Optimization of C-5 sterol desaturation

Abstract

The present invention relates to an improved method for producing 7-dehydrocholesterol (7-DHC), which is an important intermediate for the biotechnological production of vitamin D3 or its derivatives / metabolites. The invention features modified host strains expressing enzymes with improved C-5 sterol desaturase activity, and their use in methods for producing vitamin D3 or its derivatives and / or metabolites.

Description

[0001] The present invention relates to an improved method for producing 7-dehydrocholesterol (7-DHC), which is an important intermediate for the biotechnological production of vitamin D3 or its derivatives / metabolites. The invention features modified host strains expressing enzymes with improved C-5 sterol desaturase activity, and their use in methods 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 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 kidneys.

[0003] For the industrial production of vitamin D3, both chemical and biotechnological synthesis are possible (in principle). The chemical synthesis starts with the separation of cholesterol from, for example, lanolin, which is then dehydrogenated to 7-DHC, which is an important intermediate in both chemical and biotechnological synthesis. 7-DHC is converted into 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 into 7-DHC in a multi-step enzymatic process. The enzymatic conversion occurs in the endoplasmic reticulum of yeast. Excess sterols that are not needed in the cell membrane, including 7-DHC and its precursors, are toxic to yeast and are therefore stored as sterol esters in organelles (so-called liposomes) within the cell, from which they can be further separated. The balance between free sterols and sterols stored in liposomes (mainly in the form of sterol esters) is triggered by the action of several proteins (enzymes), including the action of sterol acyltransferases.

[0004] Due to the non-specific action of the sterol acyltransferase, the pool of sterol esters stored in the liposomes is relatively diverse, including, but not limited to, esters of ergosterol, yeast sterol, lanosterol, cholestanol, cholesta-5,7,24(25)-trienol, cholesta-8-enol or 7-DHC, for example. Only 7-DHC can be further processed into vitamin 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 byproducts / intermediates (including yeast sterol, lanosterol or cholestanol), in particular esters of such intermediates stored in lipid bodies.

[0006] Surprisingly, we have now discovered that the productivity of 7-DHC in host cells, in particular the ratio of 7-DHC to cholestan-7-enol and / or cholestanol, can be shifted towards 7-DHC by modifying the C-5 sterol desaturase activity within the host cells (i.e., expression of a heterologous enzyme having C-5 sterol desaturase activity), which results in a higher productivity of 7-DHC (an important intermediate in vitamin D3 production) in the host cells.

[0007] Accordingly, the present invention relates to the use of an enzyme having C-5 sterol desaturase activity in a method for producing 7-DHC, said polypeptide 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: 2 (heterologously) expressed in a suitable host cell for the production of 7-DHC, wherein the ratio of 7-DHC to by-products including lanosterol and / or cholestanol is increased by at least 5% compared to the unmodified host cell.

[0008] A polypeptide according to SEQ ID NO: 2 exhibiting C-5 sterol desaturase activity has been isolated from Pichia pastoris, including a polynucleotide encoding the polypeptide.

[0009] 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 capable of catalyzing 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. The enzymes defined herein are homologs of Saccharomyces cerevisiae ERG3 (SEQ ID NO: 8), including polypeptides encoding such polypeptides.

[0010] The terms "conversion" (or "conversion"), "enzymatic conversion" or "desaturation" in connection with the enzymatic catalysis of, for example, cholesta-7-enol to 7-DHC and / or cholesta-7,24-dienol to cholesta-5,7,24-trienol are used interchangeably herein and refer to the action of a C-5 sterol desaturase as defined herein and known in the art.

[0011] The desaturase may be used in an isolated form (e.g. in a cell-free system), or may be introduced and expressed in a suitable host cell as a heterologous enzyme or as an extra copy of an endogenous enzyme. Thus, a suitable host cell expressing one, two or more copies of a desaturase as defined herein, resulting in an increase in 7-DHC and / or an increase in the ratio of 7-DHC compared to cholestan-7-enol and / or lanosterol, is referred to herein as a genetically modified host cell. A genetically unmodified host cell or unmodified host cell as referred to herein is a corresponding host cell that carries only the endogenous C-5 sterol desaturase activity expressed by the endogenous ERG3 gene.

[0012] As used herein, the terms "zymosterol", "lanosterol", "cholestanol", "cholesta-5,8,24(25)-trienol", "cholesta-5,7,24(25)-trienol" or "7-DHC" designating the vitamin D3 intermediate include both the free form and the ester form of the compound. As used herein, a sterol mixture comprises 7-DHC and "byproducts" or intermediates, including but not limited to zymosterol, lanosterol, cholesta-8-enol, cholesta-5,8,24(25)-trienol or cholesta-5,7,24(25)-trienol.

[0013] As used herein, "cholesterol-producing yeast" is no longer able to produce ergosterol, but instead produces cholesterol products including, but not limited to, cholesta-5,7,24(25)-trienol, cholesta-5,8,24(25)-trienol, cholesta-7,24(25)-dienol, cholesta-8-enol, 7-DHC or zymosterol. Specifically, this can be achieved by introducing an erg5erg6 double knockout.

[0014] Suitable desaturases as defined herein may be obtained from different sources, such as plants, animals including humans, algae, fungi including yeast, or bacteria, preferably from fungi, in particular selected from the group consisting of Saccharomyces, Yarrowia, Klyveromyces, Schizosaccharomyces, Pichia, Candida, Penicillium, Aspergillus, Cryptococcus, Magneporte, Metarahizium and Ustilago, more preferably selected from S. cerevisiae, Yarrowia lipolytica, Kluyveromyces lactis, Schizosaccharomyces pombe. pombe, P. pastoris, C. albicans, P. roqueforti, A. nidulans, C. neoformans or U. maydis, most preferably obtained from P. pastoris.

[0015] In a preferred embodiment, the enzyme having C-5 sterol desaturase activity is obtainable from Pichia, in particular Pichia pastoris, such as the protein encoded by the polynucleotide according to SEQ ID NO: 1, more preferably, the protein is a polypeptide according to SEQ ID NO:2.

[0016] In another embodiment, the enzyme having C-5 sterol desaturase activity may be obtained from Penicillium, in particular Penicillium roqueforti, such as the protein encoded by the polynucleotide according to SEQ ID NO:3, more preferably, the protein is a polypeptide according to SEQ ID NO:4.

[0017] In one embodiment, the enzyme having C-5 sterol desaturase activity is obtainable from Schizosaccharomyces, in particular Schizosaccharomyces pombe, such as a protein encoded by the polynucleotide according to SEQ ID NO:5, more preferably, the protein is a polypeptide according to SEQ ID NO:6.

[0018] In another embodiment, an enzyme having C-5 sterol desaturase activity may be obtained from Saccharomyces, in particular Saccharomyces cerevisiae, such as a protein encoded by a polynucleotide according to SEQ ID NO: 7, more preferably, the protein is a polypeptide according to SEQ ID NO: 8 derived from UniProtKB P32352, which is expressed additionally and / or as a replacement for endogenous ERG3 when using Saccharomyces cerevisiae as a host.

[0019] Based on the sequences as disclosed herein and based on the increased accumulation of 7-DHC and / or the reduction of cholesta-7-enol and / or the reduction of cholestanol in the sterol mixture, i.e. resulting in the presence of at least 84%, such as 85%, 90%, 92%, 95%, 97% or even 100% 7-DHC in the sterol mixture, we can easily deduce other suitable genes encoding polypeptides having C-5 sterol desaturase activity as defined herein, which polypeptides can be used for the desaturation of C-5 sterols as defined herein, in particular the desaturation of cholesta-7-enol and cholesta-7,24-dienol. Thus, the present invention relates to a method for identifying novel desaturases, wherein a polypeptide having at least 44%, such as at least 48%, 50%, 52%, 60%, 70%, 80%, 90%, 92%, 95%, 98% or up to 100% identity to a polypeptide according to SEQ ID NO: 8 is used as a probe in the screening process for new C-5 sterol desaturases that preferentially produce 7-DHC over cholestanol-7-enol and / or cholestanol ene, resulting in at least about 84% 7-DHC in a sterol mixture produced by a suitable host strain. Any polypeptide having C-5 sterol desaturase activity disclosed herein can be used to produce 7-DHC, as long as the desaturase action results in at least about 84% 7-DHC in a sterol mixture based on the total amount of sterols produced, and / or an increase in the ratio of 7-DHC to cholestanol-7-enol and / or cholestanol ene.

[0020] The present invention particularly relates to the use of such novel desaturases, in particular heterologous enzymes, in a method for producing 7-DHC, wherein the production of by-products in a sterol mixture comprising cholestan-7-enol, yeast sterol, cholestan-8-enol or cholestanol is reduced to about 16% or less, such as 15%, 12%, 10%, 8%, 5%, 3% or less, based on the total amount of sterols, by the action of said desaturase as defined herein, in particular wherein the percentage of cholestan-7-enol and / or cholestanol relative to the amount of 7-DHC is reduced. The method can be performed with a suitable cholesterol-producing yeast cell expressing said heterologous desaturase, preferably wherein the gene encoding said enzyme is heterologously expressed, i.e. introduced into said host cell. 7-DHC can be further converted into vitamin D3 by (known) suitable chemical or biotechnological mechanisms. The percentage of by-products can be even further reduced when the number of copies of the ERG3 homologue to be expressed in the host cell is increased to more than one.

[0021] The terms "sequence identity", "% identity" are used interchangeably herein. For the purposes of the present invention, it is defined herein that in order to determine the sequence identity percentage of two amino acid sequences or two nucleic acid sequences, the sequences are compared to achieve the best comparison purpose. In order to optimize the comparison between the two sequences, a room can be introduced in either sequence of the two sequences being compared. Such comparisons can be performed over the full length of the sequence being compared. Alternatively, the comparison can be performed over a shorter length, for example, over about 20, about 50, about 100 or more nucleotides / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences on the reported comparison region. The Needleman and Wunsch algorithm for the comparison of two sequences can be used to determine the sequence identity percentage between two amino acid sequences or between two nucleotide sequences (Needleman, SB and Wunsch, CD (1970) J.Mol.Biol.48, 443-453). Both amino acid sequences and nucleotide sequences can be compared by this algorithm. Needleman-Wunsch algorithm has been implemented in computer program NEEDLE. For purposes of the present invention, the NEEDLE program (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6), pp. 276-277, http: / / emboss.bioinformatics.nl / ) from the EMBOSS program package is used. For protein sequences, EBLOSUM62 is used for substitution matrix. For nucleotide sequences, EDNAFULL is used. The optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5. It will be appreciated by the technician that when using different algorithms, all these different parameters will produce slightly different results, but the overall identity percentage of two sequences will not significantly change.

[0022] After alignment by the program NEEDLE as described above, the percentage of sequence identity between the query sequence and the sequence of the present invention is calculated as follows: the number of corresponding positions showing the same amino acid or the same nucleotide in the two sequences in the alignment is divided by the total length of the alignment minus the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and marked as "longest identity" in the output of the program. If the two amino acid sequences compared do not differ in any of their amino acids, they are identical or have 100% identity. With regard to the plant-derived enzymes defined herein, the skilled person is aware that the enzymes of plant origin may in fact contain chloroplast targeting signals, which will be cleaved via specific enzymes (e.g., chloroplast processing enzymes (CPE)).

[0023] ERG3 enzymes / homologues as defined herein also encompass enzymes with one or more amino acid substitutions that do not alter the activity of the enzyme, i.e., the enzyme exhibits the same properties relative to the wild-type enzyme and catalyzes the desaturation of C-5 sterols, resulting in a percentage of 7-DHC in the sterol mixture of at least about 84% (reducing cholestanol and / or cholestanol towards 7-DHC). Such mutations are also referred to as "silent mutations" that do not alter the (enzymatic) activity of the enzyme as described herein.

[0024] Depending on the host cell, the polynucleotides involved in C-5 sterol desaturation as defined herein may be optimized for expression in the corresponding host cell. The skilled person knows how to generate such modified polynucleotides. It should be understood that the polynucleotides as defined herein also include such host-optimized nucleic acid molecules, as long as they still express a polypeptide having the corresponding activity as defined herein. Examples of such host-optimized ERG3 homologues are shown, for example, in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

[0025] Thus, in one embodiment, the present invention relates to a host cell comprising a polynucleotide encoding a (heterologous) ERG3 homologue as defined herein, said polynucleotide being optimized for expression in said host cell without affecting the growth of said host cell or the expression pattern of the enzyme. In particular, a yeast, such as a cholesterol producing yeast cell, is selected from the genus Saccharomyces, such as Saccharomyces cerevisiae, wherein one, two or more copies of a polynucleotide encoding an ERG3 enzyme as defined herein are selected from polynucleotides having at least 53%, such as at least 58%, 60%, 70%, 80%, 90%, 92%, 95%, 98% or up to 100% identity to SEQ ID NO: 9, including, for example, a polypeptide according to SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.

[0026] The nucleic acid molecule according to the invention may comprise only a portion or fragment of a nucleic acid sequence provided by the invention, such as a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, for example a fragment that can be used as a probe or primer or a fragment encoding a portion of an ERG3 homologue as defined herein. The probe / primer typically comprises a substantially purified oligonucleotide, which typically comprises a nucleotide sequence region that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, more preferably about 22 or 25, even more preferably about 30, 35, 40, 45, 50, 55, 60, 65 or 75 or more consecutive nucleotides of a nucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, or a fragment or derivative thereof.

[0027] A preferred, non-limiting example of such hybridization conditions is hybridization in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 1x SSC, 0.1% SDS at 50°C, preferably at 55°C, more preferably at 60°C, and even more preferably at 65°C.

[0028] High stringency conditions include, for example, incubation at 42°C with a digoxigenin (DIG)-labeled DNA probe (prepared by using the DIG labeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solution containing or not containing 100 μg / ml salmon sperm DNA (such as DigEasyHyb solution (Roche Diagnostics GmbH)), or a solution containing 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine and 2% blocking agent (Roche Diagnostics GmbH), followed by washing the filter twice (for 5 to 15 minutes) in 2x SSC and 0.1% SDS at room temperature, and then in 0.5x SSC and 0.1% SDS or 0.1x at 65-68°C. Wash twice in SSC and 0.1% SDS (15-30 min duration).

[0029] The present invention particularly relates to the use of a heterologous enzyme having C-5 sterol desaturase activity 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, in particular a cholesterol-producing yeast cell, such as selected from Saccharomyces cerevisiae, Schizosaccharomyces, Pichia, Kluyveromyces, Hansenula 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 production of 7-DHC, an important intermediate toward vitamin D3 biosynthesis, and / or reduce accumulation of by-products.

[0031] Thus, in one embodiment, the present invention relates to a yeast strain having an altered C-5 sterol desaturase activity and in addition wherein ERG5 and ERG6 are inactivated. The yeast cell may be further modified by expressing a heterologous enzyme having C24 reductase activity, in particular selected from EC 1.3.1.72, such as a heterologous C24 reductase acting 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 a 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 cell according to the present invention can be further modified by introducing a homologue of an endogenous enzyme involved in the biosynthesis of 7-DHC, such as 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 yeast sterol, lanosterol and / or enecholestanol). Preferably, the modified host cell as defined herein comprises a heterologous ERG2, wherein the ERG2 is preferably selected from Ustilago maydis (e.g. a polypeptide derived from UniProtKB P32360).

[0033] In another embodiment, the host cell according to the present invention may be further modified in terms of sterol acyltransferase activity, in particular the activity of sterol acyltransferase isoforms Are1p and / or Are2p, comprising one or more amino acid substitutions at one or more positions corresponding to residues selected from 592 and / or 595 in the polypeptide according to SEQ ID NO: 12.

[0034] Thus, in a particular embodiment, the present invention relates to a modified yeast strain to be used in a process for producing sterols, in particular 7-DHC, wherein ERG5 and ERG6 are inactivated, optionally a heterologous enzyme having C24 reductase activity as defined herein is expressed, and an ERG3 homologue as described herein is expressed. When such a yeast strain is used, the percentage of 7-DHC present in the sterol mixture is in the range of about 84% or more, preferably for example 85%, 90%, 92%, 95%, 97% or even 100%, based on the total amount of sterols.

[0035] In a specific embodiment, the present invention relates to a method for improving a yeast cell towards 7-DHC production, wherein the modified host cell is as defined herein, i.e. expresses an ERG3 homologue as defined herein, e.g. by introducing one, two or more copies of a desaturase as defined herein, in particular a cholesterol producing yeast cell, preferably a yeast cell in which ERG5 and ERG6 are inactivated and in which optionally a heterologous enzyme with C-24 reductase activity as defined herein is expressed and / or in which ARE1 and / or ARE2 are modified as described herein and / or in which optionally a homologue of ERG2 is 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 to at least about 84%, in particular wherein the ratio of 7-DHC to by-products including cholestan-8-enol is increased by at least 2% compared to an unmodified yeast strain as defined herein (i.e. expressing only wild-type (endogenous) ERG3 activity).

[0036] In a specific embodiment, the present invention relates to a method for improving a yeast cell towards 7-DHC production, wherein the yeast cell is in particular a cholesterol-producing yeast cell, such as 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 expressed, the yeast cell for example expressing an ERG3 homologue as defined herein by introducing one, two or more copies of a desaturase as defined herein, wherein the yeast cell is improved so as to be comparable to a yeast cell expressing wild-type (endogenous) ERG3 activity. Compared to an unmodified yeast strain, the percentage of 7-DHC in the total amount of sterols produced by the yeast is increased from about 81% or less to at least about 84%, for example 85%, 90%, 92%, 95%, 97% or even 100%, and the percentage of by-products including cholestanol-7, cholestanol-enol and / or cholestan-8, and / or yeast sterol in the sterol mixture is reduced to about 16% or less based on the total amount of sterols, that is, the reduction of cholestanol-7, cholestanol-enol and / or cholestan-8, and / or yeast sterol is in the range of at least about 16% based on the total amount of sterols.

[0037] In one embodiment, the present invention relates to a method for producing a sterol mixture comprising a mixture of 7-DHC and cholestan-7-ols (cholestanols) and / or lanosterol, wherein the percentage of 7-DHC is increased by at least about 2%, such as 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, based on the total amount of sterols compared to the percentage of lanosterol / cholestanols, wherein the cholesterol producing yeast cell expresses a (heterologous) desaturase as defined herein, i.e. a polypeptide 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: 2, more preferably a polypeptide expressed by a corresponding codon-optimized polynucleotide as defined herein, such as preferably a polypeptide obtainable from Pichia pastoris, Penicillium roqueforti, or Schizosaccharomyces pombe, or Saccharomyces cerevisiae, in particular a polypeptide obtainable from Pichia pastoris.

[0038] In one embodiment, the present invention relates to a method for producing a sterol mixture comprising 7-DHC and cholestan-8-enol, wherein the ratio of 7-DHC to cholestan-8-enol based on the total amount of sterols is increased by at least about 2%, such as 3%, 4%, 5% or at least about 10%, in a cholesterol-producing yeast cell expressing a (heterologous) desaturase as defined herein, i.e. a polypeptide having at least about 45%, such as at least 50%, 52%, 60%, 70%, 80%, 90%, 92%, 95%, 98% or up to 100% identity to SEQ ID NO: 2, more preferably a polypeptide expressed by a corresponding codon-optimized polynucleotide as defined herein, such as preferably a polypeptide obtainable from Pichia pastoris.

[0039] In one embodiment, the present invention relates to a method for producing a sterol mixture comprising 7-DHC and yeast sterol, wherein the percentage of 7-DHC is increased by at least about 2%, such as 3%, 4%, 5% or at least about 10% compared to the percentage of yeast sterol based on the total amount of sterols in a cholesterol producing yeast cell expressing a (heterologous) desaturase as defined herein, i.e. a polypeptide 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: 2, more preferably a polypeptide expressed by a corresponding codon-optimized polynucleotide as defined herein, such as preferably a polypeptide obtainable from Pichia pastoris.

[0040] In one embodiment, the present invention relates to a method for producing a sterol mixture comprising 7-DHC, yeast sterol, cholestan-8-enol, lanosterol and / or cholestanol, wherein the percentage of 7-DHC is increased by at least about 2%, such as 4%, 5%, 7%, 10%, 15% or more compared to the percentage of said by-products in said sterol mixture in a cholesterol producing yeast cell expressing a (heterologous) desaturase as defined herein, i.e. a polypeptide having at least about 45%, such as at least 50%, 52%, 60%, 70%, 80%, 90%, 92%, 95%, 98% or up to 100% identity to SEQ ID NO: 2, more preferably a polypeptide expressed by a corresponding codon-optimized polynucleotide as defined herein, such as preferably a polypeptide obtainable from Pichia pastoris.

[0041] In the present invention, the increase in the percentage of 7-DHC in the sterol mixture is defined as the amount of 7-DHC produced by a host cell expressing a heterologous polypeptide having desaturase activity as defined herein, compared to a host cell expressing only an endogenous C-5 sterol desaturase (e.g., an endogenous C-5 sterol desaturase expressed by ERG3). When the host cell, such as yeast, particularly a cholesterol-producing yeast cell, is used in a sterol production process, the percentage of 7-DHC can be increased to at least about 84% based on the total amount of sterols produced by the host cell. In the present invention, "expression of an ERG3 homolog" includes the expression of additional copies of an ERG3 polypeptide, i.e., the expression of two or more copies of ERG3 (including additional copies of endogenous ERG3).

[0042] In a particular embodiment, the present invention relates to a method for producing a sterol mixture, wherein a yeast cell as described above is used, and wherein the percentage of cholestan-8-enol and / or yeast sterol and / or lanosterol and / or cholestanol present in the sterol mixture is reduced, i.e. in the range of about 16% or less based on the total amount of sterols, i.e. resulting in a higher ratio of 7-DHC in the sterol mixture.

[0043] In the method for producing the vitamin D3 precursor 7-DHC, a modified host cell capable of expressing other genes required for the biosynthesis of ERG3 homologues and vitamin D3 precursors and / or intermediates as defined herein is used. The modified host cell can be cultured in an aqueous medium supplemented with appropriate nutrients under aerobic or anaerobic conditions as known to the technician for the corresponding host cells producing cholesterol. Optionally, as known in the art, the culture is carried out in the presence of proteins and / or cofactors involved in electron transfer. The culture / growth of the host cell can be carried out in batch, fed-batch, semi-continuous or continuous mode. As known to those skilled in the art, depending on the host cell, preferably, the production of vitamin D3 and its precursors (e.g., 7-DHC) can vary. The cultivation and separation of 7-DHC and other intermediates in vitamin D3 production are described in, for example, WO2011067144 or WO2017108799.

[0044] When using a host cell as described herein, the productivity / specificity of the C-5 sterol desaturase activity can be shifted toward 7-DHC, resulting in a ratio of 7-DHC of at least about 84% of the total sterols produced by the host cell, with the titer of 7-DHC produced being as high as about 10 g / l after about 110 hours of fermentation under appropriate culture conditions.

[0045] The terms "ERG5" and "Erg5p" or "ERG6" and "Erg6p" are used interchangeably herein and refer to the polypeptides encoded by the corresponding genes erg3, erg5 and erg6.

[0046] Genes encoding ERG5, ERG6, ERG3, ARE1, ARE2 or sterol Δ24 reductase (ERG4) as used herein, the cultivation and genetic engineering of yeast cells as used herein are known and described, for example, in US Pat. No. 7,608,421.

[0047] In this context, 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 display such a methyl group at said position are not acceptable substrates for yeast ERG4.

[0048] The terms "C-8 sterol isomerase", "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. A preferred ERG2 homolog to be used in the modified host cell according to the present invention is a polypeptide having at least about 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: 14, said polypeptide exhibiting C-8 sterol isomerase activity and comprising a polynucleotide according to SEQ ID NO: 14, exhibiting C-8 sterol isomerase activity, comprising a polynucleotide encoding such a polypeptide, which can be obtained from Ustilago maydis. In particular, 1 or more copies, such as at least 1, 2, 3, 5 copies, of said ERG2 homologue are expressed in the modified host cell as defined herein.

[0049] 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 and / or product produced per defined amount of protein in a given time period and at a defined temperature. Typically, specific activity is expressed as the number of μmol of substrate consumed per minute per mg of 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 the 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. The technician knows how to measure enzyme activity, for example, by HPLC.

[0050] In connection with the present invention, it should 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).

[0051] In particular, the present invention is characterized by the following embodiments:

[0052] 1. A cholesterol-producing yeast cell comprising an enzyme having C5-sterol desaturase activity having at least about 45%, such as at least 50%, 52%, 60%, 70%, 80%, 90%, 92%, 95%, 98% or up to 100% identity to SEQ ID NO: 2 expressed (heterologously) in a suitable host cell for the production of 7-DHC, wherein the ratio of 7-DHC to by-products including lanosterol and / or cholestanol is increased by at least about 5% compared to an unmodified host cell.

[0053] 2. A cholesterol-producing yeast cell as described above, comprising an enzyme having C5-sterol desaturase activity, wherein the yeast cell produces a sterol mixture comprising at least about 84% 7-dehydrocholesterol (7-DHC), based on the total amount of sterols, preferably at least about 85%, 88%, 90%, 92%, 95%, 97%, 98% or up to 100% 7-DHC.

[0054] 3. A cholesterol producing yeast cell as described above, wherein the ratio of 7-DHC to cholester-7-enol and / or lanosterol is in the range of about 18.

[0055] 4. A cholesterol producing yeast cell as described above, wherein the ratio of 7-DHC to cholestan-7-enol and / or lanosterol is increased by at least about 5%.

[0056] 5. A cholesterol-producing yeast cell as described above, which expresses a heterologous enzyme having C5-sterol desaturase activity, said heterologous enzyme having at least about 45%, such as at least 50%, 52%, 60%, 70%, 80%, 90%, 92%, 95%, 98% or up to 100% identity to SEQ ID NO:2.

[0057] 6. A cholesterol-producing yeast cell as described above, wherein the cholesterol-producing yeast cell expresses a heterologous enzyme having C5-sterol desaturase activity, wherein the enzyme is selected from the group consisting of: Saccharomyces, such as Saccharomyces cerevisiae; Yarrowia, such as Yarrowia lipolytica; Kluyveromyces, such as Kluyveromyces lactis; Schizosaccharomyces, such as Schizosaccharomyces pombe; Pichia, such as Pichia pastoris; Candida, such as Candida albicans; Penicillium, such as Penicillium roqueforti; Aspergillus, such as Aspergillus nidulans; Cryptococcus, such as Cryptococcus neoformans; Giant Aspergillus; Metarhizium; and Ustilago, such as Ustilago maydis.

[0058] 7. A cholesterol-producing yeast cell as described above, wherein ERG5 and ERG6 are inactivated.

[0059] 8. A cholesterol-producing yeast cell as described above, wherein the yeast cell expresses 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.

[0060] 9. A cholesterol producing yeast cell as described above, wherein the yeast cell expresses a heterologous enzyme having C8-isomerase activity, preferably wherein the heterologous enzyme is obtainable from Ustilago maydis, more preferably from a polypeptide having at least about 42%, such as at least 43%, 44%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 75%, 80%, 90%, 92%, 95%, 98% or up to 100% identity with the polypeptide according to SEQ ID NO: 14.

[0061] 10. Use of a cholesterol-producing yeast cell as described above for the production of sterols, preferably for the production of a vitamin D3 precursor, more preferably for the production of 7-DHC.

[0062] 11. Use of a yeast cell for producing cholesterol as described above, wherein the 7-DHC is further converted into vitamin D3.

[0063] 12. The use as described above, wherein the 7-DHC is further converted into 25-hydroxyvitamin D3.

[0064] 13. A method for reducing the amount of cholestan-7-enol and / or lanosterol in a sterol mixture produced by a yeast cell, the method comprising expressing a heterologous enzyme having C5-sterol desaturase activity, the enzyme being selected from the group consisting of: Saccharomyces, such as Saccharomyces cerevisiae; Yarrowia, such as Yarrowia lipolytica; Kluyveromyces, such as Kluyveromyces lactis; Schizosaccharomyces, such as Schizosaccharomyces pombe; Pichia, such as Pichia pastoris; Candida, such as Candida albicans; Penicillium, such as Penicillium roqueforti; Aspergillus, such as Aspergillus nidulans; Cryptococcus, such as Cryptococcus neoformans; Aspergillus; Metarhizium; and Ustilago, such as Ustilago maydis, preferably selected from Pichia pastoris, Penicillium roqueforti, Schizosaccharomyces pombe or Saccharomyces cerevisiae.

[0065] 14. A method for producing a sterol mixture, preferably a vitamin D3 precursor, more preferably a sterol mixture having at least about 84% 7-DHC in a yeast cell, the method comprising:

[0066] (a) Inactivation of ERG5 and ERG6,

[0067] (b) expressing a heterologous enzyme selected from EC 1.3.1.72 having sterol Δ24-reductase activity towards cholesta-7,24-dienol, yeast sterol or trienol, preferably a plant or vertebrate sterol Δ24-reductase, more preferably a vertebrate sterol Δ24-reductase,

[0068] (c) expressing a heterologous enzyme having C5-sterol desaturase activity selected from the group consisting of: Saccharomyces, such as Saccharomyces cerevisiae; Yarrowia, such as Yarrowia lipolytica; Kluyveromyces, such as Kluyveromyces lactis; Schizosaccharomyces, such as Schizosaccharomyces pombe; Pichia, such as Pichia pastoris; Candida, such as Candida albicans; Penicillium, such as Penicillium roqueforti; Aspergillus, such as Aspergillus nidulans; Cryptococcus, such as Cryptococcus neoformans; Aspergillus; Metarhizium; and Ustilago, such as Ustilago maydis, preferably selected from Pichia pastoris, Penicillium roqueforti, Schizosaccharomyces pombe or Saccharomyces cerevisiae,

[0069] (d) culturing the yeast cell under conditions suitable for sterol production;

[0070] wherein the ratio of 7-DHC to cholestan-7-enol and / or lanosterol present in the sterol mixture is greater than 17.2.

[0071] The following examples are illustrative only and are not intended to limit the scope of the present invention in any way. Example

[0072] Example 1: General methods, strains and plasmids

[0073] All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York) or Ausubel et al. (1998. Current Protocols in Molecular Biology. Wiley: New York). The genotypes of the Saccharomyces cerevisiae strains and plasmids used are listed in Tables 1 and 2. The Saccharomyces cerevisiae strain Y2159 producing 7-DHC was constructed as described in Example 4. All strains listed are MATα.

[0074] Table 1: Saccharomyces cerevisiae strains.

[0075]

[0076] Table 2:Plasmids used for cloning ERG3 homologs.

[0077] Plasmids skeleton insert Oligonucleotide or source pMB7722 pMB7622 Saccharomyces cerevisiae ERG3 Synthetic Clips pMB7700 pMB7622 Pichia pastoris ERG3 Synthetic Clips pMB7721 pMB7622 Penicillium roqueforti ERG3 Synthetic Clips pMB7701 pMB7622 Schizosaccharomyces pombe ERG3 Synthetic Clips

[0078] Example 2: Cloning of various ERG3 homologs into Saccharomyces cerevisiae Y2159

[0079] All ERG3 cassettes were constructed as follows. The open reading frames were codon optimized based on the deduced amino acid sequence and synthesized using a 5'-BamHI (GGATCCatg...) site and a 3'-EcoRI site). These open reading frames were cloned by inserting the BamHI-EcoRI digested ERG3 fragment into BamHI-EcoRI digested pMB7621, which allowed targeting of the intergenic locus INT66 (approximately 769,000) on the right arm of chromosome XIII between the RKR1 gene and the GAD1 gene.

[0080] In addition to S. cerevisiae ERG3 (SEQ ID NO:7; plasmid pMB7677), the synthetic genes also included (codon-optimized) ERG3 homologs from Pichia pastoris (SEQ ID NO:9; plasmid pMB7732), ERG3 homologs from Penicillium roqueforti (SEQ ID NO:10; plasmid pMB7721), and ERG3 homologs from Schizosaccharomyces pombe (SEQ ID NO:11; plasmid pMB7681), see the sequence listing.

[0081] To test the effect of different ERG3 genes on 7-DHC production, hygromycin resistance (Hyg R ) as the selectable marker and using the strong constitutive TDH3 promoter as the control element, strain Y2159 was transformed at the INT66 locus with four different SfiI-generated fragments representing one of the four species detailed above.

[0082] After 3 days at 30°C, transformants were selected on YPD agar containing 200 mg / L hygromycin. The strains resulting from these transformations are listed above in Table 1. These strains were subsequently assayed for 7-DHC productivity and total 7-DHC sterol purity as described below.

[0083] Example 3: HPLC analysis of sterols from transformed strains

[0084] Strains were grown as follows. The strains to be tested were first inoculated onto YPD agar and incubated at 30°C for 48 hours. Two milliliters of YPD preculture were inoculated from these plates and grown on a roller wheel at 30°C for 24 hours. In a 24-well microtiter plate, 0.8 mL of YPD + 10 g / L ethanol was inoculated from the preculture to a final OD of 1.5. 600 The pH was 0.5. The microtiter plates were grown at 30°C in a humidified environment and shaken at 800 rpm on a shaker with a 3 mm orbit. 16 μl of ethanol was added to each well as a feed at 24 and 48 hours after inoculation. At 72 hours after inoculation, cells were sampled for sterol content.

[0085] Sterols were extracted from the culture and measured as follows. 80 microlitres of full fermentation broth were pipetted into 2mL Precellys tubes with glass beads. Eight hundred microlitres of saponification solution (5% KOH in ethanol) were added, and the sample was placed in a Precellys 24 homogenizer, and stirred for 3 cycles at 6500rpm, 15 seconds per cycle. Sixty microlitres of glacial acetic acid were then added, and the test tube was centrifuged at top speed for 1 minute. The sterol content of the supernatant was measured by HPLC. The results are shown in Table 3, Table 4 and Table 5.

[0086] Table 3: Ratio of 7-DHC to zymosterol in controls and strains carrying the ERG3 homolog.

[0087] strain Ratio of 7-DHC to Zymosterol SC2159-parent 18.1 Pichia pastoris ERG3 18.8

[0088] Table 3: Ratio of 7-DHC to cholestan-8-enol in controls and strains carrying an ERG3 homolog.

[0089] strain Ratio of 7-DHC to cholester-8-enol SC2159-parent 11.7 Pichia pastoris ERG3 12.1

[0090] Table 4: Ratio of 7-DHC to a mixture of lanosterol and cholestanol in controls and strains with an ERG3 homolog.

[0091] strain Ratio of 7-DHC to Lanosterol / Cholestanol SC2159-parent 17.2 Pichia pastoris ERG3 22.9 Penicillium roqueforti ERG3 19.8 Schizosaccharomyces pombe ERG3 18.1

[0092] Example 4: Construction of Y2159

[0093] Wild-type Saccharomyces cerevisiae ARE1 was synthesized by DNA2.0, with an XbaI site (TCTAGAACAAAatg...) incorporated at the 5' end and a PstI site incorporated at the 3' end. It was cloned into erg4Δ::Hyg using the unique XbaI and PstI sites. RThe deletion plasmid was then used to replace the HygR portion with LEU2 by KpnI-AgeI cloning. The result was plasmid pHyD459.

[0094] The Saccharomyces cerevisiae ARE1 mutant variant pMB7584 (F592L) was generated by ligating a PCR product produced by ARE1 and cut with BsrGI-BsaI (oligonucleotides according to SEQ ID NO: 16 and SEQ ID NO: 17) with a double-stranded oligonucleotide obtained by annealing SEQ ID NO: 19 and SEQ ID NO: 20 into pHyD459 cut with BsrGI-PstI. Similarly, the Saccharomyces cerevisiae ARE1 mutant variant pMB7585 (G595D) was generated by ligating a PCR product produced by ARE1 and cut with BsrGI-BsaI (oligonucleotides according to SEQ ID NO: 16 and SEQ ID NO: 18) with a double-stranded oligonucleotide obtained by annealing SEQ ID NO: 21 and SEQ ID NO: 22 into pHyD459 cut with BsrGI-PstI. Oligonucleotides used herein and other sequences are listed in Table 5.

[0095] Table 5: Plasmids used to construct ARE mutations. "Scer" refers to Saccharomyces cerevisiae.

[0096] Sequence Listing <110> DSM Intellectual Property Asset Management Ltd. <120> Optimization of C-5 Sterol Desaturation <130> 32960-WO-PCT <160> twenty two <170> PatentIn Version 3.5 <210> 1 <211> 1155 <212> DNA <213> Pichia pastoris <400> 1 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 acaagaagaa agatgtaaag acatggcgtt cacaagtcaa gcaggccgat 1080 tcgataagag aagacttaga gggaaaagaa gatttccgtg agtatggaac tgaggaaaaa 1140 cttaaaagca catag 1155 <210> 2 <211> 384 <212> PRT <213> Pichia pastoris (Pichia pastoris) <400> 2 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> 3 <211> 939 <212> DNA <213> Penicillium roqueforti <400> 3 atggatattt tcctggacgt tctagatact ttggtcctcg accggtgcta cgcagtactc 60 tcgccagacc caacagccat ctccaacaat gatactcaag ccactgccca tttgaaccaa 120 catgtcgggg tatattaccc tatgcagccc tcgaagtggg cggaggcaag cctctggaag 180 agagacgaca ttgccagaca agcattgtca ctgtacgtga ttatatggct tttcgcaatg 240 ataatgtacc tcctcggtag ccttctccta tatcacaccc tcttcgacaa aagactactc 300 caacacccgc gcttcctcgc acaccaagtc aagctcgaga tcaaccaagg catctccgca 360 atcccagtca tggccctcct caccgtccca ttctttctag ctgagataag gggctggtcg 420 aagctatatg atctcaccag cgactccccg ttcttcggat acaccttgct ccagtatcca 480 ttgttcatct gcttcacaga tagtggcatt tactggatac accgtggtct gcatcatcct 540 agcgtctatc gctggcttca caagccacac cataaatggg cggtgccgac tccgttcgct 600 agttatgcgt ttcaccctct ggatggatgg gcgcagagtc tcccctacca tgtttacccg 660 ttgctctttc cgttgcagaa gggagcatat ctgggactgt ttatgtttgt cactgtgtgg 720 acggtgctga ttcacgacgc tgagtacttg ccaacatcgg tggtaatcaa cggcgcttct 780 tgtcacacga tgcatcactt gtacttcaat tacaattacg ggcagtttac aacggcatgg 840 gatcgcctcg cgggaacata ccgaaagcct aagggggata gtttcatgga aggtcagcaa 900 atggatggga aagggaaact cggaggtaag tgtgaatag 939 <210> 4 <211> 312 <212> PRT <213> Penicillium roqueforti <400> 4 Met Asp Ile Phe Leu Asp Val Leu Asp Thr Leu Val Leu Asp Arg Cys 1 5 10 15 Tyr Ala Val Leu Ser Pro Asp Pro Thr Ala Ile Ser Asn Asn Asp Thr 20 25 30 Gln Ala Thr Ala His Leu Asn Gln His Val Gly Val Tyr Tyr Pro Met 35 40 45 Gln Pro Ser Lys Trp Ala Glu Ala Ser Leu Trp Lys Arg Asp Asp Ile 50 55 60 Ala Arg Gln Ala Leu Ser Leu Tyr Val Ile Ile Trp Leu Phe Ala Met 65 70 75 80 Ile Met Tyr Leu Leu Gly Ser Leu Leu Leu Tyr His Thr Leu Phe Asp 85 90 95 Lys Arg Leu Leu Gln His Pro Arg Phe Leu Ala His Gln Val Lys Leu 100 105 110 Glu Ile Asn Gln Gly Ile Ser Ala Ile Pro Val Met Ala Leu Leu Thr 115 120 125 Val Pro Phe Phe Leu Ala Glu Ile Arg Gly Trp Ser Lys Leu Tyr Asp 130 135 140 Leu Thr Ser Asp Ser Pro Phe Phe Gly Tyr Thr Leu Leu Gln Tyr Pro 145 150 155 160 Leu Phe Ile Cys Phe Thr Asp Ser Gly Ile Tyr Trp Ile His Arg Gly 165 170 175 Leu His His Pro Ser Val Tyr Arg Trp Leu His Lys Pro His His Lys 180 185 190 Trp Ala Val Pro Thr Pro Phe Ala Ser Tyr Ala Phe His Pro Leu Asp 195 200 205 Gly Trp Ala Gln Ser Leu Pro Tyr His Val Tyr Pro Leu Leu Phe Pro 210 215 220 Leu Gln Lys Gly Ala Tyr Leu Gly Leu Phe Met Phe Val Thr Val Trp 225 230 235 240 Thr Val Leu Ile His Asp Ala Glu Tyr Leu Pro Thr Ser Val Val Ile 245 250 255 Asn Gly Ala Ser Cys His Thr Met His His Leu Tyr Phe Asn Tyr Asn 260 265 270 Tyr Gly Gln Phe Thr Thr Ala Trp Asp Arg Leu Ala Gly Thr Tyr Arg 275 280 285 Lys Pro Lys Gly Asp Ser Phe Met Glu Gly Gln Gln Met Asp Gly Lys 290 295 300 Gly Lys Leu Gly Gly Lys Cys Glu 305 310 <210> 5 <211> 903 <212> DNA <213> Schizosaccharomyces pombe <400> 5 atggactacc tactcaacta tgctgaccaa tatgcgctgg attcgatata caatgctgta 60 tatccattag ctcgcgacaa tatcgttaga cagtcgatca gtttgttttt tttaacttgg 120 tttggcggta tgttcttgta tttaacattt gcgtcgcttt cctaccaatt tgtgtttgat 180 aaaagtctga tggatcaccc aaagttctta aaaaccagg tgttcatgga agttctaacg 240 gctttacaaa acttacctgg tatggcgtta ttgacggttc cgtggttttt ggctgagttg 300 catgggtaca gctacttata cgacaacatc agtgattacg gtttaaaata cttcttatgt 360 tccttacctc ttttgtcat gttctcagat tttggcattt actgggctca tcgtttcctt 420 catcaccgtt atgtataccc tcgtcttcac aaactccatc ataagtggat tatctgcact 480 ccatatgcat cccatgcttt caaatccgct gatggcttct tacaatctct tccttaccat 540 cttttcccct ttttctttcc ccttcacaag ttgacctact tggctctttt caccttgtc 600 aacttctggt ccatcatgat tcacgatggt aaatacatct ccaacaaccc catcatcaat 660 ggtgctgctc accataatgg ccatcacatt tatttcaact acaattacgg ccaattcacc 720 accctttg atcgcctcgg caactctttc cgggccccg atgaggcatg gtttgacaaa 780 gatcttcgcc aaaacgagga tgttcttcgt gtcgaattga tggagtacga ggctattcgt 840 aatgaagttg aaggtgatga tgatagagaa tacatcgcta actctgctaa gaagaaccat 900 city ​​903 <210> 6 <211> 300 <212> PRT <213> Schizosaccharomyces pombe <400> 6 Met Asp Tyr Leu Leu Asn Tyr Ala Asp Gln Tyr Ala Leu Asp Ser Ile 1 5 10 15 Tyr Asn Ala Val Tyr Pro Leu Ala Arg Asp Asn Ile Val Arg Gln Ser 20 25 30 Ile Ser Leu Phe Leu Thr Trp Phe Gly Gly Met Phe Leu Tyr Leu 35 40 45 Thr Phe Ala Ser Leu Ser Tyr Gln Phe Val Phe Asp Lys Ser Leu Met 50 55 60 Asp His Pro Lys Phe Leu Lys Asn Gln Val Phe Met Glu Val Leu Thr 65 70 75 80 Only Gln Asn and Pro Gly Met Only Thr Val Pro Trp Phe 85 90 95 Leu Ala Glu Leu His Gly Tyr Ser Tyr Leu Tyr Asp Ile Ser Asp 100 105 110 Tyr Gly Leu Lys Tyr Phe Leu Cys Ser Leu Pro Leu Phe Val Met Phe 115 120 125 Ser Asp Phe Gly Ile Tyr Trp Ala His Arg Phe Leu His His Arg Tyr 130 135 140 Val Tyr Pro Arg Leu His Lys Leu His His Lys Trp Ile Ile Cys Thr 145 150 155 160 Pro Tyr Ala Ser His Ala Phe Lys Ser Ala Asp Gly Phe Leu Gln Ser 165 170 175 Leu Pro Tyr His Leu Phe Pro Phe Phe Phe Pro Leu His Lys Leu Thr 180 185 190 Tyr Leu Ala Leu Phe Thr Phe Val Asn Phe Trp Ser Ile Met Ile His 195 200 205 Asp Gly Lys Tyr Ile Ser Asn Asn Pro Ile Ile Asn Gly Ala Ala His 210 215 220 His Asn Gly His His Ile Tyr Phe Asn Tyr Asn Tyr Gly Gln Phe Thr 225 230 235 240 Thr Leu Phe Asp Arg Leu Gly Asn Ser Phe Arg Ala Pro Asp Glu Ala 245 250 255 Trp Phe Asp Lys Asp Leu Arg Gln Asn Glu Asp Val Leu Arg Val Glu 260 265 270 Leu Met Glu Tyr Glu Ala Ile Arg Asn Glu Val Glu Gly Asp Asp Asp 275 280 285 Arg Glu Tyr Ile Ala Asn Ser Ala Lys Lys Asn His 290 295 300 <210> 7 <211> 1098 <212> DNA <213> Saccharomyces cerevisiae <400> 7 atggatttgg tcttagaagt cgctgaccat tatgtcttag acgacttgta cgctaaagtt 60 ctgcccgctt cgttggcagc taatattcct gtcaagtggc agaaattgct agggttgaac 120 agtgggttca gcaattctac gattttgcag gagactttga actccaagaa tgccgtcaaa 180 gaatgtagaa ggttctacgg gcaggtgcca ttcctgtttg atatgtcgac gacgtctttt 240 gcatcgctat tgcctcgttc cagcatcttg agagaattcc tctcactatg ggttattgtt 300 acgatctttg gtttactact ttacttattc acggctagtc tcagctacgt gtttgtgttt 360 gacaagtcga ttttcaacca tcctcgttac ttgaaaaacc aaatggcaat ggaaatcaag 420 ttggcagtca gtgctatccc atggatgtcg atgttgaccg ttccatggtt tgttatggaa 480 ttgaacggcc attctaaact atacatgaag attgattatg aaaaccacgg tgtaaggaag 540 ctcattatcg agtacttcac tttcatcttt ttcactgatt gcggtgtgta tttagcgcac 600 agatggttgc attggccaag ggtctaccgt gctctgcaca agcctcatca caagtggctg 660 gtctgcacac ctttcgcatc tcattctttc catcctgtag acgggttttt gcaatccatc 720 tcgtaccaca tctacccatt gattctgcca ttacacaagg tttcttattt gattctgttc 780 acttttgtta acttttggac tgttatgatt catgacggtc aatacctatc aaacaatcct 840 gccgtcaacg gtactgcctg ccacacggtt caccatctat atttcaacta caactacggt 900 caattcacca ctctgtggga cagactaggg ggttcttacc gtagaccaga tgactcattg 960 tttgatccta agttaagaga tgctaaggag acctgggacg ctcaagttaa ggaagttgaa 1020 catttcatca aggaggtcga aggtgatgat aatgatagaa tctatgaaaa cgacccaaat 1080 accaagaaga acaactga 1098 <210> 8 <211> 365 <212> PRT <213> Saccharomyces cerevisiae <400> 8 Put Asp Leu Val Leu Glu Val Wing Asp His Tyr Val Leu Asp Asp Leu 1 5 10 15 Tyr Ala Lys Val Leu Pro Ala Ser Leu Ala Ala Donkey Ile Pro Val Lys 20 25 30 Trp Gln Lys Leu Leu Gly Leu Asn Ser Gly Phe Ser Asn Ser Thr Ile 35 40 45 Leu Gln Glu Thr Leu Asn Ser Lys Asn Ala Val Lys Glu Cys Arg Arg 50 55 60 Phe Tyr Gly Gln Val Pro Phe Leu Phe Asp Met Ser Thr Thr Ser Phe 65 70 75 80 Ala Ser Leu Leu Pro Arg Ser Ser Ile Leu Arg Glu Phe Leu Ser Leu 85 90 95 Trp Val Ile Val Thr Ile Phe Gly Leu Leu Leu Tyr Leu Phe Thr Ala 100 105 110 Ser Leu Ser Tyr Val Phe Val Phe Asp Lys Ser Ile Phe Asn His Pro 115 120 125 Arg Tyr Leu Lys Asn Gln Met Ala Met Glu Ile Lys Leu Ala Val Ser 130 135 140 Ala Ile Pro Trp Met Ser Met Leu Thr Val Pro Trp Phe Val Met Glu 145 150 155 160 Leu Asn Gly His Ser Lys Leu Tyr Met Lys Ile Asp Tyr Glu Asn His 165 170 175 Gly Val Arg Lys Leu Ile Ile Glu Tyr Phe Thr Phe Ile Phe Phe Thr 180 185 190 Asp Cys Gly Val Tyr Leu Ala His Arg Trp Leu His Trp Pro Arg Val 195 200 205 Tyr Arg Ala Leu His Lys Pro His His Lys Trp Leu Val Cys Thr Pro 210 215 220 Phe Ala Ser His Ser Phe His Pro Val Asp Gly Phe Leu Gln Ser Ile 225 230 235 240 Ser Tyr His Ile Tyr Pro Leu Ile Leu Pro Leu His Lys Val Ser Tyr 245 250 255 Leu Ile Leu Phe Thr Phe Val Asn Phe Trp Thr Val Met Ile His Asp 260 265 270 Gly Gln Tyr Leu Ser Asn Asn Pro Ala Val Asn Gly Thr Ala Cys His 275 280 285 Thr Val His His Leu Tyr Phe Asn Tyr Asn Tyr Gly Gln Phe Thr Thr 290 295 300 Leu Trp Asp Arg Leu Gly Gly Ser Tyr Arg Arg Pro Asp Asp Ser Leu 305 310 315 320 Phe Asp Pro Lys Leu Arg Asp Ala Lys Glu Thr Trp Asp Ala Gln Val 325 330 335 Lys Glu Val Glu His Phe Ile Lys Glu Val Glu Gly Asp Asp Asn Asp 340 345 350 Arg Ile Tyr Glu Asn Asp Pro Asn Thr Lys Lys Asn Asn 355 360 365 <210> 9 <211> 1155 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae codon-optimized Pichia pastoris ERG3 <400> 9 atggatatcg ctttggaaat cttggatacc tttgttttcg acaaggttta tgctaaattg 60 ttgccaattt ccttggtcca acatttgcca gatggttattgaaaacctt gggtcatttg 120 actggtgcca acaacaccat ggaatccttg ttcggtattg ctccaaacgt tgaccaagcc 180 tctaagaacc actggttgcg tactgttaac gattccattg ctttggccag acctggtgaa 240 cgtttggtct acggtgtcaa cgctcctttg cattttttcg atgaaaccgc ttatacttac 300 gcttccatct tgggtcgttc caatatcatt cgtcaattca ctactttgat gatcttgatg 360 attttgttcg gttggggttt gtatttgtct gtcgcttctt tttcctatta ctttgttttt 420 gataaggcta ttttcaacca tccaagatac ttgaagaacc aaatgtcctt ggaaatccat 480 caagccttga ctgctattcc taccatggtt ttgttgactg ttccatggtt tttgatcgaa 540 ttgcgtggtt actctaagtt atactttgat gttaatgaat ccactggtgg ttggaaggct 600 attatttggc aaattccttg tttcattatg tttaccgatt gttgtatcta ctttattcat 660 cgttggttgc actggccatc cgtttacaag cgtttgcaca agcctcacca caagtggatt 720 gtttgtactc catttgcttc tcatgccttc catccagttg atggttatgc tcaatctttg 780 ccttaccatt tgtatggtat gttgtttcca ttgcacaagg tttcctactt gatcttgttt 840 ggtttggtca acttttggac tgttatgatc catgatggtg aatacttgtc ccgtgatcct 900 attgtcaatg gtgctgcttg tcatactgtc catcacttgt acttcaacta caattacggt 960 caattcacta cttgtggga ccgtttgggt ggttcttaca gaatgccaga caaggaattg 1020 ttcgataaga acaagaagaa agatgttaag acttggcgtt ctcaagtcaa gcaagccgac 1080 tctattagag aagacttgga aggtaaagaa gatttccgtg aatacggtac tgaagaaaaa 1140 ttgaaatcca cctaa 1155 <210> 10 <211> 939 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae-codon-optimized Penicillium roqueforti ERG3 <400> 10 atggatattt tcttggacgt tttggatact ttggtcttgg acagatgcta cgctgtcttg 60 tccccagacc caactgccat ctccaacaat gatactcaag ccactgccca tttgaaccaa 120 catgtcggtg tctattaccc tatgcaacct tccaagtggg ccgaagcctc cttgtggaag 180 cgtgatgaca ttgccagaca agccttgtcc ttgtacgtca ttatttggtt gttcgctatg 240 attatgtact tgttgggttc cttgttgttg taccatacct tgttcgacaa aagattgttg 300 caacacccaa gattcttggc ccaccaagtc aagttggaaa tcaaccaagg tatctccgct 360 atcccagtca tggccttgtt gaccgtccca ttctttttgg ctgaaatccg tggttggtcc 420 aagttgtatg acttgacctc cgactcccca ttcttcggtt acaccttgtt gcaataccca 480 ttgttcatct gcttcactga ttccggtatt tactggattc accgtggttt gcaccatcct 540 tccgtctacc gttggttgca caagccacac cataaatggg ccgtccctac ccctttcgct 600 tcttatgctt tccacccttt ggacggttgg gcccaatcct tgccatacca tgtttaccca 660 ttgttgttcc cattgcaaaa gggtgcttac ttgggtttgt tcatgtttgt cactgtctgg 720 accgtcttga tccatgacgc cgaatacttg ccaacctctg tcgttatcaa cggtgcctct 780 tgtcacacca tgcatcactt gtacttcaat tacaattacg gtcaattcac tactgcctgg 840 gaccgtttgg ccggtactta ccgtaagcct aagggtgact ctttcatgga aggtcaacaa 900 atggacggta aaggtaagtt gggtggtaag tgtgaataa 939 <210> 11 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae codon-optimized Schizosaccharomyces pombe ERG3 <400> 11 atggatgttg tcttgcaata cgctgataaa tacgtttttg acacctttta cggtaagatt 60 gctgaatcct tcgattcctc ttcctctttt gctaatactg ctgttaattc taccaccttg 120 ggtttggctg aaaaggttaa ctttgctatc acctccggtt tgttagatcg taacaatgtc 180 tggcgtcaat tcacctcctt gttcttgatc acctggatta tgggtacttt gtcttacttt 240 ttgtctgcct cttttgctta ttacgtttac tttgatcgtg aagaagccag acgtcaccct 300 aagttttga aaaaccaaga acacttggaa ttgatggttg ctttgaaaaa cttgccaggt 360 atggctatt tgaccgctcc ttggttctta gctgaaattc gtggttatgg ttatgttat 420 gataagttgg atgaatatgg ttattctat ttgttcttttt ccatcgcctt gttcttgttg 480 ttttctgatt ttttgatta ctggattcac cgtgctttgc atcatcgttg gttgtacgct 540 cctttgcata agttgcatca caaatggatt gttccaactc cttactcttc tcacgctttt 600 cattattg atggttactc tcaatccttg ccatatcata tgttcccatt tttcttccca 660 ttaaacaaat acgtttattt gttgttgttt ggttctgtta attactggac tgtcttgatc 720 cacgacggta agtacttttc taacaacgct gtcgttaatg gtgctgctca tcacgctgct 780 caccatatgt actttaacta taactatggt caattcttca ccttgtttga tcgtttgtgc 840 tcttcttaca gacaaccaga ccaagaatta ttcgatgccg aattgcgtaa cgaaaaattg 900 caagaacaac gtatccgttt catggaaact gtccaatata ccgtcgaagg taaagatgac 960 cgtacttacg cttccaagaa ggataactaa 990 <210> 12 <211> 610 <212> PRT <213> Saccharomyces cerevisiae <400> 12 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 Ile 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 Asn 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 Tyr Gln Ile Asn Tyr Pro Arg Thr Ser Arg Ile 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 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 The Lion 610 <210> 13 <211> 1833 <212> DNA <213> Saccharomyces cerevisiae <400> 13 atgacggaga ctaggattt gttgcagac gagagtttc ttagatccg cagactcaat 60 tccgcagaag ccacaacg gcattcggtc acgtacgata acgtgatcct gccacaggag 120 tccatggagg ttcgccacg gtcgtctacc acgtcgctgg tggagccagt ggagtcgact 180 tall gtcgactga long gtggactga long tall gtggactg 240 taccctgtgg acgcccacat gcaaagtac ctttcacacc tgagagca gtctcggtcg 300 aggttccacc gaaaggatgc tagcaagtat gtgtcgtttt tggggacgt gagttttgat 360 cctcgcccca cgctcctgga cagcgccatc aacgtgccct tccacgac ttcaaggt 420 ccggtgctgg agaacagct caaaattta cagttgacaa agaccagac caggccacg 480 gtgaagacta cggtgagac tacggaaa 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> 14 <211> 241 <212> PRT <213> Ustilago maydis <400> 14 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 Ile Leu 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 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 Phe <210> 15 <211> 726 <212> DNA <213> Ustilago maydis <400> 15 atggcatcgc atagaccacg cagcaacaag gctgccaatg gtgcttcgac ttcacccaaa 60 cgcagctgga taattgtctc agctgcgctc gttggcttct gcgctctcat cgccgctctc 120 gattcgatcc gatccagctt ctacatcttt gaccacaagg caatctacaa gatcgcatcg 180 actgcggtcg 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 aatacgcttt cccagctggt 480 gcgctcaagg cggagcacta ccctgccgga tcagtgcacc atcttcgccg cggaacggtc 540 aagcagtaca tgatgcctga agacggctgc tgggcgctcg agcttgctca gggctggatc 600 ccacccatgc ttccctttgg tctcgccgat gtgctcagct cgacgctcga cctgcccacc 660 tttggtatca ctgtctggat cactgcacga gaaatggttg gcaatctgct catcggcaag 720 ttttga 726 <210> 16 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 16 tgttctgtac atgctcacgt tttac 25 <210> 17 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 17 cacacggtct cacaagacaa cgttggacaa ctgc 34 <210> 18 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 cacacggtct caatcaaacg aaaagacaac gttggac 37 <210> 19 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 19 cttgtcgttt ggtgtctgtt cagggcccag tatcattatg acgttgtacc tgaccttatg 60 ACTGCA 66 <210> 20 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 20 gtcataaggt caggtacaac gtcataatga tactgggccc tgaacagaca ccaaacga 58 <210> twenty one <211> 57 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty one tgatgtctgt tcagggccca gtatcattat gacgttgtac ctgaccttat gactgca 57 <210> twenty two <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty two gtcataaggt caggtacaac gtcataatga tactgggccc tgaacagac 49

Claims

1. A cholesterol-producing yeast cell, the cholesterol-producing yeast cell expressing an enzyme with improved C5-sterol desaturase activity, expressing a heterologous enzyme selected from EC 1.3.1.72 having sterol Δ24 reductase activity, and ERG5 and ERG6 are inactivated, the enzyme with improved C5-sterol desaturase activity having 100% identity to SEQ ID NO: 2, 4 or 6 heterologously expressed in a suitable host cell for producing 7-dehydrocholesterol (7-DHC), wherein the ratio of 7-DHC to by-products including lanosterol and / or cholestanol is increased by at least 5% compared to a control host cell, the cholesterol-producing yeast cell differing from the control host cell only in that the control host cell does not express the enzyme with improved C5-sterol desaturase activity.

2. The cholesterol-producing yeast cell of claim 1, comprising an enzyme having C5-sterol desaturase activity, wherein the yeast cell produces a sterol mixture comprising at least 84% 7-DHC based on the total amount of sterols.

3. The cholesterol producing yeast cell according to claim 1 or 2, wherein the ratio of 7-DHC to cholester-7-enol and / or lanosterol is in the range of about 18.

4. The cholesterol-producing yeast cell according to claim 1 or 2, wherein the ratio of 7-DHC to cholester-7-enol and / or lanosterol is increased by at least 5%.

5. The cholesterol-producing yeast cell according to any one of claims 1 to 4, wherein the heterologous enzyme is derived from a plant or a vertebrate.

6. The cholesterol-producing yeast cell of claim 5, wherein the heterologous enzyme is derived from human, pig, dog, mouse, rat, horse or zebrafish.

7. The cholesterol-producing yeast cell according to any one of claims 1 to 6, wherein the yeast cell expresses a heterologous enzyme having C8-isomerase activity.

8. Use of the cholesterol-producing yeast cell according to any one of claims 1 to 7 for the production of sterols.

9. Use of the cholesterol-producing yeast cell according to any one of claims 1 to 7 for producing a vitamin D3 precursor.

10. Use of the cholesterol-producing yeast cell according to any one of claims 1 to 7 for producing 7-DHC.

11. Use of a cholesterol-producing yeast cell according to claim 10, wherein the 7-DHC is further converted into vitamin D3.

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

13. A method for reducing the amount of cholestan-7-enol and / or lanosterol in a sterol mixture produced by a yeast cell, the method comprising expressing in the yeast cell a heterologous enzyme having 100% identity to SEQ ID NO: 2, 4 or 6 with improved C5-sterol desaturase activity, the method comprising: (a) Inactivation of ERG5 and ERG6, (b) expressing a heterologous enzyme selected from EC 1.3.1.72 having sterol Δ24-reductase activity towards cholesta-7,24-dienol, yeast sterol or trienol, (c) cultivating the yeast cell under conditions suitable for sterol production, and (d) isolating a sterol mixture having at least about 84% 7-DHC based on total sterols.

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