The present invention is related to modified sterol acyltransferase enzymes with improved activity and/or specificity towards acylation of the vitamin D3 precursor 7-dehydrocholesterol (7-DHC) to be used in biotechnological production of vitamin D3. The invention further relates to a host strain expressing said modified enzymes and their use in a process for production of vitamin D3 or derivatives and/or metabolites thereof.

Vitamin D3 (also known as cholecalciferol or calciol) can be synthesized in the skin of mammals from provitamin D3 (also known as 7-dehydrocholesterol or 7- DHC) which is product of cholesterol biosynthesis upon exposure to UV light, whereby 7-DHC is photochemically converted into provitamin D3, which isomerizes at body temperature to the biologically active form vitamin D3. 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 kidney.

For industrial production of vitamin D3, both chemical and biotechnological synthesis is (in principle) available. Chemical synthesis starts with cholesterol isolated from e.g. wool fat which is dehydrogenated into 7-DHC, an important intermediate in both chemical and biotechnological synthesis. Through exposure by UV-light and further purification/extraction steps 7-DHC is converted into vitamin D3. Modified yeast stains can be used for biosynthesis of 7-DHC, wherein acetyl-CoA is converted in a multi-step enzymatic process into 7-DHC. Said enzymatic conversion takes place in the endoplasmatic reticulum of the yeast. Excessive amounts of sterols, including 7-DHC and precursors thereof, not required in cellular membranes, are toxic to the yeast and are thus stored as steryl esters into intracellular organelles (so-called lipid bodies) from which they can be further isolated. The equilibrium between free sterols and those stored in the lipid bodies (mainly in the form of steryl esters) is triggered via the action of several proteins (enzymes), including action of sterol acyltransferases. In yeast, particularly Saccharomyces cerevisiae, ester formation of sterols is mainly carried out by the two sterol acyltransferases Arel p and Are2p.

Due to the unspecific action of said sterol acyltransferase enzymes, the steryl ester pool which is stored within the lipid bodies is relatively diverse, including but not limited to e.g. esters of ergosterol, zymosterol, lanosterol, lathosterol, cholesta-5,7,24(25)-trienol, or 7-DHC. Since only cholesta-5,7,24(25)-trienol, a precursor for 7-DHC, and not zymosterol can be used for vitamin D3 synthesis, there is a need for either selective storage of specific esters, such as e.g. esters of 7-DHC, into the lipid bodies and/or for increasing the turnover of

intermediates of 7-DHC produced by such a yeast strains which are further converted to vitamin D3 and/or derivatives or metabolites thereof. A particular metabolite which is also in focus of the present invention is 25-hydroxyvitamin D3.

Thus, it is an ongoing task to generate host cells, such as yeast capable of producing sterols, with high productivity/specificity for 7-DHC and/or reduced accumulation of side-products/intermediates including zymosterol, lanosterol or lathosterol, in particular esters of such intermediates stored in the lipid bodies.

Surprisingly, we now found that the specificity and/or activity of the sterol acyltransferase activity in the host cell can be shifted via introduction of certain amino acid substitutions in the sequence of ARE2 and/or ARE1 , which will lead to higher productivity of the host cell towards 7-DHC as important intermediate in vitamin D3 production.

Thus, the present invention is directed to a modified enzyme with sterol acyltransferase activity, i.e. modified sterol acyltransferases, particularly activity of sterol acyltransferase isoform Arel p and/or Are2p, comprising one or more amino acid substitution(s) at (a) position(s) corresponding to residues selected from the group consisting of 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 has increased specificity for 7-DHC over side- products/intermediates including zymosterol and/or increased activity towards ester formation, including esters of 7-DHC.

The polypeptide according to SEQ ID NO: 1 , showing ARE2 activity, including polynucleotides encoding said polypeptide, has been isolated from

Saccharomyces cerevisiae. The polypeptide according to SEQ ID NO: 3, showing ARE1 activity, including polynucleotides encoding said polypeptide, has been isolated from Saccharomyces cerevisiae.

The terms "sterol acyltransferase", "acyltransferase", "ARE", "enzyme having acyltransferase activity" or just "enzyme" are used interchangeably herein and refer to enzymes [EC 2.3.1.26], i.e. acyltransferases transferring fatty acyl groups 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 different origins, including mammals, yeast or plants. Both ARE1 and ARE2 are capable of acylating sterols such as e.g. zymosterol and/or 7- DHC to the respective esters. As used herein, a "modified" enzyme, i.e. modified acyltransferase, has a preferred activity and/or specificity towards esterification of 7-DHC compared to esterification of e.g. zymosterol and/or improved formation of total sterol esters, including e.g. 7-DHC or zymosterol. Preferred acyltransferase isoforms are Are2p or Arel p. A "non-modified" sterol

acyltransferase, particularly ARE1 and ARE2, as used herein refers to the respective endogenous enzymes not carrying one or more amino acid

substitution(s) as defined herein.

As used herein, a host cell carrying a modified sterol acyltransferase activity as defined herein, particularly ARE2 and/or ARE1 comprising one or more amino acid substitution(s) as defined herein, is referred to as "modified" host cell. The respective host cell carrying a non-modified sterol acyltransferase activity, i.e. encoding the wild-type ARE2 and/or ARE1 genes, is referred to as "non-modified" host cell.

As used herein, the terms "zymosterol", "lanosterol", "lathosterol","cholesta- 5,8,24(25)-trienol", "cholesta-5,7,24(25)-trienol", or "7-DHC" specifying vitamin D3 intermediates include both the free form and the ester form of said compounds. As used herein, a sterol mix contains 7-DHC and "side-products" or intermediates, including but not limited to zymosterol, lanosterol, lathosterol, cholesta-5,8,24(25)-trienol, or cholesta-5,7,24(25)-trienol.

As used herein, a "cholesterol-producing yeast" cannot produce ergosterol anymore but cholesterol products, including, but not limited to cholesta- 5,7,24(25)-trienol, cholesta-5,8,24(25)-trienol, cholesta-7,24(25)-dienol, 7-DHC or zymosterol. Particularly, this might be achieved via introduction of erg5erg6 double-knock out.

Particularly, the modification corresponds to a modified activity of sterol acyltransferase 2 and/or 1 , i.e. ARE2 and/or ARE1 activity, comprising amino acid substitution(s), wherein preferably the at least one amino acid substitution at (a) position(s) corresponding to residues selected from the group consisting of 1 1 , 281 , 366, 442, 551 , 554, 572, 624, 626, 627, 636 and combinations thereof in the polypeptide according to SEQ ID NO: 1 corresponds to (an) amino acid substitution(s) of E1 1 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 at (a) position(s) selected from the group consisting of 1 1 , 281 , 366, 442, 551 , 554, 572, 624, 626, 627, 636, and combinations thereof have been introduced. Preferably, the enzyme having modified ARE2 and/or ARE1 activity are originated from Saccharomyces, such as S. cerevisiae.

In one embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 1 1 in the polypeptide according to SEQ ID NO: 1 , preferably substitution of glutamic acid by glycine (E1 1 G). The described amino acid substitution at a position corresponding to residue E1 1 G in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 281 , 366, 442, 551 , 554, 572, 624, 626, 627 and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Preferably, the amino acid substitution at a position corresponding to residue E1 1 G in SEQ ID NO: 1 might be combined with further substitution(s), such as amino acid substitution(s) at position(s) corresponding to F624L, G627D, D366V, C636S, and/or I442V in SEQ ID NO: 1 , more preferably are combinations of substitutions corresponding to residues E1 1 G-F624L, E1 1 G-G627D, E1 1 G-D366V-C636S, E1 1 G-D366V-G627D- C636S, E1 1 G-D366V-F624L-C636S, E1 1 G-D366V-I442V-F624L-C636S or E1 1 G- D366V-I442V-G627D-C636S in SEQ ID NO: 1 , with most preferred combinations selected from E1 1 G-D366V-C636S, E1 1 G-D366V-G627D-C636S, E1 1 G-D366V- F624L-C636S, E1 1 G-D366V-I442V-G627D-C636S or E1 1 G-D366V-I442V-F624L- C636S. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO: 1 comprising one of said amino acid substitution(s), enzyme specificity could be increased in the range of at least about 3 to 5-times compared to using a non-modified yeast as defined herein.

In one embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 281 in the polypeptide according to SEQ ID NO: 1 , preferably substitution of leucine by isoleucine (L281 I). The described amino acid substitution at a position corresponding to residue L281 I in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 1 1 , 366, 442, 551 , 554, 572, 624, 626, 627 and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO: 1 comprising one of said amino acid substitution(s), relative ester formation could be increased in the range of at least about 1 .5-times compared to using a non-modified yeast as defined herein.

In one embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 366 in the polypeptide according to SEQ ID NO: 1 , preferably substitution of aspartic acid by valine (D366V). The described amino acid substitution at a position corresponding to residue D366V in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 1 1 , 281 , 366, 442, 551 , 554, 572, 624, 626, 627, and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO: 1 comprising one of said amino acid substitution(s), enzyme specificity could be increased in the range of at least about 3-times compared to using a non- modified yeast as defined herein.

In another embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 442 in the polypeptide according to SEQ ID NO: 1 , preferably substitution of isoleucine by valine (I442V). The described amino acid substitution at a position corresponding to residue I442V in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 1 1 , 281 , 366, 551 , 554, 572, 624, 626, 627, and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Preferably, the amino acid substitution at a position corresponding to residue I442V in SEQ ID NO: 1 might be combined with further substitutions, such as amino acid substitutions at position(s) corresponding to F624L, L626F and/or G627D in SEQ ID NO: 1 , more preferably are combinations of substitutions corresponding to residues I442V- L626F, I442V-G627D, I442V-F624L-L626F or I442V-L626F-G627D in SEQ ID NO: 1 , with most preferred combinations selected from I442V-G627D, I442V-F624L- L626F or I442V-L626F-G627D. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO: 1 comprising one of said amino acid substitution(s), enzyme specificity could be increased in the range of at least about 2.2 to 4.5-times compared to using a non-modified yeast as defined herein.

In a further embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 551 in the polypeptide according to SEQ ID NO: 1 , preferably substitution of histidine by tyrosine (H551Y). The described amino acid substitution at a position corresponding to residue H551Y in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 1 1 , 281 , 366, 442, 554, 572, 624, 626, 627, and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO: 1 comprising one of said amino acid substitution(s), enzyme specificity could be increased in the range of at least about 1 .7-times compared to using a non- modified yeast as defined herein.

In one embodiment, the modified enzyme as defined herein, in particular 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 substitution of histidine by glutamine (H554Q). The described amino acid substitution at a position corresponding to residue H554Q in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 1 1 , 281 , 366, 442, 551 , 572, 624, 626, 627, and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Preferably, the amino acid substitution at a position corresponding to residue H554Q in SEQ ID NO: 1 might be combined with further substitutions, such as amino acid substitutions at position(s) corresponding to F624L, F572L and/or G627D in SEQ ID NO: 1 , more preferably are combinations of substitutions corresponding to residues H554Q- F572L-F624L or H554Q-F572L-G627D in SEQ ID NO: 1 . Using a cholesterol- producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO: 1 comprising one of said amino acid substitution(s), enzyme specificity could be increased in the range of at least about 1 .4 to over 12-times compared to using a non-modified yeast as defined herein. ln another embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 572 in the polypeptide according to SEQ ID NO: 1 , preferably substitution of phenylalanine by leucine (F572L). The described amino acid substitution at a position corresponding to residue F572L in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 1 1 , 281 , 366, 442, 551 , 554, 624, 626, 627, and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO: 1 comprising one of said amino acid substitution(s) as defined above, enzyme specificity could be increased in the range of at least about 1 .7-times compared to using a non-modified yeast as defined.

In one embodiment, the modified enzyme as defined herein, in particular 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 substitution of phenylalanine by leucine (F624L), which corresponds to substitution of F592L in the polypeptide according to SEQ ID NO: 3. The described amino acid substitution at a position corresponding to residue F624L in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e. substitutions on one or more position(s) corresponding to amino acid residue(s) 1 1 , 281 , 366, 442, 551 , 554, 572, 626, 627, and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO: 1 comprising one of said amino acid

substitution(s), enzyme specificity could be increased in the range of at least about 3.1 -times compared to using a non-modified yeast as defined herein.

In one embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 626 in the polypeptide according to SEQ ID NO: 1 , preferably substitution of leucine by phenylalanine (L626F). The described amino acid substitution at a position corresponding to residue L626F in SEQ ID NO: 1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 1 1 , 281 , 366, 442, 551 , 554, 572, 624, 627, and/or 636 in the polypeptide according to SEQ ID NO: 1 and as described herein. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO:1 comprising one of said amino acid substitution(s) as defined above, enzyme specificity could be increased in the range of at least about 2.2-times compared to using a non-modified yeast as defined herein in the range of at least about can be achieved.

In one embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 627 in the polypeptide according to SEQ ID NO:1 , preferably substitution of glycine by aspartic acid (G627D), which corresponds to substitution of G595D in the polypeptide according to SEQ ID NO:3. The described amino acid substitution at a position corresponding to residue G627D in SEQ ID NO:1 might be combined with further substitutions as defined herein, i.e. substitutions on one or more position(s) corresponding to amino acid residue(s) 11 , 281 , 366, 442, 551 , 554, 572, 624, 626, and/or 636 in the polypeptide according to SEQ ID NO:1 and as described herein. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO:1 comprising one of said amino acid

substitution(s) as defined above, enzyme specificity could be increased in the range of at least about 2.2-times compared to using a non-modified yeast as defined herein.

In one embodiment, the modified enzyme as defined herein, in particular modified ARE2 and/or ARE1 activity, comprises an amino acid substitution at a position corresponding to residue 636 in the polypeptide according to SEQ ID NO:1 , preferably substitution of cystine by serine (C636S). The described amino acid substitution at a position corresponding to residue C636S in SEQ ID NO:1 might be combined with further substitutions as defined herein, i.e.

substitutions on one or more position(s) corresponding to amino acid residue(s) 11 , 281 , 366, 442, 551 , 554, 572, 624, 626, and/or 627 in the polypeptide according to SEQ ID NO:1 and as described herein. Using a cholesterol-producing yeast carrying such modified enzymes, in particular enzymes according to SEQ ID NO:1 comprising one of said amino acid substitution(s), enzyme specificity could be increased in the range of at least about 3-times compared to using a non- modified yeast as defined herein.

A modified sterol acyltransferase according to the present invention, such as e.g. ARE2 and/or ARE1 , preferably comprises at least one amino acid

substitution on a position corresponding to F624L in the polypeptide according to SEQ ID NO:1 , leading to an enzyme specificity of about 4. This could be increased by the introduction of one or more further amino acid substitution(s), e.g. amino acid substitution on a position corresponding to F572L and/or H554Q, leading to an increase of enzyme specificity in the range of more than 12-times compared to using a non-modified yeast as defined herein.

A modified sterol acyltransferase according to another embodiment of the present invention, such as e.g. ARE2 and/or ARE1 , preferably comprises at least one amino acid substitution on a position corresponding to G627D in the polypeptide according to SEQ ID NO: 1 , leading to an enzyme specificity of more than 4. This could be increased by the introduction of one or more further amino acid substitution(s), e.g. amino acid substitution on a position corresponding to F572L and/or H554Q, leading to enzyme specificity increase of about 5-times compared to using a non-modified yeast as defined herein.

As used herein, the activity of ARE2 and/or ARE1 is modified. This might be achieved by, e.g. introducing (a) mutation(s) into the endogenous gene(s) coding for ARE2 and/or ARE1 , i.e. amino acid substitution(s) on one or more positions as described herein. The skilled person knows how to genetically manipulate a yeast cell resulting in modification of ARE2 and/or ARE1 activity. These genetic manipulations include, but are not limited to, e.g. gene replacement, gene amplification, gene disruption, transfection, transformation using plasmids, viruses, or other vectors.

The generation of a mutation into nucleic acids or amino acids, i.e. mutagenesis, may be performed in different ways, such as for instance by random or side- directed mutagenesis, physical damage caused by agents such as for instance radiation, chemical treatment, or insertion of a genetic element. The skilled person knows how to introduce mutations.

The present invention is particularly directed to the use of such modified ARE2 and/or ARE1 enzymes as defined herein in a process for production of 7-DHC, an intermediate for vitamin D3. Preferably, the modified enzymes of the present invention are introduced and/or expressed in a suitable host cell, such as yeast, preferably sterol-producing yeast, in particular cholesterol-producing yeast cell, such as selected from Saccharomyces cerevisiae, Schizosaccharomyces spp., Pichia spp., Klyuveromyces spp., Hansenula spp. or Yarrowia lipolytica, preferably S. cerevisiae. The modified host is used for production of 7-DHC, which might be further converted into vitamin D3 and/or 25-hydroxyvitamin D3. A suitable host cell might be further modified to further increase production of 7-DHC, an important intermediate towards biosynthesis of vitamin D3, and/or reduce accumulation of side-products.

Thus, in one embodiment the invention is directed to a yeast strain having modified ARE2 and/or ARE1 activity and furthermore wherein ERG5 and ERG6 are inactivated. The yeast cell might be further modified via expression of a heterologous enzyme having C24-reductase activity, particularly selected from EC 1 .3.1 .72, such as a heterologous C24-reductase that is active on cholesta- 7,24-dienol, zymosterol, or trienol (e.g. cholesta-5,7,25-trienol), preferably a plant or vertebrate sterol A24-reductase, more preferably from vertebrate source, even more preferably from human, pig, dog, mouse, rat, horse, Danio rerio or any known source, as long as it can be expressed within said yeast cell. Most preferably, the sterol A24-reductase is selected from Danio rerio, rat or human. The sequences expressing said sterol A24-reductase enzymes are publicly available, including but not limited to Um ^' ProtKB/Swiss-Prot reference Q15392, Q60HC5, Q8VCH6, Q5BQE6, Q39085 or P93472 (see e.g. W02003064650).

In another embodiment, the host cell according to the present invention might be further modified via introduction of homologs of endogenous enzymes involved in biosynthesis of 7-DHC, such as e.g. C5-sterol desaturase (ERG3) and/or C8-sterol isomerase (ERG2), resulting in increased specificity and/or productivity of 7-DHC with reduced accumulation of side-products or vitamin D3 intermediates, including but not limited to including zymosterol, lanosterol and/or lathosterol.

In a particular embodiment, the invention relates to a process for improving a host cell towards production of 7-DHC, wherein a modified host cell as defined herein, i.e. modified via introduction of one or more amino acid substitutions in sterol acyltransferases 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 wherein optionally a heterologous enzyme having C-24- reductase activity as defined herein is expressed, and/or wherein optionally homologs of endogenous ERG2 and/or ERG3 are expressed, wherein the host cell is improved such that the percentage of 7-DHC, in the total amount of sterol produced by said host cell is increased and/or the activity of the host cell towards production of sterols is increased, compared to a non-modified host cell as defined herein. In one embodiment, the present invention is directed to modified sterol acyltransferases, particularly modified ARE2 and/or ARE1 , comprising at least one or more amino acid substitution(s) as defined herein, wherein the specificity of the enzyme is increased compared to the specificity of the non-modified enzymes, leading to higher ratio of 7-DHC to side-products including e.g.

zymosterol in a sterol mix produced by a suitable host cell carrying such modified sterol acyltransferase. The ratio might be increased by at least about 1 .4-times, such as e.g. via introduction of amino acid substitutions

corresponding to H554Q in the respective ARE2 and/or ARE1 sequence, such as increased by at least 3, such as e.g. via introduction of amino acid substitutions corresponding to E1 1 G-F624L, I442V-G627D, or I442V-F624L-L626F in the respective ARE2 and/or ARE1 sequence, such as increased by at least 4-times, such as e.g. via introduction of amino acid substitutions corresponding to F624L or G627D in the respective ARE2 and/or ARE1 sequence, such as at least 5, 10 or even 12-times or more, such as e.g. via introduction of amino acid substitutions corresponding to H554Q-F572L-G627D or E1 1 G-D366V-I442V-G627D-C636S in the respective ARE2 and/or ARE1 sequence.

In one embodiment, the present invention is directed to modified sterol acyltransferases, particularly modified ARE2 and/or ARE1 , comprising at least one or more amino acid substitution(s) as defined herein, wherein the activity of the enzyme is increased compared to the activity of the non-modified enzymes, leading to higher overall production of sterols and/or steryl esters, including higher amounts of 7-DHC produced by a suitable host cell carrying such modified sterol acyltransferase. Thus, the relative ester formation, i.e. ratio of all esters to free 7-DHC might be increased by at least 1 .2-times, such as e.g. via introduction of amino acid substitutions corresponding to H55Q or H554Q-F572L in the respective ARE2 and/or ARE1 sequence, such as increased by at least 2, or 3-times, such as e.g. via introduction of amino acid substitutions corresponding to I442V-G627D or I442V-L626F-G627D in the respective ARE2 and/or ARE1 sequence. The total 7-DHC production might be increased by at least about 1 .2 to 1 .4-times via introduction of one or more amino acid substitution(s) as described herein.

Using the modified host cell, e.g. yeast, such as a sterol-producing yeast, in particular a cholesterol-producing yeast, as described herein the percentage of 7-DHC in the sterol mix could by increased by at least 40%, such as 50, 60, 70,

80, 90 % 7-DHC based on the total amount of sterols. In one aspect of the present invention, a host cell comprising modified ARE2 and/or ARE1 activity as defined herein is used in a process for production of vitamin D3 precursor 7-DHC. The modified host cell may be cultured in an aqueous medium supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person for the respective cholesterol-producing host cells. Optionally, such cultivation is in the presence of proteins and/or co-factors involved in transfer of electrons, as known in the art. The cultivation/growth of the host cell may be conducted in batch, fed- batch, semi-continuous or continuous mode. Depending on the host cell, preferably, production of vitamin D3 and precursors thereof such as 7-DHC can vary, as it is known to the skilled person. Cultivation and isolation of 7-DHC and other intermediates in production of vitamin D3 is described in e.g.

WO201 1067144 or WO2017108799. The 7-DHC might be isolated and/or optionally further purified from the sterol mix and might be further converted to vitamin D3 and/or 25-hydroxyvitamin D3 using methods known in the art.

The terms "ARE1 " and "Arel p", "ARE2" and "Are2p", "ERG5" and "Erg5p", "ERG6" and "Erg6p" are used interchangeably herein and refer to a polypeptide encoded by the respective genes are1 , are2, erg5, and erg6. For the purpose of the present invention, the cholesterol-producing yeast cell is modified such that it does show modified activity of ARE2 and/or ARE1 , e.g. carries a modification in either endogenous ARE2, ARE1 or both, leading to modified specificity of ARE2 and/or ARE1 , wherein said modification comprises the introduction of one or more amino acid substitution(s) as defined herein.

Genes encoding ERG5, ERG6, ARE1 , ARE2, ERG2, ERG3, or sterol A24-reductase (ERG4), cultivation and genetic engineering of the yeast cell as used herein are known and described in e.g. US 7608421 .

As used herein, the terms "C-24-reductase" or "A24-reductase" are used interchangeably herein. In yeast, this enzyme is encoded by erg4 and is active on the methyl-group of the carbon atom on position 24. Trienol, which does not exhibit such methyl-group on said position, is therefore not an acceptable substrate for the yeast ERG4.

The terms "C-8 sterol isomerase", "delta 8,7-isomerase", or "enzyme having C-8 sterol isomerase" are used interchangeably herein and refer to enzymes which are capable of catalyzing the conversion of cholesta-8-enol into cholesta-7-enol and/or zymosterol into cholesta-7,24-dienol. In yeast, this enzyme is encoded by erg2. In yeast, this enzyme is encoded by erg2. A preferred ERG2 homolog to be used in a modified host cell according to the present invention is a polypeptide having at least 41%, such as e.g. 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 showing C-8 sterol isomerase activity and including polynucleotides encoding such polypeptide obtainable from Ustilago maydis. Particularly, 1 or more copies, such as at least 1 , 2, 3, 5, of said ERG2 homolog are expressed in a modified host cell as defined herein.

The terms "C-5 sterol desaturase", "enzyme having C-5 sterol desaturase", "desaturase" or "ERG3-homolog" are used interchangeably herein and refer to enzymes which are capable of catalyzing the conversion of cholesta-8-enol into cholesta-7,24-dienol and/or cholesta-7-enol into cholesta-5,7,24-trienol and/or 7-DHC. In yeast, this enzyme is encoded by erg3. A preferred ERG3 homolog to be used in a modified host cell according to the present invention is a

polypeptide having at least 45%, such as e.g. at least 50, 52, 60, 70, 80, 90, 92, 95, 98 or up to 100% identity to SEQ ID NO:7 showing C-5 sterol desaturase activity and including polynucleotides encoding such polynucleotide obtainable from Pichia pastoris or Schizosaccharomyces pombe. Particularly, 1 or more copies, such as at least 1 , 2, 3, 5, of said ERG3 homolog are expressed in a modified host cell as defined herein.

The "relative ester formation" as defined herein is the ratio (all ester/ free 7- DHC)mutant/ (all ester/ free 7-DHC) _w t. Enzyme "specificity" as defined herein is the ratio (7-DHC ester/ zymosterol ester ) _mu tant/ (7-DHC ester/ zymosterol ester)wt. The "total 7-DHC production" as defined herein is the ratio (total 7- DHQmutant/ (7-DHC)wt·

As used herein, the term "specific activity" or "activity" with regards to enzymes means its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate. The specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature. Typically, specific activity is expressed in pmol substrate consumed or product formed per min per mg of protein. Typically, pmol/min is abbreviated by U (= unit). Therefore, the unit definitions for specific activity of pmol/min/(mg of protein) or U/(mg of protein) are used interchangeably throughout this document. An enzyme is active, if it performs its catalytic activity in vivo, i.e. within the host cell as defined herein or within a suitable (cell-free) system in the presence of a suitable substrate. The skilled person knows how to measure enzyme activity, such as e.g. by HPLC. With regards to the present invention, it is understood that organisms, such as e.g. 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 Code).

In particular, the present invention features the present embodiments:

(1 ) A modified enzyme as defined herein with sterol acyltransferase activity having sterol acyltransferase activity comprising one of more amino acid substitution(s) at (a) position(s) corresponding to residues selected from the group consisting of 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 substitution(s) corresponding to E11G and/or L281 I 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.

(2) The modified enzyme as defined herein and of embodiment (1 ) catalyzing the esterification of sterols comprising 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 the catalysis using the respective non-modified enzyme.

(3) The modified enzyme as defined herein and of embodiment (1 ) or (2), wherein the amino acid substitution(s) is/are selected from F624L, G627D, E11G, H554Q, I442V and combinations thereof.

(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 of embodiments (1 ), (2), (3), said host cell optionally further comprising one of more amino acid substitution(s) at (a) position(s) corresponding to residues selected from 592 and/or 595 in the polypeptide according to SEQ ID NO:3, preferably substitution corresponding to F592L and/or G595D.

(5) The host cell as defined herein and of embodiment (4) used for production of a sterol mix comprising 7-DHC and zymosterol, wherein the ratio of 7-DHC to zymosterol is increased by at least about 1.4-times compared to a host cell wherein expressing a non-modified enzyme.

(6) The host cell as defined herein and of embodiment (4) or (5), which optionally further comprises: - inactivation of ERG5 and ERG6 and/or

- expression a heterologous enzyme selected from EC 1.3.1.72 having sterol D24- reductase activity, preferably wherein the heterologous enzyme is originated from plant or vertebrate, more preferably originated from human, pig, dog, mouse, rat, horse or Danio rerio.

(7) A process for reducing the percentage of zymosterol in a sterol mix

comprising zymosterol and 7-DHC comprising cultivating a host cell as defined herein and of one of the embodiments (4), (5) or (6) under suitable conditions and optionally isolating and/or purifying the 7-DHC from the sterol mix or a process for increasing the percentage of 7-DHC in a sterol mix comprising 7-DHC and zymosterol comprising cultivating a host cell as defined herein and of one of the embodiments (4), (5) or (6)under suitable conditions and optionally isolating and/or purifying the 7-DHC from the sterol mix.

(8) A process for production of 7-DHC comprising enzymatic conversion of acetyl- CoA into a sterol mix comprising zymosterol and 7-DHC with a host as defined herein and of one of the embodiments (4), (5) or (6), wherein the percentage of 7-DHC in the sterol mix is at least 40%, with optionally further conversion of 7- DHC into vitamin D3 and/or 25-hydroxyvitamin D3.

(9) Use of a modified enzyme as defined herein and of any of embodiments (1 ), (2), (3) or a host cell as defined herein and of one of the embodiments (4), (5) or

(6) in a process for production of 7-DHC, wherein the 7-DHC is isolated from a sterol mix comprising zymosterol and 7-DHC, and wherein the ratio of 7-DHC to zymosterol is increased by at least about 1.4-times compared to a process using the respective non-modified enzyme and host cell, respectively. Figures

Figure 1. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #2 and #1 (see Table 1 ). With regards to esters of 7-DHC ("7-DHC ester") and esters of zymosterol ("Zym ester") two ester forms were detected as indicated by dark and light grey in the respective columns, free 7-DHC is indicated 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 shown by different columns. Strains were cultivated for two days with two glucose feedings in flasks without baffles. Data are mean values of 3 independent transformants each cultivated once. Figure 2. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #2 and #1 (see Table 1 ). For further details see legend to Figure 1. Strains were cultivated for four days with one glucose feeding in flasks without baffles. Data are mean values of 2 independent transformants each cultivated once.

Figure 3. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #9 and #1 (see Table 1 ). For further details see legend to Figure 1. HPLC analysis according to standard procedure. For further details see text.

Figure 4. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #24 and #20 (see Table 1 ). For further details see legend to Figure 1. HPLC analysis according to standard procedure. For further details see text.

Figure 5. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #22 and #27 (see Table 1 ). For further details see legend to Figure 1. HPLC analysis according to standard procedure. For further details see text.

Figure 6. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #20, #22, #27 and #22 (see Table 1 ). For further details see legend to Figure 1. HPLC analysis according to standard procedure. For further details see text.

Figure 7. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #34, #32, #43, #24, #20, #22, and #27 (see Table 1 ). For further details see legend to Figure 1. HPLC analysis according to standard procedure. For further details see text.

Figure 8. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #9, #35, #36, #39, and #40 (see Table 1 ). For further details see legend to Figure 1. HPLC analysis according to standard procedure. For further details see text.

Figure 9. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #27, #33, #34, #37, and #38 (see Table 1 ). For further details see legend to Figure 1. HPLC analysis according to standard procedure. For further details see text.

Figure 10. HPLC analysis of lipid extracts of ARE2 wild-type strainfWT") and Are2 variants #41 , #42, #43, #24, and #20 (see Table 1 ). For further details see legend to Figure 1. HPLC analysis according to standard procedure. For further details see text. The following examples are illustrative only and are not intended to limit the scope of the invention in any way.

Examples

Example 1 : Generation and screening of ARE2 mutants

An error prone library of 10,000 yeast clones expressing variants of

Saccharomyces cerevisiae acyltransferase 2 (ScAre2) were screened by thin layer chromatography (TLC) for improved 7-DHC content in sterol ester fraction (for wild-type sequences of ARE1 and ARE2 (see sequence listing). The screening method comprises the simultaneous extraction and separation of sterols from cells with slightly digested cell walls. The treated biomass was directly applied on the TLC plate and immersed into the solvent, which did the extraction and separation of sterol containing fractions in one step. In the sterol ester fraction the ratio of sterols with conjugated double bonds (as e.g. 7-DHC) was set into relation to sterols without conjugated double bonds by exploiting the different spectrophotometric properties of the compounds with the conjugated double bonds (e.g. ability to quench fluorescence, UV detection).

The best variants were re-screened in quintuplicates, sequenced, cultivated in shake flasks and analyzed in biological triplicates by HPLC-UV to determine the sterol and sterol ester compositions.

Plasmids containing the best variants were isolated and re-transformed into a cholesta-5,7,24-trienol producing Saccharomyces cerevisiae strain 10A (are1 are2 erg5 erg6::24R; for construction see Example 1 in WO2017108799). Mutations of variants with multiple amino acid exchanges were separated by introducing respective mutation into ARE2 by site-directed mutagenesis (silent mutations were not taken into account) to find out which mutation caused the desired effect. Strains were cultivated and analyzed by HPLC.

Example 2: HPLC-UV analysis standard procedure

Pre-cultures - 10 ml YPD with geneticin (100 pg/mL) - were inoculated with our strains of interest (3 transformants per Are2 variants) and grown at 30° C to appropriate density (24 to 48 h). For better comparison, three different transformants with the wild-type ARE2 plasmid were also inoculated, which were transformed at the same time as the variants. Main cultures of 50 mL YPD with geneticin were inoculated to OD ₆ oo 0.1 in 250 mL shake flasks without baffles and were cultivated for 3 days with 3 times glucose feeding (glucose was added to 2% final concentration after approx. 30, 45, and 60 h) with 200 rpm and 80% humidity at 30° C. 200 OD units of biomass were harvested (centrifuged for 5 min with 1600 x g and supernatant was removed) in 15 mL Greiner tubes and stored at -20° C until analysis.

For extraction, the 200 OD cell pellet was thawed, resuspended in 1 mL zymolyase solution (5 mg/mL zymolyase 20T in 50 mM KPi, pH 7, with 1 M D- sorbitol) and incubated for 15 min at 37° C (750 rpm on thermomixer). The zymolyase solution was removed after centrifugation (2500 x g, 5 min) and 3.73 mL of absolute EtOH were added to the pellet (resuspended with 1 mL by pipetting up and down carefully, then adding additional 2.73 mL). 267 pL of internal standard (cholesteryl acetate, 1 mg/mL in EtOH) were added, the cell suspension was vortexed and heated to 70° C for 1 h with mixing (750 rpm on thermomixer). After some minutes of leaving the tubes to cool down to room temperature, the cell debris was pelleted (2500 x g, 10 min at room

temperature) and 3 mL of the supernatant were transferred into Pyrex tubes which were brought to dryness under N ₂ . The lipids were taken up in 200 pL of ethyl acetate (vortexed and mixed with 750 rpm on a thermomixer at 40 °C for 15 min). The solution was centrifuged once more (2500 x g, 5 min) and transferred into a glass vial with inlay for the subsequent HPLC-UV analysis.

Lipid extracts were analyzed by HPLC with UV detection at two wavelengths (210 nm and 280 nm). Zymosterol compounds were detected at 210 nm, 7-DHC compounds were quantified at 280 nm.

Solvent: 80% EtOH 20% MeOH 0.1% TFA

Column: YMC-Pack Pro C18 RS

Method: injection volume: 10 pL

injector thermostat: 40° C

flow: 0.6 mL/min

column thermostat: 20° C

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

Standard mixtures of 7-DHC, zymosterol, cholesteryl acetate and squalene in 3 different concentrations (0.5, 1.0, and 2.0 mg/mL of each substance) were analyzed as well and standard curves were generated for each substance to calculate the concentration in pg/pL sterol in extract or pg / OD ₆ oo.

Example 3: Evaluation of ARE2 variants with regards to activity and/or

specificity For direct comparison, the wild-type ARE2 plasmid was re-transformed along with the plasmids expressing Are2 variants (see Ex. 1 ) into strain 10A and the resulting strains were analyzed (see Ex. 2) in the same run. The results of the HPLC analyses are summarized in Table 1 and Figures 1 -10. Values in the table give the fold change between strains expressing the mutant/variant compared to the wild type. The first value indicates improvements regarding the ratio between the ester fraction and the free 7-DHC fraction while the second value shows the improvement concerning the ratio of 7-DHC and zymosterol in the ester fractions. The third value is a comparison of total 7-DHC content in biomass of mutants and wild type. Some of the listed variants showed especially improvement in the ester level while others showed improvements in the 7- DHC/zymosterol ratio in the ester fraction.

Table 1A: Summary of relative ester formation of Are2 variants based on several independent experiments. "Relative ester formation" means the x-times increase in the percentage of 7-DHC based on the total amount of sterols generated using the indicated amino acids exchange instead of a wild-type ARE2.; "7-DHC- ester/zym -ester" means ratio of esters from 7-DHC towards esters of zymosterol ("zym"); "7-DHC-total/zym-total" is the ratio of total 7-DHC (free and esters) towards total zymosterol (free and esters). For more explanation, see text.

Table 1 B: Summary of specificity of Are2 variants based on several independent experiments. The number indicates the x-times increase in the percentage of 7- DHC compared to the percentage of zymosterol in the sterol mx generated using the indicated amino acids exchange instead of a wild-type ARE2. For more explanation, see text.

Table 1 C: Summary of total 7-DHC production of Are2 variants based on several independent experiments. The number indicates the x-times increase in the total amount of produced 7-DHC generated using the indicated amino acids exchange instead of a wild-type ARE2. For more explanation, see text.