Modified sterol acyltransferase

Abstract

The present invention relates to a modified sterol acyltransferase having an improved activity and / or specificity towards the acylation of the vitamin D3 precursor 7-dehydrocholesterol (7-DHC) to be used in the biotechnological production of vitamin D3. The present invention also relates to a host strain expressing the modified enzyme and the use of the host strain in a method for producing vitamin D3 or its derivatives and / or metabolites.

Description

[0001] The present invention relates to modified sterol acyltransferases having improved activity and / or specificity towards the acylation of the vitamin D3 precursor 7-dehydrocholesterol (7-DHC) to be used in the biotechnological production of vitamin D3. The present invention also relates to a host strain expressing the modified enzyme and the use of the host strain in a method for producing vitamin D3 or its derivatives and / or metabolites.

[0002] Vitamin D3 (also known as cholecalciferol or cholecalciferol) can be synthesized in mammalian skin from previtamin D3 (also known as 7-dehydrocholesterol or 7-DHC), a product of cholesterol biosynthesis upon exposure to ultraviolet light, whereby 7-DHC is photochemically converted to previtamin D3, which is isomerized to the biologically active form, vitamin D3, at body temperature. In the liver, vitamin D3 is converted to the biologically inactive 25-hydroxyvitamin D3 (also known as calcidiol / calcifediol, 25-hydroxycholecalciferol, 25-OH-D3, or HyD), which is the major circulating form of vitamin D3. Further hydroxylation occurs in the kidneys.

[0003] For the industrial production of vitamin D3, both chemical and biotechnological synthesis are feasible (in principle). Chemical synthesis begins with the isolation of cholesterol from, for example, lanolin, which is then dehydrogenated to 7-DHC, an important intermediate in both chemical and biotechnological synthesis. 7-DHC is converted to vitamin D3 by exposure to UV light and further purification / extraction steps. Modified yeast strains can be used for the biosynthesis of 7-DHC, in which acetyl-CoA is converted to 7-DHC in a multi-step enzymatic process. This enzymatic conversion occurs in the yeast endoplasmic reticulum. Excess sterols, including 7-DHC and its precursors, are unwanted in the cell membrane and are toxic to yeast. Excess sterols are therefore stored as sterol esters in intracellular organelles (so-called liposomes), from which they can be further isolated. The balance between free sterols and the sterols stored in the liposomes (primarily in the form of sterol esters) is triggered by the action of several proteins (enzymes), including sterol acyltransferases. In yeast, particularly Saccharomyces cerevisiae , sterol ester formation is primarily carried out by two sterol acyltransferases, Are1p and Are2p.

[0004] Due to the non-specific action of the sterol acyltransferase, the pool of sterol esters stored within the liposomes is relatively diverse, including, but not limited to, esters of, for example, ergosterol, zymosterol, lanosterol, enocholestanol, cholesta-5,7,24(25)-trienol, or 7-DHC. Since only the precursor of 7-DHC, cholesta-5,7,24(25)-trienol, and not zymosterol, is available for vitamin D3 synthesis, it is desirable to selectively store specific esters (e.g., esters of 7-DHC) within the liposomes and / or to increase the turnover of 7-DHC intermediates produced by such yeast strains, which are further converted to vitamin D3 and / or its derivatives or metabolites. A specific metabolite that is also the focus of the present invention is 25-hydroxyvitamin D3.

[0005] Therefore, there is an ongoing task to generate host cells (e.g., yeast) capable of producing sterols with high 7-DHC productivity / specificity for 7-DHC and / or reduced accumulation of by-products / intermediates (including zymosterol, lanosterol, or cholestanol), in particular esters of such intermediates stored in lipid bodies.

[0006] Surprisingly, we have now found that the specificity and / or activity of sterol acyltransferase activity in a host cell can be shifted by introducing certain amino acid substitutions in the sequence of ARE2 and / or ARE1, which will result in the host cell being towards a higher production of 7-DHC, an important intermediate in the production of vitamin D3.

[0007] Thus, the present invention relates to a modified enzyme having sterol acyltransferase activity, i.e. a modified sterol acyltransferase, in particular the activity of the sterol acyltransferase isoforms Are1p and / or Are2p, said modified enzyme comprising one or more amino acid substitutions at one or more positions corresponding to residues selected from the group consisting of positions 11, 281, 366, 442, 551, 554, 572, 624, 626, 627, 636 and combinations thereof in the polypeptide according to SEQ ID NO: 1, said modified enzyme having increased specificity for 7-DHC over byproducts / intermediates including yeast sterol and / or increased activity towards ester formation (including esters of 7-DHC).

[0008] A polypeptide according to SEQ ID NO: 1 showing ARE2 activity has been isolated from Saccharomyces cerevisiae, including a polynucleotide encoding the polypeptide. A polypeptide according to SEQ ID NO: 3 showing ARE1 activity has been isolated from Saccharomyces cerevisiae, including a polynucleotide encoding the polypeptide.

[0009] The terms "sterol acyltransferase," "acyltransferase," "ARE," "enzyme having acyltransferase activity," or simply "enzyme" are used interchangeably herein and refer to an enzyme [EC 2.3.1.26], i.e., an acyltransferase that transfers a fatty acyl group from one molecule to another. Such transfer or enzymatic activity can be measured by means known to the skilled person. Sterol acyltransferases have been isolated from various sources, including mammals, yeast, or plants. Both ARE1 and ARE2 are capable of acylating sterols (e.g., yeast sterol and / or 7-DHC) to the corresponding esters. As used herein, a "modified" enzyme, i.e., a modified acyltransferase, has an activity and / or specificity that is preferentially directed toward esterification of 7-DHC, as compared to, for example, esterification of yeast sterol and / or improved formation of total sterol esters comprising, for example, 7-DHC or yeast sterol. Preferred acyltransferase isoforms are Are2p or Are1p. In the present context, "unmodified" sterol acyltransferases, in particular ARE1 and ARE2, refer to the corresponding endogenous enzymes that do not carry one or more amino acid substitutions as defined herein.

[0010] Herein, host cells carrying a modified sterol acyltransferase activity as defined herein, in particular an ARE2 and / or ARE1 comprising one or more amino acid substitutions as defined herein, are referred to as "modified" host cells. The corresponding host cells carrying an unmodified sterol acyltransferase activity, i.e. encoding a wild-type ARE2 and / or ARE1 gene, are referred to as "unmodified" host cells.

[0011] As used herein, the terms "zymosterol," "lanosterol," "cholestanol," "cholesta-5,8,24(25)-trienol," "cholesta-5,7,24(25)-trienol," or "7-DHC," which designate a vitamin D3 intermediate, include both the free and ester forms of the compound. As used herein, a sterol mixture comprises 7-DHC and "byproducts" or intermediates including, but not limited to, zymosterol, lanosterol, cholesta-5,8,24(25)-trienol, or cholesta-5,7,24(25)-trienol.

[0012] In this context, "cholesterol-producing yeast" is no longer able to produce ergosterol, but instead produces cholesterol products including, but not limited to, cholesteryl-5,7,24(25)-trienol, cholesteryl-5,8,24(25)-trienol, cholesteryl-7,24(25)-dienol, 7-DHC, or zymosterol. Specifically, this can be achieved by introducing a double knockout of erg5erg6.

[0013] In particular, the modification corresponds to a modified activity of sterol acyltransferase 2 and / or 1, i.e. ARE2 and / or ARE1 activity, the modification comprising one or more amino acid substitutions, wherein preferably at least one amino acid substitution at one or more positions corresponding to a residue selected from the group consisting of position 11, 281, 366, 442, 551, 554, 572, 624, 626, 627, 636 and combinations thereof in the polypeptide according to SEQ ID NO: 1 corresponds to one or more amino acid substitutions of E11 and / or L281 and / or D366 and / or I442 and / or H551 and / or H554 and / or F572 and / or F624 and / or L626 and / or G627 and / or C636. More preferably, the modification corresponds to a modified ARE2 activity, even more preferably a modified polypeptide according to SEQ ID NO: 1, wherein at least one amino acid substitution has been introduced at one or more positions corresponding to a residue selected from the group consisting of positions 11, 281, 366, 442, 551, 554, 572, 624, 626, 627, 636 and combinations thereof. Preferably, the enzyme having modified ARE2 and / or ARE1 activity is derived from Saccharomyces, such as Saccharomyces cerevisiae.

[0014] In one embodiment, the modified enzyme as defined herein, in particular the modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 11 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of glutamic acid (E11G) by glycine. Said amino acid substitution at the position corresponding to residue E11G in SEQ ID NO: 1 may be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 281, 366, 442, 551, 554, 572, 624, 626, 627 and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). Preferably, the amino acid substitution at the position corresponding to residue E11G in SEQ ID NO: 1 may be combined with other substitutions (e.g., one or more amino acid substitutions at one or more positions corresponding to F624L, G627D, D366V, C636S and / or I442V in SEQ ID NO: 1), more preferably with residue E11G in SEQ ID NO: 1. Residues in NO: 1 are E11G-F624L, E11G-G627D, E11G-D366V-C636S, E11G-D366V-G627D-C636S, E11G-D366V-F624L-C636S, E11G-D366V-I442V-F624L-C636S, or E11G-D366V-I442V-G627D- The most preferred combination is selected from E11G-D366V-C636S, E11G-D366V-G627D-C636S, E11G-D366V-F624L-C636S, E11G-D366V-I442V-G627D-C636S or E11G-D366V-I442V-F624L-C636S. By using cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions, the enzyme specificity can be improved by at least about 3-fold to 5-fold compared to using unmodified yeast as defined herein.

[0015] In one embodiment, a modified enzyme as defined herein, in particular a modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 281 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of isoleucine for leucine (L281I). The amino acid substitution at the position corresponding to residue L281I in SEQ ID NO: 1 can be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 366, 442, 551, 554, 572, 624, 626, 627, and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). By using cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions, relative ester formation can be improved by at least about 1.5-fold compared to using unmodified yeast as defined herein.

[0016] In one embodiment, a modified enzyme as defined herein, in particular a modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 366 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of valine for aspartic acid (D366V). The amino acid substitution at the position corresponding to residue D366V in SEQ ID NO: 1 can be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 442, 551, 554, 572, 624, 626, 627, and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). By using a cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions, enzyme specificity can be improved by at least about 3-fold compared to using an unmodified yeast as defined herein.

[0017] In another embodiment, the modified enzyme as defined herein, in particular the modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 442 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of isoleucine by valine (I442V). Said amino acid substitution at the position corresponding to residue 1442V in SEQ ID NO: 1 may be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 551, 554, 572, 624, 626, 627 and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). Preferably, the amino acid substitution at the position corresponding to residue I442V in SEQ ID NO: 1 can be combined with other substitutions (e.g., amino acid substitutions at one or more positions corresponding to F624L, L626F and / or G627D in SEQ ID NO: 1), more preferably a combination of substitutions corresponding to residues I442V-L626F, I442V-G627D, I442V-F624L-L626F or I442V-L626F-G627D in SEQ ID NO: 1, wherein the most preferred combination is selected from I442V-G627D, I442V-F624L-L626F or I442V-L626F-G627D. By using cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of said one or more amino acid substitutions, the enzyme specificity can be improved in the range of at least about 2.2-fold to 4.5-fold compared to using unmodified yeast as defined herein.

[0018] In another embodiment, the modified enzyme as defined herein, in particular the modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 551 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of tyrosine for histidine (H551Y). The amino acid substitution at the position corresponding to residue H551Y in SEQ ID NO: 1 can be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 442, 554, 572, 624, 626, 627, and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). By using cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions, enzyme specificity can be improved by at least about 1.7-fold compared to using unmodified yeast as defined herein.

[0019] In one embodiment, the modified enzyme as defined herein, in particular the modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 554 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of histidine by glutamine (H554Q). Said amino acid substitution at a position corresponding to residue H554Q in SEQ ID NO: 1 may be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 442, 551, 572, 624, 626, 627 and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). Preferably, the amino acid substitution at the position corresponding to residue H554Q in SEQ ID NO: 1 can be combined with other substitutions, such as amino acid substitutions at one or more positions corresponding to F624L, F572L and / or G627D in SEQ ID NO: 1, more preferably a combination of substitutions corresponding to residues H554Q-F572L-F624L or H554Q-F572L-G627D in SEQ ID NO: 1. By using cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions, the enzyme specificity can be improved in the range of at least about 1.4-fold to more than 12-fold compared to the use of unmodified yeast as defined herein.

[0020] In another embodiment, a modified enzyme as defined herein, in particular a modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 572 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of leucine for phenylalanine (F572L). The amino acid substitution at the position corresponding to residue F572L in SEQ ID NO: 1 can be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 442, 551, 554, 624, 626, 627, and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). By using a cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions as defined above, enzyme specificity can be improved by at least about 1.7-fold compared to using an unmodified yeast as defined above.

[0021] In one embodiment, a modified enzyme as defined herein, in particular a modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 624 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of leucine for phenylalanine (F624L), which corresponds to the F592L substitution in the polypeptide according to SEQ ID NO: 3. The amino acid substitution at a position corresponding to residue F624L in SEQ ID NO: 1 can be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 442, 551, 554, 572, 626, 627, and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). By using cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions, enzyme specificity can be improved by at least about 3.1-fold compared to using unmodified yeast as defined herein.

[0022] In one embodiment, a modified enzyme as defined herein, in particular a modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 626 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of phenylalanine for leucine (L626F). The amino acid substitution at the position corresponding to residue L626F in SEQ ID NO: 1 can be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 442, 551, 554, 572, 624, 627, and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). By using a cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions as defined above, enzyme specificity can be improved by at least about 2.2-fold compared to using an unmodified yeast as defined herein.

[0023] In one embodiment, a modified enzyme as defined herein, in particular a modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 627 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of aspartic acid for glycine (G627D), which corresponds to the G595D substitution in the polypeptide according to SEQ ID NO: 3. The amino acid substitution at the position corresponding to residue G627D in SEQ ID NO: 1 can be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 442, 551, 554, 572, 624, 626, and / or 636 in the polypeptide according to SEQ ID NO: 1 as described herein). By using a cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions as defined above, enzyme specificity can be improved by at least about 2.2-fold compared to using an unmodified yeast as defined herein.

[0024] In one embodiment, a modified enzyme as defined herein, in particular a modified ARE2 and / or ARE1 activity, comprises an amino acid substitution at the position corresponding to residue 636 in the polypeptide according to SEQ ID NO: 1, preferably a substitution of cysteine ​​by serine (C636S). The amino acid substitution at the position corresponding to residue C636S in SEQ ID NO: 1 can be combined with other substitutions as defined herein (i.e., substitutions at one or more positions corresponding to amino acid residues 11, 281, 366, 442, 551, 554, 572, 624, 626, and / or 627 in the polypeptide according to SEQ ID NO: 1 as described herein). By using cholesterol-producing yeast carrying such a modified enzyme, in particular an enzyme according to SEQ ID NO: 1 comprising one of the one or more amino acid substitutions, enzyme specificity can be improved by at least about 3-fold compared to using unmodified yeast as defined herein.

[0025] The modified sterol acyltransferase according to the invention, such as ARE2 and / or ARE1, preferably comprises at least one amino acid substitution at a position corresponding to F624L in the polypeptide according to SEQ ID NO: 1, resulting in an enzyme specificity of about 4. This can be increased by introducing one or more further amino acid substitutions (e.g., amino acid substitutions at positions corresponding to F572L and / or H554Q), resulting in an increase in enzyme specificity in the range of more than 12-fold compared to the use of unmodified yeast as defined herein.

[0026] The modified sterol acyltransferase according to another embodiment of the present invention, such as ARE2 and / or ARE1, preferably comprises at least one amino acid substitution at a position corresponding to G627D in the polypeptide according to SEQ ID NO: 1, resulting in an enzyme specificity greater than 4. This can be increased by introducing one or more further amino acid substitutions (e.g., amino acid substitutions at positions corresponding to F572L and / or H554Q), resulting in an approximately 5-fold increase in enzyme specificity compared to using unmodified yeast as defined herein.

[0027] In this context, the activity of ARE2 and / or ARE1 is modified. This can be achieved, for example, by introducing one or more mutations into one or more endogenous genes encoding ARE2 and / or ARE1, i.e., by substitution of one or more amino acids at one or more positions as described herein. A skilled artisan is aware of how to genetically manipulate yeast cells to alter the activity of ARE2 and / or ARE1. These genetic manipulations include, but are not limited to, gene replacement, gene amplification, gene disruption, transfection, and transformation using plasmids, viruses, or other vectors.

[0028] The generation of mutations in nucleic acids or amino acids, i.e. mutagenesis, can be performed in different ways, such as by randomization or site-directed mutagenesis, physical damage by agents such as radiation, chemical treatment, or insertion of genetic elements. The skilled person knows how to introduce mutations.

[0029] The present invention particularly relates to the use of such modified ARE2 and / or ARE1 enzymes as defined herein in a method for producing 7-DHC, an intermediate of vitamin D3. Preferably, the modified enzyme of the present invention is introduced into and / or expressed in a suitable host cell, such as a yeast, preferably a sterol-producing yeast, in particular a cholesterol-producing yeast cell, for example, selected from Saccharomyces cerevisiae, Schizosaccharomyces spp., Pichia spp., Kluyveromyces spp., Hansenula spp. or Yarrowia lipolytica, preferably Saccharomyces cerevisiae. The modified host is used to produce 7-DHC, which can be further converted into vitamin D3 and / or 25-hydroxyvitamin D3.

[0030] Suitable host cells can be further modified to further increase the production of 7-DHC, an important intermediate towards vitamin D3 biosynthesis, and / or reduce the accumulation of by-products.

[0031] Thus, in one embodiment, the present invention relates to a yeast strain having altered ARE2 and / or ARE1 activity and in which ERG5 and ERG6 are inactivated. The yeast cell can be further modified by expressing a heterologous enzyme having C24 reductase activity, particularly selected from EC 1.3.1.72, such as a heterologous C24 reductase that acts on cholesta-7,24-dienol, yeast sterol, or trienol (e.g., cholesta-5,7,25-trienol), preferably a plant or vertebrate sterol Δ24 reductase, more preferably from a vertebrate source, even more preferably from human, pig, dog, mouse, rat, horse, zebrafish (Danio rerio), or any known source, as long as the enzyme can be expressed in the yeast cell. Most preferably, the sterol Δ24-reductase is selected from zebrafish, rat, or human. Sequences expressing such sterol Δ24 reductases are publicly available and include, but are not limited to, UniProtKB / Swiss-Prot references Q15392, Q60HC5, Q8VCH6, Q5BQE6, Q39085 or P93472 (see, for example, WO2003064650).

[0032] In another embodiment, the host cells according to the present invention can be further modified by introducing homologs of endogenous enzymes involved in the biosynthesis of 7-DHC, such as C5-sterol desaturase (ERG3) and / or C8-sterol isomerase (ERG2), resulting in increased specificity for 7-DHC and / or 7-DHC productivity and reduced accumulation of byproducts or vitamin D3 intermediates (including but not limited to zymosterol, lanosterol and / or cholestanol).

[0033] In a specific embodiment, the present invention relates to a method for improving a host cell towards 7-DHC production, wherein the modified host cell is as defined herein, i.e. modified by introducing one or more amino acid substitutions in the sterol acyltransferase ARE2 and / or ARE1 as defined herein, in particular a cholesterol-producing yeast cell, preferably a yeast cell in which ERG5 and ERG6 are inactivated and in which a heterologous enzyme having C-24 reductase activity as defined herein is optionally expressed, and / or a yeast cell in which a homolog of endogenous ERG2 and / or ERG3 is optionally expressed, wherein the host cell is improved such that the percentage of 7-DHC in the total amount of sterols produced by the host cell is increased and / or the activity of the host cell towards sterol production is increased compared to an unmodified host cell as defined herein.

[0034] In one embodiment, the present invention relates to a modified sterol acyltransferase, in particular a modified ARE2 and / or ARE1, comprising at least one or more amino acid substitutions as defined herein, wherein the specificity of the enzyme is increased compared to the specificity of the unmodified enzyme, thereby resulting in a higher ratio of 7-DHC to by-products including, for example, yeast sterols, in a sterol mixture produced by a suitable host cell carrying such modified sterol acyltransferase. This ratio may be increased by at least about 1.4-fold, for example, by introducing an amino acid substitution corresponding to H554Q in the corresponding ARE2 and / or ARE1 sequence; for example, by at least 3-fold, for example, by introducing an amino acid substitution corresponding to E11G-F624L, I442V-G627D or I442V-F624L-L626F in the corresponding ARE2 and / or ARE1 sequence; for example, by at least 4-fold, for example, by introducing an amino acid substitution corresponding to F624L or G627D in the corresponding ARE2 and / or ARE1 sequence; for example, by at least 5-fold, 10-fold or even 12-fold or more, for example, by introducing an amino acid substitution corresponding to H554Q-F572L-G627D or E11G-D366V-I442V-G627D-C636S in the corresponding ARE2 and / or ARE1 sequence.

[0035] In one embodiment, the present invention relates to a modified sterol acyltransferase, in particular a modified ARE2 and / or ARE1, comprising at least one or more amino acid substitutions as defined herein, wherein the activity of the enzyme is increased compared to the activity of the unmodified enzyme, thereby resulting in a higher total production of sterols and / or sterol esters, including a higher amount of 7-DHC produced by a suitable host cell carrying such a modified sterol acyltransferase. Thus, the relative ester formation, i.e., the ratio of all esters to free 7-DHC, can be increased by at least 1.2-fold, for example, by introducing amino acid substitutions corresponding to H55Q or H554Q-F572L into the corresponding ARE2 and / or ARE1 sequence; for example, by at least 2-fold or 3-fold, for example, by introducing amino acid substitutions corresponding to I442V-G627D or I442V-L626F-G627D into the corresponding ARE2 and / or ARE1 sequence. As described herein, total 7-DHC production can be increased by at least about 1.2-fold to 1.4-fold by introducing one or more amino acid substitutions.

[0036] By using a modified host cell, such as a yeast, such as a sterol-producing yeast, particularly a cholesterol-producing yeast, as described herein, the percentage of 7-DHC in the sterol mixture can be increased by at least 40%, such as 50%, 60%, 70%, 80%, 90% 7-DHC based on the total amount of sterols.

[0037] In one aspect of the present invention, host cells comprising modified ARE2 and / or ARE1 activity as defined herein are used in a method for producing the vitamin D3 precursor 7-DHC. The modified host cells can be cultured under aerobic or anaerobic conditions in an aqueous medium supplemented with appropriate nutrients, as known to those skilled in the art for corresponding cholesterol-producing host cells. Optionally, the culture is carried out in the presence of proteins and / or cofactors involved in electron transfer, as known in the art. The host cell culture / growth can be carried out in batch, fed-batch, semi-continuous, or continuous modes. As known to those skilled in the art, the production of vitamin D3 and its precursors (e.g., 7-DHC) can vary depending on the host cell. Cultivation and isolation of 7-DHC and other intermediates in vitamin D3 production are described, for example, in WO2011067144 or WO2017108799. 7-DHC can be isolated from the sterol mixture and / or optionally further purified, and can be further converted into vitamin D3 and / or 25-hydroxyvitamin D3 using methods known in the art.

[0038] The terms "ARE1" and "Are1p", "ARE2" and "Are2p", "ERG5" and "Erg5p", "ERG6" and "Erg6p" are used interchangeably herein and refer to the polypeptides encoded by the corresponding genes are1, are2, erg5 and erg6. For the purposes of the present invention, the cholesterol-producing yeast cell is modified such that it does exhibit modified activity of ARE2 and / or ARE1, for example, carries a modification in endogenous ARE2, ARE1 or both, resulting in a modified specificity of ARE2 and / or ARE1, wherein the modification comprises the introduction of one or more amino acid substitutions as defined herein.

[0039] Genes encoding ERG5, ERG6, ARE1, ARE2, ERG2, ERG3 or sterol Δ24 reductase (ERG4), as used herein. The culture and genetic engineering of yeast cells is known and described, for example, in US Pat. No. 7,608,421.

[0040] The terms "C-24-reductase" or "Δ24-reductase" are used interchangeably herein. In yeast, this enzyme is encoded by erg4 and is active towards a methyl group at the carbon atom at position 24. Therefore, trienols that do not exhibit such a methyl group at this position are not acceptable substrates for yeast ERG4.

[0041] The terms "C-8 sterol isomerase", "Δ8,7-isomerase" or "enzyme having C-8 sterol isomerase activity" are used interchangeably herein and refer to an enzyme capable of catalyzing the conversion of cholesta-8-enol to cholesta-7-enol and / or yeast sterol to cholesta-7,24-dienol. In yeast, the enzyme is encoded by erg2. In yeast, the enzyme is encoded by erg2. A preferred ERG2 homolog to be used in the modified host cell according to the present invention is a polypeptide having at least 41%, such as at least 44%, 45%, 48%, 49%, 53%, 56%, 60%, 70%, 80%, 90%, 92%, 95%, 98% or up to 100% identity to SEQ ID NO: 5, said polypeptide exhibiting C-8 sterol isomerase activity and comprising a polynucleotide encoding such a polypeptide, obtainable from Ustilago maydis. In particular, one or more copies, such as at least one, two, three, five copies, of said ERG2 homologue are expressed in the modified host cell as defined herein.

[0042] The terms "C-5 sterol desaturase," "enzyme having C-5 sterol desaturase activity," "desaturase," or "ERG3-homolog" are used interchangeably herein and refer to an enzyme that can catalyze the conversion of cholesta-8-enol to cholesta-7,24-dienol and / or cholesta-7-enol to cholesta-5,7,24-trienol and / or 7-DHC. In yeast, this enzyme is encoded by erg3. Preferred ERG3 homologues to be used in the modified host cells according to the present invention are polypeptides having at least 45%, such as at least 50%, 52%, 60%, 70%, 80%, 90%, 92%, 95%, 98% or up to 100% identity to SEQ ID NO: 7, said polypeptides exhibiting C-5 sterol desaturase activity and comprising a polynucleotide encoding such a polypeptide, obtainable from Pichia pastoris or Schizosaccharomyces pombe. In particular, one or more copies, such as at least one, two, three, five copies, of said ERG3 homologue are expressed in the modified host cells as defined herein.

[0043] "Relative ester formation" as defined herein is the ratio (total esters / free 7-DHC) 突变体 / (Total esters / free 7-DHC) 野生型 The "specificity" of an enzyme as defined herein is the ratio (7-DHC ester / zymosterol ester) 突变体 / (7-DHC Ester / Zymosterol Ester) 野生型 As defined herein, "total 7-DHC yield" is the ratio (total 7-DHC) 突变体 / (7-DHC)野生型 .

[0044] In this article, the term "specific activity" or "activity" about an enzyme refers to its catalytic activity, i.e., the ability of its catalysis to form a product from a given substrate. Specific activity defines the amount of substrate consumed by a protein of a given amount and / or the product produced within a given time period and at a defined temperature. Typically, specific activity is expressed as the μmol number of substrate consumed per minute per mg protein or the product formed. Typically, μmol / min is abbreviated as U (=Unit). Therefore, the specific activity unit definition of μmol / min / (mg protein) or U / (mg protein) is used interchangeably throughout this document. If an enzyme 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, the enzyme is active. Technicians know how to measure enzyme activity, for example, by HPLC.

[0045] In connection with the present invention, it is to be understood that organisms such as microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi and plants (Melbourne method).

[0046] In particular, the present invention is characterized by the following embodiments:

[0047] (1) A modified enzyme having sterol acyltransferase activity as defined herein, comprising one or more amino acid substitutions at one or more positions corresponding to residues selected from the group consisting of positions 11, 281, 366, 442, 551, 554, 572, 624, 626, 627, 636 and combinations thereof in the polypeptide according to SEQ ID NO: 1, preferably one or more amino acid substitutions corresponding to E11G and / or L281I and / or D366V and / or I442V and / or H551Y and / or H554Q and / or F572L and / or F624L and / or L626F and / or G627D and / or C636S and / or combinations thereof.

[0048] (2) A modified enzyme as defined herein and according to embodiment (1), which catalyzes the esterification of sterols including 7-dehydrocholesterol and zymosterol, wherein the ratio of 7-DHC to zymosterol in the sterol esters is increased by at least about 1.4 times compared to the ratio of 7-DHC to zymosterol in catalysis using the corresponding unmodified enzyme.

[0049] (3) The modified enzyme as defined herein and according to embodiment (1) or (2), wherein the one or more amino acid substitutions are selected from F624L, G627D, E11G, H554Q, I442V, and combinations thereof.

[0050] (4) A host cell, preferably a yeast, more preferably a sterol-producing yeast, even more preferably a cholesterol-producing yeast, comprising a modified enzyme as defined herein and according to any one of embodiments (1), (2) and (3), optionally further comprising one or more amino acid substitutions at one or more positions corresponding to residues selected from positions 592 and / or 595 in the polypeptide according to SEQ ID NO: 3, preferably substitutions corresponding to F592L and / or G595D.

[0051] (5) A host cell as defined herein and according to embodiment (4), for producing a sterol mixture comprising 7-DHC and zymosterol, wherein the ratio of 7-DHC to zymosterol is increased by at least about 1.4-fold compared to a host cell expressing an unmodified enzyme.

[0052] (6) A host cell as defined herein and according to embodiment (4) or (5), optionally further comprising:

[0053] - Inactivation of ERG5 and ERG6, and / or

[0054] - Expression of a heterologous enzyme selected from EC 1.3.1.72 having sterol Δ24-reductase activity, preferably wherein the heterologous enzyme is derived from a plant or a vertebrate, more preferably from a human, pig, dog, mouse, rat, horse or zebrafish.

[0055] (7) A method for reducing the percentage of zymosterol in a sterol mixture comprising zymosterol and 7-DHC, the method comprising culturing a host cell as defined herein and according to one of embodiments (4), (5) or (6) under suitable conditions, and optionally isolating and / or purifying 7-DHC from the sterol mixture, or a method for increasing the percentage of 7-DHC in a sterol mixture comprising 7-DHC and zymosterol, the method comprising culturing a host cell as defined herein and according to one of embodiments (4), (5) or (6) under suitable conditions, and optionally isolating and / or purifying 7-DHC from the sterol mixture.

[0056] (8) A method for producing 7-DHC, the method comprising enzymatically converting acetyl-CoA into a sterol mixture comprising yeast sterol and 7-DHC using a host cell as defined herein and according to one of embodiments (4), (5) or (6), wherein the percentage of 7-DHC in the sterol mixture is at least 40%, and optionally further converting 7-DHC into vitamin D3 and / or 25-hydroxyvitamin D3.

[0057] (9) Use of a modified enzyme as defined herein and according to any one of embodiments (1), (2), (3) or a host cell as defined herein and according to one of embodiments (4), (5) or (6) in a method for producing 7-DHC, wherein the 7-DHC is isolated from a sterol mixture comprising yeast sterol and 7-DHC, and wherein the ratio of 7-DHC to yeast sterol is increased by at least about 1.4 times compared to a method using the corresponding unmodified enzyme and host cell, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #2 and #1 (see Table 1). For esters of 7-DHC ("7-DHC Esters") and esters of zymosterol ("Zym Esters"), two ester forms were detected, as indicated by the black and light gray colors in the corresponding columns; free 7-DHC is represented in black. (A) Ratio of 7-DHC to ester formation, (B) Ratio of total 7-DHC (including free and ester forms) to total zymosterol esters, (C) Formation of free 7-DHC, 7-DHC esters, and zymosterol esters is indicated by separate columns. Strains were cultured for two days in unbaffled flasks with two glucose feeds. Data are the average of three independent transformants, each cultured once.

[0059] Figure 2HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #2 and #1 (see Table 1). For further details, see Figure 1 Legend for Figure 1. Strains were grown in unbaffled flasks with a single glucose feed for four days. Data are the average of two independent transformants, each grown once.

[0060] Figure 3 HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #9 and #1 (see Table 1). For further details, see Figure 1 HPLC analysis was performed according to standard procedures. See text for further details.

[0061] Figure 4 HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #24 and #20 (see Table 1). For further details, see Figure 1 HPLC analysis was performed according to standard procedures. See text for further details.

[0062] Figure 5 HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #22 and #27 (see Table 1). For further details, see Figure 1 HPLC analysis was performed according to standard procedures. See text for further details.

[0063] Figure 6 HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #20, #22, #27, and #22 (see Table 1). For further details, see Figure 1 HPLC analysis was performed according to standard procedures. See text for further details.

[0064] Figure 7 HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #34, #32, #43, #24, #20, #22, and #27 (see Table 1). For further details, see Figure 1 HPLC analysis was performed according to standard procedures. See text for further details.

[0065] Figure 8HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #9, #35, #36, #39, and #40 (see Table 1). For further details, see Figure 1 HPLC analysis was performed according to standard procedures. See text for further details.

[0066] Figure 9 HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #27, #33, #34, #37, and #38 (see Table 1). For further details, see Figure 1 HPLC analysis was performed according to standard procedures. See text for further details.

[0067] Figure 10 HPLC analysis of lipid extracts of ARE2 wild-type strain ("WT") and Are2 variants #41, #42, #43, #24, and #20 (see Table 1). For further details, see Figure 1 HPLC analysis was performed according to standard procedures. See text for further details.

[0068] The following examples are illustrative only and are not intended to limit the scope of the present invention in any way. Example

[0069] Example 1: Generation and screening of ARE2 mutants

[0070] An error-prone library of 10,000 yeast clones expressing a variant of the Saccharomyces cerevisiae acyltransferase 2 (ScAre2) enzyme was screened by thin-layer chromatography (TLC) for increased 7-DHC content in the sterol ester fraction relative to the wild-type sequences of ARE1 and ARE2 (see Sequence Listing). The screening method involves simultaneous extraction and isolation of sterols from cells with minimal digested cell walls. The treated biomass is applied directly to a TLC plate and immersed in a solvent, which extracts and isolates the sterol-containing fraction in a single step. Within the sterol ester fraction, the ratio of sterols with conjugated double bonds (e.g., 7-DHC) relative to sterols without conjugated double bonds is determined by exploiting the different spectrophotometric properties of compounds with conjugated double bonds (e.g., ability to quench fluorescence, UV detection).

[0071] The best variants were rescreened in quintuplicate, sequenced, grown in shake flasks, and analyzed in biological triplicate by HPLC-UV to determine the sterol and sterol ester composition.

[0072] Plasmids containing the best variants were isolated and re-transformed into the cholesta-5,7,24-trienol-producing Saccharomyces cerevisiae strain 10A (are1 are2 erg5 erg6::24R; for construction, see Example 1 in WO2017108799). Mutant variants with multiple amino acid exchanges were isolated by introducing the corresponding mutations into ARE2 via site-directed mutagenesis (silent mutations were not considered) to identify mutations that caused the desired effect. The strains were cultured and analyzed by HPLC.

[0073] Example 2: HPLC-UV analysis standard procedure

[0074] Preculture - Inoculate 10 ml of YPD containing geneticin (100 μg / mL) with the strain of interest (3 transformants per Are2 variant) and grow at 30°C to the appropriate density (24 to 48 hours). For better comparison, also inoculate three different transformants with wild-type ARE2 plasmids that were transformed at the same time as the variants. Inoculate 50 mL of the main culture of YPD containing geneticin in a 250 mL unbaffled shake flask to an OD of 0. 600 The culture medium was stirred at 200 rpm and 80% humidity at 30°C for 3 days with 3 glucose feeds (glucose was added to a final concentration of 2% after approximately 30, 45, and 60 hours). 200 OD units of biomass were harvested (centrifuged at 1600 x g for 5 minutes and the supernatant removed) in 15 mL Greiner tubes and stored at -20°C until analysis.

[0075] In order to extract, 200OD cell pellets are thawed, resuspended in 1mL lytic enzyme solution (5mg / mL lytic enzyme 20T solution in 50mM KPi, pH 7, containing 1M D-sorbitol), and hatched 15 minutes (hatched with 750rpm on thermomixer) at 37 ℃. After centrifugation (2500xg, 5 minutes), remove lytic enzyme solution, and add the anhydrous EtOH of 3.73mL (by carefully pipetting up and down and resuspending with 1mL, then adding extra 2.73mL) in precipitate. Add 267 μ L internal standards (cholesterol acetate, a solution of 1mg / mL in EtOH), the cell suspension is vortexed, and heated to 70 ℃ and continued 1 hour under mixing (on thermomixer with 750rpm). After removing the test tube for a few minutes to cool to room temperature, the cell debris was pelleted (2500 x g for 10 minutes at room temperature) and 3 mL of the supernatant was transferred to a Pyrex test tube, which was dried under N2. The lipids were absorbed in 200 μL of ethyl acetate (vortexed and mixed at 750 rpm for 15 minutes on a 40°C thermomixer). The solution was centrifuged again (2500 x g for 5 minutes) and then transferred to a glass vial with an inlay for subsequent HPLC-UV analysis.

[0076] The lipid extracts were analyzed by HPLC at two wavelengths (210 nm and 280 nm) with UV detection. Zymosterol compounds were detected at 210 nm and 7-DHC compounds were quantified at 280 nm.

[0077] solvent : 80% EtOH, 20% MeOH, 0.1% TFA

[0078] column :YMC-Pack Pro C18 RS

[0079] method :Injection volume: 10μL

[0080] Syringe thermostat: 40°C

[0081] Flow rate: 0.6mL / min

[0082] Column oven: 20°C

[0083] UV detection: 210nm, 280nm (sterols with conjugated double bonds)

[0084] A standard mixture of 7-DHC, zymosterol, cholesterol acetate, and squalene at three different concentrations (0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL of each substance) was also analyzed, and a standard curve was generated for each substance to calculate the concentration in μg / μL or μg / OD.600 The concentration of sterols in the extract is expressed as 1,000 Å.

[0085] Example 3: Evaluation of activity and / or specificity of ARE2 variants

[0086] For direct comparison, the wild-type ARE2 plasmid was re-transformed into strain 10A along with the plasmid expressing the Are2 variant (see Example 1), and the resulting strains were analyzed in the same round (see Example 2). The results of the HPLC analysis are summarized in Tables 1 and Figures 1 to 10 The values ​​in the table give the fold change between strains expressing the mutants / variants compared to the wild type. The first value indicates the improvement in the ratio between the ester fraction and the free 7-DHC fraction, while the second value shows the improvement in the ratio of 7-DHC to zymosterol in the ester fraction. The third value is a comparison of the total 7-DHC content in the biomass of the mutants and the wild type. Some of the listed variants showed particularly improved ester levels, while others showed an improvement in the 7-DHC / zymosterol ratio in the ester fraction.

[0087] Table 1A: Summary of relative ester formation of Are2 variants based on several independent experiments. "Relative ester formation" refers to the x-fold increase in the percentage of 7-DHC based on the total amount of sterols produced using the indicated amino acid exchange instead of wild-type ARE2; "7-DHC esters / zym esters" refers to the ratio of esters from 7-DHC to esters from zymosterol ("zym"); "total 7-DHC / total zym" is the ratio of total 7-DHC (free and esters) to total zymosterol (free and esters). See text for further explanation.

[0088]

[0089] Table 1B: Summary of the specificity of ARE2 variants based on several independent experiments. Values ​​represent the x-fold increase in the percentage of 7-DHC compared to the percentage of yeast sterol in sterol mx produced using the indicated amino acid exchange instead of wild-type ARE2. See text for further explanation.

[0090] serial number# One or more amino acid exchanges Specificity 1 H554Q 1.4 2 V286V-H551Y-F572L-S633S 1.7 9 H554Q-F572L 1.8 20 F624L 3.9 22 G627D 4.4 32 E11G-D366V-F624L-C636S 3.1 33 E11G-D366V-F624L-C636S 3.8 34 E11G-D366V-G627D-C636S 4.4 35 E11G-F624L 3.2 36 E11G-G627D 4.2 37 E11G-D366V-I442V-F624L-C636S 3.4 38 E11G-D366V-I442V-G627D-C636S 4.5 39 H554Q-F572L-F624L 12.2 40 H554Q-F572L-G627D 5.0 41 I442V-F624L-L626F 3.1 42 I442V-L626F-G627D 2.2 43 I442V-G627D 3.5

[0091] Table 1C: Summary of total 7-DHC production by ARE2 variants based on several independent experiments. Values ​​represent the x-fold increase in the total amount of 7-DHC produced using the indicated amino acid exchange compared to wild-type ARE2. See text for further explanation.

[0092] serial number# One or more amino acid exchanges Total 7-DHC production 1 H554Q 1.0 20 F624L 1.0 22 G627D 1.0 24 I442V-L626F 1.3 27 E11G-D366V-C636S 1.2 32 E11G-D366V-F624L-C636S 1.4 33 E11G-D366V-F624L-C636S 1.4 34 E11G-D366V-G627D-C636S 1.3 35 E11G-F624L 1.1 36 E11G-G627D 1.1 37 E11G-D366V-I442V-F624L-C636S 1.4 38 E11G-D366V-1442V-G627D-C636S 1.1 39 H554Q-F572L-F624L 1.0 40 H554Q-F572L-G627D 1.3 41 I442V-F624L-L626F 1.4 42 I442V-L626F-G627D 1.4 43 I442V-G627D 1.4 Sequence Listing <110> DSM Intellectual Property Asset Management Co., Ltd. <120> Modified sterol acyltransferase <130> 32958-WO-PCT <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 642 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Met Asp Lys Lys Lys Asp Leu Leu Glu Asn Glu Gln Phe Leu Arg Ile 1 5 10 15 Gln Lys Leu Asn Ala Ala Asp Ala Gly Lys Arg Gln Ser Ile Thr Val 20 25 30 Asp Asp Glu Gly Glu Leu Tyr Gly Leu Asp Thr Ser Gly Asn Ser Pro 35 40 45 Ala Asn Glu His Thr Ala Thr Thr Ile Thr Gln Asn His Ser Val Val 50 55 60 Ala Ser Asn Gly Asp Val Ala Phe Ile Pro Gly Thr Ala Thr Glu Gly 65 70 75 80 Asn Thr Glu Ile Val Thr Glu Glu Val Ile Glu Thr Asp Asp Asn Met 85 90 95 Phe Lys Thr His Val Lys Thr Leu Ser Ser Lys Glu Lys Ala Arg Tyr 100 105 110 Arg Gln Gly Ser Ser Asn Phe Ile Ser Tyr Phe Asp Asp Met Ser Phe 115 120 125 Glu His Arg Pro Ser Ile Leu Asp Gly Ser Val Asn Glu Pro Phe Lys 130 135 140 Thr Lys Phe Val Gly Pro Thr Leu Glu Lys Glu Ile Arg Arg Arg Glu 145 150 155 160 Lys Glu Leu Met Ala Met Arg Lys Asn Leu His His Arg Lys Ser Ser 165 170 175 Pro Asp Ala Val Asp Ser Val Gly Lys Asn Asp Gly Ala Ala Pro Thr 180 185 190 Thr Val Pro Thr Ala Ala Thr Ser Glu Thr Val Val Thr Val Glu Thr 195 200 205 Thr Ile Ile Ser Ser Asn Phe Ser Gly Leu Tyr Val Ala Phe Trp Met 210 215 220 Ala Ile Ala Phe Gly Ala Val Lys Ala Leu Ile Asp Tyr Tyr Tyr Gln 225 230 235 240 His Asn Gly Ser Phe Lys Asp Ser Glu Ile Leu Lys Phe Met Thr Thr 245 250 255 Asn Leu Phe Thr Val Ala Ser Val Asp Leu Leu Met Tyr Leu Ser Thr 260 265 270 Tyr Phe Val Val Gly Ile Gln Tyr Leu Cys Lys Trp Gly Val Leu Lys 275 280 285 Trp Gly Thr Thr Gly Trp Ile Phe Thr Ser Ile Tyr Glu Phe Leu Phe 290 295 300 Val Ile Phe Tyr Met Tyr Leu Thr Glu Asn Ile Leu Lys Leu His Trp 305 310 315 320 Leu Ser Lys Ile Phe Leu Phe Leu His Ser Leu Val Leu Leu Met Lys 325 330 335 Met His Ser Phe Ala Phe Tyr Asn Gly Tyr Leu Trp Gly Ile Lys Glu 340 345 350 Glu Leu Gln Phe Ser Lys Ser Ala Leu Ala Lys Tyr Lys Asp Ser Ile 355 360 365 Asn Asp Pro Lys Val Ile Gly Ala Leu Glu Lys Ser Cys Glu Phe Cys 370 375 380 Ser Phe Glu Leu Ser Ser Gln Ser Leu Ser Asp Gln Thr Gln Lys Phe 385 390 395 400 Pro Asn Asn Ile Ser Ala Lys Ser Phe Phe Trp Phe Thr Met Phe Pro 405 410 415 Thr Leu Ile Tyr Gln Ile Glu Tyr Pro Arg Thr Lys Glu Ile Arg Trp 420 425 430 Ser Tyr Val Leu Glu Lys Ile Cys Ala Ile Phe Gly Thr Ile Phe Leu 435 440 445 Met Met Ile Asp Ala Gln Ile Leu Met Tyr Pro Val Ala Met Arg Ala 450 455 460 Leu Ala Val Arg Asn Ser Glu Trp Thr Gly Ile Leu Asp Arg Leu Leu 465 470 475 480 Lys Trp Val Gly Leu Leu Val Asp Ile Val Pro Gly Phe Ile Val Met 485 490 495 Tyr Ile Leu Asp Phe Tyr Leu Ile Trp Asp Ala Ile Leu Asn Cys Val 500 505 510 Ala Glu Leu Thr Arg Phe Gly Asp Arg Tyr Phe Tyr Gly Asp Trp Trp 515 520 525 Asn Cys Val Ser Trp Ala Asp Phe Ser Arg Ile Trp Asn Ile Pro Val 530 535 540 His Lys Phe Leu Leu Arg His Val Tyr His Ser Ser Met Ser Ser Phe 545 550 555 560 Lys Leu Asn Lys Ser Gln Ala Thr Leu Met Thr Phe Phe Leu Ser Ser 565 570 575 Val Val His Glu Leu Ala Met Tyr Val Ile Phe Lys Lys Leu Arg Phe 580,585,590 Tyr Leu Phe Phe Phe Gln Met Leu Gln Met Pro Leu Val Ala Leu Thr 595,600,605 Asn Thr Lys Phe Met Arg Asn Arg Thr Ile Ile Gly Asn Val Ile Phe 610 615 620 Trp Leu Gly Ile Met Gly Pro Ser Val Met Cys Thr Leu Tyr Leu 625 630 635 640 Thr Phe <210> 2 <211> 1929 <212> DNA <213> Saccharomyces cerevisiae <400> 2 atggacaaga agaaggatct actggagaac gaacaatttc tccgcatcca aaagctcaac 60 gctgccgatg cgggcaaag acaatctata acagtggacg acgaggggcga actatatggg 120 ttagacacct ccggcaactc accagccaat gaacacacag ctaccacaat tacacagaat 180 cacagcgtgg tggcctcaa cggagacgtc gcattcatcc caggaacgc taccgaaggc 240 atacagaga ttgtactga agagtgatt gagaccgatg atacatgtt caacaccat 300 gtgaagactt taagctccaa agagaaggca cggtataggc aagggtccctc taactttata 360 tcgtatttcg atgatatgtc atttgaacac aggcccagta tattagatgg gtcagttaac 420 gagcccttca agaccaaatt cgtgggacct actttagaaa aggagatcag aagaagggag 480 aaagagctaa tggccatgcg caaaaattta caccaccgca agtcctcccc agatgctgtc 540 gactcagtag ggaaaaatga tggcgccgcc ccaactactg ttccaactgc cgccacctca 600 gaaacggtgg tcaccgttga aaccaccata atttcatcca atttctccgg gttgtacgtg 660 gcgttttgga tggctattgc atttggtgct gtcaaggctt tatagacta ttattaccag 720 cataatggta gcttcaagga ttcggagatc ttgaaattta tgactacgaa tttgttcact 780 gtggcatccg tagatctttt gatgtatttg agcacttatt ttgtcgttgg aatacaatac 840 tttgcaagt ggggggtctt gaaatggggc actaccggct ggatcttcac ctcaatttac 900 gagtttttgt ttgttatctt ctacatgtat ttaacagaaa acatcctaaa actacactgg 960 ctgtccaaga tcttcctttt tttgcattct ttagttttat tgatgaaaat gcattctttc 1020 gccttctaca atggctatct atggggtata aaggaagaac tacaattttc caaaagcgct 1080 cttgccaaat acaaggattc tataaatgat ccaaaagtta ttggtgctct tgagaaaagc 1140 tgtgagtttt gtagttttga attgagctct cagtctttaa gcgaccaaac tcaaaaattc 1200 ccaacaata tcagtgcaaa aagctttttt tggttcacca tgtttccaac cctaatttac 1260 caaattgaat atccaagaac taaggaaatc agatggagct acgtattaga aaagatctgc 1320 gccatcttcg gtaccatttt cttaatgatg atagatgctc aaatcttgat gtatcctgta 1380 gcaatgagag cattggctgt gcgcaattct gaatggactg gtatattgga tagattattg 1440 aaatgggttg gattgctcgt tgatatcgtc ccagggttta tcgtgatgta catcttggac 1500 ttctatttga tttgggatgc cattttgaac tgtgtggctg aattgacaag atttggcgac 1560 agatatttct acggtgactg gtggaattgt gttagttggg cagacttcag tagaatttgg 1620 aacatcccag tgcataagtt tttgttaaga catgtttacc atagttcaat gagttcattc 1680 aaattgaaca agagtcaagc aactttgatg acctttttct taagttccgt cgttcatgaa 1740 ttagcaatgt acgttatctt caagaaattg aggttttact tgttcttctt ccaaatgctg 1800 caaatgccat tagtagcttt aaaatact aaattcatga ggaacagaac cataatcgga 1860 aatgtattt tctggctcgg tatctgcatg ggaccaagtg tcatgtgtac gttgtacttg 1920 acattctaa 1929 <210> 3 <211> 610 <212> PRT <213> Saccharomyces cerevisiae <400> 3 Met Thr Glu Thr Lys Asp Leu Leu Gln Asp Glu Glu Phe Leu Lys Ile 1 5 10 15 Arg Arg Leu Asn Ser Ala Glu Ala Asn Lys Arg His Ser Val Thr Tyr 20 25 30 Asp Asn Val Ile Leu Pro Gln Glu Ser Met Glu Val Ser Pro Arg Ser 35 40 45 Ser Thr Thr Ser Leu Val Glu Pro Val Glu Ser Thr Glu Gly Val Glu 50 55 60 Ser Thr Glu Ala Glu Arg Val Ala Gly Lys Gln Glu Gln Glu Glu Glu 65 70 75 80 Tyr Pro Val Asp Ala His Met Gln Lys Tyr Leu Ser His Leu Lys Ser 85 90 95 Lys Ser Arg Ser Arg Phe His Arg Lys Asp Ala Ser Lys Tyr Val Ser 100 105 110 Phe Phe Gly Asp Val Ser Phe Asp Pro Arg Pro Thr Leu Leu Asp Ser 115 120 125 Ala Ile Asn Val Pro Phe Gln Thr Thr Phe Lys Gly Pro Val Leu Glu 130 135 140 Lys Gln Leu Lys Asn Leu Gln Leu Thr Lys Thr Lys Thr Lys Ala Thr 145 150 155 160 Val Lys Thr Thr Val Lys Thr Thr Glu Lys Thr Asp Lys Ala Asp Ala 165 170 175 Pro Pro Gly Glu Lys Leu Glu Ser Asn Phe Ser Gly Ile Tyr Val Phe 180 185 190 Ala Trp Met Phe Leu Gly Trp Ile Ala Ile Arg Cys Cys Thr Asp Tyr 195 200 205 Tyr Ala Ser Tyr Gly Ser Ala Trp Asn Lys Leu Glu Ile Val Gln Tyr 210 215 220 Met Thr Thr Asp Leu Phe Thr Ile Ala Met Leu Asp Leu Ala Met Phe 225 230 235 240 Leu Cys Thr Phe Phe Val Val Phe Val His Trp Leu Val Lys Lys Arg 245 250 255 Ile Ile Asn Trp Lys Trp Thr Gly Phe Val Ala Val Ser Ile Phe Glu 260 265 270 Leu Ala Phe Ile Pro Val Thr Phe Pro Ile Tyr Val Tyr Tyr Phe Asp 275 280 285 Phe Asn Trp Val Thr Arg Ile Phe Leu Phe Leu His Ser Val Val Phe 290,295,300 Val Met Lys Ser His Ser Phe Ala Phe Tyr Asn Gly Tyr Leu Trp Asp 305 310 315 320 Lys Gln Glu Leu Glu Tyr Ser Ser Lys Gln Leu Gln Lys Tyr Lys 325 330 335 Glu Ser Leu Ser Pro Glu Thr Arg Glu Ile Leu Gln Lys Ser Cys Asp 340 345 350 Phe Cys Leu Phe Glu Leu Asn Tyr Gln Thr Lys Asp Asp Phe Pro 355 360 365 Asn Asn Ile Ser Cys Ser Asn Phe Phe Met Phe Cys Leu Phe Pro Val 370 375 380 Leu Val Tyre Gln With Asn Tyre Pro Arg Thr Ser Arg With Arg Trp Arg 385 390 395 400 Tyr Val Leu Glu Lys Val Cys Ala Ile Ile Gly Thr Ile Phe Leu Met 405 410 415 Met Val Thr Ala Gln Phe Phe Met His Pro Val Ala Met Arg Cys Ile 420 425 430 Gln Phe His Asn Thr Pro Thr Phe Gly Gly Trp Ile Pro Ala Thr Gln 435 440 445 Glu Trp Phe His Leu Leu Phe Asp Met Ile Pro Gly Phe Thr Val Leu 450 455 460 Tyr Met Leu Thr Phe Tyr Met Ile Trp Asp Ala Leu Leu Asn Cys Val 465 470 475 480 Ala Glu Leu Thr Arg Phe Ala Asp Arg Tyr Phe Tyr Gly Asp Trp Trp 485 490 495 Asn Cys Val Ser Phe Glu Glu Phe Ser Arg Ile Trp Asn Val Pro Val 500 505 510 His Lys Phe Leu Leu Arg His Val Tyr His Ser Met Gly Ala Leu 515 520 525 His Leu Ser Lys Ser Gln Ala Thr Leu Phe Thr Phe Phe Leu Ser Ala 530 535 540 Val Phe His Glu Met Ala Met Phe Ala Ile Phe Arg Arg Val Arg Gly 545 550 555 560 Tyr Leu Phe Met Phe Gln Leu Ser Gln Phe Val Trp Thr Ala Leu Ser 565 570 575 Asn Thr Lys Phe Leu Arg Ala Arg Pro Gln Leu Ser Asn Val Val Phe 580 585 590 Ser Phe Gly Val Cys Ser Gly Pro Ser Ile Ile Met Thr Leu Tyr Leu 595 600 605 Thr Leu 610 <210> 4 <211> 1833 <212> DNA <213> Saccharomyces cerevisiae <400> 4 atgacggaga ctaaggattt gttgcaagac gaagagtttc ttaagatccg cagactcaat 60 tccgcagaag ccaacaaacg gcattcggtc acgtacgata acgtgatcct gccacaggag 120 tccatggagg tttcgccacg gtcgtctacc acgtcgctgg tggagccagt ggagtcgact 180 gaaggagtgg agtcgactga ggcggaacgt gtggcaggga agcaggagca ggaggaggag 240 taccctgtgg acgcccacat gcaaaagtac ctttcacacc tgaagagcaa gtctcggtcg 300 aggttccacc gaaaggatgc tagcaagtat gtgtcgtttt ttggggacgt gagttttgat 360 cctcgcccca cgctcctgga cagcgccatc aacgtgccct tccagacgac tttcaaaggt 420 ccggtgctgg agaaacagct caaaaattta cagttgacaa agaccaagac caaggccacg 480 gtgaagacta cggtgaagac tacggagaaa acggacaagg cagatgcccc cccaggagaa 540 aaactggagt cgaacttttc agggatctac gtgttcgcat ggatgttctt gggctggata 600 gccatcaggt gctgcacaga ttactatgcg tcgtacggca gtgcatggaa taagctggaa 660 atcgtgcagt acatgacaac ggacttgttc acgatcgcaa tgttggactt ggcaatgttc 720 ctgtgcactt tcttcgtggt tttcgtgcac tggctggtga aaaagcggat catcaactgg 780 aagtggactg ggttcgttgc agtgagcatc ttcgagttgg ctttcatccc cgtgacgttc 840 cccatttacg tctactactt tgatttcaac tgggtcacga gaatcttcct gttcctgcac 900 tccgtggtgt ttgttatgaa gagccactcg tttgcctttt acaacgggta tctttgggac 960 ataaagcagg aactcgagta ctcttccaaa cagttgcaaa aatacaagga atctttgtcc 1020 ccagagaccc gcgagattct gcaaaaaagt tgcgactttt gccttttcga attgaactac 1080 cagaccaagg ataacgactt ccccaacaac atcagttgca gcaatttctt catgttctgt 1140 ttgttccccg tcctcgtgta ccagatcaac tacccaagaa cgtcgcgcat cagatggagg 1200 tatgtgttgg agaaggtgtg cgccatcatt ggcaccatct tcctcatgat ggtcacggca 1260 cagttcttca tgcacccggt ggccatgcgc tgtatccagt tccacaacac gcccaccttc 1320 ggcggctgga tccccgccac gcaagagtgg ttccacctgc tcttcgacat gattccgggc 1380 ttcactgttc tgtacatgct cacgttttac atgatatggg acgctttatt gaattgcgtg 1440 gcggagttga ccaggtttgc ggacagatat ttctacggcg actggtggaa ttgcgtttcg 1500 tttgaagagt ttagcagaat ctggaacgtc cccgttcaca aatttttact aagacacgtg 1560 taccacagct ccatgggcgc attgcatttg agcaagagcc aagctacatt atttactttt 1620 ttcttgagtg ccgtgttcca cgaaatggcc atgttcgcca ttttcagaag ggttagagga 1680 tatctgttca tgttccaact gtcgcagttt gtgtggactg ctttgagcaa caccaagttt 1740 ctacgggcaa gaccgcagtt gtccaacgtt gtcttttcgt ttggtgtctg ttcagggccc 1800 agtatcatta tgacgttgta cctgacctta tga 1833 <210> 5 <211> 241 <212> PRT <213> Ustilago maydis <400> 5 Met Ala Ser His Arg Pro Arg Ser Asn Lys Ala Ala Asn Gly Ala Ser 1 5 10 15 Thr Ser Pro Lys Arg Ser Trp Ile Ile Val Ser Ala Ala Leu Val Gly 20 25 30 Phe Cys Ala Leu Ile Ala Ala Leu Asp Ser Ile Arg Ser Ser Phe Tyr 35 40 45 Ile Phe Asp His Lys Ala Ile Tyr Lys Ile Ala Ser Thr Ala Val Ala 50 55 60 Asn His Pro Gly Asn Ala Thr Ala Ile Phe Asp Asp Val Leu Asp Asn 65 70 75 80 Leu Arg Ala Asp Pro Lys Leu Ala Pro Tyr Ile Asn Lys Asn His Phe 85 90 95 Ser Asp Glu Ser Glu Trp Met Phe Asn Asn Ala Gly Gly Ala Met Gly 100 105 110 Ser Met Phe Ile Ile His Ala Ser Val Thr Glu Tyr Leu Ile Phe Phe 115 120 125 Gly Thr Pro Val Gly Thr Glu Gly His Thr Gly Arg His Thr Ala Asp 130 135 140 Asp Tyr Phe Asn With Thr Gly Asn Gln Tyr Ala Phe Pro Ala Gly 145 150 155 160 Ala Leu Lys Ala Glu His Tyr Pro Ala Gly Ser Val His His Leu Arg 165 170 175 Arg Gly Thr Val Lys Gln Tyr Met Met Pro Glu Asp Gly Cys Trp Ala 180 185 190 Leu Glu Leu Ala Gln Gly Trp Ile Pro Pro Met Leu Pro Phe Gly Leu 195 200 205 Ala Asp Val Leu Ser Thr Leu Asp Leu Pro Thr Phe Gly Ile Thr 210 215 220 Val Trp Ile Thr Ala Arg Glu Met Val Gly Asn Leu Leu Ile Gly Lys 225 230 235 240 Where <210> 6 <211> 726 <212> DNA <213> Ustilago maydis <400> 6 atggcatcgc atagaccacg cagcaacaag gctgccaatg gtgcttcgac ttcacccaaa 60 cgcagctgga taattgtctc agctgcgctc gttggctct gcgctctc cgccgctc 120 gattcgatcc gatccagctt ctacatcttt gaccacagg caatctaca gatcgcatcg 180 actgcggtg ccaaccatcc aggcaatgcg acggccatct ttgatgatgt cctcgacaac 240 cttcgtgccg accccaagct cgcgccttac atcaacaaga atcatttcag cgacgagtca 300 gaatggatgt tcaacaatgc cggtggtgct atgggtagca tgttcatcat tcatgcttcc 360 gtcaccgagt acctgatctt ctttggcact cccgtcggaa ccgagggtca cactggtcgt 420 cacacagccg atgactactt caacatcctt accggtaacc atacgcttt cccagctggt 480 gcgctcaagg cggagcacta ccctgccgga tcagtgcacc atcttcgccg cggaacggtc 540 aagcagtaca tgatgcctga agacggctgc tgggcgctcg agcttgctca gggctggatc 600 ccacccatgc ttccctttg tctcgccgat gtgctcagct cgacgctcga cctgcccacc 660 tttggtatca ctgtctggat cactgcacga gaaatggttg gcaatctgct catcggcaag 720 It makes 726 <210> 7 <211> 384 <212> PRT <213> Pichia pastoris (Pichia pastoris) <400> 7 Met Asp Ile Ala Leu Glu Ile Leu Asp Thr Phe Val Phe Asp Lys Val 1 5 10 15 Tyr Ala Lys Leu Leu Pro Ile Ser Leu Val Gln His Leu Pro Asp Gly 20 25 30 Tyr Leu Lys Thr Leu Gly His Leu Thr Gly Ala Asn Asn Thr Met Glu 35 40 45 Ser Leu Phe Gly Ile Ala Pro Asn Val Asp Gln Ala Ser Lys Asn His 50 55 60 Trp Leu Arg Thr Val Asn Asp Ser Ile Ala Leu Ala Arg Pro Gly Glu 65 70 75 80 Arg Leu Val Tyr Gly Val Asn Ala Pro Leu His Phe Phe Asp Glu Thr 85 90 95 Ala Tyr Thr Tyr Ala Ser Ile Leu Gly Arg Ser Asn Ile Ile Arg Gln 100 105 110 Phe Thr Thr Leu Met Ile Leu Met Ile Leu Phe Gly Trp Gly Leu Tyr 115 120 125 Leo Ser Val Ala Ser Phe Ser Tyr Tyr Phe Val Phe Asp Lys Ala Ile 130 135 140 Phe Asn His Pro Arg Tyr Leu Lys Asn Gln Met Ser Leu Glu Ile His 145 150 155 160 Gln Ala Leu Thr Ala Ile Pro Thr Met Val Leu Leu Thr Val Pro Trp 165 170 175 Phe Leu Ile Glu Leu Arg Gly Tyr Ser Lys Leu Tyr Phe Asp Val Asn 180 185 190 Glu Ser Thr Gly Gly Trp Lys Ala Ile Ile Trp Gln Ile Pro Cys Phe 195 200 205 Ile Met Phe Thr Asp Cys Cys Ile Tyr Phe Ile His Arg Trp Leu His 210 215 220 Trp Pro Ser Val Tyr Lys Arg Leu His Lys Pro His His Lys Trp Ile 225 230 235 240 Val Cys Thr Pro Phe Ala Ser His Ala Phe His Pro Val Asp Gly Tyr 245 250 255 Ala Gln Ser Leu Pro Tyr His Leu Tyr Gly Met Leu Phe Pro Leu His 260 265 270 Lys Val Ser Tyr Leu Ile Leu Phe Gly Leu Val Asn Phe Trp Thr Val 275 280 285 Met Ile His Asp Gly Glu Tyr Leu Ser Arg Asp Pro Ile Val Asn Gly 290 295 300 Ala Ala Cys His Thr Val His His Leu Tyr Phe Asn Tyr Asn Tyr Gly 305 310 315 320 Gln Phe Thr Thr Leu Trp Asp Arg Leu Gly Gly Ser Tyr Arg Met Pro 325 330 335 Asp Lys Glu Leu Phe Asp Lys Asn Lys Lys Lys Asp Val Lys Thr Trp 340 345 350 Arg Ser Gln Val Lys Gln Ala Asp Ser Ile Arg Glu Asp Leu Glu Gly 355 360 365 Lys Glu Asp Phe Arg Glu Tyr Gly Thr Glu Glu Lys Leu Lys Ser Thr 370 375 380 <210> 8 <211> 1155 <212> DNA <213> Pichia pastoris <400> 8 atggacattg ctttggagat tctagacact tttgtctttg acaaagtcta tgcaaaacta 60 ctgcccattt ctctggtgca acatttgcca gatggctatt tgaagacttt gggacatttg 120 actggtgcca acaacaccat ggaatcactg ttcggaatag ctccaaacgt tgaccaagcg 180 tctaagaacc actggctgag aacagtgaat gactctattg ccttagcccg tcccggtgag 240 cgtctggtct acggtgtcaa cgctccttta cacttttttg acgaaacagc gtatacatac 300 gcatcgatct tgggacgctc caatatcatt cgacaattca caactttgat gattctgatg 360 attctttttg gctggggttt gtatttatct gtggcttcat tttcatacta ctttgttttt 420 gataaagcca ttttcaatca cccaagatac ctcaaaaacc agatgtctct ggagatccat 480 caagcgttga ctgctatacc tacgatggtt ttgcttacag ttccatggtt tttgattgag 540 ttgcgtggat actctaaatt atactttgat gtaaatgagt ctactggagg atggaaggct 600 attatttggc aaattccttg cttcattatg tttaccgatt gttgtatcta ctttattcat 660 cgttggttgc actggccatc cgtgtataag cgtttgcaca agcctcacca caagtggatt 720 gtttgtacac cttttgctag tcatgccttc catccagttg atggttatgc acaatcacta 780 ccttaccatt tgtatggaat gttgtttcca ctacacaagg tgagctatct gatcttattt 840 gggcttgtga acttttggac tgttatgatc catgatggag aatacctgtc cagagaccct 900 atagtcaatg gagctgcttg tcatacagtg catcacctat acttcaacta caattacggc 960 cagttcacaa cactttggga ccgtcttggt ggatcataca gaatgccaga caaggaactc 1020 tttgataaga acaagagaa agatgttaaag acatggcgtt cacaagtcaa gcaggccgat 1080 tcgataagag aagacttaga gggaaaagaa gatttccgtg agtatggaac tgaggaaaaa 1140 cttaaaagca catag 1155

Claims

1. A modified enzyme of SEQ ID NO: 1 selected from EC 2.3.1.26 having sterol acyltransferase activity that catalyzes the esterification of sterols, wherein one or more amino acid substitutions are introduced at positions corresponding to residues selected from the group consisting of amino acid residues 11, 281, 366, 442, 551, 554, 572, 624, 626, 627 and 636 in the polypeptide according to SEQ ID NO: 1, wherein the one or more amino acid substitutions are selected from H554Q, L281I, F624L, G627D, H554Q-F572L, I442V-G627D, E11G-F624L, E11G-G627D, I442V-F624L-L626F, I442V-L626F-G627 D, H554Q-F572L-F624L, H554Q-F572L-G627D, E11G-D366V-F624L-C636S, E11G-D366V-G627D-C636S, V286V-H551Y-F572L-S633S, E11G-D366V-I442V-F624L-C636S, I442V-L626F, E11G-D366V-C636S and E11G-D366V-I442V-G627D-C636S.

2. The modified enzyme of claim 1, wherein the modified enzyme participates in the esterification of sterols, wherein the enzyme activity is shifted toward 7-dehydrocholesterol (7-DHC) rather than toward yeast sterol, wherein the ratio of 7-DHC to yeast sterol in the sterol mixture is increased by at least about 1.4 times compared to the corresponding ratio using the corresponding unmodified enzyme.

3. A sterol-producing yeast comprising the modified enzyme according to any one of claims 1 to 2.

4. The sterol-producing yeast of claim 3, for producing a sterol mixture comprising 7-DHC and yeast sterol, wherein the ratio of 7-DHC to yeast sterol is increased by at least about 1.4 times compared to yeast expressing an unmodified enzyme. The sterol-producing yeast according to claim 3 , wherein ERG5 and ERG6 are inactivated.

6. The sterol producing yeast of claim 3, wherein the sterol producing yeast expresses a heterologous enzyme selected from EC 1.3.1.72 having sterol Δ24 reductase activity.

7. A method for increasing the ratio of 7-DHC including free and esterified 7-DHC towards yeast sterol esters in a sterol mixture comprising yeast sterol and 7-DHC, the method comprising culturing a host cell expressing the modified enzyme according to claim 1 or 2 under suitable conditions, and optionally isolating and / or purifying the 7-DHC from the sterol mixture.

8. A method for increasing the ratio of 7-DHC esters towards yeast sterol esters in a sterol mixture comprising esters of 7-DHC and yeast sterol, the method comprising culturing a host cell of the modified enzyme according to claim 1 or 2 under suitable conditions, and optionally isolating and / or purifying the 7-DHC from the sterol mixture.

9. A method for producing 7-DHC, the method comprising enzymatically converting acetyl-CoA into a sterol mixture comprising yeast sterol and 7-DHC using the sterol-producing yeast according to claim 3, wherein the percentage of 7-DHC in the sterol mixture is at least 40%.

10. The method of claim 9, wherein the 7-DHC is also converted into vitamin D3.

11. The method according to claim 9 or 10, wherein the 7-DHC is further converted into 25-hydroxyvitamin D3.

12. Use of the modified enzyme according to any one of claims 1 to 2 or the sterol-producing yeast according to claim 3 in a method for producing 7-DHC, wherein the 7-DHC is isolated from a sterol mixture comprising yeast sterol and 7-DHC, and wherein the ratio of 7-DHC to yeast sterol is increased by at least about 1.4 times compared to a method using the corresponding unmodified enzyme and yeast, respectively.

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