A novel mutant acetyltransferase, SB-ATF

Abstract

The present invention relates to a process for converting a low activity enzyme involved in the acetylation of retinoids into a high activity enzyme, specifically a process for converting an enzyme highly active in the acetylation of retinol to retinyl acetate.

Description

Detailed Description of the Invention

[0001] The present invention relates to a process for converting a low activity enzyme involved in the acetylation of retinoids into a high activity enzyme, specifically a process for converting an enzyme highly active in the acetylation of retinol to retinyl acetate.

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

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

[0004] Generally, biological systems that produce retinoids are industrially intractable and / or produce the compounds at such low levels that isolating them on an industrial scale is not economically feasible. There are several reasons for this, among them instability of intermediates / retinoids and / or relatively high accumulation of by-products.

[0005] One way to address the stability issue is to produce the product in the form of an acetylated product. For example, the acetylation of carotenoids, such as astaxanthin or zeaxanthin, by the action of Atf1 (SbATF1) from Saccharomyces bayanus has been reported (WO 2014096992), resulting in up to 90% acetylation of zeaxanthin by weight. However, when this enzyme is used to acetylate retinoids, specifically retinol, the acetylation rate is only approximately 10% of the total retinoids (see, for example, WO 2019058001), far too low to establish a sustainable and economically viable industrial process for the bioproduction of vitamin A.

[0006] Therefore, there is a strong need to improve the enzymes used to acetylate retinoids.

[0007] Surprisingly, the present inventors have now identified amino acid positions in fungal acetyltransferase enzymes (ATFs), such as those from Saccharomyces bayanus and other yeasts, that are crucial for the formation of acetylated retinoids, specifically the conversion of retinol to retinyl acetate. Modification of specific amino acids resulted in increased formation of retinyl acetate compared to the respective wild-type enzymes, e.g., an increase of at least about 10% when compared to the acetylation of retinol using the respective unmodified enzymes, e.g., wild-type ATF1 from Saccharomyces bayanus as set forth 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 a fungal ATF, comprising the introduction of at least one modification, e.g., an amino acid substitution, e.g., 1 to 9, e.g., at least 2 amino acid substitutions, at a position specified herein. Upon introduction of the specified amino acid substitutions, the unmodified ATF, particularly a fungal ATF, such as ATF1 from Saccharomyces, e.g., ATF1 from Saccharomyces bayanus (SbATF) according to SEQ ID NO: 1, is modified or transformed into a modified or mutant ATF with increased activity, and when such modified enzyme is expressed in a suitable expression system comprising a suitable host cell as specified herein, the percentage of retinyl acetate obtained from the acetylation of retinol can be increased by at least about 10%, e.g., by more than 700%, compared to the percentage of retinyl acetate obtained using the respective unmodified or wild-type enzyme.

[0009] Specifically, the present invention relates to a method for modifying an enzyme, preferably a fungal enzyme, that catalyzes the acetylation of retinoids, specifically the acetylation of retinol to retinyl acetate, 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, of a polypeptide according to SEQ ID NO: 1, wherein the introduction of the one or more amino acid substitutions results in a reduced activity towards the formation of retinyl acetate compared to the activity using the respective unmodified enzyme, including the enzyme according to SEQ ID NO: 1. and / or a combination thereof of the polypeptide according to SEQ ID NO: 1, such that the amino acid substitutions comprise, for example, 1 to 9 amino acid substitutions, different from the corresponding amino acid residues at positions specified herein, preferably 34, 35, 346, 371, 373, 419, 434, 437, 478 and / or a combination thereof, i.e., L34, Y35, F346, V371, F373, N419, M434, R437, W478 and a combination thereof, respectively, of SEQ ID NO: 1.

[0010] More specifically, the present invention relates to a method for modifying an enzyme, preferably a fungal enzyme, that catalyzes the acetylation of retinoids, specifically the acetylation of retinol to retinyl acetate, comprising introducing at least two amino acid substitutions at positions selected from amino acid residues 34 and 434 of a polypeptide according to SEQ ID NO: 1, such that the introduction of said at least two amino acid substitutions increases the activity for the formation of retinyl acetate by at least about 10%, such as at least about 25, 20, 25, 30, 35, 40, 45%, or more, compared to the activity using the respective unmodified enzyme, including the enzyme according to SEQ ID NO: 1. , 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%, for example in the range of 10 to 760%, and preferably the amino acid residues at positions corresponding to amino acid residues 34 and 434 of SEQ ID NO: 1 are different from L34 and M434, more preferably the amino acid substitutions at positions corresponding to L34 and M434 of the polypeptide according to SEQ ID NO: 1 are selected from L34F and M434V.

[0011] As used herein, an unmodified retinol acetyltransferase, as defined herein, having "low activity" for retinol acetylation means a percentage of retinyl acetate in the range of about 10% by weight or less, based on the total retinoids produced in a suitable retinoid-producing host cell expressing the unmodified enzyme. An engineered enzyme, as defined herein, having "enhanced activity" for retinol acetylation means a percentage of retinyl acetate in the range of about 11% to 86% by weight, based on the total retinoids produced in a suitable retinoid-producing host cell expressing the engineered enzyme. That is, the engineered enzyme is generated by introducing one or more amino acid substitutions, as defined herein, that convert an enzyme with low (enzymatic or acetylating) activity into an enzyme with increased (enzymatic or acetylating) activity. As shown, the activity towards acetylation of retinol can be increased by at least about 10%, such as 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 provides a process for producing a retinol acetyltransferase having increased activity for the acetylation of retinol to retinyl acetate, comprising: (a) providing a fungal enzyme involved in the acetylation of retinol, specifically an ATF derived from the genus Saccharomyces, wherein the unmodified enzyme expressed in a suitable host cell and under suitable culture conditions is capable of producing retinyl acetate from retinol in the range of up to 10% by weight based on total retinoids; (b) introducing one or more amino acid substitutions, e.g., at least 1-9, preferably at least 2, into the enzyme of (a) to generate a modified enzyme involved in the acetylation of retinol, wherein the ability to convert retinol to retinyl acetate is increased by at least about 10%, e.g., at least 10-760%, when compared to the acetylation of the respective unmodified enzyme of step (a). Regarding processes involving: The one or more, such as at least two, amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of positions 34, 35, 346, 371, 373, 419, 434, 437, 478 of SEQ ID NO: 1 or a combination thereof, wherein the substituted amino acids are different from L34, Y35, F346, V371, F373, N419, M434, R437, W478 and / or a combination thereof, and preferably the at least two amino acid substitutions are located at positions corresponding to positions 34 and 434 of the polypeptide according to SEQ ID NO: 1.

[0013] In one embodiment, the present invention relates to an engineered enzyme, in particular a fungal enzyme, involved in the acetylation of retinol to retinyl acetate, comprising one or more modifications, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, such as at least 2, amino acid substitutions, wherein said amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of positions 34, 35, 346, 371, 373, 419, 434, 437, 478 and combinations thereof of the polypeptide according to SEQ ID NO: 1, and the substituted amino acid residues are different from the amino acids corresponding to L34, Y35, F346, V371, F373, N419, M434, R437, W478 and combinations thereof of the enzyme according to SEQ ID NO: 1, and preferably the substituted amino acid residue is selected from the group consisting of L34, Y35, F346, V371, F373, N419, M434, R437, W478 and combinations thereof of the enzyme according to SEQ ID NO: 1. , Y35, F346, V371, F373, N419, M434, R437, W478 and combinations thereof, and more preferably, the substituted amino acid residue introduced at a position corresponding to position 34, 35 or 478 of SEQ ID NO:1 is preferably phenylalanine, the substituted amino acid residue introduced at a position corresponding to position 346 or 419 of SEQ ID NO:1 is preferably leucine, the substituted amino acid residue introduced at a position corresponding to position 371 of SEQ ID NO:1 is preferably isoleucine, the substituted amino acid residue introduced at a position corresponding to position 373 of SEQ ID NO:1 is preferably alanine, and / or the substituted amino acid residue introduced at a position corresponding to position 434 or 437 of SEQ ID NO:1 is preferably valine.

[0014] Specifically, modified enzymes according to the present invention and as defined herein are selected from polypeptides having at least about 20%, such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO: 1, and include the following: L34, Y35, F346, V371, F373, N419, M434, R437 or comprises one or more amino acid substitutions at positions selected from 34, 35, 346, 371, 373, 419, 434, 437 and / or 478 of SEQ ID NO: 1 which differ from W478, preferably the one or more amino acid substitutions are selected from the group consisting of L34F, Y35F, V371I, M434V, N419L, F346L, R437V, W478F, F373A, and combinations thereof.

[0015] Use of such modified enzymes in a process for producing retinoids, expressed, particularly heterologously expressed, in a suitable host cell, particularly a fungal host cell capable of producing retinol, results in at least about a 10% increase in retinyl acetate produced by acetylation of retinol, compared to a process using the same conditions but using the respective or corresponding wild-type enzyme, such as the ATF enzyme according to SEQ ID NO: 1. Specifically, use of such modified enzymes results in retinyl acetate titers ranging up to 86% by weight based on total retinoids, compared to use of the respective unmodified enzyme, which has retinyl acetate titers ranging up to 10% by weight based on total retinoids.

[0016] The terms "acetyltransferase," "retinol acetyltransferase," "enzyme with retinol acetylation activity," "ATF," or "ATF1" are used interchangeably herein to refer to enzymes in the EC classification [EC 2.3.1.84] that can catalyze the conversion of retinol to retinyl acetate and include both naturally occurring enzymes and enzymes synthetically produced with the aid of artificial intelligence. The terms "SbATF" and "SbATF1" are used interchangeably herein. An example of such an enzyme is set forth in SEQ ID NO: 1. As defined herein, an ATF that can catalyze the acetylation of retinol to retinyl acetate at a rate of about 10% or less of retinyl acetate based on total retinoids is referred to herein as an "unmodified" or "wild-type" ATF, such as SbATF according to SEQ ID NO: 1.

[0017] A "modified" ATF, specifically based on an "unmodified" ATF, as defined herein, such as an enzyme having at least about 20% identity to SEQ ID NO: 1 as defined herein, exhibits increased activity, i.e., increased formation of retinyl acetate by conversion of retinol to the extent of at least 10% based on total retinoids, compared to the formation of retinyl acetate using the respective / corresponding wild-type enzyme, e.g., an enzyme according to SEQ ID NO: 1.

[0018] In one embodiment, the modified ATF of the present invention contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, for example at least 2, amino acid substitutions compared to the corresponding wild-type ATF, and one or more of the amino acids corresponding to L34, Y35, F346, V371, F373, N419, M434, R437, and / or W478 of the polypeptide according to SEQ ID NO: 1 are replaced, resulting in an increase in enzymatic activity for the formation of retinyl acetate of at least about 10% compared to the respective unmodified ATF, for example, an ATF according to SEQ ID NO: 1, specifically an increase in the proportion of retinyl acetate of about 10% to 86% by weight, or even more, based on total retinoids.

[0019] The present invention includes a method for converting / transforming a low activity enzyme (for the acetylation of retinol to retinyl acetate), also referred to herein as an unmodified enzyme, into a modified enzyme having increased activity for the acetylation of retinol to retinyl acetate, as defined herein. An example of such an unmodified enzyme is an enzyme having at least about 20% identity to SEQ ID NO: 1, which exhibits 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, specifically ATF1 (SbATF1) isolated from Saccharomyces bayanus, which comprises the enzyme encoded by the polynucleotide according to SEQ ID NO: 2.

[0020] 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 bayanus, 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 retinol conversion / acetylation is in the range of 10% by weight or less based on total retinoid, and are also suitable for fungal enzymes, particularly enzymes obtained from yeast, particularly [NSD [EHC]-Hx(3)-D-[GA] (a motif in Prosite syntax, see https: / / prosite.expasy.org / scanprosite / scanprosite_doc.html), preferably [NHx(3)-D-[GA], more preferably NHCMCDG (where "x" represents any amino acid, and the central histidine is part of the binding pocket of the enzyme and corresponds to positions N190 to G196 of the polypeptide according to SEQ ID NO: 1).

[0021] Suitable unmodified enzymes include those from the genus Saccharomyces, such as S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. pastorianus, S. arboricola, and the like; those from the genus Candida, such as C. dubliniensis, C. The yeast may be selected from, but is not limited to, yeasts such as C. albicans, C. intermedia, C. auris, C. lusitaniae, etc., Wickerhamomyces, such as W. anomalus, and Aspergillus, such as A. ellipticus.

[0022] The modified ATFs as defined herein are capable of converting retinol to retinyl acetate at a conversion rate that is specifically at least about 10% increased compared to the conversion of retinol to retinyl acetate using the respective / corresponding unmodified enzyme, e.g., the enzyme according to SEQ ID NO: 1, as obtained by expressing the modified ATFs under suitable culture conditions, including, but not limited to, culture in glucose, galactose, xylose, with or without ethanol.

[0023] Enzymes as defined herein are used to convert retinol to retinyl acetate, and the substrate (i.e., retinol) can be either cis-retinol, trans-retinol, or a mixture of cis- / trans-retinol in any possible ratio. Preferably, the retinol mixture used as the substrate has a high proportion of trans-retinol, e.g., at least about 65-98% by weight of the trans isomer, based on the total retinol in the host cell. Acetylation of such retinol mixtures containing at least about 65-98% by weight of trans-retinol results in the production of retinyl acetate containing approximately the same proportions of trans-retinyl acetate and cis-retinyl acetate, based on the total retinyl acetate produced by the host cell.

[0024] In one particular embodiment, the present invention relates to the conversion of retinol to retinyl acetate using a suitable host cell, as defined herein, containing and expressing a modified enzyme, as defined herein, wherein the retinol is a mixture of trans-retinol and cis-retinol, and the proportion of trans-retinol is in the range of at least about 65-98% by weight of trans-retinol based on total retinol.

[0025] The terms "conversion," "enzymatic conversion," "acetylation," or "enzymatic acetylation," in relation to enzymatic catalysis of retinol, are used interchangeably herein and refer to the action of a modified or unmodified ATF in catalyzing the conversion of retinol to retinyl acetate, which, upon expression of said ATF, results in a percentage of retinyl acetate based on total retinoids present / produced by a suitable host cell, and an increase to retinyl acetate of 86% or more by weight based on total retinoids can be achieved using modified ATFs as defined herein.

[0026] Suitable host cells according to the present invention include fungal host cells as well as microbial host cells, such as, for example, E. coli. As used herein, the term "fungal host cell" specifically includes yeast cells, which are retinol-producing host cells, specifically retinyl acetate-producing host cells, e.g., retinyl acetate-producing fungal host cells, including, but not limited to, yeast cells of the genus Yarrowia or Saccharomyces, such as Yarrowia lipolytica or Saccharomyces cerevisiae.

[0027] The modified ATF enzyme may be used in isolated form (e.g., in a cell-free system) or may be expressed in a suitable host cell, such as, for example, a retinol-producing host cell, particularly a fungal host cell as defined herein. The enzyme may be expressed as an endogenous enzyme or as a heterologous enzyme. Preferably, the modified enzyme as described herein is introduced and expressed as a heterologous enzyme in a suitable host cell, such as, for example, a retinol-producing host cell, particularly a fungal host cell as defined herein.

[0028] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 34 of a polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with phenylalanine, e.g., by substituting leucine with any amino acid other than leucine (e.g., L34F in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity to SbATF as set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises an amino acid substitution at a position corresponding to residue 434 of a polypeptide according to SEQ ID NO: 1, specifically by substituting valine for methionine at the position corresponding to M434 of SEQ ID NO: 1. 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 the acetylation of retinol to retinyl acetate, as compared to a corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0029] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 35 of a polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with phenylalanine, e.g., by substituting tyrosine with any other amino acid other than tyrosine (e.g., Y35F in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity to SbATF as set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises two amino acid substitutions at positions corresponding to residues 34 and 434 of a polypeptide according to SEQ ID NO: 1, specifically, a substitution of leucine with phenylalanine at position corresponding to L34 of SEQ ID NO: 1, and a substitution of methionine with valine at position corresponding to M434 of SEQ ID NO: 1. Use of such a modified enzyme comprising the above mutations 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 the acetylation of retinol to retinyl acetate, compared to a corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0030] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 346 of the polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with leucine, e.g., by substituting phenylalanine with leucine, resulting in the substitution of the original residue with any other amino acid other than phenylalanine (e.g., F346L in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity to SbATF as set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises two amino acid substitutions at positions corresponding to residues 34 and 434 of the polypeptide according to SEQ ID NO: 1, specifically, a substitution of phenylalanine for leucine at position L34 of SEQ ID NO: 1, and a substitution of valine for methionine at position M434 of SEQ ID NO: 1. Use of such a modified enzyme comprising the above mutations 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 the acetylation of retinol to retinyl acetate, compared to a corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0031] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 371 of a polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with isoleucine, e.g., by substituting valine with any other amino acid other than valine (e.g., V371I in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity to SbATF as set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises two amino acid substitutions at positions corresponding to residues 34 and 434 of a polypeptide according to SEQ ID NO: 1, specifically, a substitution of phenylalanine for leucine at position corresponding to L34 of SEQ ID NO: 1, and a substitution of valine for methionine at position corresponding to M434 of SEQ ID NO: 1. Use of such a modified enzyme comprising the mutations 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 the acetylation of retinol to retinyl acetate, compared to a corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0032] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 373 of a polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with alanine, e.g., by substituting phenylalanine with alanine, resulting in the substitution of the original residue with any other amino acid other than phenylalanine (e.g., F373A in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity to SbATF as set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises two amino acid substitutions at positions corresponding to residues 34 and 434 of a polypeptide according to SEQ ID NO: 1, specifically, a substitution of phenylalanine for leucine at position corresponding to L34 of SEQ ID NO: 1, and a substitution of valine for methionine at position corresponding to M434 of SEQ ID NO: 1. Use of such a modified enzyme comprising the above mutations 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 the acetylation of retinol to retinyl acetate, as compared to a corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0033] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 419 of the polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with leucine, e.g., by substituting asparagine with any other amino acid other than asparagine (e.g., N419L in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity to SbATF as set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises two amino acid substitutions at positions corresponding to residues 34 and 434 of the polypeptide according to SEQ ID NO: 1, specifically, a substitution of phenylalanine for leucine at the position corresponding to L34 of SEQ ID NO: 1, and a substitution of valine for methionine at the position corresponding to M434 of SEQ ID NO: 1. Use of such a modified enzyme comprising the above mutations 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 the acetylation of retinol to retinyl acetate, compared to a corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0034] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 434 of the polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with valine, e.g., by substituting methionine with any other amino acid other than methionine (e.g., M434V in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity to SbATF as set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises an amino acid substitution at a position corresponding to residue 34 of the polypeptide according to SEQ ID NO: 1, specifically by substituting phenylalanine for leucine at position corresponding to L34 of SEQ ID NO: 1. 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 the acetylation of retinol to retinyl acetate, compared to a corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0035] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 437 of a polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with valine, e.g., by substituting arginine with any other amino acid other than arginine (e.g., R437V in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity to SbATF as set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises two amino acid substitutions at positions corresponding to residues 34 and 434 of a polypeptide according to SEQ ID NO: 1, specifically, a substitution of phenylalanine for leucine at a position corresponding to L34 of SEQ ID NO: 1, and a substitution of valine for methionine at a position corresponding to M434 of SEQ ID NO: 1. Use of such a modified enzyme comprising the mutations 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 the acetylation of retinol to retinyl acetate, compared to a corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0036] In one embodiment, a modified ATF enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 478 of the polypeptide according to SEQ ID NO: 1, specifically by replacing the original amino acid with phenylalanine, e.g., by substituting tryptophan with phenylalanine, resulting in the substitution of the original residue with any other amino acid other than tryptophan (e.g., W478F in SEQ ID NO: 1). The modified enzyme may be derived from an ATF having at least about 20% identity with SbATF set forth in SEQ ID NO: 1. Preferably, such a modified enzyme further comprises two amino acid substitutions at positions corresponding to residues 34 and 434 of the polypeptide according to SEQ ID NO: 1, specifically by substituting phenylalanine for leucine at the position corresponding to L34 of SEQ ID NO: 1, and by substituting valine for methionine at the position corresponding to M434 of SEQ ID NO: 1. Use of such modified enzymes containing the mutations in fermentation processes using a suitable carbon source, such as glucose, results in at least about a 10-700% increase in the acetylation of retinol to retinyl acetate compared to the corresponding process using the respective wild-type enzyme, e.g., the enzyme according to SEQ ID NO: 1.

[0037] As used herein, the term "original amino acid" refers to an amino acid residue present in a wild-type or unmodified ATF that is to be replaced by another amino acid residue in such a way that the activity of the modified enzyme for acetylating retinol as defined herein is increased.

[0038] The present invention relates to a process for introducing at least about 1 to 9, e.g., at least 2, amino acid substitutions into an ATF, particularly a fungal ATF, e.g., an ATF according to SEQ ID NO: 1, or an ATF having at least about 20% identity thereto, wherein the activity of the unmodified ATF as defined herein is increased by at least about 10% compared to the proportion of retinyl acetate in the range of up to about 10% by weight obtained in a system / host cell using the respective unmodified ATF, e.g., an ATF according to SEQ ID NO: 1, i.e., the activity of the unmodified ATF is increased by at least about 10% (to result in the production of up to 86% by weight of retinyl acetate based on total retinoids) by acetylation of retinol to retinyl acetate; and 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 acetylating retinol to retinyl acetate.

[0039] Thus, in some embodiments, the modified enzymes described herein comprise two amino acid substitutions, specifically at residues corresponding to positions L34 and M434 of the polypeptide according to SEQ ID NO: 1, and more specifically, the amino acid substitutions comprise the introduction of a phenylalanine at the position corresponding to L34 and a valine at the position corresponding to M434 of the polypeptide according to SEQ ID NO: 1, e.g., amino acid substitutions corresponding to L34F and M434V of the polypeptide according to SEQ ID NO: 1. Use of such modified enzymes can result in an increase in retinyl acetate of at least about 150% compared to the rate of retinyl acetate using the respective unmodified ATF, e.g., the enzyme according to SEQ ID NO: 1.

[0040] In some embodiments, the modified enzyme as described herein comprises three amino acid substitutions, specifically located at residues corresponding to positions L34 and M434 of the polypeptide according to SEQ ID NO: 1, in combination with amino acid substitutions at positions corresponding to F346, V371, N419, R437 or W478, more specifically comprising introducing a phenylalanine at a position corresponding to L34 and a valine at a position corresponding to M434 of the polypeptide according to SEQ ID NO: 1 in combination with introducing a leucine at a position corresponding to F346, an isoleucine at a position corresponding to V371, a leucine at a position corresponding to N419, or a valine at a position corresponding to R437 of the polypeptide according to SEQ ID NO: 1, for example comprising amino acid substitutions corresponding to L34F and M434V in combination with F346L, V371I, N419L or R437V of the polypeptide according to SEQ ID NO: 1. Use of such modified enzymes can result in an increase in retinyl acetate of at least about 60-550% compared to the rate of retinyl acetate using the respective unmodified ATF, for example, an enzyme according to SEQ ID NO:1.

[0041] In some embodiments, the modified enzyme as described herein comprises four amino acid substitutions, specifically located at residues corresponding to positions L34 and M434 of the polypeptide according to SEQ ID NO: 1, in combination with amino acid substitutions at positions corresponding to Y35, F346, V371, N419, R437 or W478 of the polypeptide according to SEQ ID NO: 1, more specifically comprising introducing a phenylalanine at a position corresponding to L34 and a valine at a position corresponding to M434 of the polypeptide according to SEQ ID NO: 1 in combination with introducing a phenylalanine at a position corresponding to Y35, a leucine at a position corresponding to F346, an isoleucine at a position corresponding to V371, a leucine at a position corresponding to N419, a valine at a position corresponding to R437 or a phenylalanine at a position corresponding to W478 of the polypeptide according to SEQ ID NO: 1, for example, Y35F, F346L, V371I, N419L of the polypeptide according to SEQ ID NO: 1. The modified enzymes may include amino acid substitutions corresponding to L34F and M434V in combination with R437V or W478F, including but 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 retinyl acetate of at least about 230-670% compared to the rate of retinyl acetate using the respective unmodified ATF, for example an enzyme according to SEQ ID NO: 1.

[0042] In some embodiments, the modified enzyme as described herein comprises five amino acid substitutions, specifically located at residues corresponding to positions L34, M434, and V371 of the polypeptide according to SEQ ID NO: 1, in combination with amino acid substitutions at positions corresponding to Y35, F346, N419, R437, or W478 of the polypeptide according to SEQ ID NO: 1, more specifically, a phenylalanine at a position corresponding to L34 of the polypeptide according to SEQ ID NO: 1, in combination with introducing a phenylalanine at a position corresponding to Y35, a leucine at a position corresponding to F346, a leucine at a position corresponding to N419, a valine at a position corresponding to R437, or a phenylalanine at a position corresponding to W478 of the polypeptide according to SEQ ID NO: 1. and introducing a valine at the position corresponding to M434, for example, amino acid substitutions corresponding to L34F and M434V in combination with Y35F, F346L, N419L, R437V or W478F of the polypeptide according to SEQ ID NO: 1, including, but not limited to, L34F_Y35F_V371I_M434V_N419L or L The amino acid substitutions include 34F_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 retinyl acetate of at least about 300-760% compared to the rate of retinyl acetate using the respective unmodified ATF, for example, an enzyme according to SEQ ID NO: 1.

[0043] In some embodiments, the modified enzyme as described herein comprises six amino acid substitutions, specifically located at residues corresponding to positions L34, Y35, V371, M434 and N419 of the polypeptide according to SEQ ID NO: 1, in combination with amino acid substitutions at positions corresponding to F346, R437 or W478, more specifically introducing a phenylalanine at position L34 of the polypeptide according to SEQ ID NO: 1, a valine at position corresponding to R437, or a phenylalanine at position corresponding to Y35 of the polypeptide according to SEQ ID NO: 1, in combination with introducing a leucine at position corresponding to F346, a valine at position corresponding to R437, or a phenylalanine at position corresponding to W478 of the polypeptide according to SEQ ID NO: 1. at position corresponding to V371, an isoleucine at position corresponding to V371, a valine at position corresponding to M434, and a leucine at position corresponding to N419, for example including amino acid substitutions corresponding to L34F, Y35F, V371I, M434V and N419L in combination with F346L, R437V or W478L of the polypeptide according to 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 retinyl acetate of at least about 130-600% compared to the rate of retinyl acetate using the respective unmodified ATF, for example, the enzyme according to SEQ ID NO: 1.

[0044] In some embodiments, the modified enzyme as described herein comprises eight amino acid substitutions, specifically located at residues corresponding to positions L34, Y35, V371, M434, N419, F346, R437, and W478 of the polypeptide according to SEQ ID NO: 1, more specifically comprising introducing a phenylalanine at a position corresponding to L34, a phenylalanine at a position corresponding to Y35, an isoleucine at a position corresponding to V371, a valine at a position corresponding to M434, a leucine at a position corresponding to N419, a leucine at a position corresponding to F346, a valine at a position corresponding to R437, and a phenylalanine at a position corresponding to W478 of the polypeptide according to SEQ ID NO: 1, e.g., comprising amino acid substitutions corresponding to L34F_Y35F_V371I_M434V_N419L_F346L_R437V_W478F. Use of such modified enzymes can result in an increase in retinyl acetate of at least about 700% compared to the rate of retinyl acetate using the respective unmodified ATF, eg, an enzyme according to SEQ ID NO:1.

[0045] In some embodiments, the modified enzyme as described herein comprises nine amino acid substitutions, specifically located at residues corresponding to positions L34, Y35, V371, M434, N419, F346, R437, W478, and F373 of the polypeptide according to SEQ ID NO: 1, and more specifically comprising introducing a phenylalanine at a position corresponding to L34, a phenylalanine at a position corresponding to Y35, an isoleucine at a position corresponding to V371, a valine at a position corresponding to M434, a leucine at a position corresponding to N419, a leucine at a position corresponding to F346, a valine at a position corresponding to R437, a phenylalanine at a position corresponding to W478, and an alanine at a position corresponding to F373 of the polypeptide according to SEQ ID NO: 1, for example, comprising amino acid substitutions corresponding to L34F_Y35F_V371I_M434V_N419L_F346L_R437V_W478F_F373A. Use of such modified enzymes can result in an increase in retinyl acetate of at least about 10% compared to the rate of retinyl acetate using the respective unmodified ATF, eg, an enzyme according to SEQ ID NO:1.

[0046] The engineered host cells as described herein are capable of converting retinol to retinyl acetate under suitable culture conditions, including, but not limited to, culture on glucose, galactose, or xylose, with or without ethanol, and with at least about a 10% or greater increase in conversion compared to the conversion by the respective unmodified enzyme, e.g., the polypeptide according to SEQ ID NO: 1, as obtained by expressing the engineered ATF in a suitable host cell, e.g., selected from fungal host cells, e.g., Yarrowia or Saccharomyces, or microbial host cells, e.g., Escherichia coli, with a 10-760% or greater increase in production of retinyl acetate, at a percentage of retinyl acetate ranging from 86% by weight based on total retinoids. Suitable conditions can be, for example, culture in fed-batch fermentation for 80, 90, 100, 110, 120, or 130 hours.

[0047] The modified host cell as defined herein preferably contains one or more copies of the modified ATF as defined herein, and the ATF is preferably heterologously expressed in the modified host cell. The modification to obtain the host cell as defined herein results in more copies of a gene and / or protein, such as more copies of the modified ATF, that have a preference for the formation of retinyl acetate as defined herein, and includes at least about a 10% increase in the conversion of retinol to retinyl acetate compared to a process using the respective unmodified ATF, such as SbATF1 according to SEQ ID NO: 1 (FIG. 1), as obtained by expressing the unmodified ATF under suitable culture conditions, including, but not limited to, culture in glucose, galactose, or xylose, with or without ethanol, and may include the use of a strong promoter, suitable transcriptional and / or translational enhancers, or the introduction of one or more gene copies into the retinol-producing host cell, particularly a fungal host cell, resulting in increased accumulation of the respective enzyme over a given period of time. The techniques to be used depending on the host cell are known to those skilled in the art. Increases and decreases in gene expression can be measured by a variety of methods, such as, for example, Northern, Southern, or Western blot techniques known in the art.

[0048] Mutations in nucleic acids or amino acids, i.e., mutagenesis, may be performed by a variety of methods, such as random or site-directed mutagenesis, physical damage caused by agents (e.g., radiation, chemical treatment, or insertion of genetic elements), etc. Methods for introducing mutations are known to those skilled in the art.

[0049] Thus, the present invention relates to a retinol-producing host cell, particularly a fungal host cell, as described herein, comprising an expression vector or polynucleotide encoding a modified ATF as described herein integrated into the chromosomal DNA of the host cell. Such a retinol-producing host cell, particularly a fungal host cell, comprising a heterologous polynucleotide in an expression vector or a heterologous polynucleotide integrated into the chromosomal DNA encoding a modified ATF as described herein is referred to as a recombinant cell or modified host cell. A retinol-producing host cell, particularly a fungal host cell, may contain one or more copies of a gene encoding a modified ATF as defined herein, including a mutation as defined herein, thereby resulting in overexpression of such a gene encoding a modified ATF as defined herein. Increased gene expression can be measured by various methods, such as, for example, Northern, Southern, or Western blot techniques known in the art.

[0050] The present invention specifically relates to the use of such novel modified ATF enzymes, particularly in processes for producing retinyl acetate under conditions that reduce the amount of other retinyl esters, particularly long-chain retinyl esters. Methods for creating such conditions are known to those skilled in the art (see, for example, WO2021136689 or WO2022090548). Retinyl acetate can be further converted to vitamin A by the action of suitable (known) chemical or biotechnological mechanisms.

[0051] The terms "sequence identity" and "% identity" are used interchangeably herein. For purposes of the present invention, aligning sequences for optimal comparison purposes is defined herein as determining the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. Gaps may be introduced into either of the two sequences being compared to optimize the alignment between the two sequences. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, alignments may be performed over shorter lengths, for example, over about 20, about 50, about 100, or more nucleic acids / bases or amino acids. Sequence identity is the percentage of perfect matches between the two sequences over the reported alignment region. The percent sequence identity between two amino acid sequences or two nucleotide sequences may be determined using the Needleman-Wunsch algorithm (Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48, 443-453) for aligning the two sequences. Both amino acid sequences and nucleotide sequences can be aligned using this algorithm. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used as the substitution matrix. For nucleotide sequences, EDNAFULL is used. Optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5.Those skilled in the art will understand that all of these different parameters will produce slightly different results, but the overall percentage identity of the two sequences will not change significantly when using different algorithms.

[0052] After alignment by the program NEEDLE as described above, the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: [the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences] divided by [the total length of the alignment after subtracting the total number of gaps in the alignment]. Identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is displayed as "longest identity" in the program output. Both amino acid sequences being compared are identical, or have 100% identity, if they do not differ in any of their amino acids.

[0053] The modified ATF as defined herein also includes enzymes with 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 retinyl acetate in a composition of about 86% by weight or more (e.g., 11 to 86% by weight) based on the total amount of retinoids. Such mutations do not alter the (enzymatic) activity of the enzymes of the present invention and are also called "silent mutations."

[0054] Expression of the enzyme / polynucleotide encoding one of the modified enzymes as defined herein can be achieved in any host system, including a (micro)organism, that is suitable for producing retinoids (including retinol) and that is capable of expressing a nucleic acid encoding one of the enzymes as disclosed herein, including functional equivalents or derivatives as described herein. Examples of suitable retinol-producing host (micro)organisms are bacteria, algae, fungi such as yeast, plant or animal cells. Preferred bacteria include, for example, Escherichia coli (Escherichia coli), and the like. coli), Streptomyces, Pantoea (Erwinia), Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, ococcus, Mixococcus, Brevibacterium, Bradyrhizobium, Gordonia, Dietzia, Muricauda, ​​Sphingomonas, Synochocystis, and Paracoccus, such as Paracoccus zeaxanthinifaciens.Preferred eukaryotic microorganisms, specifically fungi such as yeasts, include those of the genus Saccharomyces, such as Saccharomyces cerevisiae, Aspergillus, such as Aspergillus niger, Pichia, such as Pichia pastoris, Hansenula, such as Hansenula polymorpha, Kluyveromyces, such as Kluyveromyces lactis, and Phycomyces brackensis. blakesleanus), Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, for example Blakeslea trispora, or Yarrowia, for example Yarrowia lipolytica. Expression in fungal host cells, such as Yarrowia or Saccharomyces, or in Escherichia, more preferably Yarrowia lipolytica or Saccharomyces cerevisiae, is particularly preferred.

[0055] Depending on the host cell, the polynucleotide as defined herein for the acetylation of retinol may be optimized for expression in each host cell.Methods for producing such further modified polynucleotides are known to those skilled in the art.It is understood that the polynucleotide as defined herein also encompasses nucleic acid molecules optimized for such hosts, as long as it still expresses a polypeptide having the respective activity as defined herein.

[0056] Thus, in one embodiment, the present invention relates to a retinol-producing host cell, in particular a fungal host cell, comprising a polynucleotide encoding a modified ATF enzyme as defined herein, optimized for expression in said host cell. In particular, the retinol-producing host cell, in particular the fungal host cell, is a yeast, such as Saccharomyces cerevisiae or Yarrowia lipolytica. lipolytica), e.g., from the genus Yarrowia or Saccharomyces, and encoding a modified ATF enzyme as defined herein, is selected from polynucleotides that express a modified polypeptide comprising one or more amino acid substitutions in a sequence having at least 20%, such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO: 1, e.g., the introduction of one or more amino acid substitutions at positions corresponding to residues selected from the group consisting of L34, Y35, V371, M434, N419, F346, R437, W478, F373, and combinations thereof, and are preferably highly conserved as defined herein. The modified ATFs comprise a consensus active site or "Prosite motif" (the motif is in the Prosite syntax as defined at https: / / prosite.expasy.org / scanprosite / scanprosite_doc.html) of at least seven amino acid residues selected from the partial amino acid sequence, i.e., [NSDEHC]-Hx(3)-D-[GA] (where "x" represents any amino acid), corresponding to positions N190 to G196 of the polypeptide according to SEQ ID NO: 1, and the host cells produce at least about 10% increased retinyl acetate compared to host cells expressing the respective unmodified enzyme, e.g., the enzyme according to SEQ ID NO: 1, as obtained by expressing the modified ATF under suitable culture conditions, including, but not limited to, culture on glucose, galactose, or xylose.

[0057] In the context of the present invention, organisms such as microorganisms, fungi, algae, or plants are also understood to include synonyms or basonyms of such species having the same physiological properties as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). Thus, for example, the Lachancea mirantina strain is a synonym of the Zygosaccharomyces sp. IFO 11066 strain originating from Japan.

[0058] The present invention relates to a process for producing retinyl acetate, wherein retinyl acetate is produced by acetylation of retinol as disclosed herein (specifically, as at least 65% trans-retinol) by the action of a modified ATF enzyme as described herein, wherein the acetylation enzyme is preferably heterologously expressed in a suitable host cell under suitable conditions as described herein. The produced retinyl acetate may be isolated and, optionally, further purified from the culture medium and / or host cell. The acetylated retinoid defined herein may be used as a building block in a multi-step process that yields vitamin A. Vitamin A may be isolated and, optionally, further purified from the culture medium and / or host cell as known in the art.

[0059] Preferably, acetylation of retinol using a modified ATF as described herein can result in a percentage of retinyl acetate in the range of at least about 11-86% by weight based on the total retinoids, i.e., total retinyl acetate, present in the retinoid mixture produced by the host cell, such as obtained by expressing the modified enzyme under suitable culture conditions, including, but not limited to, culture on glucose, galactose, or xylose. In a more preferred embodiment, a retinol mixture having a percentage of trans-retinol of at least about 65% is used as a substrate for acetylation by the modified enzyme as defined herein.

[0060] Host cells capable of producing retinol, i.e., microorganisms, algae, fungi, animal, or plant cells, may also be capable of producing β-carotene, which may further be enzymatically converted to retinal, which may then be converted to retinol. Those skilled in the art know which genes to use / express for the biosynthesis of β-carotene and / or the biological conversion of β-carotene to retinol. Such host cells, which are further capable of expressing the modified ATF as defined herein and / or additional genes required for the biosynthesis of vitamin A, can be cultured under aerobic or anaerobic conditions in aqueous media supplemented with appropriate nutrients, as known to those skilled in the art for the respective retinol-producing host cells. Optionally, such culture is carried out in the presence of proteins and / or cofactors involved in electron transfer, as known in the art. Suitable carbon sources for the purposes of the present invention may be selected from glucose, fructose, raffinose, lactose, galactose, glycerol, xylose, arabinose, sucrose, or maltose, with or without ethanol, specifically glucose, galactose, or xylose. Specific culture conditions may include batch and feed runs, with concentrations of 5% (w / v) glucose and 1% ethanol (w / v) used in the batch phase and 100% (w / v) used in the feed phase. Cultivation / growth of the host cells may be carried out under suitable culture conditions in batch, fed-batch, semi-continuous, or continuous mode, specifically fed-batch mode, for periods of 80, 90, 100, 110, 120, or 130 hours. Depending on the host cell, production of retinoids, such as vitamin A, their precursors and / or derivatives, e.g., retinal, retinol, retinyl esters, specifically retinyl acetate, may be varied as known to those skilled in the art. The cultivation and isolation of beta-carotene and retinoid-producing host cells selected from the genera Yarrowia and Saccharomyces is described, for example, in WO2008042338.Methods are described, for example, in US Patent Application Publication No. 20070166782, for the production of beta-carotene and retinoids in host cells selected from E. coli.

[0061] Specifically, fermentations using suitable retinoid-producing host strains as defined herein that express modified ATFs as described herein are cultured in a two-phase system, with the retinoid (including, but not limited to, retinyl acetate) recovered in a suitable lipophilic phase from which it is subsequently isolated. Specific conditions and lipophilic solvents are disclosed in WO2022090548 or WO2022090549.

[0062] In some embodiments, the present invention relates to two-phase fermentation using a lipophilic solvent, such as isopar or corn oil, as the second phase in addition to known solvents including Drakeol®, silicones, or n-dodecane (see Jang et al., Microbial Cell Factories 10:59, 2011).

[0063] As used herein, the term "specific activity" or "activity" with respect to an enzyme refers to its catalytic activity, i.e., its ability to catalyze the formation of a product from a given substrate. Specific activity defines the amount of substrate consumed and / or product produced per defined amount of protein in a given time and at a defined temperature. Typically, specific activity is expressed in μmol of substrate consumed or product formed per mg of protein per minute. μmol / min is typically abbreviated as U (=unit). Thus, the unit definitions of specific activity μmol / min / (mg of protein) or U / (mg of protein) are used interchangeably throughout this specification. An enzyme is active when it performs its catalytic activity in vivo, i.e., in a host cell as defined herein, or in a suitable (cell-free) system in the presence of a suitable substrate. Methods for measuring enzyme activity are known to those of skill in the art, and analytical methods for assessing the ability of suitable ATFs as defined herein, specifically Atf1, to convert retinol to produce retinyl acetate are known in the art, for example, as described in Example 4 of WO2014096992. In summary, titers of products such as retinyl acetate, retinol, trans-retinal, cis-retinal, beta-carotene, etc. can be measured by HPLC.

[0064] With regard to suitable host cells that contain specific enzymes involved in the biosynthesis of beta-carotene and that are expressed and active in vivo, thereby resulting in the production of carotenoids, such as beta-carotene, both genes and methods for producing carotenoid-producing host cells are known in the art, see, for example, WO 2006102342. Depending on the carotenoid to be produced, different genes may be involved.

[0065] As used herein, a "retinol-producing host cell" is a host cell in which the respective polypeptides are expressed and active in vivo, thereby resulting in the production of retinoids, e.g., vitamin A and its precursors, including retinol, by enzymatically converting beta-carotene via retinal to retinol. These polypeptides include modified ATFs as defined herein. Methods for generating vitamin A pathway genes and retinoid-producing host cells are known in the art. The term retinoid includes retinol, which is used as a substrate for modified acetyltransferases as defined herein.

[0066] As used herein, retinoids include beta-carotene degradation products, also known as apocarotenoids, including, but not limited to, retinal, retinoic acid, retinol, retinoic acid methoxide, retinyl acetate, retinyl esters, 4-keto-retinoids, 3-hydroxy-retinoids, or combinations thereof. As used herein, long-chain retinyl esters are defined as hydrocarbon esters of retinol and fatty acids, where the fatty acid contains at least about 8 carbon atoms, e.g., 9, 10, 12, 13, 15, or 20 carbon atoms, and up to about 26 carbon atoms, e.g., 25, 22, or 21 carbon atoms or less, and preferably up to about 6 unsaturated bonds, e.g., 0, 1, 2, 4, 5, or 6 unsaturated bonds. Fatty acids in long-chain retinyl esters include, but are not limited to, linoleic acid, oleic acid, or palmitic acid. The biosynthesis of retinoids is described, for example, in WO2008042338.

[0067] As used herein, "retinal" is known by its IUPAC name of (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenal. Retinal is referred to interchangeably herein as retinaldehyde or vitamin A aldehyde, and includes both cis- and trans-isoforms, such as 11-cis retinal, 13-cis retinal, trans-retinal, and all-trans-retinal.

[0068] As used herein, the term "carotenoid" is well known in the art. Carotenoids include long 40-carbon conjugated isoprenoid polyenes formed naturally by linking two 20-carbon geranylgeranyl diphosphate molecules. These include, but are not limited to, phytoene, lycopene, and carotenes, such as β-carotene, which can be oxidized at the 4-keto or 3-hydroxy position to yield canthaxanthin, zeaxanthin, or astaxanthin. The biosynthesis of carotenoids is described, for example, in International Publication No. WO2006102342.

[0069] "Vitamin A," as used herein, refers to any chemical form of vitamin A found in aqueous solutions, solids, and formulations, including retinol, retinyl acetate, and retinyl esters, including retinoic acid, whether undissociated in the free acid form or dissociated as an anion.

[0070] Specifically, the present invention relates to the following embodiments (1) to (14).

[0071] (1) A method for modifying an enzyme, preferably a fungal enzyme, that catalyzes the acetylation of retinoids, specifically the acetylation of retinol to retinyl acetate, comprising introducing at least one amino acid substitution into 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, of a polypeptide according to SEQ ID NO: 1, wherein the introduction of the one or more amino acid substitutions increases the activity for the formation of retinyl acetate by at least about 10%, for example, in the range of 10 to 760%, compared to the activity using the respective unmodified enzyme, including the enzyme according to SEQ ID NO: 1.

[0072] (2) The method according to embodiment (1), wherein the amino acid residue substituted at position 34 of SEQ ID NO: 1 is not leucine, and / or the amino acid residue substituted at position 35 of SEQ ID NO: 1 is not tyrosine, and / or the amino acid residue substituted at position 346 or 373 of SEQ ID NO: 1 is not phenylalanine, and / or the amino acid residue substituted at position 371 of SEQ ID NO: 1 is not valine, and / or the amino acid residue substituted at position 419 of SEQ ID NO: 1 is not asparagine, and / or the amino acid residue substituted at position 434 of SEQ ID NO: 1 is not methionine, and / or the amino acid residue substituted at position 437 of SEQ ID NO: 1 is not arginine, and / or the amino acid residue substituted at position 478 of SEQ ID NO: 1 is not tryptophan.

[0073] (3) The method according to embodiment (1) or (2), wherein the amino acid residue introduced at the position corresponding to position 34, 35 or 478 of SEQ ID NO: 1 is phenylalanine, and / or the amino acid residue introduced at the position corresponding to position 346 or 419 of SEQ ID NO: 1 is leucine, and / or the amino acid residue introduced at the position corresponding to position 371 of SEQ ID NO: 1 is isoleucine, and / or the amino acid residue introduced at the position corresponding to position 373 of SEQ ID NO: 1 is alanine, and / or the amino acid residue introduced at the position corresponding to position 434 or 437 of SEQ ID NO: 1 is valine.

[0074] (4) The method of embodiment (1), (2), or (3), 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 L34, Y35, F346, V371, F373, N419, M434, R437, W478, and / or combinations thereof, preferably, the modified enzyme comprises one or more amino acid substitutions selected from the group consisting of L34F, Y35F, V371I, M434V, N419L, F346L, R437V, W478F, F373A, and combinations thereof.

[0075] (5) The method according to embodiment (1), (2), (3) or (4), wherein the introduction of one or more amino acid substitutions increases the proportion of retinyl acetate based on total retinoids in a process comprising expression of the modified enzyme in a suitable host cell to about 86% by weight compared to the proportion using the respective unmodified enzyme, including the enzyme according to SEQ ID NO: 1.

[0076] (6) A process for producing a retinol acetyltransferase, preferably an acetyltransferase of the class [EC 2.3.1.84], having increased activity for the acetylation of retinol to retinyl acetate, comprising: (a) providing a fungal enzyme involved in the acetylation of retinol, specifically an ATF derived from the genus 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 retinyl acetate from retinol, based on the total retinoid produced by the host cell; (b) introducing one or more amino acid substitutions, e.g., at least 1-9 amino acid substitutions, into the enzyme of (a) to generate a modified enzyme involved in the acetylation of retinol, wherein the ability to convert retinol to retinyl acetate is increased by at least about 10%, e.g., by at least 10-760%, when compared to the acetylation of the respective unmodified enzyme of step (a). Includes; the one or more amino acid substitutions are made at positions corresponding to amino acid residues selected from the group consisting of positions 34, 35, 346, 371, 373, 419, 434, 437, 478 of SEQ ID NO: 1, wherein the substituted amino acids are different from L34, Y35, F346, V371, F373, N419, M434, R437, W478 and / or combinations thereof, and wherein the amino acid residue introduced at position 34, 35 or 478 of SEQ ID NO: 1 is preferably phenylalanine, and / or the amino acid residue introduced at position 346 or 419 of SEQ ID NO: 1 is preferably leucine, and / or the amino acid residue introduced at position 371 of SEQ ID NO: 1 is preferably isoleucine, and / or the amino acid residue introduced at position 373 of SEQ ID NO: 1 is preferably alanine, and / or the amino acid residue introduced at position 434 or 437 of SEQ ID NO: 1 is preferably valine.

[0077] (7) The process according to embodiment (6), wherein the acetyltransferase of step (a) is selected from yeast ATFs.

[0078] (8) A modified enzyme, particularly a fungal enzyme, involved in the acetylation of retinol to retinyl acetate, comprising one or more modifications, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, wherein the amino acid substitutions are 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 1 and a polypeptide comprising a sequence having up to 8, 99% or 100% identity thereto, wherein the substituted amino acid residues are located at positions corresponding to amino acid residues selected from the group consisting of positions 34, 35, 346, 371, 373, 419, 434, 437, 478, and combinations thereof of the enzyme according to SEQ ID NO: 1, and the substituted amino acid residues are different from the amino acids corresponding to positions L34, Y35, F346, V371, F373, N419, M434, R437, W478, and combinations thereof of the enzyme according to SEQ ID NO: 1. the substituted amino acid residues correspond to residues selected from the group consisting of L34, Y35, F346, V371, F373, N419, M434, R437, W478 of the polypeptide according to SEQ ID NO: 1 and combinations thereof, specifically, the substituted amino acid residue introduced at a position corresponding to position 34, 35 or 478 of SEQ ID NO: 1 is preferably phenylalanine, the substituted amino acid residue introduced at a position corresponding to position 346 or 419 of SEQ ID NO: 1 is preferably leucine, the substituted amino acid residue introduced at a position corresponding to position 371 of SEQ ID NO: 1 is preferably isoleucine, the substituted amino acid residue introduced at a position corresponding to position 373 of SEQ ID NO: 1 is preferably alanine, and / or the substituted amino acid residue introduced at a position corresponding to position 434 or 437 of SEQ ID NO: 1 is preferably valine.

[0079] (9) A polypeptide having at least about 20%, for example, 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, wherein the polypeptides are selected from polypeptides having at least about 20%, for example, 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, and are different from L34, Y35, F346, V371, F373, N419, M434, R437, or W478. 10. The modified enzyme of embodiment (8), comprising one or more amino acid substitutions at positions selected from the group consisting of: 1) L34F, 2) Y35F, 3) V371I, 4) M434V, 5) N419L, 6) F346L, 7) R437V, 8) W478F, 9) F373A, and / or 10) .

[0080] (10) A modified enzyme described in embodiment (8) or (9), in which the enzymatic activity for the formation of retinyl acetate is increased by at least about 10% compared to the respective unmodified ATF, for example, an ATF according to sequence number 1, specifically, the proportion of retinyl acetate is increased to about 86% by weight based on total retinoids.

[0081] (11) The modified enzyme according to embodiment (8), (9) or (10), comprising amino acid substitutions corresponding to positions 34 and 434 of SEQ ID NO: 1, preferably the amino acid substitutions corresponding to L34F_M434V of the polypeptide according to SEQ ID NO: 1.

[0082] (12) A process for producing retinyl acetate, comprising the step of acetylating retinol by the action of an enzyme according to embodiment (8), (9), (10) or (11).

[0083] (13) The process according to embodiment (12), wherein a suitable retinol-producing host cell, preferably selected from the genus Yarrowia, Saccharomyces or Escherichia coli, is transformed with and expresses the enzyme according to embodiment (8), (9), (10) or (11).

[0084] (14) The process of embodiment (12) or (13), wherein the percentage of retinyl acetate produced under suitable culture conditions is about 86% by weight, based on the total retinoids produced by the host cell. [Brief explanation of the drawings]

[0085] [Figure 1] 1 is the amino acid sequence of Saccharomyces bayanus ATF1 (SbATF; SEQ ID NO: 1), with residues selected for amino acid substitution as described in this application shown in bold / underlined.

[0086] The following examples are merely illustrative and are not intended to limit the scope of the present invention in any way. The contents of all references, patent applications, patents, and published patent applications cited throughout this application, in particular WO2014096992, WO2019058001, WO2021136689, WO2022090548, WO2008042338, US Patent Application Publication No. 20070166782, WO2022090549, WO2006102342, WO2020141168, and WO2016172282, are incorporated herein by reference.

[0087] [Example] Example 1: General methods, strains, and plasmids All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989), or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998).

[0088] Shake Plate Assay (Yarrowia). To test the transforming activity of mutants, typically 200 μl of 0.25% yeast extract, 0.5% peptone (0.25X YP) was inoculated with 10 μl of freshly grown Yarrowia and overlaid with 200 μl of mineral oil (Isopar M, Exxon Mobile) containing 2% oleic acid as the carbon source in the mineral oil phase. Transformants were grown in 24-well plates (Microplate Devices 24 Deep Well Plates, Whatman 7701-5102), covered with mat seals (Analytical Sales and Services Inc. Plate Mats 24010CM), sterile sealed with Qiagen Airpore Tape Sheets (19571), and shaken at 30°C and 800 RPM for 4 days in an Infors Multitron multiplate shaker. The mineral oil fractions were removed from the shaker plate wells and analyzed by UPLC reversed-phase column with a photodiode array detector. This method is also used in Example 2.

[0089] DNA transformation. Yarrowia lipolytica strains were transformed after overnight growth on YPD plates. 50 μl of cells were scraped from the plates and transformed by incubating 1 μg of transforming DNA (typically linear DNA for integrative transformation) in 500 μl of 40% PEG 3550MW, 100 mM lithium acetate, 50 mM dithiothreitol, 5 mM Tris-Cl (pH 8.0), and 0.5 mM EDTA at 40°C for 30 minutes and then plated directly onto selective media. Alternatively, for selection with dominant antibiotic markers, cells were grown in YPD liquid medium at 30°C for 4 hours before plating onto selective media. Saccharomyces strains were transformed from log-phase YPD-grown cells using the lithium acetate method and expanded by subculturing YPD cultures overnight. 10 cells per transformation were used. 8 Cells were harvested and resuspended in a final volume of 500 μL in a mixture containing 40% PEG 3350 (MW), 100 mM lithium acetate, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 5 μg of sheared salmon sperm DNA, and 2 μg of linearized transforming DNA. This mixture was incubated at 30°C for 1 hour, followed by incubation at 42°C for 30 minutes. Cells were then pelleted, resuspended in liquid YPD medium, and grown at 30°C for 3 hours or at 22°C overnight to allow expression of the HygR antibiotic resistance gene before plating on selective medium containing 100 μg / ml hygromycin. Most of the DNA sequences used herein have been codon-optimized for expression in the respective host systems and are shown in the Sequence Listing.

[0090] DNA Molecular Biology. Plasmid MB10362 (SEQ ID NO: 3), containing the DrBCO, LmATF, and FfRDH expression systems, was synthesized using GenScript (Piscataway, NJ, USA). Plasmid MB10362 contains both the "URA3" and "HOM3" markers for selection in Yarrowia lipolytica transformation. To achieve clean gene insertion by random, non-homologous end-ligation of this gene with the SfiI marker, the MB10362 plasmid fragment, or any other plasmid of interest listed in Table 1, was purified by gel electrophoresis and a Qiagen gel purification column. Clones were confirmed by sequencing. Typically, genes are synthesized using GenScript (Piscataway, NJ) by introducing amino acid substitutions listed in Table 1. Transformants were screened for homoserine auxotrophy and subsequently sequenced using primers flanking the HOM3 sequence to select for clean frameshifts for forward progression. Expression of mutant ATFs in Saccharomyces cerevisiae is described in Example 1 of WO2020141168.

[0091] Sequences. The wild-type SbATF (polynucleotide according to SEQ ID NO: 2) and the plasmids used to express modified enzymes containing specific amino acid substitutions, with codon-optimized sequences for expression in Yarrowia lipolytica or Saccharomyces cerevisiae specified, are listed in Table 1 and / or the Sequence Listing. Figure 1 shows the amino acid sequence of wild-type SbATF, with the residues selected for modification according to Table 1 specified.

[0092] [Table 1]

[0093] [Table 2]

[0094] UPLC reversed-phase retinol method. For rapid screening, this method does not separate cis isomers, but only the major functional groups. Samples were injected using a Waters Acquity UPLC with PDA detection (or similar) along with an autosampler. Retinoids were resolved using an Acquity UPLC HSS T3 1.8um P / N 186003539. The mobile phase consisted of either 1000 mL of hexane, 30 mL of isopropanol, or 0.1 mL of acetic acid for retinoid-related compounds. The flow rate for each was 0.6 mL / min. The column temperature was 20 °C. The injection volume was 5 μL. The detector was a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 2.

[0095] [Table 3]

[0096] [Table 4]

[0097] Assay Method: The method assays for retinyl acetate and quantifies retinol and retinal relative to retinyl acetate using the indicated response factors. To prepare a stock solution using a volumetric flask, retinyl acetate was dissolved in THF at approximately 200 μg / ml. Using a volumetric flask, 20x, 50x, and 100x dilutions of the stock solution were made with 50 / 50 methanol / MTBE. The UV absorbance of retinyl acetate becomes nonlinear fairly rapidly, so care must be taken to stay within the linear range. Therefore, lower concentrations may be more appropriate. Retinyl palmitate can also be used as a calibrator for retinyl esters. The retinyl acetate peak is at approximately 3 minutes, while the retinyl esters (long-chain retinyl esters) peak at approximately 3.5 minutes.

[0098] Sample preparation. Samples were prepared using various methods depending on the conditions. For whole broth or washed broth samples, the broth was weighed into a Precellys® tube and mobile phase was added. Briefly, 25 μl of thoroughly mixed broth and 975 μl of THF were added to a 2 ml Precellys® tube. The sample was then processed in a Precellys® homogenizer (Bertin Corp, Rockville, MD, USA) at the highest setting three times according to the manufacturer's instructions, typically 3 × 15 × 7500 tpms. For washed pellets, the sample was spun in a 1.7 ml tube in a microcentrifuge at 10,000 rpm for 1 minute, the broth was decanted, 1 ml of water was added, mixed, pelleted, and decanted back to the original volume. The mixture was pelleted again, placed back in the appropriate amount of mobile phase, and processed by Precellys® bead beating. For analysis of the silicone oil fraction, samples were spun at 4000 RPM for 10 min, the oil was decanted using a positive displacement pipette (Eppendorf, Hauppauge, NY, USA), the top portion discarded, diluted in mobile phase mixed by vortexing, and the retinoid concentration determined by UPLC analysis.

[0099] Fermentation conditions for Yarrowia. Fermentation was preferably the same as described above using a silicone oil overlay and stirred tank, preferably glucose, in a benchtop reactor with a total volume of 0.5-5 L (see WO2016172282). Generally, the same results were observed in a fed-batch stirred tank reactor, demonstrating the utility of the system for producing retinoids through increased productivity. Preferably, fermentation was batched with 5% glucose, with 20% silicone oil added after the dissolved oxygen had fallen below about 20%, and feeding resumed to achieve 20% dissolved oxygen throughout the entire feed program.

[0100] Example 2: Production of retinyl acetate in Yarrowia lipolytica expressing mutant SbATF To express heterologous ATFs in Yarrowia lipolytica as a host, strain ML15710 (see Example 5 in WO 2016172282) was transformed with plasmid MB9287 (see Example 1 in WO 2022090548) and a lip2 lip3 lip8 mutant derivative was isolated. This derivative was selected on 5-fluorouracil acid to isolate a uracil auxotroph, designated strain ML18667-new. This strain was transformed with the plasmids listed in Table 1 above, each consisting of the indicated ATF allele, DrBCO, and FfRDH12. Transformants of ML18667-new containing the SfiI-linearized plasmids from Table 1 were selected for uracil prototrophy. Transformants were grown in shake plates as described in Example 1, and the percentage of retinyl acetate using the mutant ATFs relative to the percentage of retinyl acetate using the reference SbATF expressed in plasmid MB10362 (set at 100%) (retinyl acetate / total retinoids) is shown in Table 3.

[0101] [Table 5]

[0102] The proportion of retinyl acetate based on total retinoids introducing the double mutant (see Table 1) could be increased to more than 26% by weight compared to the process using the enzyme according to SEQ ID NO: 1.

[0103] [Table 6]

[0104] The proportion of retinyl acetate based on total retinoids introducing the double mutant (see Table 1) could be increased by 66% by weight compared to the process using the enzyme according to SEQ ID NO: 1.

[0105] [Table 7]

[0106] The proportion of retinyl acetate based on total retinoids introduced with the four mutants (see Table 1) could be improved by up to 77% by weight compared to the process using the enzyme according to SEQ ID NO: 1.

[0107] [Table 8]

[0108] The proportion of retinyl acetate based on total retinoids introduced with the four mutants (see Table 1) could be improved by 86% by weight compared to the process using the enzyme according to SEQ ID NO: 1.

[0109] [Table 9]

[0110] The proportion of retinyl acetate based on total retinoids introduced with six mutants (see Table 1) could be improved by up to 80% by weight compared to the process using the enzyme according to SEQ ID NO: 1.

[0111] [Table 10]

[0112] The proportion of retinyl acetate based on total retinoids introduced with the four mutants (see Table 1) could be improved by up to 80% by weight compared to the process using the enzyme according to SEQ ID NO: 1.

[0113] [Table 11]

[0114] The proportion of retinyl acetate based on total retinoids introduced with the four mutants (see Table 1) could be improved by up to 11% by weight compared to the process using the enzyme according to SEQ ID NO: 1.

Claims

1. 1. A method for modifying an enzyme, preferably a fungal enzyme, that catalyzes the acetylation of retinoids, specifically the acetylation of retinol to retinyl acetate, comprising introducing at least two amino acid substitutions at positions selected from amino acid residues 34 and 434 of a polypeptide according to SEQ ID NO: 1, wherein the introduction of said at least two amino acid substitutions increases the activity towards the formation of retinyl acetate by at least about 10% compared to the activity using the respective unmodified enzyme, including the enzyme according to SEQ ID NO: 1, and wherein said amino acid residues at positions corresponding to amino acid residues 34 and 434 of SEQ ID NO: 1 are different from L34 and M434.

2. 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, of the polypeptide according to SEQ ID NO: 1, wherein introducing the one or more amino acid substitutions increases the activity for the formation of retinyl acetate by at least about 10%, for example, in the range of 10 to 760%, compared to the activity using a respective unmodified enzyme, including the enzyme according to SEQ ID NO:

1.

3. 3. The method of claim 1, wherein the amino acid residue substituted at position 35 of SEQ ID NO: 1 is not tyrosine, and / or the amino acid residue substituted at position 346 or 373 of SEQ ID NO: 1 is not phenylalanine, and / or the amino acid residue substituted at position 371 of SEQ ID NO: 1 is not valine, and / or the amino acid residue substituted at position 419 of SEQ ID NO: 1 is not asparagine, and / or the amino acid residue substituted at position 437 of SEQ ID NO: 1 is not arginine, and / or the amino acid residue substituted at 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 introduced at a position corresponding to position 34, 35 or 478 of SEQ ID NO: 1 is phenylalanine, and / or the amino acid residue introduced at a position corresponding to position 346 or 419 of SEQ ID NO: 1 is leucine, and / or the amino acid residue introduced at a position corresponding to position 371 of SEQ ID NO: 1 is isoleucine, and / or the amino acid residue introduced at a position corresponding to position 373 of SEQ ID NO: 1 is alanine, and / or the amino acid residue introduced at a position corresponding to position 434 or 437 of SEQ ID NO: 1 is valine.

5. 5. The method of 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 amino acid substitutions L34F and M434V.

6. 1. A process for producing a retinol acetyltransferase, preferably an acetyltransferase of the class [EC 2.3.1.84], having increased activity for the acetylation of retinol to retinyl acetate, comprising: (a) providing a fungal enzyme involved in the acetylation of retinol, specifically an ATF derived from the genus 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 retinyl acetate from retinol, based on the total retinoid produced by the host cell; (b) introducing at least two amino acid substitutions at positions corresponding to L34 and M434 of the polypeptide according to SEQ ID NO: 1 to generate a modified enzyme involved in the acetylation of retinol, and further introducing at least 1 to 7 amino acid substitutions into the enzyme of (a), which increases the ability to convert retinol to retinyl acetate by at least about 10%, such as by at least 10 to 760%, when compared to the acetylation of the respective unmodified enzyme of step (a). Including; the at least one or seven amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of positions 35, 346, 371, 373, 419, 437, 478 of SEQ ID NO: 1, wherein the substituted amino acids are different from Y35, F346, V371, F373, N419, R437, W478 and / or combinations thereof, and wherein the amino acid residue introduced at position 34, 35 or 478 of SEQ ID NO: 1 is preferably phenylalanine, and / or the amino acid residue introduced at position 346 or 419 of SEQ ID NO: 1 is preferably leucine, and / or the amino acid residue introduced at position 371 of SEQ ID NO: 1 is preferably isoleucine, and / or the amino acid residue introduced at position 373 of SEQ ID NO: 1 is preferably alanine, and / or the amino acid residue introduced at position 434 or 437 of SEQ ID NO: 1 is preferably valine.

7. 7. The process of claim 6, wherein the acetyltransferase of step (a) is selected from yeast ATFs.

8. A modified enzyme, particularly a fungal enzyme, involved in the acetylation of retinol to retinyl acetate, comprising at least two modifications, e.g., amino acid substitutions located at positions corresponding to amino acid residues 34 and 434 of a polypeptide according to SEQ ID NO: 1, and further comprising at least 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, wherein the amino acid substitutions are at least about 20%, e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 1109, 1111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 14 and a combination thereof of a polypeptide comprising a sequence of 5, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity, wherein the substituted amino acid residues are located at positions corresponding to amino acid residues selected from the group consisting of L34, Y35, F346, V371, F373, N419, M434, R437, W478 and their combinations of the enzyme according to SEQ ID NO:

1. the substituted amino acid residue is different from an amino acid corresponding to a combination thereof, and preferably the substituted amino acid residue corresponds to a residue selected from the group consisting of L34, Y35, F346, V371, F373, N419, M434, R437, W478 of the polypeptide according to SEQ ID NO: 1, and a combination thereof; specifically, the substituted amino acid residue introduced at a position corresponding to position 34, 35 or 478 of SEQ ID NO: 1 is preferably phenylalanine; the substituted amino acid residue introduced at a position corresponding to position 346 or 419 of SEQ ID NO: 1 is preferably leucine; the substituted amino acid residue introduced at a position corresponding to position 371 of SEQ ID NO: 1 is preferably isoleucine; the substituted amino acid residue introduced at a position corresponding to position 373 of SEQ ID NO: 1 is preferably alanine; and / or the substituted amino acid residue introduced at a position corresponding to position 434 or 437 of SEQ ID NO: 1 is preferably valine.

9. 1, wherein the polypeptides are selected from polypeptides 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, and ... The modified enzyme of claim 8, comprising one or more amino acid substitutions at positions selected from 346, 371, 373, 419, 434, 437 and / or 478, preferably wherein the one or more amino acid substitutions are selected from the group consisting of L34F, Y35F, V371I, M434V, N419L, F346L, R437V, W478F, F373A, and combinations thereof.

10. A modified enzyme as described in claim 8 or 9, wherein the activity of the enzyme for the formation of retinyl acetate is increased by at least about 10% compared to the respective unmodified ATF, such as an ATF according to sequence number 1, and specifically the proportion of retinyl acetate is increased to about 86% by weight based on total retinoids.

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

1.

12. A process for producing retinyl acetate, comprising the step of acetylating retinol by the action of an enzyme according to any one of claims 8 to 11.

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

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

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