Novel mutant of acetyltransferase SB-ATF

Abstract

The invention relates to a method for converting a low-activity enzyme participating in acetylation of retinoid into a high-activity enzyme, in particular to a method for converting the low-activity enzyme into the high-activity enzyme for acetylating retinol into retinol acetate.

Description

[0001] The present invention relates to a method for converting a low-activity enzyme involved in retinoid acetylation into a high-activity enzyme. The enzyme catalyzing retinoid acetylation is particularly a high-activity enzyme for acetylation of retinol to retinol acetate.

[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 or intermediates, including microbial conversion steps, have been investigated, which would be more economical and ecological.

[0004] Typically, biological systems that produce retinoids are industrially difficult to handle and / or produce compounds at such low levels that their isolation on an industrial scale is not economically viable. There are several reasons for this, the most important of which are the instability of the intermediates / retinoids and / or the relatively high accumulation of by-products.

[0005] One way to address the instability problem is to produce acetylated product forms. Acetylation of carotenoids (e.g., astaxanthin or zeaxanthin) has been previously reported, for example, by the action of Atf1 (SbATF1) from Saccharomyces pasteurianus (WO2014096992), where, for example, acetylation of zeaxanthin is in the range of up to 90 wt%. However, when the enzyme is used in the acetylation of retinoids, in particular retinol, the percentage of acetylation is only in the range of about a maximum of 10% based on the total retinoid (see, for example, WO2019058001), which is too low to establish a sustainable and economically viable industrial process for the biological production of vitamin A.

[0006] Therefore, there is a strong need for improved enzymes for the acetylation of retinoids.

[0007] Surprisingly, we have now been able to identify amino acid positions in fungal acetyltransferases (ATFs), such as those from Saccharomyces bayanus and other yeasts, that are critical for the formation of acetylated retinoids, particularly the conversion of retinol to retinyl acetate. Modification of certain amino acids results in increased formation of retinyl acetate compared to the corresponding wild-type enzyme, for example, an increase of at least about 10% when compared to the acetylation of retinol using the corresponding unmodified enzyme (e.g., wt-ATF1 from Saccharomyces bayanus as shown in SEQ ID NO: 1).

[0008] Thus, in one embodiment, the present invention relates to a method for modifying an enzyme involved in / catalyzing the acetylation of retinoids, more particularly fungal ATF, comprising introducing at least one modification (e.g., amino acid substitution) at a position specified herein, for example comprising 1 to 9, for example at least 2, amino acid substitutions. Upon introduction of the specified amino acid substitutions, the unmodified ATF, in particular the fungal ATF (e.g., ATF1 from Saccharomyces spp., for example, ATF1 from Saccharomyces pastorianus (SbATF) according to SEQ ID NO: 1), is modified or converted into a modified or mutated ATF having increased activity, wherein when such modified enzyme is expressed in a suitable expression system (including a suitable host cell, as specified herein), the percentage of retinol acetate obtained from the acetylation of retinol can be increased by at least about 10%, for example, by more than 700%, compared to the percentage of retinol acetate obtained using the corresponding unmodified or wild-type enzyme.

[0009] In particular, the present invention relates to a method for modifying an enzyme that catalyzes the acetylation of retinoids, in particular an enzyme that catalyzes the acetylation of retinol to retinyl acetate, preferably a fungal enzyme, comprising introducing at least one amino acid substitution at one or more positions selected from the group consisting of amino acid residues 34, 35, 346, 371, 373, 419, 434, 437, 478, and combinations thereof, in a polypeptide according to SEQ ID NO: 1, wherein after introduction of said one or more amino acid substitutions, the activity of forming retinyl acetate is increased by at least about 10%, such as in the range of 10-760%, compared to the activity of the corresponding unmodified enzyme using the enzyme according to SEQ ID NO: 1, preferably wherein at least one modification, such as an amino acid substitution, such as comprising 1 to 9 amino acid substitutions, at a position specified herein, preferably at one or more positions selected from the group consisting of amino acid residues 34, 35, 346, 371, 373, 419, 434, 437, 478, and combinations thereof, in a polypeptide according to SEQ ID NO: 1. The amino acid residues corresponding to 34, 35, 346, 371, 373, 419, 434, 437, 478 and / or combinations thereof in the polypeptide of NO: 1 are different from L34, Y35, F346, V371, F373, N419, M434, R437, W478 and combinations thereof, i.e., correspond to the corresponding amino acid residues in SEQ ID NO: 1.

[0010] More specifically, the present invention relates to a method for modifying an enzyme that catalyzes the acetylation of a retinoid, in particular an enzyme that acetylates retinol to retinyl acetate, preferably a fungal enzyme, comprising introducing at least two amino acid substitutions at positions 34 and 434 of an amino acid residue in a polypeptide selected from the group consisting of SEQ ID NO: 1, wherein after the introduction of the at least two amino acid substitutions, the enzyme is modified using a method comprising modifying the enzyme according to SEQ ID NO: 1. %, 350%, 380%, 400%, 430%, 450%, 480%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, e.g., in the range of 10% to 760%, compared to the activity of the corresponding unmodified enzyme of SEQ ID NO:1, preferably wherein in the range of 10% to 760% the activity of the retinyl acetate forming enzyme corresponding to SEQ ID NO:1 is increased by at least about 10%, such as at least about 25%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, 180%, 200%, 230%, 250%, 280%, 300%, 330%, 350%, 380%, 400%, 430%, 450%, 480%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, The amino acid residues at positions 34 and 434 of NO: 1 are different from L34 and M434, more preferably wherein the amino acid substitutions corresponding to positions L34 and M434 in the polypeptide according to SEQ ID NO: 1 are selected from L34F and M434V.

[0011] As used herein, an unmodified retinol acetylase as defined herein having "low activity" for the acetylation of retinol means that the percentage of retinol acetate is in the range of about 10 wt % or less based on the total retinoid produced in a suitable retinoid-producing host cell expressing the unmodified enzyme. A modified enzyme as defined herein having "increased activity" for the acetylation of retinol means that the percentage of retinol acetate is from about 11 wt % to 86 wt % based on the total retinoid produced in a suitable retinoid-producing host cell expressing the modified enzyme, i.e., the modified enzyme is produced by converting an enzyme with low (enzyme or acetylation) activity to an enzyme with increased (enzyme or acetylation) activity as defined herein by introducing one or more amino acid substitutions as defined herein. As shown, the acetylation activity of retinol can be increased by at least about 10%, for example, by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750% or more.

[0012] More specifically, the present invention relates to a method for producing a retinol acetylase enzyme having increased activity for the acetylation of retinol to retinol acetate, the method comprising:

[0013] (a) providing a fungal enzyme involved in the acetylation of retinol, in particular an ATF derived from Saccharomyces, preferably SbATF, wherein the unmodified enzyme expressed in a suitable host cell and under suitable culture conditions is capable of producing 10 wt% or less of retinyl acetate based on total retinoids;

[0014] (b) further introducing one or more amino acid substitutions, e.g., at least one to nine, preferably at least two, amino acid substitutions, into the enzyme of (a) to generate a modified enzyme that participates in the acetylation of retinol, wherein the ability to convert retinol to retinol acetate is increased by at least about 10%, e.g., at least 10 to 760%, when compared to the acetylation activity of the corresponding unmodified enzyme of step (a);

[0015] wherein the one or more (e.g., at least two) amino acid substitutions are at positions corresponding to amino acid residues selected from positions 34, 35, 346, 371, 373, 419, 434, 437, 478, and combinations thereof in SEQ ID NO: 1, and wherein the substitute amino acid is different from L34, Y35, F346, V371, F373, N419, M434, R437, W478, and / or combinations thereof, preferably wherein the at least two amino acid substitutions are at positions corresponding to positions 34 and 434 in the polypeptide according to SEQ ID NO: 1.

[0016] In one embodiment, the present invention relates to a modified enzyme, in particular a fungal enzyme, involved in the acetylation of retinol to retinol acetate, comprising one or more amino acid substitutions, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9 amino acid substitutions, such as at least 2 amino acid substitutions, at positions corresponding to amino acid residues selected from positions 34, 35, 346, 371, 373, 419, 437, 478 and combinations thereof in a polypeptide according to SEQ ID NO: 1, and wherein the substituted amino acid residue is different from the amino acid corresponding to L34, Y35, F346, V371, F373, N419, M434, R437, W478 and combinations thereof in a polypeptide according to SEQ ID NO: 1, preferably wherein the amino acid residue to be substituted corresponds to the amino acid residue selected from positions 34, 35, 346, 371, 373, 419, 437, 478 and combinations thereof in a polypeptide according to SEQ ID NO: 1. NO: 1 in the polypeptide of residues selected from L34, Y35, F346, V371, F373, N419, M434, R437, W478 and combinations thereof, more preferably wherein the substituted amino acid residue to be introduced at the position corresponding to position 34, 35 or 478 in SEQ ID NO: 1 is preferably phenylalanine, wherein the substituted amino acid residue to be introduced at the position corresponding to position 346 or 419 in SEQ ID NO: 1 is preferably leucine, wherein the substituted amino acid residue to be introduced at the position corresponding to position 371 in SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at the position corresponding to position 373 in SEQ ID NO: 1 is preferably alanine, and / or wherein the substituted amino acid residue to be introduced at the position corresponding to position 434 or 437 in SEQ ID NO: 1 is preferably valine.

[0017] In particular, the modified enzyme according to the invention and as defined herein is selected from a polypeptide having at least about 20% identity to SEQ ID NO: 1, 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, contained in a polypeptide selected from the group consisting of SEQ ID NO: 1. One or more amino acid substitutions different from L34, Y35, F346, V371, F373, N419, M434, R437 or W478 at positions 34, 35, 346, 371, 373, 419, 434, 437 and / or 478 in NO: 1, preferably wherein the one or more amino acid substitutions are selected from L34F, Y35F, V371I, M434V, N419L, F346L, R437V, W478F, F373A and combinations thereof.

[0018] The use of such a modified enzyme 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, results in an increase of at least about 10% in the production of retinol acetate via retinol acetylation compared to a method using the same conditions but using the respective or corresponding wild-type enzyme (e.g., the ATF enzyme according to SEQ ID NO: 1). In particular, the use of such a modified enzyme results in a titer of retinol acetate in the range of up to 86% by weight based on total retinoid compared to the use of the respective unmodified enzyme where the titer is in the range of a maximum of 10% by weight of retinol acetate based on total retinoid.

[0019] The terms "acetyltransferase," "retinol acetylase," "enzyme having retinol acetylation activity," "ATF," or "ATF1" are used interchangeably herein and refer to an enzyme of EC class [EC 2.3.1.84] that is capable of catalyzing the conversion of retinol to retinyl acetate, including naturally occurring enzymes and enzymes produced synthetically with the aid of artificial intelligence. The terms "SbATF" and "SbATF1" are used interchangeably herein. An example of such an enzyme is shown in SEQ ID NO: 1. As defined herein, an ATF that is capable of catalyzing the acetylation of retinol to retinyl acetate and having a percentage of retinyl acetate based on total retinoids of about 10% or less is referred to herein as "unmodified" ATF or "wild-type" ATF, such as SbATF according to SEQ ID NO: 1.

[0020] 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 as defined herein, shows an increase in the formation of retinol acetate from retinol by at least 10%, based on total retinoids and compared to the formation of retinol acetate using the corresponding / corresponding wild-type enzyme (e.g. an enzyme according to SEQ ID NO: 1).

[0021] In one embodiment, the modified ATF according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 amino acid substitutions compared to the corresponding wild-type ATF, such as, for example, at least 2 amino acid substitutions, wherein one or more amino acids corresponding to L34, Y35, F346, V371, F373, N419, M434, R437 and / or W478 in the polypeptide according to SEQ ID NO: 1 are exchanged, resulting in an increase of at least about 10% in the enzymatic activity towards retinyl acetate formation compared to the corresponding unmodified ATF (e.g., ATF according to SEQ ID NO: 1), in particular an increase in the percentage of retinyl acetate based on total retinoids from about 10 wt.-% to 86 wt.-% or even more.

[0022] The present invention includes methods for converting / converting a low-activity enzyme (with respect to the acetylation of retinol to retinyl acetate) into a modified enzyme having increased activity for the acetylation of retinol to retinyl acetate as defined herein, also referred to herein as an unmodified enzyme. Examples of such unmodified enzymes are enzymes having at least about 20% identity to SEQ ID NO: 1, which exhibit about 10% acetylation activity, i.e., when expressed in a suitable retinol-producing host cell, the percentage of retinyl acetate obtained from the conversion / acetylation of retinol is in the range of 10% by weight or less based on total retinoids, in particular ATF1 isolated from Saccharomyces pastorianus (SbATF1), including the enzyme encoded by the polynucleotide according to SEQ ID NO: 2.

[0023] 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, including SbATF1 isolated / derived from Saccharomyces pasteurianus) are defined as enzymes that exhibit about 10% acetylation activity, i.e., when expressed in a suitable retinol-producing host cell, the percentage of retinyl acetate obtained from the conversion / acetylation of retinol based on total retinoids is in the range of 10% by weight or less, and are capable of converting true retinol to acetylated retinol. An enzyme obtained from a bacterial enzyme, in particular an enzyme obtainable from yeast, in particular comprising at least 7 highly conserved partial amino acid sequences selected from the group consisting of [NSDEHC]-Hx(3)-D-[GA] (motif in Prosite syntax, see: https: / / prosite.expasy.org / scanprosite / scanprosite_doc.html), preferably [NHx(3)-D-[GA], more preferably NHCMCDG, wherein "x" represents any amino acid and the central histidine is part of the binding pocket of the enzyme, corresponding to positions N190 to G196 in the polypeptide according to SEQ ID NO: 1.

[0024] Suitable unmodified enzymes can be selected from yeast, including but not limited to Saccharomyces, such as S. bayanus, S. cerevisia, S. kudriavzevii, S. mikatae, S. pastorianus, S. arboricola, Candida, such as C. dubliniensis, C. albicans, C. intermedia, C. auris, C. lusitaniae, Wickerhamomyces, such as W. anomalus, Aspergillus, such as A. ellipticus.

[0025] 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 corresponding / corresponding unmodified enzyme (e.g. the enzyme according to SEQ ID NO: 1), for example being obtainable 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.

[0026] 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.

[0027] 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 %.

[0028] 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 certain percentage of retinol acetate based on the total retinoids present / produced by a suitable host cell when expressing the ATF, wherein an increase of retinol acetate to 86 wt. % or more based on the total retinoids can be achieved using a modified ATF as defined herein.

[0029] Suitable host cells according to the present invention include fungal host cells and microbial host cells, such as E. coli. As used herein, the term "fungal host cell" particularly includes yeast cells, wherein the cell is a retinol-producing host cell, particularly a retinyl acetate-producing host cell, such as a retinyl 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.

[0030] 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.

[0031] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at the position corresponding to residue 34 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being replaced by any amino acid other than leucine, in particular by exchanging the original amino acid with phenylalanine, for example, in particular by replacing leucine with phenylalanine (e.g., L34F in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with SbATF as shown in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises an amino acid substitution at the position corresponding to residue 434 in the polypeptide according to SEQ ID NO: 1, in particular, a substitution of methionine at the position corresponding to M434 in SEQ ID NO: 1 by valine. The use of such a modified enzyme comprising the mutation in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase of at least about 10% to more than 700% in retinol acetate produced from the acetylation of retinol, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0032] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 35 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being substituted by any other amino acid except tyrosine, in particular by exchanging the original amino acid with phenylalanine, for example in particular via replacing tyrosine with phenylalanine (e.g. Y35F in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with the SbATF shown in SEQ ID NO: 1. Preferably, this modified enzyme further comprises an amino acid substitution at a position corresponding to residues 34 and 434 in the polypeptide according to SEQ ID NO: 1, in particular the leucine at a position corresponding to L34 in SEQ ID NO: 1 is substituted by phenylalanine and the methionine at a position corresponding to M434 in SEQ ID NO: 1 is substituted by valine. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase in retinyl acetate production from acetylation of retinol by at least about 10% to over 700%, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0033] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 346 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being substituted by any other amino acid except phenylalanine, in particular by exchanging the original amino acid with leucine, for example in particular via substitution of phenylalanine with leucine (e.g. F346L in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with the SbATF shown in SEQ ID NO: 1. Preferably, this modified enzyme further comprises an amino acid substitution at a position corresponding to residues 34 and 434 in the polypeptide according to SEQ ID NO: 1, in particular substitution of leucine at a position corresponding to L34 in SEQ ID NO: 1 by phenylalanine and substitution of methionine at a position corresponding to M434 in SEQ ID NO: 1 by valine. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase in retinyl acetate production from acetylation of retinol by at least about 10% to over 700%, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0034] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at the position corresponding to residue 371 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being substituted by any other amino acid except valine, in particular by exchanging the original amino acid with isoleucine, such as in particular via substitution of valine with isoleucine (e.g. V371I in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with the SbATF shown in SEQ ID NO: 1. Preferably, this modified enzyme further comprises an amino acid substitution at the position corresponding to residues 34 and 434 in the polypeptide according to SEQ ID NO: 1, in particular substitution of leucine at the position corresponding to L34 in SEQ ID NO: 1 by phenylalanine and substitution of methionine at the position corresponding to M434 in SEQ ID NO: 1 by valine. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase in retinyl acetate production from acetylation of retinol by at least about 10% to over 700%, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0035] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 373 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being substituted by any other amino acid except phenylalanine, in particular by exchanging the original amino acid with alanine, for example in particular via replacing phenylalanine with alanine (e.g. F373A in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with the SbATF shown in SEQ ID NO: 1. Preferably, this modified enzyme further comprises an amino acid substitution at a position corresponding to residues 34 and 434 in the polypeptide according to SEQ ID NO: 1, in particular the leucine at a position corresponding to L34 in SEQ ID NO: 1 is substituted by phenylalanine and the methionine at a position corresponding to M434 in SEQ ID NO: 1 is substituted by valine. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase in retinyl acetate production from acetylation of retinol by at least about 10% to over 700%, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0036] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at the position corresponding to residue 419 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being substituted by any other amino acid than asparagine, in particular by exchanging the original amino acid with leucine, for example in particular via substitution of asparagine with leucine (e.g. N419L in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with the SbATF shown in SEQ ID NO: 1. Preferably, this modified enzyme further comprises an amino acid substitution at the position corresponding to residues 34 and 434 in the polypeptide according to SEQ ID NO: 1, in particular substitution of leucine at the position corresponding to L34 in SEQ ID NO: 1 by phenylalanine and substitution of methionine at the position corresponding to M434 in SEQ ID NO: 1 by valine. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase in retinyl acetate production from acetylation of retinol by at least about 10% to over 700%, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0037] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at the position corresponding to residue 434 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being replaced by any other amino acid except methionine, in particular by exchanging the original amino acid with valine, for example, in particular by replacing methionine with valine (e.g., M434V in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with the SbATF shown in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises an amino acid substitution at the position corresponding to residue 34 in the polypeptide according to SEQ ID NO: 1, in particular, a substitution of leucine at the position corresponding to L34 in SEQ ID NO: 1 by phenylalanine. The use of such a modified enzyme comprising the mutation in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase of at least about 10% to more than 700% in retinol acetate produced from the acetylation of retinol, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0038] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 437 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being substituted by any other amino acid except arginine, in particular by exchanging the original amino acid with valine, for example in particular via replacing arginine with valine (e.g. R437V in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with the SbATF shown in SEQ ID NO: 1. Preferably, this modified enzyme further comprises an amino acid substitution at a position corresponding to residues 34 and 434 in the polypeptide according to SEQ ID NO: 1, in particular the leucine at a position corresponding to L34 in SEQ ID NO: 1 is substituted by phenylalanine and the methionine at a position corresponding to M434 in SEQ ID NO: 1 is substituted by valine. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase in retinyl acetate production from acetylation of retinol by at least about 10% to over 700%, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0039] In one embodiment, the modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 478 in the polypeptide according to SEQ ID NO: 1, resulting in the original residue being substituted by any other amino acid except tryptophan, in particular by exchanging the original amino acid with phenylalanine, for example in particular via substitution of tryptophan with phenylalanine (e.g. W478F in SEQ ID NO: 1). The enzyme to be modified may be derived from an ATF having at least about 20% identity with the SbATF shown in SEQ ID NO: 1. Preferably, this modified enzyme further comprises an amino acid substitution at a position corresponding to residues 34 and 434 in the polypeptide according to SEQ ID NO: 1, in particular substitution of the leucine at the position corresponding to L34 in SEQ ID NO: 1 by phenylalanine and substitution of the methionine at the position corresponding to M434 in SEQ ID NO: 1 by valine. Use of such modified enzymes comprising the mutations in a fermentation process using a suitable carbon source (e.g., glucose) results in an increase in retinyl acetate production from acetylation of retinol by at least about 10% to over 700%, compared to a corresponding process using the corresponding wild-type enzyme (e.g., an enzyme according to SEQ ID NO: 1).

[0040] As used herein, the term "original amino acid" refers to an amino acid residue present in wild-type or unmodified ATF that is to be substituted with another amino acid residue such that the activity of the modified enzyme towards retinol acetylation is increased as defined herein.

[0041] The present invention relates to a method for introducing at least about 1 to 9, for example at least 2, amino acid substitutions into an ATF (particularly a fungal ATF, for example an ATF according to SEQ ID NO: 1 or having at least about 20% identity thereto), wherein the activity of the unmodified ATF as described herein is increased by at least about 10%, i.e., the acetylation of retinol to retinol acetate is increased by at least about 10% (resulting in the production of up to 86 wt % retinol acetate based on total retinoids), compared to the percentage of retinol acetate obtained in the system / host cell using the corresponding unmodified ATF (for example an ATF according to SEQ ID NO: 1) in the range of up to about 10 wt %, wherein a single amino acid substitution as described herein can be combined with one or more amino acid substitutions to increase the activity of the enzyme for the acetylation of retinol to retinol acetate.

[0042] Thus, in some embodiments, the modified enzyme as described herein comprises two amino acid substitutions, particularly at residues corresponding to positions L34 and M434 in the polypeptide according to SEQ ID NO: 1, more particularly comprising the introduction of a phenylalanine at the position corresponding to L34 and a valine at the position corresponding to M434 in the polypeptide according to SEQ ID NO: 1, such as amino acid substitutions corresponding to L34F and M434V in the polypeptide according to SEQ ID NO: 1. The use of such a modified enzyme can result in an increase in retinyl acetate of at least about 150% compared to the percentage of retinyl acetate using the corresponding unmodified ATF (e.g., the enzyme according to SEQ ID NO: 1).

[0043] In some embodiments, the modified enzyme as described herein comprises three amino acid substitutions, in particular at the residues corresponding to positions L34 and M434, in combination with an amino acid substitution at a position corresponding to F346, V371, N419, R437 or W478 in the polypeptide according to SEQ ID NO: 1, more particularly comprising the introduction of a phenylalanine at the position corresponding to L34 in the polypeptide according to SEQ ID NO: 1 and a valine at the position corresponding to M434 in the polypeptide according to SEQ ID NO: 1, in combination with the introduction of a leucine at the position corresponding to F346, an isoleucine at the position corresponding to V371, a leucine at the position corresponding to N419 or a valine at the position corresponding to R437 in the polypeptide according to SEQ ID NO: 1, for example the amino acid substitutions L34F and M434V with F346L, V371I, N419L or R437V in the polypeptide according to SEQ ID NO: 1. Use of such modified enzymes can result in an increase in retinol acetate of at least about 60 to 550% compared to the percentage of retinol acetate using a corresponding unmodified ATF (eg, the enzyme according to SEQ ID NO: 1).

[0044] In some embodiments, the modified enzyme as described herein comprises four amino acid substitutions, in particular at residues corresponding to positions L34 and M434, in combination with amino acid substitutions at positions corresponding to Y35, F346, V371, N419, R437 or W478 in the polypeptide according to SEQ ID NO: 1, more particularly comprising the introduction of a phenylalanine at the position corresponding to L34 and a valine at the position corresponding to M434 in the polypeptide according to SEQ ID NO: 1, in combination with the introduction of a phenylalanine at the position corresponding to Y35, a leucine at the position corresponding to F346, an isoleucine at the position corresponding to V371, a leucine at the position corresponding to N419, a valine at the position corresponding to R437 or a phenylalanine at the position corresponding to W478 in the polypeptide according to SEQ ID NO: 1, e.g. Amino acid substitutions of L34F and M434V in combination with Y35F, F346L, V371I, N419L, R437V or W478F in the polypeptide of ID NO: 1, include but are not limited to the amino acid substitutions L34F_Y35F_V371I_M434V or L34F_V371I_M434V_N419L or L34F_V371I_M434V_F346L or L34F_M434V_N419L_F346L or L34F_V371I_M434V_F437V or L34F_M434V_N419L_R437V or L34F_M434V_N419L_W478F. Use of such modified enzymes can result in an increase in retinol acetate of at least about 230 to 670% compared to the percentage of retinol acetate using a corresponding unmodified ATF (eg, the enzyme according to SEQ ID NO: 1).

[0045] In some embodiments, the modified enzyme as described herein comprises 5 amino acid substitutions, in particular at the residues corresponding to positions L34, M434 and V371, in combination with an amino acid substitution at a position corresponding to Y35, F346, N419, R437 or W478 in the polypeptide according to SEQ ID NO: 1, more particularly comprising the introduction of a phenylalanine at the position corresponding to L34 in the polypeptide according to SEQ ID NO: 1 and a valine at the position corresponding to M434 in the polypeptide according to SEQ ID NO: 1, in combination with the introduction of a phenylalanine at the position corresponding to Y35 in the polypeptide according to SEQ ID NO: 1, a leucine at the position corresponding to F346, a leucine at the position corresponding to N419, a valine at the position corresponding to R437 or a phenylalanine at the position corresponding to W478 in the polypeptide according to SEQ ID NO: 1, e.g. Amino acid substitutions of L34F and M434V in combination with Y35F, F346L, N419L, R437V or W478F in the polypeptide of ID NO: 1, include but are not limited to the amino acid substitutions L34F_Y35F_V371I_M434V_N419L or L34F_V371I_M434V_N419L_F436L or L34F_Y35F_V371I_M434V_R437V or L34F_V371I_M434V_N419L_R437V or L34F_Y35F_V371I_M434V_W478F or L34F_V371I_M434V_N419L_W478F. Use of such modified enzymes can result in an increase in retinol acetate of at least about 300 to 760% compared to the percentage of retinol acetate using a corresponding unmodified ATF (eg, the enzyme according to SEQ ID NO: 1).

[0046] In some embodiments, the modified enzyme as described herein comprises 6 amino acid substitutions, in particular at the residues corresponding to positions L34, Y35, V371, M434 and N419, in combination with an amino acid substitution at a position corresponding to F34, R437 or W478 in the polypeptide according to SEQ ID NO: 1, more particularly comprising the introduction of a phenylalanine at the position corresponding to L34, a phenylalanine at the position corresponding to Y35, an isoleucine at the position corresponding to V371, a valine at the position corresponding to M434 and a leucine at the position corresponding to N419 in the polypeptide according to SEQ ID NO: 1, in combination with the introduction of a leucine at the position corresponding to F346, a valine at the position corresponding to R437 or a phenylalanine at the position corresponding to W478 in the polypeptide according to SEQ ID NO: 1, e.g. Amino acid substitutions of L34F, Y35F, V371I, M434V, and N419L in combination with F346L, R437V, or W478F in the polypeptide of SEQ ID NO: 1, including but not limited to the amino acid substitutions L34F_Y35F_V371I_M434V_N419L_F346L, or L34F_Y35F_V371I_M434V_N419L_R437V, or L34F_Y35F_V371I_M434V_N419L_W478F. Use of such modified enzymes can result in an increase in retinol acetate of at least about 130 to 600% compared to the percentage of retinol acetate using a corresponding unmodified ATF (e.g., an enzyme according to SEQ ID NO: 1).

[0047] In some embodiments, the modified enzyme as described herein comprises 8 amino acid substitutions, in particular at the residues corresponding to positions L34, Y35, V371, M434, N419, F346, R437 and W478 in the polypeptide according to SEQ ID NO: 1, more particularly comprising the introduction of a phenylalanine at the position corresponding to L34, a phenylalanine at the position corresponding to Y35, an isoleucine at the position corresponding to V371, a valine at the position corresponding to M434, a leucine at the position corresponding to N419, a leucine at the position corresponding to F346, a valine at the position corresponding to R437 and a phenylalanine at the position corresponding to W478 in the polypeptide according to SEQ ID NO: 1, for example the amino acid substitutions corresponding to L34F_Y35F_V371I_M434V_N419L_F346L_R437V_W478F. Use of such modified enzymes can result in an increase in retinol acetate of at least about 700% compared to the percentage of retinol acetate using a corresponding unmodified ATF (eg, the enzyme according to SEQ ID NO: 1).

[0048] In some embodiments, the modified enzyme as described herein comprises 9 amino acid substitutions, particularly at residues corresponding to positions L34, Y35, V371, M434, N419, F346, R437, W478 and F373 in the polypeptide according to SEQ ID NO: 1, more particularly at residues corresponding to positions L34, Y35, V371, M434, N419, F346, R437, W478 and F373 in the polypeptide according to SEQ ID NO: 1. In the polypeptide of SEQ ID NO: 1, a phenylalanine is introduced at a position corresponding to L34, a phenylalanine is introduced at a position corresponding to Y35, an isoleucine is introduced at a position corresponding to V371, a valine is introduced at a position corresponding to M434, a leucine is introduced at a position corresponding to N419, a leucine is introduced at a position corresponding to F346, a valine is introduced at a position corresponding to R437, a phenylalanine is introduced at a position corresponding to W478, and an alanine is introduced at a position corresponding to F373, such as amino acid substitutions corresponding to L34F_Y35F_V371I_M434V_N419L_F346L_R437V_W478F_F373A. Use of such a modified enzyme can result in an increase in retinyl acetate by at least about 10% compared to the percentage of retinyl acetate when using a corresponding unmodified ATF (e.g., an enzyme according to SEQ ID NO: 1).

[0049] The modified host cells described herein are capable of converting retinol to retinol acetate with an increase in conversion rate of at least about 10% or more, particularly an increase in retinol acetate production of 10% to 760% or more, and more particularly an increase in the percentage of retinol acetate based on total retinoids of up to 86% by weight, as can be obtained, for example, by expressing a modified ATF under suitable culture conditions, including but not limited to culture on xylose, glucose, galactose, and a suitable host cell, for example, selected from fungal host cells (including Yarrowia or Saccharomyces) or other microbial host cells (e.g., E. coli), in the presence or absence of ethanol. Suitable conditions can be culture in a fed-batch fermentation for, for example, 80, 90, 100, 110, 120, 130 hours.

[0050] 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 a 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, the modification (including the use of a corresponding unmodified ATF (e.g. according to SEQ ID NO: 1 ( Figure 1 ) compared to the method of SbATF1) of ) , the conversion rate of retinol to retinyl acetate is increased by at least about 10%, 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) 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 host cell producing retinol, particularly a fungal host cell, resulting in increased accumulation of the corresponding enzyme within a given time. The skilled person knows 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.

[0051] 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.

[0052] Therefore, the present invention relates to production retinol host cell, particularly fungal host cell as described herein, it comprises expression vector or the polynucleotide of the modified ATF of coding as described herein, and it has been integrated in the chromosomal DNA of host cell.This type of production retinol host cell, particularly fungal host cell, that is included in the expression vector or is integrated into the chromosomal DNA of the heterologous polynucleotide in the modified ATF of coding as described herein is referred to as recombinant or modified host cell.The host cell that produces retinol, particularly fungal host cell, can contain one or more copies of the gene of the ATF of modification as defined herein of coding, it comprises sudden change as defined herein, causes the overexpression of this type of gene of the modified ATF as defined herein of coding.The increase of gene expression can be measured by various methods, such as Northern, Southern or Western blotting techniques known in the art.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Modified ATF enzymes as defined herein also include enzymes that carry additional amino acid substitutions that do not alter the enzymatic activity, i.e., enzymes that exhibit the same properties as the enzymes defined herein and catalyze the conversion of retinol to retinol acetate, forming about 86% or more (e.g., 11-86% by weight) of retinol acetate based on the total amount of retinoids. Such mutations are also referred to as "silent mutations" that do not alter the (enzymatic) activity of the enzyme according to the invention.

[0057] 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.

[0058] 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.

[0059] 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. In particular, the retinol-producing host cell, in particular the fungal host cell, is selected from yeast, such as Yarrowia or Saccharomyces, such as Saccharomyces cerevisiae or Yarrowia lipolytica, wherein the polynucleotide encoding the modified ATF enzyme as defined herein is selected from polynucleotides 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, for example introducing one or more amino acid substitutions at positions as defined herein corresponding to residues selected from positions L34, Y35, V371, M434, N419, F346, R437, W478, F373 and combinations thereof, and preferably comprising a highly conserved partial amino acid sequence, i.e. a common active site or selected from the group consisting of residues corresponding to the residues according to SEQ ID NO: 1. NO: 1, wherein the host cell produces retinol acetate that is increased by at least about 10% compared to a host cell expressing the corresponding unmodified enzyme (e.g., an enzyme according to SEQ ID NO: 1), the unmodified enzyme being obtainable, for example, by expressing the modified ATF under suitable culture conditions, including but not limited to culture on glucose, galactose or xylose.

[0060] 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.

[0061] 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.

[0062] Preferably, acetylation of retinol by using a modified ATF as described herein can result in a retinol acetate percentage in the range of at least about 11 to 86 wt % based on total retinoids (i.e., retinol acetate present in the retinoid mixture produced by the host cell), 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 a trans-retinol percentage of at least about 65% is used as a substrate for acetylation via a modified enzyme as defined herein.

[0063] 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.

[0064] 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.

[0065] 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).

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Specifically, the present invention relates to the following embodiments (1) to (14):

[0074] (1) A method for modifying an enzyme (preferably a fungal enzyme) that catalyzes the acetylation of a retinoid (particularly retinol) to retinyl acetate, the method comprising introducing at least one amino acid substitution at one or more positions selected from amino acid residues 34, 35, 346, 371, 373, 419, 434, 437, 478, and combinations thereof, in a polypeptide according to SEQ ID NO: 1, wherein after introduction of the one or more amino acid substitutions, the activity of forming retinyl acetate is increased by at least about 10%, for example, in the range of 10-760%, compared to the activity of the corresponding unmodified enzyme using the enzyme according to SEQ ID NO: 1.

[0075] (2) The method according to embodiment (1), wherein the amino acid residue to be substituted at the position corresponding to position 34 of SEQ ID NO: 1 is not leucine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 35 of SEQ ID NO: 1 is not tyrosine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 346 or 373 of SEQ ID NO: 1 is not phenylalanine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 371 of SEQ ID NO: 1 is not valine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 419 of SEQ ID NO: 1 is not asparagine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 434 of SEQ ID NO: 1 is not methionine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 437 of SEQ ID NO: 1 is not arginine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 478 of SEQ ID NO: 1 is not tryptophan.

[0076] (3) The method according to embodiment (1) or (2), wherein the amino acid residue to be introduced at the position corresponding to position 34, 35 or 478 of SEQ ID NO: 1 is phenylalanine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 346 or 419 of SEQ ID NO: 1 is leucine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 371 of SEQ ID NO: 1 is isoleucine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 373 of SEQ ID NO: 1 is alanine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 434 or 437 of SEQ ID NO: 1 is valine.

[0077] (4) The method according to any one of embodiments (1), (2) or (3), wherein the enzyme having at least 20% identity with SEQ ID NO: 1 is modified by introducing one or more amino acid substitutions at positions corresponding to amino acid residues L34, Y35, F346, V371, F373, N419, M434, R437, W478 and / or combinations thereof, preferably, wherein the modified enzyme comprises one or more amino acid substitutions selected from L34F, Y35F, V371I, M434V, N419L, F346L, R437V, W478F, F373A and combinations thereof.

[0078] (5) The method according to embodiment (1), (2), (3) or (4), wherein after introduction of one or more amino acid substitutions, the percentage of retinyl acetate based on total retinoids is increased to about 86 weight % compared to the percentage using the corresponding unmodified enzyme comprising the enzyme according to SEQ ID NO: 1, in a method comprising expressing the modified enzyme in a suitable host cell.

[0079] (6) A method for producing a retinol acetylase, preferably an acetyltransferase of the [EC 2.3.1.84] class, having increased activity for the acetylation of retinol to retinol acetate, the method comprising:

[0080] (a) providing a fungal enzyme involved in the acetylation of retinol, in particular ATF derived from Saccharomyces, wherein the unmodified enzyme expressed in a suitable host cell and under suitable culture conditions is capable of producing up to 10% by weight of retinol acetate based on the total retinoids produced by the host cell;

[0081] (b) further introducing one or more amino acid substitutions, e.g., at least one to nine amino acid substitutions, into the enzyme of (a) to generate a modified enzyme that participates in the acetylation of retinol, wherein the ability to convert retinol to retinol acetate is increased by at least about 10%, e.g., at least 10 to 760%, when compared to the acetylation by the corresponding unmodified enzyme of step (a); wherein the at least one or more amino acid substitutions are at positions corresponding to amino acid residues selected from positions 34, 35, 346, 371, 373, 419, 434, 437, 478 in SEQ ID NO: 1, and wherein the substituted amino acid residue is different from L34, Y35, F346, V371, F373, N419, M434, R437, W478, and / or a combination thereof, wherein the amino acid residue to be introduced at the position corresponding to position 34, 35 or 478 in SEQ ID NO: 1 is preferably phenylalanine, and / or wherein the amino acid residue ... The amino acid residue to be introduced at the position corresponding to position 346 or 419 of SEQ ID NO: 1 is preferably leucine, and / or the amino acid residue to be introduced at the position corresponding to position 371 of SEQ ID NO: 1 is preferably isoleucine, and / or the amino acid residue to be introduced at the position corresponding to position 373 of SEQ ID NO: 1 is preferably alanine, and / or the amino acid residue to be introduced at the position corresponding to position 434 or 437 of SEQ ID NO: 1 is preferably valine.

[0082] (7) The method according to embodiment (6), wherein the acetyltransferase in step (a) is selected from yeast ATF.

[0083] (8) A modified enzyme, particularly a fungal enzyme, involved in the acetylation of retinol to retinol acetate, comprising one or more modifications, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9 amino acid substitutions, at positions selected from the group consisting of amino acid residues in positions 34, 35, 346, 371, 373, 419, 434, 437, 478, and combinations thereof, in a polypeptide corresponding to a sequence in accordance with SEQ ID NO: 1 or having at least about 20% identity, 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, and wherein the substituted amino acid residue is different from the amino acid residue in the polypeptide corresponding to the sequence in accordance with SEQ ID NO: 1. NO: 1, preferably wherein the amino acid residue to be substituted corresponds to a residue selected from the group consisting of L34, Y35, F346, V371, F373, N419, M434, R437, W478 and combinations thereof in the polypeptide according to SEQ ID NO: 1, in particular wherein the substituted amino acid residue to be introduced at a position corresponding to position 34, 35 or 478 of SEQ ID NO: 1 is preferably phenylalanine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 346 or 419 of SEQ ID NO: 1 is preferably leucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 371 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 372 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 373 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 374 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 375 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 376 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 377 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 378 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 379 of SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at a position corresponding to position 377 of SEQ ID NO: The substituted amino acid residue to be introduced at position 373 in NO: 1 is preferably alanine, and / or the substituted amino acid residue to be introduced at the position corresponding to position 434 or 437 in SEQ ID NO: 1 is preferably valine.

[0084] (9) The modified enzyme according to embodiment (8), which is selected from a polypeptide having at least about 20% identity to SEQ ID NO: 1, 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, which is contained in a polypeptide selected from SEQ ID NO: One or more amino acid substitutions different from L34, Y35, F346, V371, F373, N419, M434, R437 or W478 at positions 34, 35, 346, 371, 373, 419, 434, 437 and / or 478 in NO: 1, preferably wherein the one or more amino acid substitutions are selected from L34F, Y35F, V371I, M434V, N419L, F346L, R437V, W478F, F373A and combinations thereof.

[0085] (10) The modified enzyme according to embodiment (8) or (9), wherein the enzyme activity for forming retinyl acetate is increased by at least about 10% compared to the corresponding unmodified ATF, e.g., the ATF according to SEQ ID NO: 1, in particular the percentage of retinyl acetate based on total retinoids is increased to about 86% by weight.

[0086] (11) The modified enzyme according to embodiment (8), (9) or (10), comprising amino acid substitutions corresponding to positions 34 and 434 in SEQ ID NO: 1, preferably wherein the amino acid substitutions correspond to L34F_M434V in the polypeptide according to SEQ ID NO: 1.

[0087] (12) A method for producing retinol acetate, comprising the step of acetylation of retinol by the action of the enzyme according to embodiment (8), (9), (10) or (11).

[0088] (13) A method according to embodiment (12), wherein a suitable retinol-producing host cell, preferably selected from the genus Yarrowia, Saccharomyces or Escherichia coli, is transformed and expressed with an enzyme according to embodiment (8), (9), (10) or (11).

[0089] (14) The method of embodiment (12) or (13), wherein the percentage of retinyl acetate produced under suitable culture conditions is about 86 wt % based on the total retinoids produced by the host cell.

[0090] Attached photos

[0091] Figure 1: Amino acid sequence of Saccharomyces pasteurianus ATF1 (SbATF; SEQ ID NO: 1), wherein the residues selected for amino acid substitutions as described in this application are marked in bold / underlined.

[0092] 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

[0093] Example 1: General methods, strains and plasmids

[0094] 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).

[0095] 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.

[0096] 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.

[0097] DNA molecular biology. Plasmid MB10362 (SEQ ID NO: 3) containing the DrBCO, LmATF and FfRDH expression systems was synthesized at Genscript (Piscataway, NJ, USA). Plasmid MB10362 contains "URA3" and "HOM3" markers for selection in Yarrowia lipolytica transformation. For clean gene insertion by random non-homologous end joining of genes and markers, the SfiI plasmid fragment of MB10362 or other plasmids of interest in Table 1 was purified by gel electrophoresis and Qiagen gel purification columns. Clones were verified by sequencing. Typically, genes were synthesized at GenScript (Piscataway, NJ) and amino acid substitutions according to Table 1 were introduced. Transformants were screened for homoserine auxotrophy and subsequently sequenced using primers flanking the HOM3 sequence, and clean frameshifts were selected for forward movement. Expression of mutant ATFs in Saccharomyces cerevisiae is described in Example 1 of WO2020141168.

[0098] sequence. Plasmids for expressing wild-type SbATF (polynucleotide according to SEQ ID NO: 2) and modified enzymes comprising specific amino acid substitutions are listed in Table 1 and / or the Sequence Listing, with codon-optimized sequences for expression in Yarrowia lipolytica or Saccharomyces cerevisiae being specifically indicated. Figure 1 The wild-type SbATF amino acid sequence is shown with specific indication of residues selected for modification according to Table 1.

[0099] Table 1: List of plasmids used to construct strains carrying unmodified or modified heterologous Yarrowia lipolytica codon-optimized ATF genes from pasteurian yeast as inserts. All inserts are based on SbATF according to SEQ ID NO: 1. See the text for more details.

[0100]

[0101]

[0102] 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.

[0103] 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.

[0104]

[0105]

[0106] Table 2B: UPLC method gradient, solvent A: water; solvent B: acetonitrile; solvent C: methanol; solvent D: tert-butyl methyl ether.

[0107] 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

[0108] 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.

[0109] 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.

[0110] 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.

[0111] Example 2: Production of retinyl acetate in Yarrowia lipolytica expressing mutant SbATF

[0112] 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 ATF (retinol acetate / total retinoids) relative to the percentage of retinol acetate using the reference SbATF expressed on plasmid MB10362 (set to 100%) is shown in Table 3.

[0113] Table 3A: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of a modified ATF comprising two amino acid substitutions at positions corresponding to the corresponding positions in SbATF according to SEQ ID NO: 1. "Purity" refers to the weight % of retinyl acetate based on total retinoids produced by the host organism. See text or Table 1 for more details.

[0114]

[0115]

[0116] By introducing the double mutant (see Table 1 ), the percentage of retinyl acetate based on total retinoids could be increased to more than 26% by weight compared to the process using the enzyme according to SEQ ID NO: 1 .

[0117] Table 3B: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of a modified ATF comprising three amino acid substitutions at positions corresponding to the corresponding positions in SbATF according to SEQ ID NO: 1. See text or Table 1 for more details.

[0118] plasmids retAc[%] purity[%] MB10362 100 10 MB10364 475 47.5 MB10366 290 29.0 MB10369 375 37.5 MB10381 161 16.1 MB10387 655 65.5

[0119] By introducing the double mutant (see Table 1 ), the percentage of retinyl acetate based on total retinoids could be increased up to 66% by weight compared to the process using the enzyme according to SEQ ID NO: 1 .

[0120] Table 3C: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of a modified ATF comprising four amino acid substitutions at positions corresponding to the corresponding positions in SbATF according to SEQ ID NO: 1. See text or Table 1 for more details.

[0121]

[0122]

[0123] By introducing 4 mutations (see Table 1 ), the percentage of retinyl acetate based on total retinoids could be increased to up to 77% by weight compared to the process using the enzyme according to SEQ ID NO: 1 .

[0124] Table 3D: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of a modified ATF comprising five amino acid substitutions at positions corresponding to the corresponding positions in SbATF according to SEQ ID NO: 1. See text or Table 1 for more details.

[0125] plasmids retAc[%] purity[%] MB10362 100 10 MB10368 399 39.9 MB10373 316 31.6 MB10383 591 59.1 MB10385 776 72.6 MB10389 860 86.0 MB10391 784 78.4

[0126] By introducing 4 mutations (see Table 1 ), the percentage of retinyl acetate based on total retinoids could be increased to up to 86% by weight compared to the process using the enzyme according to SEQ ID NO: 1 .

[0127] Table 3E: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of a modified ATF comprising six amino acid substitutions at positions corresponding to the corresponding positions in SbATF according to SEQ ID NO: 1. See text or Table 1 for more details.

[0128]

[0129]

[0130] By introducing 6 mutations (see Table 1 ), the percentage of retinyl acetate based on total retinoids could be increased to up to 80% by weight compared to the process using the enzyme according to SEQ ID NO: 1 .

[0131] Table 3F: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of a modified ATF comprising eight amino acid substitutions at positions corresponding to the corresponding positions in SbATF according to SEQ ID NO: 1. See text or Table 1 for more details.

[0132] plasmids retAc[%] purity[%] MB10362 100 10 MB10393 802 80.2

[0133] By introducing 4 mutations (see Table 1 ), the percentage of retinyl acetate based on total retinoids could be increased to up to 80% by weight compared to the process using the enzyme according to SEQ ID NO: 1 .

[0134] Table 3G: Acetylation of retinol to retinyl acetate ("retAc") as enhanced by the action of a modified ATF comprising 9 amino acid substitutions at positions corresponding to the corresponding positions in SbATF according to SEQ ID NO: 1. See text or Table 1 for more details.

[0135] plasmids retAc[%] purity[%] MB10362 100 10 MB10394 112 11.2

[0136] By introducing 4 mutations (see Table 1 ), the percentage of retinyl acetate based on total retinoids could be increased to up to 11 wt.-% compared to the process using the enzyme according to SEQ ID NO: 1 .

Claims

1. A method for modifying an enzyme that catalyzes the acetylation of retinoids, in particular an enzyme, preferably a fungal enzyme, that catalyzes the acetylation of retinol to retinol acetate, the method comprising introducing at least two amino acid substitutions at positions selected from amino acid residues 34 and 434 into a polypeptide according to SEQ ID NO: 1, wherein after introduction of the at least two amino acid substitutions, the activity of forming retinol acetate is increased by at least about 10% compared to the activity of the corresponding unmodified enzyme using the enzyme according to SEQ ID NO: 1, and wherein the amino acid residues at the positions corresponding to amino acid residues 34 and 434 in SEQ ID NO: 1 are different from L34 and M434.

2. The method of claim 1 , further comprising introducing at least one amino acid substitution at one or more positions selected from the group consisting of amino acid residues 35, 346, 371, 373, 419, 437, 478, and combinations thereof in the polypeptide according to SEQ ID NO: 1 , wherein after introducing the one or more amino acid substitutions, the activity of forming retinyl acetate is increased by at least about 10%, e.g., in the range of 10 to 760%, compared to the activity of the corresponding unmodified enzyme using the enzyme according to SEQ ID NO:

1.

3. The method according to claim 1 or 2, wherein the amino acid residue to be substituted at the position corresponding to position 35 of SEQ ID NO: 1 is not tyrosine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 346 or 373 of SEQ ID NO: 1 is not phenylalanine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 371 of SEQ ID NO: 1 is not valine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 419 of SEQ ID NO: 1 is not asparagine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 437 of SEQ ID NO: 1 is not arginine, and / or wherein the amino acid residue to be substituted at the position corresponding to position 478 of SEQ ID NO: 1 is not tryptophan.

4. The method according to any one of claims 1 to 3, wherein the amino acid residue to be introduced at the position corresponding to position 34, 35 or 478 of SEQ ID NO: 1 is phenylalanine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 346 or 419 of SEQ ID NO: 1 is leucine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 371 of SEQ ID NO: 1 is isoleucine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 373 of SEQ ID NO: 1 is alanine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 434 or 437 of SEQ ID NO: 1 is valine.

5. The method according to any one of claims 1 to 4, wherein the enzyme having at least 20% identity to SEQ ID NO: 1 is modified by introducing one or more amino acid substitutions at positions corresponding to amino acid residues Y35, F346, V371, F373, N419, R437, W478 and / or combinations thereof, preferably wherein the modified enzyme comprises one or more amino acid substitutions selected from the group consisting of Y35F, V371I, N419L, F346L, R437V, W478F, F373A and combinations thereof in combination with the amino acid substitutions L34F and M434V.

6. A method for producing a retinol acetylase, preferably an acetyltransferase of the [EC 2.3.1.84] class, having increased activity for the acetylation of retinol to retinol acetate, the method comprising: (a) providing a fungal enzyme involved in the acetylation of retinol, in particular ATF derived from Saccharomyces, wherein the unmodified enzyme expressed in a suitable host cell and under suitable culture conditions is capable of producing up to 10% by weight of retinol acetate based on the total retinoids produced by the host cell; (b) introducing at least two amino acid substitutions at positions corresponding to L34 and M434 in the polypeptide according to SEQ ID NO: 1, and further introducing at least one to seven amino acid substitutions in the enzyme of (a) to generate a modified enzyme that participates in the acetylation of retinol, wherein the ability to convert retinol to retinol acetate is increased by at least about 10%, e.g., at least 10 to 760%, when compared to acetylation by the corresponding unmodified enzyme of step (a); wherein the at least 1 to 7 amino acid substitutions are at positions corresponding to amino acid residues selected from positions 35, 346, 371, 373, 419, 437, 478 in SEQ ID NO: 1, and wherein the substituted amino acid is different from Y35, F346, V371, F373, N419, R437, W478 and / or a combination thereof, wherein the amino acid residue to be introduced at the position corresponding to position 34, 35 or 478 in SEQ ID NO: 1 is preferably phenylalanine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 346 or 419 of SEQ ID NO: 1 is preferably leucine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 371 of SEQ ID NO: 1 is preferably isoleucine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 373 of SEQ ID NO: 1 is preferably alanine, and / or wherein the amino acid residue to be introduced at the position corresponding to position 434 or 437 of SEQ ID NO: 1 is preferably valine.

7. method according to claim 6, wherein the acetyltransferase of step (a) is selected from yeast ATF.

8. A modified enzyme, in particular a fungal enzyme, involved in the acetylation of retinol to retinol acetate, comprising at least two modifications, such as amino acid substitutions at positions corresponding to amino acid residues 34 and 434 in the polypeptide according to SEQ ID NO: 1, and further comprising at least 1, 2, 3, 4, 5, 6, 7 amino acid substitutions at positions corresponding to amino acid residues 34 and 434 in the polypeptide according to SEQ ID NO: 1 or in conjunction with SEQ ID NO: NO: 1 at a position selected from the group consisting of amino acid residues at positions 35, 346, 371, 373, 419, 437, 478, and combinations thereof, in a polypeptide having a sequence with at least about 20% identity, 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, and wherein the substituted amino acid residue is different from the amino acid corresponding to L34, Y35, F346, V371, F373, N419, M434, R437, W478, and combinations thereof in a polypeptide according to SEQ ID NO: 1, preferably wherein the amino acid residue to be substituted corresponds to the amino acid residue at position 35, 346, 371, 373, 419, 437, 478, and combinations thereof, in a polypeptide according to SEQ ID NO: 1 373, N419, M434, R437, W478, and combinations thereof in the polypeptide of SEQ ID NO: 1, in particular wherein the substituted amino acid residue to be introduced at the position corresponding to position 34, 35 or 478 of SEQ ID NO: 1 is preferably phenylalanine, wherein the substituted amino acid residue to be introduced at the position corresponding to position 346 or 419 in SEQ ID NO: 1 is preferably leucine, wherein the substituted amino acid residue to be introduced at the position corresponding to position 371 in SEQ ID NO: 1 is preferably isoleucine, wherein the substituted amino acid residue to be introduced at the position corresponding to position 373 in SEQ ID NO: 1 is preferably alanine, and / or wherein the substituted amino acid residue to be introduced at the position corresponding to position 434 or 437 in SEQ ID NO: 1 is preferably valine.

9. The modified enzyme of claim 8, selected from a polypeptide having at least about 20% identity to SEQ ID NO: 1, 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, contained in a polypeptide selected from the group consisting of SEQ ID NO: One or more amino acid substitutions different from L34, Y35, F346, V371, F373, N419, M434, R437 or W478 at positions 34, 35, 346, 371, 373, 419, 434, 437 and / or 478 in NO: 1, preferably wherein the one or more amino acid substitutions are selected from L34F, Y35F, V371I, M434V, N419L, F346L, R437V, W478F, F373A and combinations thereof.

10. The modified enzyme according to claim 8 or 9, wherein the enzymatic activity for forming retinyl acetate is increased by at least about 10% compared to the corresponding unmodified ATF, for example the ATF according to SEQ ID NO: 1, in particular the percentage of retinyl acetate based on total retinoids is increased to about 86% by weight.

11. The modified enzyme according to any one of claims 8 to 10, comprising an amino acid substitution corresponding to L34F_M434V in the polypeptide according to SEQ ID NO:

1. 12 . A method for producing retinol acetate, comprising the step of acetylating retinol through the action of the enzyme according to claim 8 .

13. The method according to claim 12, wherein a suitable retinol-producing host cell, preferably selected from Yarrowia, Saccharomyces or Escherichia coli, is transformed with and expresses the enzyme according to any one of claims 8 to 11.

14. The method of claim 12 or 13, wherein the percentage of retinyl acetate produced under suitable culture conditions is about 86% by weight based on total retinoids produced by the host cell.

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