Novel acetyltransferase

Abstract

The present invention relates to the production of retinyl acetate, which is produced by the enzymatic conversion of retinol, said process comprising the use of modified enzymes with improved activity.

Description

Detailed Description of the Invention

[0001] The present invention relates to the production of retinyl acetate, which is produced by the enzymatic conversion of retinol, said process comprising the use of modified enzymes with improved activity.

[0002] Retinyl acetate is an important intermediate or precursor for producing retinoids, specifically vitamin A. Retinoids, including vitamin A, are one of the most important and essential nutrients for humans and animals, and must be supplied through the diet. Retinoids promote health, particularly with regard to vision, the immune system, and growth.

[0003] Current chemical production methods for retinoids, specifically vitamin A and its precursors, have several undesirable features, such as high energy consumption, complex purification steps, and / or unwanted by-products. Therefore, alternative approaches to producing retinoids (specifically vitamin A and its precursors), including microbial conversion steps, which are not only more economical but also more ecological, have been investigated over the past few decades.

[0004] Generally, biological systems that produce retinoids are industrially intractable and / or produce the compounds at such low levels that isolating them on an industrial scale is not economically feasible for several reasons, including the instability of retinoids in such 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 Saccharomyces bayanus has been reported (WO 2014096992), with up to 90% acetylation of zeaxanthin. However, these acetyltransferase enzymes typically have different substrate specificities for different alcohol substrates, determined by the local structural environment of the alcohol functional group on the molecule being acetylated. For example, the hydroxyl group acetylated in carotenoids, such as zeaxanthin, is located on the β-ionone ring structure, whereas the hydroxyl group acetylated in retinol is not located on the ionone ring structure but on the other end of the terminal CH2 carbon of the polyene chain of the molecule. Because of these differences in the local molecular context of the acetylated hydroxyl group, it is very difficult to predict the acetylation of retinol from data on carotenoid acetylation.

[0006] For the acetylation of retinoids, the enzyme LmATF1, an acetyltransferase from the genus Lachancea, specifically Lachanceae mirantina, is particularly useful for acetylating retinol to retinyl acetate. Using wild-type LmATF from a retinol-producing strain of Yarrowia lipolytica, up to 40% retinyl acetate by weight (based on total retinoids) could be obtained. By substituting specific amino acids, increases of over 80% by weight were achieved (see WO2020141168).

[0007] However, for such enzymatic processes to be used on an industrial scale, further improvements in efficiency for the production of retinyl acetate are required.

[0008] Surprisingly, the present inventors have now identified amino acid positions in fungal acetyltransferases, specifically the ATF enzyme from Lachancea mirantina, as disclosed in WO 2019058001, that are crucial for the formation of acetylated retinoids, specifically the conversion of retinol to retinyl acetate. Modification of specific amino acids resulted in increased formation of retinyl acetate compared to the respective wild-type enzymes, e.g., by at least about 10-20% when compared to the acetylation of retinol using the respective unmodified enzymes, e.g., wild-type ATF1 from Lachancea mirantina, as disclosed in WO 2019058001. Specifically, the rate of retinyl acetate can be even more improved compared to the best enzymes known to date, such as those disclosed in WO 2020141168.

[0009] Specifically, the present invention relates to modified enzymes involved in the acetylation of retinol to retinyl acetate in suitable retinol-producing host cells at a rate of at least about 81% retinyl acetate based on total retinoids, and methods for producing said modified enzymes, specifically fungal enzymes comprising one or more modifications, e.g., 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 to SEQ ID NO: 1 (FIG. 1) or SEQ ID NO: 3 (FIG. 2), The one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of positions 68, 451, 452, 473, 483, 512 of the polypeptide according to SEQ ID NO: 1, and combinations thereof, specifically including at least one amino acid substitution at positions corresponding to A451, T473, and / or L483 of the polypeptide according to SEQ ID NO: 1, and introduction of said amino acid substitutions increases the proportion of retinyl acetate by at least 10 to 20% by weight compared to a process using the same conditions but using the respective or corresponding wild-type enzyme, for example, the ATF enzyme according to SEQ ID NO: 1.

[0010] The use of such modified enzymes in a process for producing retinoids, expressed, particularly heterologously expressed, in a suitable host cell, particularly a fungal host cell capable of producing retinol, results in an increase in retinyl acetate in the range of at least about 10% based on the total retinoids present / produced by the modified host cell, compared to a process using the same conditions but using the ATF enzyme according to SEQ ID NO: 1.

[0011] The terms "acetyltransferase," "retinol acetyltransferase," "enzyme with retinol acetylation activity," "ATF," or "ATF1" are used interchangeably herein and refer to enzymes in the EC classification [EC 2.3.1.84] that can catalyze the conversion of retinol to retinyl acetate, specifically with 30-90% acetylation by weight based on total retinoids, and include both naturally occurring enzymes and enzymes produced synthetically with the aid of artificial intelligence. As used herein, such enzymes are referred to as "unmodified" ATFs. An example of such an unmodified enzyme is the enzyme (LmATF1) shown in SEQ ID NO: 1 or 3, isolated from or derived from Lachancea mirantina, as shown, for example, in Figures 1 and 2.

[0012] A "modified" ATF, specifically based on an "unmodified" ATF as defined herein, such as an enzyme having at least about 20% identity to SEQ ID NO: 1 or SEQ ID NO: 3, exhibits an increase in the formation of retinyl acetate from the conversion of retinol, for example, an increase of at least 10% on a total retinoid basis compared to the formation of retinyl acetate using an enzyme specifically according to SEQ ID NO: 1.

[0013] Suitable unmodified enzymes, including 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 LmATF isolated / derived from Lachancea mirantina, include the NHx(3)-D-[GA] (motif defined at https: / / prosite.expasy.org / scanprosite / scanprosite_doc.html) and wherein "x" means any amino acid and the central histidine is part of the binding pocket of the enzyme; and preferably the seven amino acid motif is selected from NHCSSDG, NHCLCDG or NHILKDG, more particularly NHCSSDG, which corresponds to positions N218 to G224 of the polypeptide according to SEQ ID NO: 1.

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

[0015] Enzymes as defined herein are used to convert retinol to retinyl acetate, and the substrate (i.e., retinol) can be either cis-retinol, trans-retinol, or a mixture of cis- / trans-retinol in any possible ratio. Preferably, the retinol mixture used as the substrate has a high proportion of trans-retinol, e.g., at least about 65-98% by weight of the trans isomer, based on the total retinol in the host cell. Acetylation of such retinol mixtures containing at least about 65-98% by weight of trans-retinol results in the production of retinyl acetate containing approximately the same proportions of trans-retinyl acetate and cis-retinyl acetate, based on the total retinyl acetate produced by the host cell.

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

[0017] The terms "conversion," "enzymatic conversion," "acetylation," or "enzymatic acetylation," in relation to 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 retinyl acetate, which, upon expression of said ATF, results in a percentage of retinyl acetate relative to total retinoids present / produced by a suitable host cell, and an increase in retinyl acetate of at least 10% relative to 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 as well as cells derived from, for example, E. coli. As used herein, the term "fungal host cell" specifically includes yeast cells, which are retinol-producing host cells, specifically retinyl acetate-producing host cells, e.g., retinyl acetate-producing fungal host cells, including, but not limited to, cells of the genus Yarrowia or Saccharomyces, such as Yarrowia lipolytica or Saccharomyces cerevisiae.

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

[0020] In one embodiment, the modified ATF enzyme as defined herein used to produce retinyl acetate as defined herein comprises an amino acid substitution at a position corresponding to residue 68 of a polypeptide according to SEQ ID NO: 1 or 3, resulting in a leucine at said residue, such as a substitution of glutamine with leucine (Q68L). The modified enzyme may be derived from a species of Lachancea, such as L. mirantina, L. fermentati, preferably L. mirantina. Use of such modified enzymes comprising said mutations in fermentation processes using a suitable carbon source, such as glucose, results in an increase of at least about 10%, such as 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, in retinyl acetate based on total retinoids from acetylation of retinol, compared to a corresponding process using an enzyme according to SEQ ID NO: 1. Furthermore, this mutation may be combined with additional mutations as defined herein, such as one or more amino acid substitutions at positions corresponding to residues 451 and / or 452 and / or 473 and / or 483 and / or 512 of the polypeptide according to SEQ ID NO: 1 or 3.

[0021] In one embodiment, the modified ATF enzyme as defined herein used to produce retinyl acetate as defined herein comprises an amino acid substitution at a position corresponding to residue 451 of the polypeptide according to SEQ ID NO: 1 or 3, resulting in said residue being replaced with a leucine or methionine, such as a substitution of alanine with leucine (A451L) or alanine with methionine (A451M). The modified enzyme may be derived from a species of Lachancea, such as L. mirantina, L. fermentati, preferably L. mirantina. Use of such modified enzymes comprising said mutations in fermentation processes using a suitable carbon source, such as glucose, results in an increase of at least about 20%, such as 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, in retinyl acetate based on total retinoids from acetylation of retinol, compared to the corresponding process using the enzyme according to SEQ ID NO: 1. Furthermore, this mutation may be combined with additional mutations as defined herein, such as one or more amino acid substitutions at positions corresponding to residues 68 and / or 452 and / or 473 and / or 483 and / or 512 of the polypeptide according to SEQ ID NO: 1 or 3.

[0022] In one embodiment, the modified ATF enzyme as defined herein used to produce retinyl acetate as defined herein comprises an amino acid substitution resulting in a phenylalanine at a position corresponding to residue 452 of a polypeptide according to SEQ ID NO: 1 or 3, such as, for example, a substitution of leucine with phenylalanine (L452F). The modified enzyme may be derived from a species of Lachancea, such as, for example, L. mirantina, L. fermentati, preferably L. mirantina. Use of such modified enzymes comprising said mutations in fermentation processes using a suitable carbon source, such as glucose, results in an increase of at least about 20%, such as 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, in retinyl acetate based on total retinoids from acetylation of retinol, compared to the corresponding process using the enzyme according to SEQ ID NO: 1. Furthermore, this mutation may be combined with additional mutations as defined herein, such as one or more amino acid substitutions at positions corresponding to residues 68 and / or 451 and / or 473 and / or 483 and / or 512 of the polypeptide according to SEQ ID NO: 1 or 3.

[0023] In one embodiment, a modified ATF enzyme as defined herein used to produce retinyl acetate as defined herein comprises an amino acid substitution at a position corresponding to residue 473 of a polypeptide according to SEQ ID NO: 1 or 3 resulting in a leucine or alanine at said residue, such as, for example, a substitution of threonine with leucine (T473L) or threonine with alanine (T473A). The modified enzyme may be derived from a species of Lachancea, such as, for example, L. mirantina, L. fermentati, preferably L. mirantina. Use of such modified enzymes comprising said mutations in fermentation processes using a suitable carbon source, such as glucose, results in an increase of at least about 20%, such as 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, in retinyl acetate based on total retinoids from acetylation of retinol, compared to the corresponding process using the enzyme according to SEQ ID NO: 1. Furthermore, this mutation may be combined with additional mutations as defined herein, such as one or more amino acid substitutions at positions corresponding to residues 68 and / or 451 and / or 452 and / or 483 and / or 512 of the polypeptide according to SEQ ID NO: 1 or 3.

[0024] In one embodiment, the modified ATF enzyme as defined herein used to produce retinyl acetate as defined herein comprises an amino acid substitution resulting in a phenylalanine at a position corresponding to residue 512 of a polypeptide according to SEQ ID NO: 1 or 3, such as, for example, a substitution of asparagine with phenylalanine (N512F). The modified enzyme may be derived from a species of Lachancea, such as, for example, L. mirantina, L. fermentati, preferably L. mirantina. Use of such modified enzymes comprising said mutations in fermentation processes using a suitable carbon source, such as glucose, results in an increase of at least about 20%, such as 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 acetylation of retinol, compared to the corresponding process using the enzyme according to SEQ ID NO: 1. Furthermore, this mutation may be combined with additional mutations as defined herein, such as one or more amino acid substitutions at positions corresponding to residues 68 and / or 451 and / or 452 and / or 473 and / or 483 of the polypeptide according to SEQ ID NO: 1 or 3.

[0025] Host cells as described herein are capable of converting retinol to retinyl acetate at a conversion rate that is at least about 10-20% or more increased relative to the conversion by the respective enzyme according to SEQ ID NO: 1, as obtained by expressing the engineered ATF in a suitable host cell, such as a fungal host cell, including, for example, a Yarrowia or Saccharomyces species, or a microbial host cell, such as, for example, Escherichia coli, under suitable culture conditions, including, but not limited to, culture on glucose, galactose, or xylose, with or without ethanol, at a conversion rate that is at least about 10-20% or more increased relative to the production of retinyl acetate (based on the total amount of retinoid produced by the host cell). Suitable conditions can be, for example, culture in fed-batch fermentation for 80, 90, 100, 110, 120, or 130 hours.

[0026] Preferably, the modified host cell as defined herein comprises one or more copies of a modified ATF as defined herein, and the ATF is heterologously expressed in the modified host cell. The modification to obtain the host cell as defined herein results in more copies of a gene and / or protein, e.g., more copies of the modified ATF, that selects for the formation of retinyl acetate as defined herein, and includes at least about a 10-20% increase in conversion relative to the total amount of retinoid produced by the host cell relative to the production of retinyl acetate, as obtained by expressing unmodified ATF under suitable culture conditions, including, but not limited to, culture on glucose, galactose, or xylose, with or without ethanol, compared to a process using LmATF1 according to SEQ ID NO: 1 (FIG. 1). This may include the use of a strong promoter, suitable transcriptional and / or translational enhancers, or the introduction of more than one gene copy into a retinol-producing host cell, particularly a fungal host cell, resulting in increased accumulation of the respective enzyme over a given time period. The techniques used depending on the host cell are known to those skilled in the art. Increases and decreases in gene expression can be measured by a variety of methods, such as, for example, Northern, Southern, or Western blot techniques known in the art.

[0027] Mutations in nucleic acids or amino acids, i.e., mutagenesis, may be performed by a variety of methods, such as random or site-directed mutagenesis, physical damage caused by agents (e.g., radiation, chemical treatment, or insertion of genetic elements), etc. Methods for introducing mutations are known to those skilled in the art.

[0028] Thus, the present invention relates to a retinol-producing host cell, particularly a fungal host cell, as described herein, comprising an expression vector or polynucleotide encoding a modified ATF as described herein integrated into the chromosomal DNA of the host cell. Such a retinol-producing host cell, particularly a fungal host cell, comprising a heterologous polynucleotide in an expression vector or a heterologous polynucleotide integrated into the chromosomal DNA encoding a modified ATF as described herein, is referred to as a recombinant cell or modified host cell. A retinol-producing host cell, particularly a fungal host cell, may contain one or more copies of a gene encoding a modified ATF as defined herein, comprising a mutation as defined herein, thereby resulting in overexpression of the gene encoding the modified ATF as defined herein, particularly the Atf1 enzyme. Increased gene expression can be measured by various methods, such as, for example, Northern, Southern, or Western blot techniques known in the art.

[0029] The present invention specifically relates to the use of such novel modified ATF enzymes, particularly in processes for producing retinyl acetate under conditions that reduce the amount of other retinyl esters, particularly long-chain retinyl esters. Methods for creating such conditions are known to those skilled in the art (see, for example, WO2021136689 or WO2022090548). Retinyl acetate can be further converted to vitamin A by the action of suitable (known) chemical or biotechnological mechanisms.

[0030] The terms "sequence identity" and "% identity" are used interchangeably herein. For purposes of the present invention, aligning sequences for optimal comparison purposes is defined herein as determining the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. Gaps may be introduced into either of the two sequences being compared to optimize the alignment between the two sequences. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, alignments may be performed over shorter lengths, for example, over about 20, about 50, about 100, or more nucleic acids / bases or amino acids. Sequence identity is the percentage of perfect matches between the two sequences over the reported alignment region. The percent sequence identity between two amino acid sequences or two nucleotide sequences may be determined using the Needleman-Wunsch algorithm (Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48, 443-453) for aligning the two sequences. Both amino acid sequences and nucleotide sequences can be aligned using this algorithm. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used as 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.Those skilled in the art will understand that all of these different parameters will produce slightly different results, but the overall percentage identity of the two sequences will not change significantly when using different algorithms.

[0031] After alignment by the program NEEDLE as described above, the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: [the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both 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 by using the NOBRIEF option and is displayed as "longest identity" in the program output. Both amino acid sequences being compared are identical, or have 100% identity, if they do not differ in any of their amino acids.

[0032] Modified ATFs as defined herein also include enzymes with additional amino acid substitutions that do not alter the enzymatic activity, i.e., that exhibit the same properties as the enzymes defined herein and catalyze the conversion of retinol to retinyl acetate as described herein. Such mutations do not alter the (enzymatic) activity of the enzymes according to the present invention and are also called "silent mutations."

[0033] Expression of the enzyme / polynucleotide encoding one of the modified enzymes as defined herein can be achieved in any host system, including a (micro)organism, that is suitable for producing retinoids (including retinol) and that is capable of expressing a nucleic acid encoding one of the enzymes as disclosed herein, including functional equivalents or derivatives as described herein. Examples of suitable retinol-producing host (micro)organisms are bacteria, algae, fungi such as yeast, plant or animal cells. Preferred bacteria include, for example, Escherichia coli (Escherichia coli), and the like. coli), Streptomyces, Pantoea (Erwinia), Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, ococcus, Mixococcus, Brevibacterium, Bradyrhizobium, Gordonia, Dietzia, Muricauda, ​​Sphingomonas, Synochocystis, and Paracoccus, such as Paracoccus zeaxanthinifaciens.Preferred eukaryotic microorganisms, specifically fungi such as yeasts, include those of the genus 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, and Phycomyces brackensis. blakesleanus), Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, for example Blakeslea trispora, or Yarrowia, for example Yarrowia lipolytica. Expression in fungal host cells, such as Yarrowia or Saccharomyces, or in Escherichia, more preferably Yarrowia lipolytica or Saccharomyces cerevisiae, is particularly preferred.

[0034] Depending on the host cell, the polynucleotide as defined herein for acetylating retinol may be optimized for expression in each host cell.Methods for producing such further modified polynucleotides are known to those skilled in the art.It is understood that the polynucleotide as defined herein also encompasses nucleic acid molecules optimized for such hosts, as long as it still expresses a polypeptide having the respective 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, optimized for expression in said host cell and used to produce retinyl acetate.In particular, the retinol-producing host cell, in particular the fungal host cell, is a yeast, such as Saccharomyces cerevisiae or Yarrowia lipolytica. lipolytica), for example, from the genus Yarrowia or Saccharomyces, and encoding a modified ATF enzyme as defined herein, is selected from polynucleotides that express a modified polypeptide comprising one or more amino acid substitutions in a sequence having at least 20%, such as 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, such as the introduction of one or more amino acid substitutions at positions corresponding to residues selected from the group consisting of 68, 451, 452, 473, 483, 512, and combinations thereof, as described herein, specifically comprising at least one amino acid substitution at positions corresponding to A451, T473, and / or L483 of the polypeptide according to SEQ ID NO: 1 (FIG. 1), wherein the introduction of said amino acid substitutions results in a decrease in the rate of retinyl acetate. , at least 10-20% by weight increase compared to a process using the same conditions but using the respective or corresponding wild-type enzyme, including the ATF enzyme according to SEQ ID NO: 1, and preferably comprising a consensus active site or "Prosite motif" (the motif is in the Prosite syntax as defined at https: / / prosite.expasy.org / scanprosite / scanprosite_doc.html) of at least seven amino acid residues selected from a highly conserved partial amino acid sequence, i.e., NHx(3)-DG (where "x" represents any amino acid), corresponding to positions N218 to G224 of the polypeptide according to SEQ ID NO: 1, wherein the host cell produces at least about 10% increased retinyl acetate compared to a host cell expressing the enzyme according to SEQ ID NO: 1, as obtained by expressing the modified ATF under suitable culture conditions, including, but not limited to, culture on glucose, galactose, or xylose.

[0036] In the context of the present invention, organisms such as microorganisms, fungi, algae, or plants are also understood to 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). Thus, for example, the Lachancea mirantina strain is a synonym of the Zygosaccharomyces sp. IFO 11066 strain originating from Japan.

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

[0038] Preferably, acetylation of retinol using the modified ATF as described herein can result in an increased titer of retinyl acetate, e.g., at least about 50-92% by weight retinyl acetate based on total retinoids, i.e., a percentage in the range of at least 50-92%, e.g., 55, 60, 65, 70, 75, 80, 85, 90% or more acetylated retinoid, i.e., retinyl acetate, based on the total retinoids present in a retinoid mixture produced by a host cell, such as 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 is used as a substrate for acetylation by a modified enzyme as defined herein.

[0039] Host cells capable of producing retinol, i.e., microorganisms, algae, fungi, animal, or plant cells, may also be capable of producing β-carotene, which may further be enzymatically converted to retinal, which may then be converted to retinol. Those skilled in the art know which genes to use / express for the biosynthesis of β-carotene and / or the biological conversion of β-carotene to retinol. Such host cells, which are further capable of expressing the modified ATF as defined herein and / or additional genes required for the biosynthesis of vitamin A, can be cultured under aerobic or anaerobic conditions in aqueous media supplemented with appropriate nutrients, as known to those skilled in the art for the respective retinol-producing host cells. Optionally, such culture 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, with or without ethanol, specifically glucose, galactose, or xylose. Specific culture conditions may include batch and feed runs, with concentrations of 5% (w / v) glucose and 1% ethanol (w / v) used in the batch phase and 100% (w / v) used in the feed phase. Cultivation / growth of the host cells may be carried out under suitable culture conditions in batch, fed-batch, semi-continuous, or continuous mode, specifically fed-batch mode, for periods of 80, 90, 100, 110, 120, or 130 hours. Depending on the host cell, production of retinoids, such as vitamin A, their precursors and / or derivatives, e.g., retinal, retinol, retinyl esters, specifically retinyl acetate, may be varied as known to those skilled in the art. The cultivation and isolation of beta-carotene with retinoid-producing host cells selected from the genera Yarrowia and Saccharomyces is described, for example, in WO2008042338.Methods are described, for example, in US Patent Application Publication No. 20070166782, for the production of beta-carotene and retinoids in host cells selected from E. coli.

[0040] Specifically, fermentations using suitable retinoid-producing host strains as defined herein that express modified ATFs as described herein are cultured in a two-phase system, with the retinoid (including, but not limited to, retinyl acetate) recovered in a suitable lipophilic phase from which it is subsequently isolated. Specific conditions and lipophilic solvents are disclosed in WO2022090548 or WO2022090549.

[0041] In some embodiments, the present invention relates to two-phase fermentation using a lipophilic solvent, such as isopar or corn oil, as the second phase in addition to known solvents including Drakeol®, silicones, or n-dodecane (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 per defined amount of protein in a given time and at a defined temperature. Typically, specific activity is expressed in μmol of substrate consumed or product formed per mg of protein per minute. μmol / min is typically abbreviated as U (=unit). Thus, the unit definitions of specific activity μmol / min / (mg of protein) or U / (mg of protein) are used interchangeably throughout this specification. An enzyme is active when it performs 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. Methods for measuring enzyme activity are known to those of skill in the art, and analytical methods for assessing the ability of suitable ATFs as defined herein, specifically Atf1, to convert retinol to produce retinyl acetate are known in the art, for example, as described in Example 4 of WO2014096992. In summary, product titers such as retinyl acetate, retinol, trans-retinal, cis-retinal, and beta-carotene can be measured by HPLC.

[0043] With regard to suitable host cells that contain specific enzymes involved in the biosynthesis of beta-carotene and that are expressed and active in vivo, thereby resulting in the production of carotenoids, such as beta-carotene, both genes and methods for producing carotenoid-producing host cells are known in the art, 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 respective polypeptides are expressed and active in vivo, thereby resulting in the production of retinoids, e.g., vitamin A and its precursors, including retinol, by enzymatically converting beta-carotene via retinal to retinol. These polypeptides include modified ATFs as defined herein. Methods for generating vitamin A pathway genes and retinoid-producing host cells are known in the art. The term retinoid includes retinol, which is used as a substrate for modified acetyltransferases as defined herein.

[0045] As used herein, retinoids include beta-carotene degradation products, also known as apocarotenoids, including, but not limited to, retinal, retinoic acid, retinol, retinoic acid methoxide, retinyl acetate, retinyl esters, 4-keto-retinoids, 3-hydroxy-retinoids, or combinations thereof. As used herein, long-chain retinyl esters are defined as hydrocarbon esters of retinol and fatty acids, where the fatty acid contains at least about 8 carbon atoms, e.g., 9, 10, 12, 13, 15, or 20 carbon atoms, and up to about 26 carbon atoms, e.g., 25, 22, or 21 carbon atoms or less, and preferably up to about 6 unsaturated bonds, e.g., 0, 1, 2, 4, 5, or 6 unsaturated bonds. 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, for example, in WO2008042338.

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

[0047] As used herein, the term "carotenoid" is well known in the art. Carotenoids include long 40-carbon conjugated isoprenoid polyenes formed naturally by linking two 20-carbon geranylgeranyl diphosphate 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 position to yield canthaxanthin, zeaxanthin, or astaxanthin. The biosynthesis of carotenoids is described, for example, in International Publication No. WO2006102342.

[0048] "Vitamin A," as used herein, refers to any chemical form of vitamin A found in aqueous solutions, solids, and formulations, including retinol, retinyl acetate, and retinyl esters, including retinoic acid, whether undissociated in the free acid form or dissociated as an anion.

[0049] Specifically, the present invention relates to the following embodiments (1) to (14): (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 ATF1 of Lachancea mirantina according to SEQ ID NO: 1 or SEQ ID NO: 3, wherein the modified enzyme comprises the seven amino acid motif NHx(3)-D-[GA] (wherein "x" represents any amino acid), wherein the motif corresponds to positions N218 to G224 of the polypeptide according to SEQ ID NO: 1, and wherein the modified acetyltransferase comprises one or more amino acid substitutions at positions corresponding to Q68, A451, L452, T473, L483, and / or N512 of the polypeptide according to SEQ ID NO: 1, and wherein the modified enzyme is used to acetylate retinol to retinyl acetate at a rate of at least about 81% retinyl acetate based on total retinoids.

[0050] (2) The modified enzyme according to embodiment (1), wherein the glutamine at position 68 of SEQ ID NO: 1 is substituted with leucine, and / or the alanine at position 451 of SEQ ID NO: 1 is substituted with leucine or methionine, and / or the leucine at position 452 of SEQ ID NO: 1 is substituted with phenylalanine, and / or the threonine at position 473 of SEQ ID NO: 1 is substituted with leucine or alanine, and / or the leucine at position 483 of SEQ ID NO: 1 is substituted with methionine, and / or the asparagine at position 512 of SEQ ID NO: 1 is substituted with phenylalanine.

[0051] (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 of the unmodified polypeptide according to SEQ ID NO: 3.

[0052] (4) The modified enzyme according to embodiment (3), wherein the histidine at position 69 of SEQ ID NO: 1 is substituted with alanine, asparagine, or serine, and / or the valine at position 407 of SEQ ID NO: 1 is substituted with isoleucine, and / or the glycine at position 409 of SEQ ID NO: 1 is substituted with alanine, and / or the serine at position 480 of SEQ ID NO: 1 is substituted with glutamic acid, phenylalanine, leucine, methionine, or glutamine, and / or the isoleucine at position 484 of SEQ ID NO: 1 is substituted with leucine.

[0053] (5) The modified enzyme according to embodiment (1), (2), (3) or (4), wherein the percentage of retinyl acetate based on total retinoids obtained from catalytic acetylation of retinol is increased by at least 8% compared to the catalytic acetylation reaction using the corresponding unmodified enzyme according to SEQ ID NO: 1.

[0054] (6) The modified enzyme of embodiment (1), (2), (3), (4), or (5), expressed in a retinol-producing host cell expressing genes involved in catalyzing retinal to retinol and / or beta-carotene to retinal.

[0055] (7) A retinoid-producing host cell expressing the enzyme according to embodiment (1), (2), (3), (4), (5) or (6).

[0056] (8) The host cell according to embodiment (7), which is a fungal host cell.

[0057] (9) The host cell according to embodiment (8), wherein the host cell is selected from the genus Yarrowia or Saccharomyces.

[0058] (10) The host cell according to any one of embodiments (7), (8), or (9), further expressing enzymes involved in the mevalonate pathway and / or the carotenoid pathway for producing beta-carotene, retinal, and retinol.

[0059] (11) The host cell according to embodiment (10), wherein the enzyme that catalyzes the conversion of β-carotene to retinal is β-carotene oxygenase that selectively produces trans-retinal at a rate of at least 95% trans-retinal based on the total retinoids, including cis-retinal and trans-retinal.

[0060] (12) A process for producing retinoids, including retinal, retinol, and retinyl acetate, comprising culturing the host cell of embodiment (7), (8), (9), (10), or (11) under suitable culture conditions comprising a carbon source selected from glucose, fructose, raffinose, lactose, galactose, glycerol, xylose, arabinose, sucrose, maltose, ethanol, or a mixture thereof, and in the presence of a lipophilic substance, wherein the percentage of retinyl acetate produced during the process is at least 81% based on total retinoids.

[0061] (13) The process according to embodiment (12), wherein the lipophilic substance is selected from synthetic or natural oils or isoparaffins.

[0062] (14) The process according to embodiment (12) or (13), wherein the proportion of retinyl acetate based on total retinoids is increased by at least 8 to 500% compared to a process using ATF1 according to SEQ ID NO: 1. [Brief explanation of the drawings]

[0063] [Figure 1] 1 is the amino acid sequence of ATF1 (LmATF; SEQ ID NO: 1) from Lachancea mirantina, where the residues selected for amino acid substitution as described in this application are shown in bold / underlined, and the 10th amino acid residues are shown in bold. [Figure 2]Amino acid sequence of ATF1 (LmATF*; SEQ ID NO: 3) from Lachancea mirantina, where residues selected for amino acid substitution as described in this application are shown in bold / underlined, with the 10th amino acid residues shown in bold.

[0064] 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 published patent applications cited throughout this application, in particular WO2014096992, WO2019058001, WO2021136689, WO2022090548, WO2008042338, US Patent Application Publication No. 20070166782, WO2022090549, WO2006102342, WO2020141168, and WO2016172282, are incorporated herein by reference.

[0065] [Example] 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).

[0066] Shake Plate Assay (Yarrowia). To test the transforming activity of mutants, typically 200 μl of 0.25% yeast extract, 0.5% peptone (0.25X YP) was inoculated with 10 μl of freshly grown Yarrowia and overlaid with 200 μl of mineral oil (Isopar M, Exxon Mobile) containing 2% oleic acid as the carbon source in the mineral oil phase. Transformants were grown in 24-well plates (Microplate Devices 24 Deep Well Plates, Whatman 7701-5102), covered with mat seals (Analytical Sales and Services Inc. Plate Mats 24010CM), sterile sealed with Qiagen Airpore Tape Sheets (19571), and shaken at 30°C and 800 RPM for 4 days in an Infors Multitron multiplate shaker. The mineral oil fraction was removed from the shaker plate wells and analyzed by UPLC reversed-phase column with a photodiode array detector. This method is also used in Example 2.

[0067] DNA transformation. Yarrowia lipolytica strains were transformed after overnight growth on YPD plates. 50 μl of cells were scraped from the plates and transformed by incubating 1 μg of transforming DNA (typically linear DNA for integrative transformation) in 500 μl of 40% PEG 3550MW, 100 mM lithium acetate, 50 mM dithiothreitol, 5 mM Tris-Cl (pH 8.0), and 0.5 mM EDTA at 40°C for 30 minutes and then plated directly onto selective media. Alternatively, for selection with dominant antibiotic markers, cells were grown in YPD liquid medium at 30°C for 4 hours before plating onto selective media. Saccharomyces strains were transformed from log-phase YPD-grown cells using the lithium acetate method and expanded by subculturing YPD cultures overnight. 10 cells per transformation were used. 8Cells were harvested and resuspended in a final volume of 500 μL in a mixture containing 40% PEG 3350 (MW), 100 mM lithium acetate, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 5 μg of sheared salmon sperm DNA, and 2 μg of linearized transforming DNA. This mixture was incubated at 30°C for 1 hour, followed by incubation at 42°C for 30 minutes. Cells were then pelleted, 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 before plating on selective medium containing 100 μg / ml hygromycin. Most of the DNA sequences used herein have been codon-optimized for expression in the respective host systems and are shown in the Sequence Listing.

[0068] DNA Molecular Biology. Plasmids MB10306 (SEQ ID NO: 5) and MB10569 (SEQ ID NO: 6), containing the DrBCO, LmATF, and FfRDH expression systems, were synthesized using GenScript (Piscataway, NJ, USA). Plasmid MB10306 contains both the "URA3" and "HOM3" markers for selection in Yarrowia lipolytica transformation. To ensure clean gene insertion by random, non-homologous end-ligation of this gene with the SfiI marker, the desired plasmid fragment (MB10306 or other plasmids listed in Table 1) was purified by gel electrophoresis and a Qiagen gel purification column. Clones were verified by sequencing. Typically, genes are synthesized using GenScript (Piscataway, NJ) by introducing amino acid substitutions (see "Mutant" column) according to Table 1. Transformants were screened for homoserine auxotrophy and subsequently sequenced using primers flanking the HOM3 sequence to select clean frameshifts for forward progression. Expression of mutant ATFs in Saccharomyces cerevisiae is described in Example 1 of WO2020141168.

[0069] Sequences. Plasmids containing LmATF and LmATF*, respectively, according to SEQ ID NOs: 2 and 4 (the polynucleotide according to SEQ ID NO: 4 corresponds to the nucleic acid encoding LmATF1 from L. mirantina set forth in SEQ ID NO: 2 in WO2020141168), with codon-optimized sequences for expression in Yarrowia lipolytica or Saccharomyces cerevisiae, as well as modified enzymes containing specific amino acid substitutions, are listed in Table 1 and / or the Sequence Listing.

[0070] [Table 1]

[0071] [Table 2]

[0072] UPLC reversed-phase retinol method. For rapid screening, this method does not separate cis isomers, but only the major functional groups. Samples were injected using a Waters Acquity UPLC with PDA detection (or similar) along with an autosampler. Retinoids were resolved using an Acquity UPLC HSS T3 1.8um P / N 186003539. The mobile phase consisted of either 1000 mL of hexane, 30 mL of isopropanol, or 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.

[0073] [Table 3]

[0074] [Table 4]

[0075] Assay Method: The method assays for retinyl acetate and quantifies retinol and retinal relative to retinyl acetate using the indicated response factors. To prepare a stock solution using a volumetric flask, retinyl acetate was dissolved in THF at approximately 200 μg / ml. Using a volumetric flask, 20x, 50x, and 100x dilutions of the stock solution were made with 50 / 50 methanol / MTBE. The UV absorbance of retinyl acetate becomes nonlinear fairly rapidly, so care must be taken to stay within the linear range. Therefore, lower concentrations may be more appropriate. Retinyl palmitate can also be used as a calibrator for retinyl esters. The retinyl acetate peak is at approximately 3 minutes, while the retinyl esters (long-chain retinyl esters) peak at approximately 3.5 minutes.

[0076] Sample preparation. Samples were prepared using various methods depending on the conditions. For whole broth or washed broth samples, the broth was weighed into a Precellys® tube and mobile phase was added. Briefly, 25 μl of thoroughly mixed broth and 975 μl of THF were added to a 2 ml Precellys® tube. The sample was then processed in a Precellys® homogenizer (Bertin Corp, Rockville, MD, USA) at the highest setting three times according to the manufacturer's instructions, typically 3 × 15 × 7500 tpms. For washed pellets, the sample was spun in a 1.7 ml tube in a microcentrifuge at 10,000 rpm for 1 minute, the broth was decanted, 1 ml of water was added, mixed, pelleted, and decanted back to the original volume. The mixture was pelleted again, placed back in the appropriate amount of mobile phase, and processed by Precellys® bead beating. For analysis of the silicone oil fraction, samples were spun at 4000 RPM for 10 min, the oil was decanted using a positive displacement pipette (Eppendorf, Hauppauge, NY, USA), the top portion discarded, diluted in mobile phase mixed by vortexing, and the retinoid concentration determined by UPLC analysis.

[0077] Fermentation conditions for Yarrowia. Fermentation was preferably the same as described above using a silicone oil overlay and stirred tank, preferably glucose, in a benchtop reactor with a total volume of 0.5-5 L (see WO2016172282). Generally, the same results were observed in a fed-batch stirred tank reactor, demonstrating the utility of the system for producing retinoids through increased productivity. Preferably, fermentation was batched with 5% glucose, with 20% silicone oil added after the dissolved oxygen had fallen below about 20%, and feeding resumed to achieve 20% dissolved oxygen throughout the entire feed program.

[0078] Example 2: Production of retinyl acetate in Yarrowia lipolytica expressing mutant LmATF To express heterologous ATFs in Yarrowia lipolytica as a host, strain ML15710 (see Example 5 in WO 2016172282) was transformed with plasmid MB9287 (see Example 1 in WO 2022090548) and a lip2 lip3 lip8 mutant derivative was isolated. This derivative was selected on 5-fluorouracil acid to isolate a uracil auxotroph, designated strain ML18667-new. This strain was transformed with the plasmids listed in Table 1 above, each consisting of the indicated ATF allele, DrBCO, and FfRDH12. Transformants of ML18667-new containing the SfiI-linearized plasmids from Table 1 were selected for uracil prototrophy. Transformants were grown in shake plates as described in Example 1, and the percentage of retinyl acetate using the mutant ATFs relative to the percentage of retinyl acetate using the reference LmATF expressed in plasmid MB10603 (set at 100%; SEQ ID NO: 5) or LmATF* expressed in plasmid MB10569 (retinyl acetate / total retinoids) is shown in Table 3.

[0079] [Table 5]

[0080] [Table 6]

[0081] Each of the mutations shown increased the percentage of retinyl acetate from 8% to over 560% when compared to the reference sequence according to SEQ ID NO:1.

[0082] [Table 7]

[0083] Each of the mutations shown improved the percentage of retinyl acetate from 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 at a rate of at least about 81% retinyl acetate based on total retinoids, said enzyme being derived from a Lachancea milantina (Lachancea milanthina) enzyme according to SEQ ID NO: 1 or SEQ ID NO:

3. mirantina) ATF1, wherein the modified enzyme comprises a seven amino acid motif N-H-x(3)-D-[GA], where "x" represents any amino acid, corresponding to positions N218 to G224 of the polypeptide according to SEQ ID NO: 1, and the modified acetyltransferase comprises at least one amino acid substitution at a position corresponding to A451, T473, and / or L483 of the polypeptide according to SEQ ID NO: 1, wherein introduction of the amino acid substitution increases the rate of retinyl acetate by at least 10-20% by weight compared to a method using the same conditions but using the respective or corresponding wild-type enzyme comprising the ATF enzyme according to SEQ ID NO:

1.

2. The modified enzyme of claim 1, further comprising at least one amino acid substitution at positions corresponding to Q68, L452, and / or N512 of the unmodified polypeptide according to SEQ ID NO: 1, wherein the modified enzyme is used to acetylate retinol to retinyl acetate at a rate of at least about 81% retinyl acetate based on total retinoids.

3. 3. The modified enzyme of claim 1 or 2, wherein the glutamine at position 68 of SEQ ID NO: 1 is substituted with leucine, and / or the alanine at position 451 of SEQ ID NO: 1 is substituted with leucine or methionine, and / or the leucine at position 452 of SEQ ID NO: 1 is substituted with phenylalanine, and / or the threonine at position 473 of SEQ ID NO: 1 is substituted with leucine or alanine, and / or the leucine at position 483 of SEQ ID NO: 1 is substituted with methionine, and / or the asparagine at position 512 of SEQ ID NO: 1 is substituted with 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 of the polypeptide according to SEQ ID NO: 1, wherein the histidine at position corresponding to 69 of SEQ ID NO: 1 is substituted with alanine, asparagine or serine, and / or the valine at position corresponding to 407 of SEQ ID NO: 1 is substituted with isoleucine, and / or the glycine at position corresponding to 409 of SEQ ID NO: 1 is substituted with alanine, and / or the serine at position corresponding to 480 of SEQ ID NO: 1 is substituted with glutamic acid, phenylalanine, leucine, methionine or glutamine, and / or the isoleucine at position corresponding to 484 of SEQ ID NO: 1 is substituted with leucine.

5. The following amino acid substitutions: 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_A451 5. The modified enzyme of any one of claims 1 to 4, comprising at least one of the following positions: _L483M_L452F, T473L_A451M_L483M_L452F, LmATF_T473A_A451L_L483M_L452F_Q68L_N512F, T473A_A451M_L483M_L452F_Q68L_N512F, T473L_A451M_L483M_L452F_Q68L_N512F, wherein said positions correspond to amino acid residues in a polypeptide according to SEQ ID NO:

1.

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

1.

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

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

9. 9. The host cell of 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 for producing beta-carotene, retinal and retinol.

11. The host cell of claim 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 rate of at least 95% trans-retinal based on all retinoids including cis-retinal and trans-retinal.

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

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

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

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