Novel method

Abstract

The present invention relates to the fermentative production of hydroxylated derivatives of vitamin D3 in oleaginous host cells, in particular hosts selected from Yarrowia lipolytica.

Description

[0001] This invention relates to the fermentation production of hydroxylated derivatives of vitamin D3 in oil-producing host cells, particularly in hosts selected from Yarrowia lipolytica.

[0002] Vitamin D3 (also known as cholecalciferol or cholecalciferol) can be synthesized in mammalian skin from provitamin D3 (also known as 7-dehydrocholesterol or 7-DHC), a product of cholesterol biosynthesis upon exposure to ultraviolet (UV) light, whereby 7-DHC is photochemically converted to provitamin D3, which is isomerized at body temperature to the biologically active form, vitamin D3. In the liver, vitamin D3 is converted to the biologically active 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 at the 1α-position to produce 1α,25-dihydroxyvitamin D3 (also known as calcitriol) occurs primarily in the kidneys.

[0003] For the industrial production of vitamin D3, both chemical and biotechnological synthesis are (in principle) available, whereby starting materials are converted into pre-vitamin D3 (7-DHC) through a multi-step process and further processed into various forms of vitamin D3. Chemical synthesis begins with the isolation of cholesterol from, for example, lanolin, which is then dehydrogenated to 7-DHC, followed by ultraviolet light exposure and further purification / extraction / conversion steps to produce vitamin D3 or its hydroxylated form. Alternatively, modified yeast strains can be used to produce vitamin D3 precursors, including 7-DHC, which are then isolated and further converted into various forms of vitamin D3.

[0004] However, several challenges must be addressed before using yeast as the putative production strain in such biological processes: First, yeast, particularly the genus *Saccharomyces*, tends to store excess sterols such as 7-DHC and other intermediates in intracellular organelles (so-called liposomes), thus requiring further isolation and / or purification steps and resulting in relatively low yields of the target product. The balance between free sterols and sterols stored in liposomes (in the form of sterols or sterol esters) is triggered by the action of several proteins, such as, for example, sterol acyltransferases. Due to the non-specific action of these enzymes, the library of sterol esters stored in liposomes is relatively diverse, including but not limited to esters of, for example, ergosterol, yeast sterol, lanosterol, enolanol, cholesterol-5,7,24(25)-trienol, cholesterol-8-enol, or 7-DHC.

[0005] Second, sterol biosynthesis in yeast, including the genus *Saccharomyces*, is a multi-step process, resulting in a mixture of compounds, with only 7-DHC available for conversion into vitamin D3 or its hydroxylated derivatives. Therefore, to obtain sufficient production of the 7-DHC precursor cholesterol-5,7,24(25)-trienol, yeast cells must be modified by introducing a double knockout in the genes encoding ERG5 and ERG6 to block the competitive accumulation of ergosterol and enhance the flux to 7-DHC.

[0006] Third, in addition to the desired production of cholesterol-5,7,24(25)-trienol, yeast and other fungal cells will still accumulate more or less equivalent amounts of byproducts, such as yeast sterols, thereby reducing the yield and purity of the desired product and requiring further cumbersome and expensive purification steps.

[0007] Furthermore, clinical studies have demonstrated that supplementation with hydroxylated vitamin D3, such as calcidiol, is up to five times more effective in increasing blood levels of 25-hydroxylated vitamin D3 compared to supplementation with an equivalent dose of cholecalciferol (see, for example, Graeff-Armas et al., J Nutr. 150 (1):73-81, 2020).

[0008] Therefore, there is a strong need to develop a sustainable and cost-effective fermentation method for the production of 25-hydroxylated vitamin D3, thereby avoiding chemical hydroxylation and / or cumbersome purification steps, in which the flux toward 7-DHC available in the free sterol pool will be kept at a high level, but the accumulation of unwanted byproducts will be kept at a low level.

[0009] Surprisingly, we have now discovered that oil-producing yeasts, particularly Yersinia lipolytica, are ideal host organisms for the fermentation production of hydroxylated derivatives of vitamin D3, especially 25-hydroxylated 7-DHC (HyDHC), which will be used for further conversion to 25-hydroxylated vitamin D3 (HyD).

[0010] Compared to the production of 25-hydroxylated vitamin D3 in Saccharomyces cerevisiae, we were able to obtain higher yields and higher purity after introducing genetic modifications as defined herein (see, for example, disclosed in WO2011067144, in which cholesterol 25-hydroxylase from wild boar (S. scrofa) has been integrated; see SEQ ID NO: 27 in WO2011067144).

[0011] Specifically, the present invention relates to the production of the 25-hydroxylated form of vitamin D3, wherein a host cell (such as an oleogenous yeast, preferably Yersinia lipophila) that produces a mixture of sterols containing 7-DHC is genetically modified by introducing an enzyme having sterol C-25 hydroxylase activity, thereby enabling 7-DHC to be C25-hydroxylated to HyDHC.

[0012] Furthermore, surprisingly, the expression of heterologous enzymes that catalyze the acetylation of 7-DHC or its hydroxylated forms, particularly in host cells that produce 7-DHC, preferably in Yersinia lipolytica, of which enzymes with acetyltransferase 2 activity, can further enhance the production of sterols, including 7-DHC and hydroxylated forms of vitamin D3, as well as the purity of such hydroxylated products.

[0013] The terms “sterol” or “sterol mixture” are used interchangeably herein and refer to a mixture containing precursors, intermediates, and derivatives of the vitamin D3 biosynthetic pathway, such as, for example, cholesterol-5,7,24(25)-trienol, yeast sterol, lanosterol, encholestanol, cholesterol-5,8,24(25)-trienol, 7-DHC, HyDHC, HyD, including various amounts of acetylated forms of such compounds. In particular, the sterol mixture produced according to the invention contains hydroxylated forms of vitamin D3, particularly at least about 70-80% of hydroxylated 7-DHC, including HyDHC and HyDHC-acetate, based on total sterols in the mixture produced from recombinant host cells under the conditions defined herein.

[0014] As used herein, terms such as “precursor” or “intermediate” are used interchangeably and refer to all possible biochemical entities in the process from starting material (carbon source) (such as, for example, selected from the group consisting of glucose, vegetable oil, ethanol, acetyl-CoA, and mixtures thereof) toward a target product (such as, for example, vitamin D3). An important intermediate in the vitamin D3 biosynthetic pathway is 7-DHC.

[0015] As used herein, the terms "derivative of vitamin D3" or "form of vitamin D3" refer to acetylated and / or hydroxylated forms of vitamin D3, such as, for example, HyD. It also includes, but is not limited to, derivatives of 7-DHC, such as, for example, HyDHC, 7-DHC-acetate, or HyDHC-acetate. Therefore, HyDHC or HyDHC-acetate are intermediates of HyD, which is referred to herein as vitamin D3 or intermediates of vitamin D3 forms.

[0016] The suitable host cell according to the invention can be any lipogenic yeast, i.e., a yeast strain that exhibits an lipogenic phenotype under certain conditions, wherein the host cell is preferably selected from Yeastia lipolytica, the host cell being genetically modified such that the host cell is able to produce sterols containing at least about 70-80% 7-DHC based on total sterols, and the host cell is also able to express heterologous enzymes involved in the 25-hydroxylation and / or acetylation of various forms of vitamin D3, particularly 7-DHC. Besides *Yarrowia lipolytica*, suitable host cells could be selected from lipogenic yeasts, such as *Cystobasidium oligophagum*, *Candida bombicola*, *Lipomyces starkeyi*, *Lipomyces doorenjongii*, *Lipomyces kockii*, *Lipomyces lipofer*, *Lipomyces soligophaga*, *Lipomyces spencermartinsiae*, *Lipomyces tetrasporus*, *Leucosporidium creatinivorum*, *Cyberlindnera saturnus*, *Wickerhamomyces ciferrii*, *Aureobasidium pullulans*, and *Trichosporon*. Fermentans, Trichosporon cutaneum, Trichosporon dermatis, Geotrichum histeridarum, Geotrichum vulgare, Magnusiomyces magnusii, Pichia, Apiotrichum brassicae, Candida curvata, Candida aff.Candida tenuis, Candida krusei, Candida tropicalis, Candida guilliermondii, Schwanniomyces occidentalis, Kluyveromyces sp., Kurtzmaniella cleridarum, Sporidiobolus johnsonii, Sporidiobolus pararoseus, Sporidiobolus ruineniae, Sporidiobolus salmonicolor, Sporobolomyces bannaensis, Sporobolomyces carnicolor, Rhodosporidium fluviale, Rhodosporidium babuye Strains of *Rhodotorula babjevae*, *Rhodotorula aurantiaca*, *Rhodotorula glacialis*, *Rhodotorula bogoriensis*, *Rhodotorula diobovata*, *Rhodotorula glutinis*, *Rhodotorulaterpenoidalis*, *Rhodotorula minuta*, *Rhodotorulatoruloides*, *Cryptococcus neoformans*, *Toluraspora delbrueckii*, and *Rhodosporidiobolus colostri* are listed (for further information, see Savador Lopéz et al., Yeast; 39:553-606, 2022). Another suitable host cell may be selected from sterol-producing *Saccharomyces cerevisiae*, including but not limited to host cells based on *Saccharomyces cerevisiae* strains ATCC 200062, ATCC 2345, BY4741, and RF11, provided that, in the case of *Saccharomyces cerevisiae*, the endogenous enzyme expressing acetyltransferase 2 (ATF2) is preferably inactive and / or replaced by a heterologous or mutated ATF2 homolog.

[0017] The terms “oil-producing yeast” or “yeast strain exhibiting an oil-producing phenotype under certain conditions”, used interchangeably herein, refer to certain strains capable of accumulating large amounts of lipids intracellularly or extracellularly, wherein the term “lipid” is defined as a condensation of all or part of isoprene units or ketoacyl thioesters, and wherein at least about 20% of the cell dry weight is accumulated in the form of lipids. Such oil-producing yeasts intended to be used as host cells for carrying out the present invention can be natural or genetically engineered strains, such as those acquired through artificial means (e.g., mutagenesis) to produce oil.

[0018] The terms “host cell” and “host strain” are used interchangeably in this article.

[0019] As used herein, the term "enzyme having cholesterol 25 hydroxylase activity," "enzyme involved in hydroxylation," "enzyme catalyzing the hydroxylation of a substrate," or "enzyme catalyzing the C25-hydroxylation of a substrate" preferably includes cholesterol 25 hydroxylases that catalyze the monohydroxylation of C25 in 7-DHC, more preferably enzymes isolated from vertebrates (such as from humans or mice as described in US6562609, or from other vertebrate sources including rats or pigs as described in WO2011067144) (see in particular SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30 and Table 15 in the cited references).

[0020] Preferably, the host cell, as defined herein, contains cholesterol 25-hydroxylase (CH25H) having at least about 60%, such as 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with the enzyme (SsCH25H) from wild boar (Sus scrofa) according to SEQ ID NO: 1, obtainable via expression of the polynucleotide according to SEQ ID NO: 2. Useful enzymes according to the invention involved in the hydroxylation of vitamin D3 precursors (such as 7-DHC) may also include enzymes having equivalent activity to the enzyme according to SEQ ID NO: 1, but isolated or derived from other natural sources, or even synthesized with the aid of artificial intelligence. To achieve the expression and therefore activity of such enzymes, codon optimization may be necessary depending on the appropriate host cell.

[0021] As used herein, the terms "enzyme with acetyltransferase 2 activity," "enzyme involved in acetylation," or "enzyme that catalyzes acetylation," as defined and used interchangeably herein, preferably include alcohol acetyltransferase 2, more preferably enzymes isolated from yeasts, particularly *Saccharomyces cerevisiae* (Nagasawa et al., Biosci Biotechnol Biochem 62 (10:1852-7, 1998)). It includes enzymes that exhibit catalytic activity equivalent to *Saccharomyces cerevisiae* ATF2 in the acetylation of 7-DHC or HyDHC.

[0022] Preferably, the host cell, as defined herein, contains acetyltransferase 2 (ATF2), which has at least about 60%, such as 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with the enzyme from *Saccharomyces cerevisiae* according to SEQ ID NO:3, and can be obtained by expression of the polynucleotide according to SEQ ID NO:4. The useful enzymes according to the invention involved in the acetylation of vitamin D3 forms (such as 7-DHC or HyDHC) may also include enzymes having equivalent activity to the enzyme according to SEQ ID NO:3, but isolated or derived from other natural sources, or even synthesized with the aid of artificial intelligence. To achieve the expression and therefore the activity of such enzymes, codon optimization may be required depending on the appropriate host cell.

[0023] As used herein, the term "heterologous" in relation to the expression of polynucleotides or polypeptides (including enzymes) means the expression of non-endogenous polynucleotides (i.e., polynucleotides not originally / naturally expressed in the species in question). Such heterologous polynucleotides can be introduced into the corresponding host cells according to known methods, thereby producing "genetically modified" or "recombinant" host cells. Techniques for generating such genetically modified host cells, including host cells expressing heterologous enzymes as defined herein, preferably selected from host cells of *Yarrowia lipolytica* expressing heterologous CH25H or ATF2, are known in the art and depend on the given host cell. Preferably, such heterologous nucleic acid molecules are codon-optimized for expression in the corresponding host cells.

[0024] This invention relates to a genetically modified host cell, particularly an oleogenetic yeast, preferably *Yarrowia lipolytica*, capable of producing sterols having at least about 70-80% 7-DHC based on total sterols, the host cell comprising a heteropolynucleotide sequence expressing an enzyme having at least 60% identity with a polypeptide as defined herein according to SEQ ID NO: 1, the enzyme catalyzing the hydroxylation of 7-DHC to HyDHC, thereby producing a sterol mixture having at least about 70-80% HyDHC, and thus, under the conditions defined herein, a sterol mixture produced by the recombinant host cell containing at least about 70%, 75%, 78%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 97%, or 98% HyDHC based on total sterols.

[0025] In particular, the present invention relates to a genetically modified host cell, particularly an oil-producing yeast, preferably *Yersinia lipophila*, capable of producing sterols having at least about 70-80% of 7-DHC based on total sterols, the host cell comprising a heteropolynucleotide sequence expressing an enzyme having at least about 60% identity with the polynucleotide as defined herein according to SEQ ID NO: 3, the enzyme catalyzing the acetylation of 7-DHC to 7-DHC acetate, thereby producing a mixture of sterols having at least about 70-80% acetylated 7-DHC, thus, under the conditions defined herein, the mixture produced by the recombinant host cell contains at least about 70%, 75%, 78%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 97%, or 98% of acetylated 7-DHC based on total sterols.

[0026] More specifically, the present invention relates to a genetically modified host cell, particularly an oleogenous yeast, preferably *Yarrowia lipolytica*, capable of producing sterols having at least about 70-80% 7-DHC based on total sterols, said host cell comprising a heteropolynucleotide sequence expressing an enzyme having at least 60% identity with the polypeptide catalyzing 7-DHC hydroxylation according to SEQ ID NO: 1; and further comprising expressing an acetylated polypeptide catalyzing 7-DHC and / or HyDHC according to SEQ ID NO: 1. The polynucleotide of 3 has a heteropolynucleotide sequence of at least about 60% identity with the enzyme, thereby producing a mixture of hydroxylated vitamin D3 forms, particularly at least about 70-80% of hydroxylated 7-DHC (including HyDHC and HyDHC-acetate), and thus a mixture of sterols based on total sterols of at least about 70%, 75%, 78%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 97%, and 98% of hydroxylated 7-DHC (including HyDHC and HyDHC-acetate) in a mixture produced from recombinant host cells under the conditions defined herein.

[0027] Host cells suitable for producing the 25-hydroxylated and / or acetylated forms of 7-DHC, as defined herein, may contain additional genetic modifications, such as the inactivation of endogenous genes encoding ERG5 and ERG6. Those skilled in the art know how to generate such modifications; see, for example, Bean et al., Nature Communications, 13:2882, 2022; https: / / doi.org / 10.1038 / s41467-022-30570-7).

[0028] Host cells suitable for producing the 25-hydroxylated and / or acetylated form of 7-DHC, as defined herein, may contain additional genetic modifications, such as, for example, the introduction of a heteropolynucleotide encoding sterol δ24-reductase (S24R), preferably derived from mammalian or plant-derived S24R, which is optionally codon-optimized for host-specific expression. Those skilled in the art know how to generate such host strains expressing the heterologous S24R enzyme; see, for example, WO2003064650. Suitable examples of publicly available S24R sequences may include UniProtKB / Swiss-Prot references Q15392, Q60HC5, Q8VCH6, Q5BQE6, Q39085, or P93472. Preferably, S24R is selected from a polypeptide having at least about 60%, such as 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity with the rat-derived enzyme (RnS24R) according to SEQ ID NO: 5, and can be obtained via expression of a polynucleotide according to SEQ ID NO: 6.

[0029] Host cells suitable for producing the 25-hydroxylated and / or acetylated form of 7-DHC, as defined herein, may contain additional genetic modifications, such as, for example, modifications to the endogenous gene encoding HMG1, thereby producing a truncated version (tHMG) of the protein. Preferably, tHMG is selected from polypeptides having at least about 60%, such as 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identity with the polypeptide according to SEQ ID NO: 7, and can be obtained via expression of the polynucleotide according to SEQ ID NO: 8. Those skilled in the art know how to generate such host strains expressing the truncated HMG1 (tHMG1) enzyme, see, for example, WO2008130372.

[0030] Therefore, the present invention relates to a genetically modified host cell, particularly an oleogenous yeast, preferably *Yarrowia lipophila*, capable of producing sterols having at least about 70-80% 7-DHC based on total sterols, wherein the host cell further comprises genetic modifications, wherein: (1) Introduce and express an enzyme that catalyzes the C25-hydroxylation of 7-DHC, preferably an enzyme having at least 60% identity with SEQ ID NO: 1. (2) Introducing and expressing an enzyme that catalyzes the acetylation of 7-DHC and / or HyDHC, preferably an enzyme having at least 60% identity with SEQ ID NO: 3; and optionally (3) Inactivate, preferably knock out, the endogenous genes encoding ERG5 and ERG6; and / or (4) Introducing and expressing a polynucleotide encoding sterol δ24-reductase (S24R), preferably S24R of plant or mammalian origin, more preferably a polynucleotide of an enzyme having at least 60% identity with SEQ ID NO:5; and / or (5) The endogenous gene encoding HMG1, preferably having at least 60% identity with the enzyme in SEQ ID NO: 7, is truncated and expressed; The modified host cells produce a mixture of sterols comprising the form of hydroxylated vitamin D3, particularly having at least about 70-80% of hydroxylated 7-DHC (including HyDHC and HyDHC-acetate), thus forming a mixture of sterols based on total sterols of at least about 70%, 75%, 78%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 97%, and 98% hydroxylated 7-DHC (including HyDHC and HyDHC-acetate).

[0031] This invention relates to an improved biosynthetic method toward the 25-hydroxylated form of vitamin D3, the method comprising fermenting, under suitable culture conditions and standard conditions as defined herein, a recombinant host cell, particularly an oleogenous yeast, preferably a recombinant Yersinia lipolytica, to obtain a sterol mixture containing the hydroxylated form of vitamin D3, particularly having at least about 70-80% of hydroxylated 7-DHC (including HyDHC and HyDHC-acetate), thus comprising at least about 70%, 75%, 78%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 97%, or 98% of hydroxylated 7-DHC (including HyDHC and HyDHC-acetate) based on total sterols in the mixture produced by the recombinant host cell.

[0032] As used herein, fermentation or culture of recombinant host cells under “standard conditions” as defined herein means fermentation in a batch, fed-batch, semi-continuous, or continuous manner, as known to those skilled in the art and described elsewhere. Preferably, the method described herein is carried out in a fed-batch manner, optionally with the addition of glucose and / or ethanol to the feed. Depending on the recombinant host cells as described herein, fermentation can be carried out for a period of 100 to 240 hours, preferably 160 to 200 hours, more preferably 110 to 125 hours, at 22-30°C, preferably 24-26°C.

[0033] Suitable carbon sources for use in this invention may be selected from straight-chain alkanes, free fatty acids, including triglycerides, and particularly vegetable oils, such as those selected from the group consisting of, for example, oils derived from corn, soybean, olive, sunflower, low-erucic acid rapeseed, cottonseed, rapeseed, sesame, safflower, grapeseed, or mixtures thereof, including the corresponding free fatty acids, such as, for example, oleic acid, palmitic acid, or linoleic acid. Suitable carbon sources may also be selected from ethanol, methanol, glycerol, or glucose, and mixtures of one or more of the above carbon sources. The production of sterols as defined herein, including 25-hydroxylated 7-DHC derivatives as described herein, using suitable carbon sources and suitable culture conditions is known in the art.

[0034] In a preferred embodiment, the recombinant host cell described herein is selected from Yersinia lipolytica, and the host cell is cultured under fed-batch conditions using soybean oil and optionally ethanol as carbon sources, wherein the culture is carried out at 24-30°C for approximately 110-240 hours.

[0035] In one embodiment, the recombinant host cells described herein are selected from yeast species in which endogenous ATF2 is inactivated, more preferably Saccharomyces cerevisiae, said host cells being cultured in a fed-batch manner using a tandem fed-batch culture, said tandem fed-batch comprising glucose as a carbon source and glucose and ethanol fed in parallel, wherein the culture is carried out at 24-30°C for approximately 110-240 hours.

[0036] In one embodiment, the present invention relates to a fermentation method as described herein, the fermentation method further comprising deacetylation of the sterol mixture having at least about 70-80% hydroxylated 7-DHC (including HyDHC and HyDHC-acetate), optionally combined with further separation and / or purification steps comprising washing steps using isopropanol, heptane, hexane, ethanol, acetone, or similar substances as known in the art. Deacetylation of HyDHC-acetate is performed according to standard methods known in the art, including saponification, etc., to cleave acetyl residues.

[0037] In one embodiment, a sterol mixture containing at least about 70-80% HyDHC, as described above, is converted into a composition containing 25-hydroxyvitamin D3 (HyD) by known methods, such as by applying ultraviolet light or LED.

[0038] Therefore, the present invention specifically provides a method for producing a composition containing HyD, the method comprising: (1) Provide a recombinant host cell capable of producing 7-DHC, wherein the host cell expresses a heterologous enzyme involved in the hydroxylation of 7-DHC and / or the acetylation of 7-DHC or HyDHC as defined herein. (2) The recombinant host cells were cultured under standard fermentation conditions to enable the production of a mixture of sterols having at least about 70-80% of the form of 25-hydroxylated vitamin D3. (3) Separating and optionally purifying the 25-hydroxylated form of vitamin D3 from the fermentation broth, including deacetylation and washing, crystallization and / or filtration steps with a suitable solvent (such as, for example, isopropanol, hexane, heptane, acetone and / or ethanol) to obtain a sterol mixture having at least about 70-80% HyDHC, such as a mixture produced from recombinant host cells under conditions as defined herein, based on total sterols of at least about 75%, 78%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 97%, 98%, or 99%.

[0039] (4) By means of methods known in the art, including, for example, crystallization and photochemical steps, converting HyDHC to HyD to produce a composition having at least about 90%, such as, for example, 92%, 94%, 95%, 97%, 98%, 99%, or 100% HyD (w / w) in such compositions.

[0040] In one aspect, the present invention provides compositions comprising hydroxylated vitamin D3, such as compositions comprising, for example, at least about 70-80% HyDHC based on total sterols in such compositions, said compositions being prepared according to the methods described herein, preferably produced by means of Yersinia lipolytica as the host cell.

[0041] In another aspect, the present invention provides compositions comprising the form of hydroxylated vitamin D3, such as compositions comprising, for example, at least about 90% (w / w) of HyD, said compositions being prepared according to the methods described herein.

[0042] In one embodiment, the composition thus generated as described above, containing at least 90% (w / w) HyD, is further used in various applications in the food, feed, pharmaceutical, or personal care fields in the form of capsules, powders, liquids, etc. Those skilled in the art know how to produce such forms for the respective applications.

[0043] Compositions comprising the hydroxylated form of vitamin D3 as described herein are produced using the recombinant host cell method as described herein, wherein the formation of unwanted byproducts is kept to a minimum, including but not limited to esterification intermediates of trienols, yeast sterols, and / or 7-DHC.

[0044] Therefore, according to one aspect, the present invention provides a method as described herein, said method producing a sterol mixture comprising hydroxylated vitamin D3 form, particularly having at least about 70-80% of hydroxylated 7-DHC (including HyDHC and HyDHC-acetate) and trienols, particularly about 1.8%, 1.7%, 1.6%, 1.5%, 1.2%, 1.0%, 0.8%, 0.5%, 0.3%, 0.09%, 0.07%, 0.05%, 0.03%, 0.02% or less of trienols, all of which are based on total sterols in the mixture, wherein the maximum value of trienols in the sterol mixture produced as described herein is about less than 0.04 g / kg fermentation broth.

[0045] According to another aspect, the present invention provides a method as described herein, which produces a sterol mixture comprising hydroxylated vitamin D3, particularly having at least about 70-80% of hydroxylated 7-DHC (including HyDHC and HyDHC-acetate) and yeast sterols, particularly about 0.02%, 0.01%, 0.001% or less of yeast sterols, all of which are based on the total sterols in the mixture. Preferably, in the composition comprising hydroxylated vitamin D3 as described herein, yeast sterols are undetectable by standard methods known in the art, wherein the maximum value of yeast sterols in the sterol mixture produced as described herein is about less than 0.2 g / kg fermentation broth.

[0046] According to another aspect, the present invention provides a method as described herein, which produces a sterol mixture comprising a hydroxylated form of vitamin D3, particularly having at least about 70-80% of hydroxylated 7-DHC (including HyDHC and HyDHC-acetate) and 7-DHC esterification intermediates, particularly 7-DHC-palmitate (if palm oil is used as a carbon source) or 7-DHC-oleate (if oleic acid is used as a carbon source), and particularly about 0.05%, 0.03%, 0.02%, 0.03% or less of the 7-DHC esterification intermediates, all of which are based on the total sterols in the mixture. Preferably, in the compositions comprising the hydroxylated form of vitamin D3 as described herein, the esterified 7-DHC intermediates are undetectable by standard methods known in the art.

[0047] In one embodiment, the present invention relates to a method for increasing the fermentation yield of HyDHC in a sterol mixture, the method comprising culturing recombinant host cells selected from *Yersinia lipolytica* as defined herein, such as in fed-batch culture using glucose as feed and culturing under suitable conditions for about 110 h to 120 h, wherein the titer of HyDHC can be increased by at least about 20-60%, such as at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, under fed-batch conditions with tandem feed as described in, for example, WO2011067144, using recombinant *Saccharomyces cerevisiae* expressing the enzyme according to SEQ ID NO: 1 and expressing endogenous ATF2 for 110-120 h, compared to such methods.

[0048] In one embodiment, the present invention relates to a method for increasing the fermentation yield of 25-hydroxylated vitamin D3 derivatives as defined herein, particularly increasing the yield of HyDHC, the method comprising introducing and expressing a heterologous enzyme having CH25H activity and ATF2 (particularly ScATF2) activity in an oil-producing yeast, particularly *Yarrowia lipolytica*, wherein, when expressing the heterologous ATF2, the yield is increased by more than 60% compared to host cells expressing the corresponding natural endogenous ATF2. Preferably, such a method comprises culturing recombinant host cells selected from *Yarrowia lipolytica* as defined herein, such as in fed-batch culture using glucose as feed under suitable conditions for about 110 h to 120 h, wherein the titer of HyDHC can be increased by more than 60% compared to such methods, as described in, for example, WO2011067144, using fed-batch conditions with tandem feed and expressing the enzyme according to SEQ ID NO:1 and endogenous ATF2 for 110-120 h.

[0049] In one embodiment, the present invention relates to a method for increasing the yield of 7-DHC in the production of 25-hydroxylated vitamin D3 derivatives as defined herein, the method comprising culturing recombinant host cells selected from the genus *Yersinia* as defined herein, such as fed-batch culture with glucose as feed under suitable conditions for about 110 to 120 h, wherein the titer of 7-DHC can be increased by at least about 150-300% compared to such methods as described in, for example, WO2011067144, using recombinant *Saccharomyces cerevisiae* expressing the enzyme according to SEQ ID NO: 1 and expressing endogenous ATF2 under fed-batch conditions with tandem feed.

[0050] In one embodiment, the present invention relates to a method for increasing sterol fermentation yield in the production of 25-hydroxylated vitamin D3 derivatives as defined herein, the method comprising culturing recombinant host cells selected from *Yersinia lipolytica* as defined herein, such as in fed-batch culture using glucose as feed and culturing under suitable conditions for about 110 h to 120 h, wherein the sterol potency can be increased by at least about 50-86%, such as at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, in fed-batch culture with tandem feed for about 110-120 h, compared to such methods as described in, for example, WO2011067144, using recombinant *Saccharomyces cerevisiae* expressing the enzyme according to SEQ ID NO: 1 and expressing endogenous ATF2 for 110-120 h.

[0051] In one embodiment, the present invention relates to a method for increasing sterol production in the production of 25-hydroxylated vitamin D3 derivatives as defined herein, the method comprising fermenting an oil-producing host cell expressing an enzyme with CH25H activity, particularly *Yersinia lipolytica* as defined herein, the method comprising introducing and expressing a heterologous enzyme with ATF2 activity, resulting in a yield increase of greater than 86% after expression of the heterologous ATF2 compared to host cells expressing the corresponding natural endogenous ATF2. Preferably, such a method comprises culturing recombinant host cells selected from *Yersinia lipolytica* as defined herein, such as in fed-batch culture using glucose as feed under suitable conditions for about 110 h to 120 h, wherein the sterol potency can be increased by at least about 150-200% compared to such methods as described in, for example, WO2011067144, using fed-batch conditions with tandem feed and expressing the enzyme according to SEQ ID NO: 1 and endogenous ATF2 for 110-120 h.

[0052] Regarding this invention, it should be understood that organisms, such as animals, microorganisms, fungi, algae, or plants, also include synonyms or base names of such species having the same physiological properties, as defined by the International Code of Zoological Nomenclature, the International Code of Nomenclature of Prokaryotes, the International Code of Nomenclature for Algae, Fungi and Plants (Melbourne Code), or the International Commission on the Taxonomy of Fungi (ICTF).

[0053] This invention is particularly relevant to the following embodiments (1) to (14): (1) A sterol-producing oleogen host cell that expresses a heterologous enzyme that catalyzes the C25-hydroxylation of 7-dehydrocholesterol (7-DHC).

[0054] (2) The host cell according to embodiment (1), wherein the enzyme is a C25 hydroxylase (CH25H) having at least about 60% identity with SEQ ID NO: 1.

[0055] (3) The host cell according to embodiment (1) or (2) expresses a heterologous enzyme that catalyzes the acetylation of 7-DHC and / or 25-hydroxy-dehydrocholesterol (HyDHC).

[0056] (4) The host cell according to embodiment (3), wherein the enzyme is acetyltransferase 2 (ATF2) having at least about 60% identity with SEQ ID NO: 3.

[0057] (5) The host cell according to embodiments (1), (2), (3) and / or (4) is selected from Yersinia lipolytica.

[0058] (6) The host cell according to embodiments (1), (2), (3), (4) and / or (5) produces a mixture of sterols in the 25-hydroxylated form of vitamin D3, comprising at least about 70-80% of vitamin D3.

[0059] (7) The host cell according to embodiment (6), wherein the 25-hydroxylated form of vitamin D3 is HyDHC.

[0060] (8) A method for producing the 25-hydroxylated form of vitamin D3, the method comprising culturing, under suitable culture conditions, sterol-producing host cells according to embodiments (1), (2), (3), (4), (5), (6) and / or (7).

[0061] (9) The method according to embodiment (8) is used to produce a sterol mixture containing HyDHC, the method comprising: (a) Providing an oil-producing host cell that produces sterols, said host cell being capable of producing at least 70-80% 7-DHC based on the total sterols produced by said host cell. (b) Introducing and expressing a heterologous enzyme that catalyzes the C25-hydroxylation of 7-DHC to HyDHC. (c) Introducing and expressing heterologous enzymes that catalyze the acetylation of 7-DHC or HyDHC to 7-DHC-acetate or HyDHC-acetate. (d) Culture the recombinant host cells under suitable culture conditions to produce a sterol mixture containing at least about 70-80% of 25-hydroxylated vitamin D3 forms (including HyDHC and HyDHC-acetate), optionally (e) Deacetylate the sterol mixture containing at least 70-80% of the 25-hydroxylated form of vitamin D3 (including HyDHC and HyDHC-acetate) in a sterol mixture having at least about 70-80% HyDHC.

[0062] (10) A method for reducing the percentage of yeast sterols during the production of HyDHC by fermentation of host cells according to embodiments (1), (2), (3), (4), (5), (6) and / or (7), the method comprising introducing and expressing a heterologous enzyme having Saccharomyces cerevisiae ATF2 activity, wherein the yeast sterols are reduced to a percentage of 0.02% or less based on the total sterols produced by the host cells.

[0063] (11) A method for increasing the percentage of sterols produced by fermentation in an oil-producing host cell expressing an enzyme with CH25H activity, the method comprising introducing and expressing a heterologous enzyme with ATF2 activity, wherein the percentage of sterols is increased by more than about 86% when the heterologous ATF2 is expressed compared to a host cell expressing the corresponding natural endogenous ATF2.

[0064] (12) A method for increasing the percentage of fermented HyDHC produced in oil-producing host cells expressing an enzyme with CH25H activity, the method comprising introducing and expressing a heterologous enzyme with ATF2 activity, wherein the percentage of sterols is increased by more than about 60% when expressing the heterologous ATF2 compared to host cells expressing the corresponding natural endogenous ATF2.

[0065] (13) The method according to embodiment (8) or (9), the method further comprising: (f) Separate and further purify the fermentation-produced sterol mixture, said sterol mixture comprising at least about 70-80% HyDHC based on total sterols, and (g) Photochemically convert HyDHC to 25-hydroxyvitamin D3 (HyD).

[0066] (14) A composition comprising at least about 90% HyD (w / w) of a hydroxylated form of vitamin D3, wherein the HyD is produced by means of the method according to embodiment (13).

[0067] Example Example 1: General methods, strains and analysis All basic molecular biology and DNA manipulation procedures described herein are generally performed in accordance with Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds.), Current Protocols in Molecular Biology. Wiley: New York (1998).

[0068] DNA molecular biology. The plasmids listed in Table 1 of the Genscript (Piscataway, NJ, USA) and the sequence listing used to construct recombinant Yarrowia were synthesized. The plasmid DNA used for integration transformation was digested with a suitable restriction digest, sized by agarose gel electrophoresis, and purified using a Qiagen gel purification column (catalog number 28706X4). Whole-genome sequencing and assembly were performed by Genewiz (South Plainfield, NJ, USA).

[0069] Table 1: Plasmids and corresponding genotypes used to construct Yersinia lipolytica that produces HyDHC. See the text for more details.

[0070] Construction of recombinant strains.Using the methods described herein, *Yarrowia lipolytica* strain constructs are listed in Table 2. Strain ML326 (MATA ura3 leu2 lys1) was transformed with BstEII-cut MB6075 and selected on uracil-reduced medium to generate ML10288, which integrates MB6075 at the ERG5 locus via homologous recombination, as confirmed by sequence analysis. Isolates lacking the chromosomal ERG5 gene were identified by selection on medium containing fluoroorotic acid (FOA); ML10370 is one such isolate. ML10370 was transformed with BstEII-linearized MB6133 and selected on uracil-reduced medium to generate strain ML10603, which integrates MB6133 at the ERG6 locus via homologous recombination, as confirmed by sequence analysis. This strain maintains a functional copy of ERG6. ML10603 was subsequently transformed with the ERG6-containing plasmid MB6174 [CEN ARS ERG6 NATR] and selected on norsinolate-containing medium to prepare strain ML10735. Sequence analysis identified an isolate ML10738 with a chromosome erg6 deletion in its genome from the FOA-resistant derivatives of ML10735. Strain ML10738 was sequentially transformed with MB6267 and MB6274, each digested with HinDIII and XbaI, respectively, to integrate sterol-25-hydroxylase via selection on leucine-reduced medium and sterol-24-reductase via selection on uracil-reduced medium, resulting in strain ML11781. This strain was then transformed with MB6403 digested with PciI and selected on lysine-reduced medium to produce strain MB12184. The ERG6 covering plasmid MB6174 of MB12184 was screened for loss (by identification of noroside-sensitive colonies) to obtain the HyDHC-producing strain ML12297. Strain ML12280 was isolated from ML12184 by FOA selection. This uracil auxotroph was transformed with MB6273, which had been digested with XbaI and HindIII, and selected on uracil-reduced medium. The plasmid MB6174 of the transformants was screened for loss (by identification of noroside-sensitive colonies) to generate strain ML12298.

[0071] Yeast strain SC1394 was constructed by crossing two strains, SC1234α and SC1251a. In short, the resulting strain was diploid and homozygous, with are1 removed and replaced with a (p-)S24R driven by the HIS3 and PGK promoters, terminated by a (t-)PGK terminator labeled with TRP1. The are2 gene was heterozygous in the wild type, and PGK1p-CH25OH CYC1t- was labeled with TRP1. The erg5 gene was heterozygous for the null allele of erg5, and erg5 was replaced with CYC1t-CH25OH-PGK1p. The erg6 gene was heterozygous for the null allele of erg5, and erg6 was replaced with TDH3p-S24R2-PGKt labeled with a standard hygromycin resistance marker. A duplication exists at the ADE2 locus, resulting in circular integration of the CYCt-CH25OH-PGKp and wild-type alleles. The synthetic gene used was constructed using reverse translation of amino acid sequences from the most commonly used codons in the yeast genus to assemble the ORF from: sterol-42-reductase S24R1 from brown rat (Rattus Norvegicus), S24R2 from zebrafish (Danio rerio), and sterol-25-hydroxylase CH25OH from wild boar. (See also WO2017108799 and US20120231495 for the construction). The complete genotypes are shown in Table 2.

[0072] Table 2. Strain modifications as described in the text. Vectors involved in the modifications are specified using the symbol ^vector# to associate the changes with the vectors and operations described in the text.

[0073] Microtiter plate (MTP) determination. To test small-scale strain production, 10 µl of freshly grown yeast and up to 10% of the final volume of carbon source, such as 2% glucose, 2% glucose + 1% oleic acid, or other carbon source combinations (e.g., glucose, ethanol, linoleic acid, or oleic acid), are typically inoculated with 200 µl of 0.25% yeast extract and 0.5% peptone (0.25x YP). The strain is grown in 24-well plates (24-well microplate device, Whatman 7701-5102), covered with sealing pads (Analytical Sales and Services Inc., Plate Mats 24010CM), aseptically sealed with Qiagen porous film sheets (19571), and shaken at 800 RPM for 4 days at 30°C in a Multitron multiplate shaker (Infors HT). The cell suspension is collected and extracted as described below.

[0074] Sample preparation. Sterol extraction from the entire fermentation broth was performed using saponification as follows. Eighty μL of yeast fermentation broth (from MTP or other fermentation conditions) was added to a 2 mL Precellys tube (Bertin Instruments, Rockville MD) along with 860 μL of saponification solution consisting of 5% potassium hydroxide in ethanol. The sample was agitated at 6500 RPM for 3 cycles, each cycle lasting 15 seconds, using a Precellys cell lyser. The sample was briefly centrifuged, and 60 μL of glacial acetic acid was added and vortexed for 15 seconds to thoroughly mix. The sample was then centrifuged at 15000 x g for 2 minutes. The supernatant was then analyzed by HPLC-UV. Esters (e.g., HyDHC-ethyl ester, 7-DHC-oleate ester, etc.) were sampled; potassium hydroxide was omitted above.

[0075] C18 UPLC chromatography. As shown in Table 3, discrete sterols were determined by monitoring UV absorbance at 282 nm. Specifically, the instrument was calibrated using pure standards measured at 282 nm and adjusted using Beer's Law and published extinction coefficients. In some cases, no 5-7 diene chromophores were found at 282 nm, therefore these molecules, such as yeast sterols, were scanned at 212 nm. Samples were processed with Precellys to extract total sterols. Samples were run on an Agilent 1260 Infinity instrument using a YMC propack C18RS at 20°C, a 150 × 4.6 mm (3 µm) column, and an injection volume of 10 µl. The instrument was run at a flow rate of 1 ml / min for 30 minutes. Two solvents were used to form a gradient: solvent A was 96% (v / v) methanol and 4% water and 0.1% methane-sulfonic acid, and solvent B was 100% ethanol and 0.1% methane-sulfonic acid. The gradient began at 95% solvent A and 5% solvent B. At 12 minutes, the gradient transitioned to 65% solvent A and 35% solvent B. From 22 minutes onwards, the gradient transitioned to 40% solvent A and 60% solvent B, and then returned to the initial conditions at 25 minutes.

[0076] Table 3: List of analytes for C18 UPLC chromatography. “RT” refers to retention time. See the text for more details.

[0077] Series feeding scheme. Fermentation of the *Saccharomyces cerevisiae* strain was performed using a fed-batch tandem feeding scheme, which consisted of a short glucose batching phase followed by parallel glucose and ethanol feeding. The scheme is described below.

[0078] Seed flasks were prepared from plate cultures or frozen vials. Seed cultures were grown at 30°C in a floor shaker at 250 RPM for approximately 24 hours. The fermenter was inoculated at 10%, for example, 45 mL of seed culture into a 450 mL starting batch volume. Operating parameters were as follows: pH 5.7 controlled with ammonium hydroxide only, gas flow: 1 VVM, agitation maintained at maximum temperature: 30°C. The composition of the culture medium is shown in Tables 4A to 4D.

[0079] Feeding was performed using glucose and ethanol, all calculated for an initial volume of 500 mL post-inoculation. The feed consisted of 650 g / L glucose (total feed of 27 g glucose) fed at a rate of 0.9 mL / h between 2 and 48 hours and 130 mL of pure ethanol fed at the rate of 5 hours.

[0080] Table 4A: Shake flask culture media for seed flask preparation. This is an example for a 1L fermenter, but can be applied to any fermenter size. See the text for more details.

[0081] Table 4B: Sterol trace metal solutions for use in shake flask culture media and primary fermenter batch culture media.

[0082] Table 4C: Sterol vitamin solution to be used in shake flask culture medium.

[0083] Table 4D: Batch Culture Medium for Main Fermenter. This is an example for a 1L fermenter, but it can be applied to any fermenter size.

[0084] Table 5: Yeast Ethanol Feeding Protocol. Feeding begins at hour 5, with ramps between all given set points. See the text for more details.

[0085] Feeding batching scheme using oil-fed yeast (Yersinia genus). Fermentation of *Yarrowia lipolytica* strain was performed using a fed-batch feeding protocol, which consisted of adding soybean oil to maintain a dissolved oxygen setpoint of 20%. The protocol is described below.

[0086] Seed flasks were prepared from plate cultures or frozen vials. Seed cultures were grown at 30°C in a floor shaker at 250 RPM for approximately 24 hours. The fermenter was inoculated at 10%, for example, 45 mL of seed culture into a 450 mL starting batch volume. Operating parameters were as follows: pH 5.5 controlled with only 28% (w / w) ammonium hydroxide, gas flow: 1.17 VVM, agitation maintained at maximum temperature: 30°C. The composition of the culture medium is shown in Tables 6A to 6D.

[0087] Feeding was performed using a programmed control system that added soybean oil. Once the batch carbon was depleted, as demonstrated by a sudden increase in dissolved oxygen levels, soybean oil was added using a peristaltic pump. The program was designed to activate the pump whenever dissolved oxygen exceeded a 20% saturation setpoint and to shut it off whenever dissolved oxygen fell below that setpoint. This dissolved oxygen-based feeding strategy was maintained for a duration of 240 hours of EFT (Estimated Run Time).

[0088] Table 6A: Shake flask culture media for seed flask preparation. This is an example for a 1L fermenter, but can be applied to any fermenter size. See the text for more details.

[0089] Table 6B: Batch culture medium for primary fermenter. This is an example for a 1L fermenter, but it can be applied to any fermenter size.

[0090] Table 6C: Trace metal solutions to be used in batch culture media in the primary fermenter.

[0091] Example 2: HyDHC production of *Yersinia* relative to *Saccharomyces* Fermentation was performed using Saccharomyces cerevisiae strain SC1394 and Yersinia spp. strains ML12297 and ML12298 as described in Example 1. Total sterols and biomass, as well as HyDHC, 7-DHC, yeast sterols, and other sterols, were calculated from the fermentation broths of strains ML12297, ML12298, and SC1394. After 115-120 hours of fermentation, SC1394 showed a HyDHC yield of at least approximately 0.7 g / kg, and yeast sterols and 7-DHC levels in SC1394 were detected to be less than 0.3 g / kg. The results are shown in Table 7.

[0092] Table 7: Sterol composition analysis of Sc1394, ML12297, and ML12298. The amounts of HyDHC, 7-DHC, yeast sterols, and sterols obtained from fermentation of SC1394 were set to 100. "Natural" refers to the strain expressing natural Saccharomyces cerevisiae ATF2 (ScATF2), i.e., SC1394. "Subtracted" indicates that the strain, i.e., ML12297, does not express any ATF2, and "Added" indicates that the strain, i.e., ML12298, expresses heterologous SCATF2. "ND" means undetectable, and "zym" means yeast sterols. See the text for more details.

[0093] Using *Yersinia* strains that produce HyDHC as host strains nearly doubled the total sterol production, and increased intermediates such as 7-DHC and HyDHC by at least 180% and 60%, respectively. These figures were further increased using strains expressing heterologous SCATF2. Interestingly, compared to *Yersinia* strains, the formation of impurities such as yeast sterols was completely eliminated in hosts selected from *Yersinia*.

Claims

1. A sterol-producing oleogen host cell, said host cell expressing a heterologous enzyme that catalyzes the hydroxylation of the carbon atom at position 25 of 7-dehydrocholesterol (7-DHC).

2. The sterol-producing host cell according to claim 1, wherein the host cell produces a mixture of sterols comprising at least about 70-80% of the 25-hydroxylated form of vitamin D3.

3. The host cell according to claim 1 or 2, wherein the enzyme exhibits at least about 60% identity with the C25 hydroxylase (CH25H) according to SEQ ID NO:

1.

4. The host cell according to any one of claims 1 to 3, wherein the host cell further expresses a heterologous enzyme that catalyzes the acetylation of 7-DHC and / or 25-hydroxy-dehydrocholesterol (HyDHC).

5. The host cell according to claim 4, wherein the enzyme catalyzing the acetylation of 7-DHC and / or HyDHC exhibits at least about 60% identity with acetyltransferase 2 (ATF2) according to SEQ ID NO:

3.

6. The host cell according to any one of claims 1 to 5, wherein the host cell is selected from Yersinia lipolytica.

7. The host cell according to claim 1, 3, 4, 5 or 6, wherein the host cell produces a mixture of sterols comprising at least about 70-80% of the 25-hydroxylated form of vitamin D3.

8. The host cell according to claim 7, wherein the 25-hydroxylated form of vitamin D3 is HyDHC.

9. A method for producing the 25-hydroxylated form of vitamin D3, the method comprising culturing a host cell that produces sterols according to any one of claims 1 to 8 under suitable culture conditions.

10. The method of claim 9 for producing a sterol mixture comprising HyDHC, the method comprising: (a) Providing an oil-producing host cell that generates sterols, said host cell being capable of producing at least 70-80% 7-DHC based on the total sterols produced by said host cell. (b) Introducing and expressing a heterozyme that catalyzes the hydroxylation of the carbon atom at position 25 of 7-DHC to HyDHC. (c) Introducing and expressing heterologous enzymes that catalyze the acetylation of 7-DHC to 7-DHC-acetate and / or the acetylation of HyDHC to HyDHC-acetate; and (d) The recombinant host cells are cultured under suitable culture conditions to produce a mixture of sterols containing at least about 70-80% of 25-hydroxylated vitamin D3, including HyDHC and HyDHC-acetate.

11. The method according to claim 10, further comprising: (e) Deacetylate the sterol mixture containing at least 70-80% of the form of 25-hydroxylated vitamin D3, which comprises at least about 70-80% HyDHC.

12. A method for reducing the percentage of yeast sterols in a method for producing HyDHC using host cells fermented according to any one of claims 1 to 8, the method comprising introducing and expressing a heterozyme that exhibits catalytic activity equivalent to that of Saccharomyces cerevisiae ATF2 for the acetylation of 7-DHC or HyDHC, wherein the percentage of yeast sterols is reduced to 0.02% or less based on the total sterols produced by the host cells.

13. A method for increasing the percentage of sterols produced by fermentation in an oil-producing host cell expressing an enzyme with CH25H activity, the method comprising introducing and expressing a heterologous enzyme that exhibits catalytic activity equivalent to Saccharomyces cerevisiae ATF2 for the acetylation of 7-DHC or HyDHC, wherein the percentage of sterols increases by more than about 86% after expression of the heterologous ATF2 compared to host cells expressing the corresponding natural endogenous ATF2.

14. A method for increasing the percentage of HyDHC produced by fermentation in an oil-producing host cell expressing an enzyme with CH25H activity, the method comprising introducing and expressing a heterologous enzyme that exhibits catalytic activity equivalent to Saccharomyces cerevisiae ATF2 for the acetylation of 7-DHC or HyDHC, wherein the percentage of HyDHC increases by more than about 60% after expression of the heterologous ATF2 compared to host cells expressing the corresponding natural endogenous ATF2.

15. The method according to claim 11, wherein the method further comprises: (f) Separate and further purify the fermentation-produced sterol mixture, said sterol mixture comprising at least about 70-80% HyDHC based on total sterols, and (g) Photochemically convert HyDHC to 25-hydroxyvitamin D3 (HyD).

16. A composition comprising at least about 90% (w / w) of a hydroxylated form of vitamin D3, wherein the HyD is produced via the method of claim 15.

17. The composition of claim 16, wherein the HyD is produced by means of Yersinia lipolyticis as a host cell.

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