218 to G224 in the polypeptide according to SEQ ID NO: 1 (said motif is defined in Prosite syntax as defined in https: / / prosite.expasy.org / scanprosite / scanprosite_doc.html) and wherein "x" represents any amino acid, said host cell produces retinol acetate, and is similar to expressing the polypeptide according to SEQ ID NO: 1. The host cell of the enzyme NO:1 increases by at least about 10%, and the unmodified enzyme is, for example, obtainable by expressing the modified ATF under suitable culture conditions, including but not limited to culture on glucose, galactose or xylose.

[0036] In connection with the present invention, it is understood that organisms (e.g., microorganisms, fungi, algae, or plants) also include synonyms or substantial synonyms 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). Thus, for example, strain Lachancea mirantina is a synonym for strain Zygosaccharomyces sp. IFO 11066 originating from Japan.

[0037] The present invention relates to a method for producing retinyl acetate, wherein retinyl acetate is produced by acetylation of retinol as disclosed herein (particularly at least 65% trans-retinol) by the action of a modified ATF enzyme as described herein, wherein the acetylase is preferably heterologously expressed in a suitable host cell under suitable conditions as described herein. The retinyl acetate produced can be isolated from the culture medium and / or the host cell and optionally further purified. The acetylated retinoids defined herein can be used as building blocks in a multi-step process for producing vitamin A. As known in the art, vitamin A can be isolated from the culture medium and / or the host cell and optionally further purified.

[0038] Preferably, acetylation of retinol by using a modified ATF as described herein can result in an increase in the titer of retinol acetate, for example, at least about 50 to 92% by weight of retinol acetate based on total retinol, i.e., based on the total retinoids present in the retinoid mixture produced by the host cell, the percentage of acetylated retinoid (i.e., retinol acetate) is in the range of at least about 50% to 92%, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more, such as can be obtained by expressing the modified enzyme under suitable culture conditions, including but not limited to culture on glucose, galactose or xylose. In a more preferred embodiment, a retinol mixture having at least about 65% trans-retinol percentage is used as a substrate for acetylation via a modified enzyme as defined herein.

[0039] Host cells (i.e., microorganisms, algae, fungi, animals, or plants) capable of producing retinol may also be capable of producing β-carotene, which can be further enzymatically converted to retinal, which can be further converted to retinol. The skilled person knows which genes are used / expressed for the biosynthesis of β-carotene and / or the bioconversion of β-carotene to retinol. Such host cells, further capable of expressing modified ATFs as defined herein and / or other genes required for vitamin A biosynthesis, can be cultured in an aqueous medium supplemented with appropriate nutrients under aerobic or anaerobic conditions, as known to those skilled in the art for corresponding retinol-producing host cells. Optionally, such cultivation is carried out in the presence of proteins and / or cofactors involved in electron transfer, as known in the art. Suitable carbon sources for the purposes of the present invention may be selected from glucose, fructose, raffinose, lactose, galactose, glycerol, xylose, arabinose, sucrose, or maltose (in the presence or absence of ethanol), in particular from glucose, galactose, or xylose. Specific culture conditions may include batch and fed-feed operation, with a glucose concentration of 5% (w / v) and an ethanol concentration of 1% (w / v) in the batch phase and a concentration of 100% (w / v) in the fed phase. The host cell culture / growth can be carried out in batch, fed-batch, semi-continuous, or continuous mode under appropriate culture conditions, in particular in fed-batch mode for 80, 90, 100, 110, 120, or 130 hours. Depending on the host cell, the production of retinoids (e.g., vitamin A), their precursors and / or derivatives (e.g., retinal, retinol, retinyl esters, in particular retinyl acetate) may vary, as known to those skilled in the art. The cultivation and isolation of host cells selected from the genera Yarrowia and Saccharomyces for producing β-carotene and retinoids is described, for example, in WO2008042338. Methods for producing β-carotene and retinoids in host cells selected from Escherichia coli are described, for example, in US20070166782.

[0040] In particular, fermentation using a suitable retinoid-producing host strain expressing a modified ATF as described herein as defined herein is cultured in a two-phase system, wherein the retinoid (including but not limited to retinyl acetate) is collected in a suitable lipophilic phase and subsequently separated from the suitable lipophilic phase. Specific conditions and lipophilic solvents are disclosed in WO2022090548 or WO2022090549.

[0041] In some embodiments, the present invention relates to a two-phase fermentation using a lipophilic solvent as the second phase, wherein the lipophilic solvent comprises, in addition to In addition to known solvents such as silicone or n-dodecane, such as isopars or corn oil (see Jang et al., Microbial Cell Factories 10:59, 2011).

[0042] As used herein, the term "specific activity" or "activity" with respect to an enzyme refers to its catalytic activity, i.e., its ability to catalyze the formation of a product from a given substrate. Specific activity defines the amount of substrate consumed and / or product produced within a given time period and per a defined amount of protein at a defined temperature. Typically, specific activity is expressed as μmol of product formed or consumed per mg of protein per minute. Typically, μmol / min is abbreviated as U (= unit). Therefore, the unit definitions of specific activity of μmol / min / (mg protein) or U / (mg protein) are used interchangeably in this document. An enzyme is active if it exerts its catalytic activity in vivo, i.e., in a host cell as defined herein or in a suitable (cell-free) system in the presence of a suitable substrate. Technicians know how to measure enzyme activity, and analytical methods for evaluating the ability of suitable ATFs, particularly Atf1 defined herein, to convert retinol to produce retinyl acetate are known in the art, such as described in Example 4 of WO2014096992. Briefly, the titers of products such as retinyl acetate, retinol, trans-retinal, cis-retinal, β-carotene, etc. can be measured by HPLC.

[0043] Genes and methods for producing carotenoid-producing host cells are known in the art for suitable host cells that contain specific enzymes involved in β-carotene biosynthesis and are expressed and active in vivo, resulting in the production of carotenoids (e.g., β-carotene), see, for example, WO 2006102342. Depending on the carotenoid to be produced, different genes may be involved.

[0044] As used herein, a "retinol-producing host cell" is a host cell in which the corresponding polypeptide is expressed and active in vivo, resulting in the production of retinoids, such as vitamin A and its precursors, including retinol, through the enzymatic conversion of β-carotene to retinol via retinal. These polypeptides include modified ATF as defined herein. Genes of the vitamin A pathway and methods for generating retinoid-producing host cells are known in the art. The term "retinoid" includes retinol, which serves as a substrate for the modified acetylase enzymes defined herein.

[0045] Retinoids used herein include beta-carotene cleavage products, also known as apocarotenoids (apocarotenoids), including but not limited to retinal, retinoic acid, retinol, retinoic acid methoxylate (retinoicmethoxide), retinyl acetate, retinyl esters, 4-keto-retinoids, 3-hydroxy-retinoids or combinations thereof. Long-chain retinyl esters used herein are defined as hydrocarbon esters of retinol and fatty acids, wherein the fatty acid is composed of at least about 8, such as 9, 10, 12, 13, 15 or 20 carbon atoms and up to about 26, such as 25, 22, 21 or less carbon atoms, preferably up to about 6 unsaturated bonds, such as 0, 1, 2, 4, 5, 6 unsaturated bonds. The fatty acids in long-chain retinyl esters include but are not limited to linoleic acid, oleic acid or palmitic acid. The biosynthesis of retinoids is described in, for example, WO2008042338.

[0046] As used herein, "retinal" is known by the IUPAC name (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenal. It is referred to interchangeably herein as retinal or vitamin A aldehyde and includes cis- and trans-isomers, such as 11-cis retinal, 13-cis retinal, trans-retinal, and all-trans-retinal.

[0047] The term "carotenoid," as used herein, is well known in the art. It includes long, 40-carbon conjugated isoprenoid polyenes that are formed in nature by the linking of two 20-carbon geranylgeranyl pyrophosphate molecules. These include, but are not limited to, phytoene, lycopene, and carotenes, such as β-carotene, which can be oxidized at the 4-keto or 3-hydroxy positions to produce canthaxanthin, zeaxanthin, or astaxanthin. The biosynthesis of carotenoids is described, for example, in WO2006102342.

[0048] As used herein, "vitamin A" can be any chemical form of vitamin A found in aqueous solutions, solids, and formulations, and includes retinol, retinyl acetate, and retinyl esters. It also includes retinoic acid, e.g., undissociated, in its free acid form, or dissociated into anions.

[0049] Specifically, the present invention relates to the following embodiments (1) to (14):

[0050] (1) A modified acetyltransferase [EC 2.3.1.84] having increased catalytic activity for the acetylation of retinol, wherein the enzyme is based on an enzyme having at least 20% identity to Lachancea mirantina ATF1 according to SEQ ID NO: 1 or SEQ ID NO: 3, the modified enzyme comprising a 7 amino acid motif NHx(3)-D-[GA], wherein "x" represents any amino acid, and wherein the motif corresponds to positions N218 to G224 in the polypeptide according to SEQ ID NO: 1, the modified acetyltransferase comprising at least one amino acid substitution at positions corresponding to A451, L452, T473, L483 and / or N512 in the unmodified polypeptide according to SEQ ID NO: 1, the modified enzyme being useful for the acetylation of retinol to retinyl acetate, wherein the percentage of retinyl acetate based on total retinoids is at least about 81%.

[0051] (2) The modified enzyme according to embodiment (1), wherein the glutamine at the position corresponding to 68 in SEQ ID NO: 1 is replaced by leucine, and / or wherein the alanine at the position corresponding to 451 in SEQ ID NO: 1 is replaced by leucine or methionine, and / or wherein the leucine at the position corresponding to 452 in SEQ ID NO: 1 is replaced by phenylalanine, and / or wherein the threonine at the position corresponding to 473 in SEQ ID NO: 1 is replaced by leucine or alanine, and / or wherein the leucine at the position corresponding to 483 in SEQ ID NO: 1 is replaced by methionine, and / or wherein the asparagine at the position corresponding to 512 in SEQ ID NO: 1 is replaced by phenylalanine.

[0052] (3) The modified enzyme according to embodiment (1) or (2), further comprising one or more amino acid substitutions at positions corresponding to amino acid residues selected from H69, V407, G409, S480 and / or I484 in the unmodified polypeptide according to SEQ ID NO: 3.

[0053] (4) The modified enzyme according to embodiment (3), wherein the histidine at position corresponding to 69 in SEQ ID NO: 1 is substituted by alanine, asparagine or serine, and / or wherein the valine corresponding to 407 in SEQ ID NO: 1 is substituted by isoleucine, and / or wherein the glycine corresponding to 409 in SEQ ID NO: 1 is substituted by alanine, and / or wherein the serine corresponding to 480 in SEQ ID NO: 1 is substituted by glutamic acid, phenylalanine, leucine, methionine or glutamine, and / or wherein the isoleucine corresponding to 484 in SEQ ID NO: 1 is substituted by leucine.

[0054] (5) The modified enzyme of embodiment (1), (2), (3) or (4), wherein the percentage of retinyl acetate based on total retinoids obtained from the catalyzed acetylation of retinol is increased by at least 8% compared to the acetylation reaction catalyzed by the corresponding unmodified enzyme according to SEQ ID NO: 1.

[0055] (6) The modified enzyme of embodiment (1), (2), (3), (4) or (5), which is expressed in a retinol-producing host cell that expresses a gene involved in the catalysis of retinal to retinol and / or β-carotene to retinal.

[0056] (7) A retinoid-producing host cell expressing the enzyme according to embodiment (1), (2), (3), (4), (5) or (6).

[0057] (8) The host cell according to embodiment (7), which is a fungal host cell.

[0058] (9) The host cell according to embodiment (8), which is selected from the genus Yarrowia or Saccharomyces.

[0059] (10) The host cell according to embodiment (7), (8) or (9), further expressing enzymes involved in the mevalonate pathway and / or the carotenoid pathway to produce β-carotene, retinal and retinol.

[0060] (11) The host cell of embodiment (10), wherein the enzyme that catalyzes the conversion of β-carotene to retinal is a β-carotene oxygenase that selectively produces trans-retinal at a percentage of at least 95% based on total retinoids comprising cis- and trans-retinal.

[0061] (12) A method for producing a retinoid comprising retinal, retinol and retinyl acetate, comprising culturing the host cell according to embodiment (7), (8), (9), (10) or (11) under suitable culture conditions with a carbon source selected from glucose, fructose, raffinose, lactose, galactose, glycerol, xylose, arabinose, sucrose, maltose, ethanol or a mixture thereof, wherein the percentage of retinyl acetate produced during the method based on total retinoids is at least 81%.

[0062] (13) The method according to embodiment (12), wherein the lipophilic substance is selected from synthetic or natural oils or isoparaffins.

[0063] (14) The method according to embodiment (12) or (13), wherein the percentage of retinyl acetate based on total retinoids is increased by at least 8% to 500% compared to the method using ATF1 according to SEQ ID NO: 1.

[0064] Attached photos

[0065] Figure 1 : Amino acid sequence of Lachancea mirantina ATF1 (LmATF; SEQ ID NO: 1), wherein residues selected for amino acid substitutions as described in this application are marked in bold / underlined, and every tenth amino acid residue is marked in bold.

[0066] Figure 2 : Amino acid sequence of Lachancea mirantina ATF1 (LmATF*; SEQ ID NO: 3), wherein residues selected for amino acid substitutions as described in the present application are marked in bold / underlined, and every tenth amino acid residue is marked in bold.

[0067] The following examples are merely illustrative and are not intended to limit the scope of the present invention in any way. The contents of all references, patent applications, patents and disclosed patent applications cited throughout this application are incorporated herein by reference, particularly WO2014096992, WO2019058001, WO2021136689, WO2022090548, WO2008042338, US20070166782, WO2022090549, WO2006102342, WO2020141168 and WO2016172282. Example

[0068] Example 1: General methods, strains and plasmids

[0069] All basic molecular biology and DNA manipulation procedures described herein are generally 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).

[0070] Shake plate assay (Yarrowia spp.).In order to test the conversion activity of mutant, usually 200 μ l of 0.25% yeast extract, 0.5% peptone (0.25 * YP) and 10 μ l freshly grown Yarrowia yeast are inoculated, and covered with 200 μ l of mineral oil (Isopar M, Exxon Mobile) with 2% oleic acid in the mineral oil phase as a carbon source. Transformant is grown in 24-well plates (Microplate Devices 24 Deep Well Plates Whatman 7701-5102), covered with a pad seal (Analytical Sales and Services Inc. Plate Mats 24010cm), aseptically sealed with Qiagen Airpore tape (19571), and shaken 4 days at 30 ℃, 800RPM in an Infors multi-plate shaking table (Multitron). From the shaking plate wells, take out the mineral oil fraction, and analyze by UPLC reversed-phase column with a photodiode array detector. This method is also used in Example 2.

[0071] DNA transformation. Yarrowia lipolytica strain is transformed from overnight growth on YPD plate medium. 50 μ l cells are scraped from the plate and transformed by incubating in 500 μ l with 1 μ g transforming DNA (linear DNA typically used for integration transformation), 40% PEG 3550MW, 100mM lithium acetate, 50mM dithiothreitol, 5mM Tris-Cl pH 8.0, 0.5mM EDTA at 40° C. for 30 minutes and directly plated onto selective medium, or in the case of dominant antibiotic marker selection, cells are grown on YPD liquid medium at 30° C. for 4 hours and then plated onto selective medium. Yeast strains are transformed from cells grown in exponential phase YPD using the lithium acetate method, which are grown by subculture of overnight YPD culture. Harvest 10 8 Cells / transformation were resuspended in a mixture containing 40% PEG 3350 (MW), 100mM lithium acetate, 10mM Tris-Cl pH 8.0, 1mM EDTA, 5 μg sheared salmon sperm DNA and 2 μg linearized transformed DNA in a final volume of 500 μL. The mixture was incubated at 30°C for 1 hour and then at 42°C for 30 minutes. The cells were then precipitated and resuspended in liquid YPD medium and grown at 30°C for 3 hours or at 22°C overnight to allow expression of the HygR antibiotic resistance gene and then plated on a selective culture medium containing 100 μg / ml hygromycin. Most of the DNA sequences used herein are codon-optimized for expression in the corresponding host system and as shown in the sequence table.

[0072] DNA molecular biology.Plasmids MB10306 (SEQ ID No: 5) and MB10569 (SEQ ID NO: 6) containing DrBCO, LmATF and FfRDH expression systems were synthesized at Genscript (Piscataway, NJ, USA). Plasmid MB10306 contains " URA3 " and " HOM3 " markers for selection in Yarrowia lipolytica transformation. In order to insert clean genes by random non-homologous end joining of genes and markers, the SfiI plasmid fragment of interest (the SfiI plasmid fragment of MB10306 or the SfiI plasmid fragment of other plasmids in Table 1) was purified by gel electrophoresis and Qiagen gel purification columns. Cloning was verified by sequencing. Typically, genes were synthesized at GenScript (Piscataway, NJ) and amino acid substitutions ("mutations" column) according to Table 1 were introduced. Transformants were screened for homoserine auxotrophy and subsequently sequenced using primers located on both sides of the HOM3 sequence, and clean frameshifts were selected to move forward. Expression of mutant ATF in Saccharomyces cerevisiae is described in Example 1 of WO2020141168.

[0073] sequence. Plasmids comprising the corresponding LmATF and LmATF* according to SEQ ID NO: 2 and 4 (the polynucleotide according to SEQ ID NO: 4 corresponds to the nucleic acid encoding LmATF1 derived from L. mirantina as shown in SEQ ID NO: 2 in WO2020141168) and modified enzymes comprising specific amino acid substitutions are listed in Table 1 and / or in the sequence listing, wherein the codon-optimized sequences for expression in Yarrowia lipolytica or Saccharomyces cerevisiae are particularly indicated.

[0074] Table 1: List of plasmids used to construct strains carrying unmodified or modified heterologous Yarrowia lipolytica codon-optimized ATF genes from Lachancea mirantina as inserts. With the exception of MB10569, MB10597, and MB10599 (based on LmATF*), all inserts are based on LmATF according to SEQ ID NO: 1. See the text for more details.

[0075]

[0076]

[0077] UPLC reversed-phase retinol method.For rapid screening, this method does not separate cis isomers, only the major functional groups. A Waters Acquity UPLC with PDA detection (or similar) with an autosampler was used to inject the samples. Retinoids were separated using an Acquity UPLC HSS T3 1.8um P / N 186003539. The mobile phase consisted of 1000 mL of hexane, 30 mL of isopropanol, and 0.1 mL of acetic acid for retinoid-related compounds. The flow rate for each was 0.6 mL / min. The column temperature was 20°C. The injection volume was 5 μL. The detector was a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 2.

[0078] Table 2A: List of analytes used in the reversed-phase retinol method. The sum of all added intermediates gives the total amount of retinoids. β-Carotene* can be detected at 325 nm and interferes with retinyl ester quantification, so care must be taken to observe the carotene peak and not include it in the retinoid quantification. "N / A" indicates "not available." See the text for more details.

[0079]

[0080]

[0081] Table 2B: UPLC method gradient, solvent A: water; solvent B: acetonitrile; solvent C: methanol; solvent D: tert-butyl methyl ether.

[0082] Time [minutes] ％A ％B ％C ％D Flow rate [ml / min] Pressure [psi / bar] 0 50 50 0 0 0.5 Maximum 9500-14000 0.5 50 50 0 0 0.5 1.0 0 50 50 0 0.5 1.25 0 0 100 0 0.5 3.25 0 0 5 95 0.5 3.5 0 0 5 95 0.5 4.0 0 0 100 0 0.5 4.25 0 50 50 0 0.5 4.5 50 50 0 0 0.5

[0083] Method calibration. The method is calibrated on retinyl acetate to quantify retinol and retinal using the specified response factors relative to retinyl acetate. Retinyl acetate is dissolved in THF at ~200 μg / ml as a stock solution using a volumetric flask. Using a volumetric flask, x20, x50, and x100 dilutions of the stock solution in 50 / 50 methanol / MTBE are prepared. The UV absorbance of retinyl acetate becomes nonlinear fairly quickly, so care must be taken to stay within the linear range. Therefore, lower concentrations may be better. Retinyl palmitate can also be used as a retinyl ester calibrant. The peak for retinyl acetate is at approximately 3 minutes, and the peak for retinyl esters (long chain retinyl esters) is at approximately 3.5 minutes.

[0084] Sample preparation. Samples were prepared by various methods depending on the conditions. For whole broth or washed broth samples, the broth was placed in tube, weigh it, and add mobile phase. In a tube, 25 μl of well-mixed fermentation broth and 975 μl of THF were added. The homogenizer (Bertin Corp, Rockville, MD, USA) was processed at the highest setting 3X according to the manufacturer's instructions, typically 3 × 15 × 7500 TPMS. For the washed pellet, the sample was spun at 10,000 rpm for 1 minute in a microcentrifuge in a 1.7 ml tube, the fermentation broth was decanted, 1 ml of water was added, mixed, precipitated and decanted, and adjusted to the original volume. The mixture was pelleted again and placed in an appropriate amount of mobile phase and passed through To analyze the silicone oil fraction, samples were spun at 4000 RPM for 10 minutes and the oil was decanted from the top by a positive displacement pipette (Eppendorf, Hauppauge, NY, USA) and diluted into the mobile phase which was mixed by vortexing and the retinoid concentration was measured by UPLC analysis.

[0085] Fermentation conditions of Yarrowia yeast. Fermentation is carried out under the same conditions as previously described, preferably using a silicone oil blanket and a stirred tank, preferably glucose in a benchtop reactor with a total volume of 0.5 L to 5 L (see WO2016172282). In general, the same results are observed with a batch fed stirred tank reactor with increased productivity, demonstrating the utility of this system for the production of retinoids. Preferably, batch fermentation is carried out with 5% glucose, after the dissolved oxygen drops below about 20%, and the feed is resumed to achieve 20% dissolved oxygen throughout the feeding procedure.

[0086] Example 2: Production of retinyl acetate in Yarrowia lipolytica expressing mutant LmATF

[0087] In order to express heterologous ATF in Yarrowia lipolytica as a host, strain ML15710 (see Example 5 in WO2016172282) was transformed with plasmid MB9287 (see Example 1 in WO2022090548) to isolate lip2 lip3 lip8 mutant derivatives. This derivative was selected on 5-fluoroorotic acid to isolate a uracil auxotrophic strain, designated as strain ML18667-new. The strain was transformed with the plasmids listed in Table 1 above, each consisting of a specified ATF allele, DrBCO, and FfRDH12. ML18667-new transformants with the SfiI linearized plasmid from Table 1 were selected for uracil prototrophy. Transformants were grown in shake plates as described in Example 1, and the percentage of retinol acetate using the mutant ATFs (retinol acetate / total retinoids) relative to the percentage of retinol acetate using the reference LmATF expressed on plasmid MB10603 (set as 100%; SEQ ID NO: 5) or the reference LmATF* expressed on plasmid MB10569 is shown in Table 3.

[0088] Table 3A: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of modified ATFs (all based on LmATF according to SEQ ID NO: 2). See text or Table 1 for more details.

[0089]

[0090]

[0091] Each of the indicated mutations increased the percentage of retinyl acetate by 8% to over 560% when compared to the reference sequence according to SEQ ID NO: 1.

[0092] Table 3B: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of modified ATFs (all based on LmATF* according to SEQ ID NO: 4). See text or Table 1 for more details.

[0093] plasmids retAc[%] MB10596 100 MB10597 156 MB10599 177

[0094] Each of the indicated mutations increased the percentage of retinyl acetate by 56% to 77% when compared to the reference sequence according to SEQ ID NO: 3.

Claims

1. A modified acetyltransferase [EC 2.3.1.84] having increased catalytic activity for the acetylation of retinol in a suitable retinol-producing host cell, wherein the percentage of retinyl acetate based on total retinoids is at least about 81%, wherein the enzyme is based on an enzyme having at least 20% identity to Lachancea mirantina ATF1 according to SEQ ID NO: 1 or SEQ ID NO: 3, the modified enzyme comprising a 7 amino acid motif NHx(3)-D-[GA], wherein "x" represents any amino acid, and wherein the motif corresponds to positions N218 to G224 in the polypeptide according to SEQ ID NO: 1, the modified acetyltransferase comprising at least one amino acid substitution at a position corresponding to A451, T473 and / or L483 in the polypeptide according to SEQ ID NO: 1, wherein after introduction of the amino acid substitution, the modified acetyltransferase is comparable to the acetylation of retinol using the same conditions but using the corresponding or corresponding wild-type enzyme, comprising the enzyme according to SEQ ID NO: Compared to the ATF enzyme method of No. 1, the percentage of retinyl acetate is increased by at least 10-20% by weight.

2. The modified enzyme of claim 1 , further comprising at least one or more amino acid substitutions corresponding to positions Q68, L452 and / or N512 in the unmodified polypeptide according to SEQ ID NO: 1, wherein the modified enzyme is for acetylation of retinol to retinyl acetate, the percentage of retinyl acetate based on total retinoids being at least about 81%.

3. The modified enzyme according to claim 1 or 2, wherein the glutamine at the position corresponding to 68 in SEQ ID NO: 1 is substituted by leucine, and / or wherein the alanine at the position corresponding to 451 in SEQ ID NO: 1 is substituted by leucine or methionine, and / or wherein the leucine at the position corresponding to 452 in SEQ ID NO: 1 is substituted by phenylalanine, and / or wherein the threonine at the position corresponding to 473 in SEQ ID NO: 1 is substituted by leucine or alanine, and / or wherein the leucine at the position corresponding to 483 in SEQ ID NO: 1 is substituted by methionine, and / or wherein the asparagine at the position corresponding to 512 in SEQ ID NO: 1 is substituted by phenylalanine.

4. The modified enzyme according to any one of claims 1 to 3, further comprising one or more amino acid substitutions at positions corresponding to amino acid residues selected from H69, V407, G409, S480 and / or I484 in the polypeptide according to SEQ ID NO: 1, wherein the histidine at position corresponding to 69 of SEQ ID NO: 1 is substituted by alanine, asparagine or serine, and / or wherein the valine at position corresponding to 407 of SEQ ID NO: 1 is substituted by isoleucine, and / or wherein the glycine at position corresponding to 409 of SEQ ID NO: 1 is substituted by alanine, and / or wherein the serine at position corresponding to 480 of SEQ ID NO: 1 is substituted by glutamic acid, phenylalanine, leucine, methionine or glutamine, and / or wherein the isoleucine at position corresponding to 484 of SEQ ID NO: 1 is substituted by leucine.

5. The modified enzyme according to any one of claims 1 to 4, comprising at least one of the following amino acid substitutions, wherein the positions correspond to amino acid residues in the polypeptide according to SEQ ID NO: 1: T473A_A451L, T473A_A451M, T473A_L483M, A451L_L483M, A451M_L483M, T473L_L483M, T473A_A451L_L483M, T473L_A451L_L483M, T473A_A451M_L483M, T473L_A451M_L483M, T473A_A451L_L483M_L452F, T473L_A451L_L483M_L452F, T473A_A451M_L483M_L452F, T473L_A451M_L483M_L452F, LmATF_T473A_A451L_L 483M_L452F_Q68L_N512F, T473A_A451M_L483M_L452F_Q68L_N512F, T473L_A451M_L483M_L452F_Q68L_N512F.

6. The modified enzyme according to any one of claims 1 to 5, wherein the percentage of retinyl acetate based on total retinoids obtained from the catalyzed acetylation of retinol is increased by at least 10 to 20% compared to the acetylation reaction catalyzed using the corresponding unmodified enzyme according to SEQ ID NO:

1.

7. The modified enzyme according to any one of claims 1 to 6, which is expressed in a retinol-producing host cell expressing a gene involved in the catalysis of retinal to retinol and / or β-carotene to retinal.

8. A retinoid-producing host cell expressing the enzyme according to any one of claims 1 to 7.

9. The host cell according to claim 8, which is a fungal host cell, preferably selected from the genus Yarrowia or Saccharomyces.

10. The host cell according to any one of claims 7 to 9, further expressing enzymes involved in the mevalonate pathway and / or the carotenoid pathway to produce β-carotene, retinal and retinol.

11. The host cell of claim 10, wherein the enzyme that catalyzes the conversion of beta-carotene to retinal is a beta-carotene oxygenase that selectively produces trans-retinal at a percentage of at least 95% based on total retinoids comprising cis- and trans-retinal.

12. A method for producing a retinoid comprising retinal, retinol and retinyl acetate, comprising culturing the host cell according to any one of claims 7 to 11 under suitable culture conditions with a carbon source selected from glucose, fructose, raffinose, lactose, galactose, glycerol, xylose, arabinose, sucrose, maltose, ethanol or a mixture thereof, wherein the percentage of retinyl acetate produced during the method based on total retinoids is at least 81%.

13. The method according to claim 13, wherein the lipophilic substance is selected from synthetic or natural oils or isoparaffins.

14. The method of claim 12 or 13, wherein the percentage of retinyl acetate based on total retinoids is increased by at least 10% to 500% compared to a method using ATF1 according to SEQ ID NO: 1.

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