WO2022051613A1

WO2022051613A1 - Production of hydroxylated polyunsaturated fatty acids

Info

Publication number: WO2022051613A1
Application number: PCT/US2021/049049
Authority: WO
Inventors: Martin SCHÜRMANN; Peter Jan Leonard Mario Quaedflieg; Paul Frederick Mugford
Original assignee: Dsm Ip Assets B.V.
Priority date: 2020-09-04
Filing date: 2021-09-03
Publication date: 2022-03-10

Abstract

A method for producing and purifying hydroxylated polyunsaturated fatty acid (PUFA) derivatives is provided. The method includes reacting a PUFA, preferably eicosapentaenoic acid (EPA) and an enzyme, preferably a P450 enzyme, followed by purification via filtration and/orextraction and/or chromatography. Use of the resulting hydroxylated PUFA derivative, preferably 18-hydroxyeicosapentaenoic acid (18-HEPE) in medicines or food applications, preferably in infant food and/or formula is also provided.

Description

PRODUCTION OF HYDROXYLATED POLYUNSATURATED FATTY ACIDS

[0001] The present disclosure relates to processes for producing and purifying polyunsaturated fatty acid (PUFA) derivatives. The process includes reacting a PUFA, preferably eicosapentaenoic acid (EP A) and an enzyme, preferably a cytochrome P450 enzyme, optionally followed by purification via filtration and/or extraction and/or chromatography.

BACKGROUND

[0002] The inflammatory response is an integral part of the innate immune mechanism, triggered in response to a real or perceived threat to tissue homeostasis, with the primary aim of neutralizing infectious agents and initiating repair to damaged tissues. Inflammation is a finite process that resolves as soon as the threat of infection has abated and the damaged tissue is sufficiently repaired.

[0003] Resolution of inflammation involves apoptosis and subsequent clearance of activated inflammatory cells. Chronic inflammation is a characteristic feature in virtually all inflammatory diseases, and derangement of the processes usually involved in resolution of inflammation is an underlying feature of chronic inflammatory conditions. In other words, failed resolution of acute inflammation leads to chronic inflammation.

[0004] A recently discovered class of compounds, naturally biosynthesized from the omega- 3 fatty acids EPA and DHA, promote the resolution of inflammation. The derivatives consist of resolvins, protectins, and maresins; collectively, called specialized pro-resolving mediators.

[0005] Resolvins are metabolic byproducts of omega-3 fatty acids, primarily eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as well as docosapentaenoic acid (DPA) and clupanodonic acid. As autacoids similar to hormones acting on local tissues, resolvins are of interest for their involvement in promoting restoration of normal cellular function following the inflammation that occurs after tissue injury. Resolvins belong to the class of PUFA metabolites known as specialized pro-resolving mediators (SPMs), which are signaling molecules for the resolution of inflammation.

[0006] SPMs have been shown to alleviate or otherwise aid in the management of chronic inflammation resulting from or associated with metabolic syndrome, stroke, wound healing, eczema, inflammatory bowel disease, and asthma, atherosclerosis, cancer, kidney disease and cystic fibrosis. These lipids confer the benefit of shortening recovery times and improving health outcomes in patients suffering from inflammation.

[0007] The beneficial effects of n-3 PUFAs, such as EP A, DHA, and DPA, were first recognized in the late 1960s with epidemiological evidence obtained among the Inuit population, who consumed a diet rich in n-3 PUFAs and exhibited a low incidence of myocardial infarction. [0008] In the body, omega-3 fatty acids form lipid derivatives that promote the resolution of inflammation and shorten healing times. However, the relationship between the dose of oral omega-3 supplements and the resulting SPM levels in the body is not yet established and it is unlikely that such supplements would provide the same results as direct delivery of SPMs.

[0009] One application area for delivery of SPMs is wound healing. SPMs could be incorporated in one of the many types of wound dressings, or as an ingredient in a topical skin cream. In mice, topically applied resolvins have been observed to accelerate wound closure, thereby reducing healing time by 30%. Shortened healing times might also reduce infections and overuse of antibiotics.

[0010] Similarly, SPMs may be delivered in medical coatings, such as in a surgical mesh having a coating comprising fish oil omega-3.

[0011] As it is unclear whether the body can efficiently convert omega-3 fatty acids to active SPM levels, there is an unmet need for pharmaceuticals, nutraceuticals, dietary supplements, food products, and topically applied products (e.g., ointments, coatings, or creams) which deliver SPMs directly.

[0012] There is an ongoing need for new products that directly deliver these lipid derivatives capable of reducing the time needed to resolve inflammation. Thus, there remains a need to provide improved processes for the production and purification of PUFA derivatives, in particular hydroxylated PUFAs, at lower costs and higher efficiency, yield, and/or purity.

[0013] The solution to this technical problem is provided by the embodiments characterized below.

[0014] 18-HEPE is a mono-hydroxylated omega-3 polyunsaturated fatty acid derivative and intermediate to certain SPMs which have been found to be effective for resolving chronic inflammation. Early lactating women have quite high levels of monohydroxylated PUFAs such as 18-HEPE in their milk. Accordingly, there is a need for infant food and/or formula comprising PUFA derivatives such as 18-HEPE. BRIEF SUMMARY

[0015] The present application provides methods for producing and purifying hydroxylated PUFA derivatives. In particular, the method of the present invention includes biocatalytic synthesis and purification of the resulting hydroxylated PUFA derivative product to remove contaminants and obtain the desired hydroxylated PUFA derivative.

[0016] In one embodiment, the present application provides a method for the production of one or more hydroxylated polyunsaturated fatty acid derivatives comprising reacting a polyunsaturated fatty acid (PUFA) with a P450 enzyme or an enzyme that is at least 70% homologous thereto, whereby one or more hydroxylated PUFAs are produced.

[0017] In some embodiments, the desired hydroxylated PUFA derivative to be obtained is 18-HEPE, and is obtained by carrying out a hydroxylation reaction with EPA and a cytochrome P450 enzyme (P450).

[0018] In some embodiments, the hydroxylated PUFA derivative is purified from the reaction mixture by chromatography. Following synthesis, a reaction mixture containing the desired hydroxylated PUFA derivative is applied to the desired chromatography. Optionally, before the chromatography step, the reaction mixture is subjected to one or more of the following: i) decolorization; ii) filtration and optionally , washing of the solids filtrated; and/or iii) extraction with a water-immiscible organic solvent; and/or iv) drying.

[0019] In some embodiments, the hydroxylation reaction is carried out in the presence of molecular oxygen as an oxidant.

[0020] In some embodiments, the oxygen used as oxidant in the hydroxylation reaction is originating from air or oxygen enriched air dispersed into the reaction.

[0021] In some embodiments, the oxygen used as oxidant in the hydroxylation reaction is originating from pure oxygen dispersed into the reaction.

[0022] It will be understood that the steps of the method of the present invention may be performed in any order. In some embodiments, one or more steps of the method of the present invention may be performed more than once, for instance the extraction with a water-immiscible organic solvent. In a preferred embodiment, the steps of the present invention are performed in the order listed above.

[0023] Also provided is the product obtained according to the method of the present invention.

[0024] In some embodiments, the product obtained according to the method of the present invention is in a food, supplement, or pharmaceutical composition. The pharmaceutical composition can contain a pharmaceutically acceptable carrier.

[0025] In some embodiments, the product obtained according to the method of the present invention can be used in a food product. A food product is any food for non-human animal or human consumption and includes both solid and liquid compositions. A food product can be an additive to animal or human foods. Foods include, but are not limited to, common foods; liquid products, including milks, beverages, therapeutic drinks, and nutritional drinks; functional foods; supplements; nutraceuticals; infant formulas, including formulas for pre-mature infants; foods for pregnant or nursing women; foods for adults; geriatric foods; and animal foods.

[0026] In some embodiments, the product obtained according to the method of the present invention can be used as a topical application or as a precursor for ingredients, e.g., SPMs, for use in topical applications.

[0027] In some embodiments, the product obtained according to the method of the present invention can be used as a precursor for delivery of certain ingredients, e.g., SPMs. One application area for delivery of SPMs is wound healing. SPMs could be incorporated in one of the many types of wound dressings. Similarly, SPMs may be delivered in medical coatings, such as in a surgical mesh.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be made to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements.

[0029] FIG. 1 shows the results of a hydroxylation reaction between EPA and 8 g of biocatalyst.

[0030] FIG. 2 shows the results of a hydroxylation reaction between EPA and 4 g of biocatalyst.

[0031] FIG. 3 shows the effect of increased biocatalyst amount on the amount of 18-HEPE formed according to the method of the present invention.

[0032] FIGs. 4A and 4B show the effect of increased EPA amount on the amount of 18- HEPE formed according to the method of the present invention.

[0033] FIG. 5 shows the typical time course of a one-liter scale hydroxylation reaction of EPA with P450-BM3 variant C4 (SEQ ID NO:3) with pure molecular oxygen as an oxidant.

[0034] FIG. 6 shows the results of an 18-HEPE product isolated from a one-liter scale hydroxylation reaction of EPA with P450.

[0035] FIG. 7 shows the separation conditions upon screening by thin-layer chromatography.

[0036] FIG. 8 shows the results obtained via thin-layer chromatography of a test column.

[0037] FIG. 9 shows a chromatogram of the test column.

[0038] FIG. 10 shows a chromatogram of the final reaction conditions.

[0039] FIG. 11 shows the SDS-PAGE analysis of cell-free extracts of the E. coli strains expressing the target P450s

[0040] FIGs. 12A and 12B show the time course of EPA conversion (FIG. 12 A) through hydroxylation by the P450 enzymes to 18-HEPE (FIG. 12B).

[0041] FIG. 13 shows the development of 18-HEPE vs. HEPE regio-isomer ratio over time in the individual P450 reactions on EPA.

[0042] FIG. 14 shows a chromatogram of racemic 18-HEPE at a concentration of 0.04 mg/ml. DETAILED DESCRIPTION

[0043] Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims.

[0044] It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.

[0045] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0046] The term “product obtained according to the method of the present invention”, as used herein, refers to the product of the reaction of the present method of a selected hydroxylated polyunsaturated fatty acid (PUFA) with a P450 enzyme.

[0047] In one embodiment, the present application provides methods for the production of one or more hydroxylated polyunsaturated fatty acid derivatives comprising reacting a polyunsaturated fatty acid (PUFA) with a P450 enzyme or an enzyme that is at least 70% homologous thereto, whereby one or more hydroxylated PUFAs are produced.

[0048] In some embodiments, the selected PUFA is EPA.

[0049] In some embodiments, the hydroxylated PUFA product is an EPA derivative having a single hydroxy group at carbons 5, 12, or 18 from the carboxyl group end.

[0050] In some embodiments, the hydroxylated PUFA product is 18-HEPE.

[0051] The terms “eicosapentaenoic acid” and “EPA”, as used herein, refer to any of the following compounds having the molecular formula C20H30O2 (CAS registry no. 10417-94-4):

[0052] The term “monohydroxylated EPA”, as used herein, refers to an EPA derivative having a single hydroxy group at carbons 5, 12, or 18 from the carboxyl group end. [0053] The term “18-HEPE”, as used herein, refers to the following compound having CAS registry no. 141110-17-0 and CAS registry no. 312516-11-3:

[0054] The present inventors have found that PUFAs can be reliably and efficiently converted to hydroxylated PUFA derivatives via a hydroxylation reaction catalyzed by a P450 enzyme.

[0055] Enzymes produced from the cytochrome P450 genes are involved in the formation (synthesis) and breakdown (metabolism) of various molecules and chemicals within cells. Cytochrome P450 enzymes play a role in the synthesis of many molecules including steroid hormones, certain fats (cholesterol and other fatty acids), and acids used to digest fats (bile acids). Additionally, cytochrome P450 enzymes metabolize external substances, such as medications that are ingested, and internal substances, such as toxins that are formed within cells. There are approximately 60 cytochrome P450 genes in humans.

[0056] Cytochrome P450 enzymes are primarily found in liver cells but are also located in cells throughout the body. Within cells, cytochrome P450 enzymes are located in a structure involved in protein processing and transport (endoplasmic reticulum) and the energy-producing centers of cells (mitochondria). The enzymes found in mitochondria are generally involved in the synthesis and metabolism of internal substances, while enzymes in the endoplasmic reticulum usually metabolize external substances, primarily medications and environmental pollutants. [0057] Common variations (polymorphisms) in cytochrome P450 genes can affect the function of the enzymes. The effects of polymorphisms are most prominently seen in the breakdown of medications. Depending on the gene and the polymorphism, drugs can be metabolized quickly or slowly. If a cytochrome P450 enzyme metabolizes a drug slowly, the drug stays active longer and less is needed to get the desired effect. A drug that is quickly metabolized is broken down sooner and a higher dose might be needed to be effective.

Cytochrome P450 enzymes may account for 70 percent to 80 percent of the enzymatic activity involved in drug metabolism. [0058] Each cytochrome P450 gene is named with CYP, indicating that it is part of the cytochrome P450 gene group. The gene is also given a number associated with a specific group within the gene group, a letter representing the gene's subgroup, and a number assigned to the specific gene within the subgroup. For example, the cytochrome P450 gene that is in group 27, subgroup A, gene 1 is written as CYP27A1.

[0059] Diseases caused by mutations in cytochrome P450 genes typically involve the buildup of substances in the body that are harmful in large amounts or that prevent other necessary molecules from being produced.

[0060] Examples of genes in this gene group: CYP1B1, CYP2C9, CYP2C19, CYP2R1, CYP4V2, CYP7B1, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP21A2, CYP24A1, CYP27A1, and CYP27B1.

[0061] The HUGO Gene Nomenclature Committee (HGNC) provides an index of gene groups and their member genes.

[0062] In addition to the P450 enzymes encoded and present in human cells, these enzymes can also be found in plants, fungi and prokaryotes such as Archaea and Bacteria, for example in Bacteria of the gastro-intestinal tract (microbiome) or free-living water or soil bacteria as e.g., of the Bacillus species.

[0063] In particular, the present inventors have found that variants of a P450 enzyme can convert EP A to primarily 18-HEPE (/.< ., the major monohydroxylated product) via a hydroxylation reaction. The present inventors screened a library of P450 wild-type and variant enzymes to determine the P450 most productive and selective for this reaction, and determined the appropriate reaction conditions for a multigram scale reaction.

[0064] The terms “P450 enzyme” or “P450 variant” or “P450”, as used herein, refer to any enzyme belonging to the class of cytochrome P450s (CYPs), which oxidize molecules.

[0065] In an aspect, the method of the present invention may employ any wild-type P450 enzyme or variant thereof.

[0066] In an aspect, the P450 enzyme used in the reaction of the present invention may be obtained from any source.

[0067] In some embodiments, the P450 enzyme used in the reaction of the present invention may be obtained from a microbial organism, such as Escherichia coli (E. coli). [0068] In some embodiments, the P450 enzyme used in the reaction of the present invention may be obtained from a microbial organism which has been genetically modified.

[0069] In an aspect, the method of the present invention may employ any of the P450 sequences of SEQ ID NOs: 1, 2, 3, or 4 and/or can include sequences that are 70% or more homologous thereto, or 75% or more homologous thereto, or 80% or more homologous thereto, or 85% or more homologous thereto, or 90% or more homologous thereto, or 95% or more homologous thereto, or 96%, 97%, 98% or 99% or more homologous thereto:

* The amino acid sequence does not contain Methionine in the first position as the positions of the amino acid exchanges in P450-BM3 are usually given based on the sequence lacking the Methionine of the start codon because it is removed in the biosynthesis of this enzyme.

[0070] In some embodiments, the enzyme has an amino acid sequence consisting of any of the sequences of SEQ ID NOs: 1, 2, 3 or 4.

[0071] In some embodiments, the enzyme has an amino acid sequence comprising any of the sequences of SEQ ID NOs: 1, 2, 3 or 4.

[0072] In some embodiments, the enzyme has an amino acid sequence comprising the sequence of SEQ ID NO: 1.

[0073] In some embodiments, the enzyme has an amino acid sequence consisting of the sequence of SEQ ID NO: 1.

[0074] In some embodiments, the enzyme has an amino acid sequence comprising the sequence of SEQ ID NO:2.

[0075] In some embodiments, the enzyme has an amino acid sequence consisting of the sequence of SEQ ID NO:2. [0076] In some embodiments, the enzyme has an amino acid sequence comprising the sequence of SEQ ID NO:3.

[0077] In some embodiments, the enzyme has an amino acid sequence consisting of the sequence of SEQ ID NO:3.

[0078] In some embodiments, the enzyme has an amino acid sequence comprising the sequence of SEQ ID NO:4.

[0079] In some embodiments, the enzyme has an amino acid sequence consisting of the sequence of SEQ ID NO:4.

[0080] The hydroxylation reaction of the present invention is carried out in the presence of an oxidant.

[0081] In some embodiments, the hydroxylation reaction of the present invention is carried out in the presence of molecular oxygen as the oxidant.

[0082] In some embodiments, the oxygen used as oxidant in the hydroxylation reaction is originating from air or oxygen enriched air dispersed into the reaction.

[0083] In some embodiments, the oxygen used as oxidant in the hydroxylation reaction is originating from pure oxygen dispersed into the reaction.

[0084] In some embodiments, the hydroxylation reaction of the present invention is carried out in the presence of D-glucose.

[0085] In an aspect, a crude oil product is obtained from reacting the PUFA and the P450 enzyme.

[0086] In an aspect, the PUFA and P450 enzyme may be present in any amount or concentration.

[0087] In an aspect, the PUFA and P450 enzyme may be reacted in any molar ratio.

[0088] In some embodiments, the PUFA and P450 enzyme are reacted in a ratio PUFA:P450 of from 1 : 100 to 100: 1, or from 1 :75 to 75: 1, or 1 :50 to 50: 1 on a weight to weight basis.

[0089] In some embodiments, the hydroxylation reaction is carried out in a buffered reaction mixture. In an aspect, any buffer may be used.

[0090] In some embodiments, the product obtained according to the method of the present invention is a racemic mixture.

[0091] In some embodiments, the product obtained according to the method of the present invention is primarily an (A) enantiomer. In an aspect, the product obtained is at least 60% (A) enantiomer, or at least 70% (R) enantiomer, or at least 80% (R) enantiomer, or at least 90% (R) enantiomer, or at least 95% (R) enantiomer, or at least 98% (A) enantiomer.

[0092] In some embodiments, the product obtained according to the method of the present invention is primarily an (5) enantiomer. In an aspect, the product obtained is at least 60% (5) enantiomer, or at least 70% (5) enantiomer, or at least 80% (S) enantiomer, or at least 90% (5) enantiomer, or at least 95% (5) enantiomer, or at least 98% (S) enantiomer.

[0093] In some embodiments, the process of the present invention includes subjecting the reaction mixture to chromatography to purify the desired hydroxylated PUFA derivative, such as 18-HEPE, from impurities, other small molecules, and enzyme fragments. Flash chromatography is preferably performed.

[0094] In some embodiments, the reaction mixture is subjected to chromatography, preferably flash chromatography, after one or more filtration steps. In some embodiments, the reaction mixture is subjected to one or more filtration steps selected from filtration over a Buchner filter, centrifugation, guard filtration, ultrafiltration, and/or one or more nanofiltration steps prior to performing chromatography, preferably flash chromatography, straight phase chromatography, reversed phase chromatography or high-performance liquid chromatography (HPLC).

[0095] In some embodiments, the straight phase chromatography performed is silica column chromatography.

[0096] In some embodiments, the reaction mixture obtained from the hydroxylation reaction of the present invention is titrated with an acid prior to extraction, followed by purification by chromatography.

[0097] In some embodiments, the reaction mixture obtained from the hydroxylation reaction of the present invention is titrated with an acid to a pH of 8 or less, to a pH of 7 or less, to a pH of 6 or less, to a pH of 5 or less, to a pH of 4 or lower, prior to extraction, followed by purification by chromatography

[0098] In an aspect, any suitable eluent or combination of eluents may be used during chromatography.

[0099] In some embodiments, one or more eluents may be selected from water, tetrahydrofuran, acetone, ethyl acetate, di chloromethane, ethanol, chloroform, methyl tert-butyl ether and/or cyclohexane. In some embodiments, two or more eluents may be used in combination in different ratios.

[00100] In some embodiments, one or more eluents may be used without any modifier.

[00101] In some embodiments, one or more eluents may be used with an acidic modifier.

[00102] In an aspect, the acidic modifier may be acetic acid or formic acid.

[00103] In some embodiments, the process of the present invention includes one or more decolorization steps. Decolorization can be performed by any suitable means. For example, decolorization can be performed by treatment of the enzyme reaction product with activated carbon. The one or more decolorization steps may be performed at any point during the process of the invention. In a preferred embodiment, decolorization is performed after chromatography. [00104] The resulting solution containing the desired hydroxylated PUFA derivative, such as 18-HEPE, may be subjected to drying. In some embodiments, the drying comprises evaporation, freeze drying, drying under vacuum, or any combination thereof.

[00105] The resulting solution containing the desired hydroxylated PUFA derivative, such as 18-HEPE, may be concentrated.

[00106] The hydroxylated PUFA derivatives obtained via the process of the present invention are free of contaminants and are suitable for use in human food applications. In some embodiments, the obtained hydroxylated PUFA derivatives are used in infant food, infant formula, and/or infant supplements. In a preferred embodiment, the obtained hydroxylated PUFA derivatives are used in human infant food, human infant formula, and/or human infant supplements.

[00107] In other embodiments, the hydroxylated PUFA derivatives obtained via the process of the present invention are used in compositions, such as, for example, dietary supplements or pharmaceutical composition for use in prevention or treatment of certain conditions or diseases, e.g., as a treatment for a chronic inflammatory condition.

[00108] The following examples are offered to illustrate, but not to limit, the claimed invention. EXAMPLES

[00109] EXAMPLE 1 - 30-mL Scale Reactions

[00110] The reaction of EP A with the biocatalyst towards (5)- 18-HEPE was optimized and scaled up for the production of gram quantities. The present inventors established a workup procedure for obtaining a crude oil containing more than 1 g of fS')- l 8-HEPE. Said oil would then be used in a later chromatographic purification step to achieve pure material.

[00111] To make the most efficient use of the available biocatalyst amounts, several small- scale experiments were applied to determine the minimal biocatalyst loading that still produces the product quantities and concentrations required for an efficient product isolation procedure. [00112] Two experiments on a 30-ml scale were performed to investigate the relationship between biocatalyst concentration and the amount of 18-HEPE yielded thereby.

[00113] In both experiments, the biocatalyst was added over time and air was used as the source of oxygen.

[00114] In reaction 1 (see FIG. 1), 8 g biocatalyst was added, and a resulting 27 mg 18-HEPE was obtained. In reaction 2 (see FIG. 2), when 4 g biocatalyst was added, significantly less 18- HEPE was obtained.

[00115] The biocatalyst used was a cell-free extract of a batch E. coli fermentation in which the P450 gene for P450-BM3 variant F87A/A328V of SEQ ID NO:3 was expressed. To obtain such a cell-free extract (CFE), cells from this fermentation were harvested, resuspended in twice the volume of the cell wet weight of 100 mM potassium phosphate (KPi) buffer (pH 7.5) and homogenized in a Microfluidics M-l 10P homogenizer.

[00116] Table 1 sets forth the amounts and conditions of reactions 1 and 2 as depicted in FIGs. 1 and 2:

Table 1

[00117] FIG. 3 shows the amount of 18-HEPE obtained from reactions 1 and 2 plotted against the amount of biocatalyst added over time. As is apparent from FIG. 3, the two respective reactions with 8 g and 4 g of biocatalyst are superimposable and therefore reproducible. The slope in the linear portion is 4.46 mg/g, meaning that for each gram of biocatalyst used, 4.46 mg 18-HEPE was obtained. Furthermore, the linearity ends at about 6 g of biocatalyst. The addition of biocatalyst amounts higher than 6 g would thus lead to a lower slope and consequently, a less efficient use of the P450 biocatalyst than would be achieved with 6 g biocatalyst.

[00118] EXAMPLE 2 - 30-mL Scale Optimization

[00119] In three parallel experiments at a 30-mL scale, the EPA concentration was varied to find the optimal concentration for scale-up and sample preparation. The biocatalyst loading was fixed to 18% (w/w) in each reaction, based on the findings above.

[00120] An increased initial amount of EPA had a positive effect on the 18-HEPE formation (see FIG. 4A). An increase of the initial EPA concentration to 30 mM had a lower impact. Based on the results depicted in FIGs. 4A and 4B, the present inventors found that a semi-optimized initial amount would therefore be 20 mM EPA. As in reactions 1 and 2, air was again used as the source of oxygen. [00121] Table 2 sets forth the amounts and conditions of reactions 3-5 as depicted in FIGs. 4A and 4B:

Table 2

[00122] EXAMPLE 3 - One-Liter Scale-Up

[00123] The previous findings from biocatalyst and substrate loading optimization were applied in a scale-up. As the biocatalyst amount was limiting, loading was reduced slightly to 16.6% (w/w).

[00124] Instead of air as the oxygen source (as it was applied on 30-mL scale) pure, pressurized oxygen from a gas bottle was used. The oxygen was applied by a sintered frit to create small bubbles and consequently a large gas surface was created, thereby increasing the gas/ oxygen transfer rate.

[00125] For process safety reasons (to avoid combustible mixtures in the gas phase) the oxygen concentration in the headspace was monitored and a nitrogen blanket was applied (100 ml/min). The standard oxygen aeration rate was initially 30 mL/min and was reduced over time to keep the oxygen concentration in the headspace below 21% (/.< ., below ambient).

[00126] The reaction was then worked up by adding 230 g ethyl acetate, titration to pH 4 with 10% (w/w) sulfuric acid (typically about 16 g). Then 36 g dicalite (filter aid) was added and the suspension was filtered over a dicalite pre-coated 100 mm Buchner filter (pore size 3), which typically took 30 minutes. The cake was washed twice with 300 mL ethyl acetate, which was also used each time to extract the water layer.

[00127] Table 3 sets forth the amounts and conditions of a typical one-liter scale reaction:

Table 3

[00128] The time course of EP A conversion and 18-HEPE formation of the one-liter scale reaction was comparable to the small-scale experiments (see FIG. 5). Additionally, the typical ratio of approximately 3 mol 18-HEPE to 1 mol of other HEPE regio-isomers was found by HPLC analysis. Accordingly, the scale-up thus described herein was successful.

[00129] EXAMPLE 4 - Purification

[00130] At the end of the P450 hydroxylation reaction as described in Example 3, 237 g ethyl acetate was added to the reactor and the pH was reduced to 4 by addition of 10% (w/w) sulfuric acid.

[00131] 36 g dicalite (filter aid) was added and the suspension was filtered over a dicalite precoated Buchner funnel (100 mm diameter and pore size 3) applying 200 mbar vacuum. [00132] Filtration took about 30 min. The filter cake was washed with 300 ml ethyl acetate and the filtrate was used to extract the water layer once again. The filter cake was thereafter washed once more with 300 ml ethyl acetate, which was again used to extract the water layer.

[00133] All organic layers were pooled and concentrated on a rotary evaporator. The brown clear oil had a mass of 3.55 g, which represented 75.9% of the initially added EPA mass of 4.68 g-

[00134] However, HPLC analysis showed no EPA substrate, HEPE products or other detectable by-products remaining in the water layer (see FIG. 6). All organics could therefore be extracted by the above procedure.

[00135] By applying this total recipe 7 times a total amount of 25.5 g oil containing about 1.3 g 18-HEPE (by HPLC) could be produced.

[00136] EXAMPLE 5 - Purification of the 18-HEPE Product Via Flash Chromatography

[00137] The crude 18-HEPE oil from Example 4 was purified by silica column chromatography in 9 batches. For this purpose, a Grace Reveleris X2 flash chromatography system was used.

[00138] Prior to purification, the optimal conditions had been determined by thin layer chromatography (TLC) analysis (see FIG. 7). Hence, several eluent combinations (i.e., tetrahydrofuran (THF), acetone, ethyl acetate (EtOAc), di chloromethane (DCM), ethanol (EtOH), chloroform (CHCh) or methyl tert-butyl ether (MTBE) in cyclohexane) in different ratios, with and without acidic modifier (/.< ., acetic acid (AcOH) or formic acid) were tested. [00139] A combination of MTBE in cyclohexane with 0.1 vol% AcOH as acidic modifier resulted in the best separation (see FIG. 7, red box).

[00140] A test batch/column was run to confirm and translate the separation conditions from TLC to flash chromatography. Due to the difficult separation of the desired product vs. more polar side products (see FIG. 8), faint spots on TLC below the product spot), a dry loader was selected for sample preparation.

[00141] To the crude material was added double the amount of Florisil (Florisil® 30-60 mesh for chromatography - magnesium silicate, activated) as adsorbent. The sticky crude 18-HEPE oil was dissolved/suspended in di chloromethane. After evaporating to dryness, the absorbed substrate was obtained as a yellow powder.

[00142] A dry loader cartridge was charged with the yellow powder, serving as a pre-column and thus improving separation. As eluents a gradient from 10% MTBE in cyclohexane to 80% THF in cyclohexane was used (see FIG. 9). An 80 g silica cartridge was loaded with 1 g of crude mixture resulting a in ratio 1 :80 substrate/silica.

[00143] To improve separation of the product from the starting material as well as from the side-products, the linear gradient was modified into a step gradient.

[00144] The gradient depicted in FIG. 10 led to a superior separation.

[00145] In total, three flash chromatography column purifications were performed with the 25.5 g oil containing about 1.3 g 18-HEPE synthesized in Example 4. The fractions containing 18-HEPE of each run were pooled, concentrated by rotary evaporation to 2.18 g yellow oil and analyzed for their purity by HPLC. For these pooled batches, purities of 88.4%, 86.9% and 84.2% were obtained.

[00146] EXAMPLE 6 - Comparison of P450 wild-type and variant enzymes in EPA hydroxylation

[00147] 4 P450-BM3 (homologue) variants enzymes were compared to P450-BM3 wild-type, with the latter reproducibly showing full conversion of EPA but no HEPE formation in the small scale reactions.

Table 4: P450 enzymes and variants selected for reaction optimization (BM3 wild-type as control)

[00148] E. coli strains expressing these 5 P450s were cultivated in shake flasks. Cell-free extracts were prepared as described above, but with using ultrasound probe instead of a homogenizer. The expression level of all 5 P450s was good to excellent (FIG. 11 Error! Reference source not found.), the wild-type and variants of P450-BM3 (CYP102A1) expression clearly better than the variant of CYP102A7.

[00149] The enzymatic reactions were monitored in time to study the course of the reaction. Conditions and protocol used were similar to before: EPA (as stock solution in methanol or dosed directly when larger volumes were used): 5 mM EPA in reaction (max. 5% methanol), 20% (v/v) glucose/Glucose Dehydrogenase cofactor-regeneration mix, 30-ml scale pH stat reactor set-up, pH = 7.5, 28°C with top-cooler, stirring rate of 1500 rpm, aeration of 10 ml air/min, enzyme/substrate ratio of E:S = 7 (on weight basis).

[00150] Samples were taken and diluted 5x with water/acetonitrile (25/75), shaken, centrifuged and analyzed with HPLC.

[00151] While the fastest conversion of EPA was observed with P450-BM3 wild-type (F4), this conversion was not matched by the formation of 18-HEPE or HEPE type of products (FIG. 12A). The EPA decrease in the other reactions was slower and sometimes fluctuating due to analytical inaccuracies. 18-HEPE formation was clearly highest for the B4 (F87A/A328I) and C4 (F87A/A328V) double mutant of P450-BM3, while the single mutant Bl (F87A alone) gave clearly lower 18-HEPE concentrations (FIG. 12B).

[00152] Looking at the ratio of 18-HEPE vs the other formed HEPE regio-isomers, B4 (F87A/A328I) and C4 (F87A/A328V) clearly outperformed the other P450 enzymes. After 60 min reaction time a constant 18-HEPE to 18-HEPE isomers ratio of 2.7: 1 and 3.0: 1, respectively, was established in the reactions with B4 (F87A/A328I) and C4 (F87A/A328V), while none of the other P450 enzymes exceeded a ratio of more than 1.5:1 (FIG. 13).

[00153] It can be concluded from this example that enzymes B4 (F87A/A328I) and C4 (F87A/A328V) performed best in 18-HEPE formation. [00154] EXAMPLE 7 - Determination of the enantioselectivity of EPA hydroxylation

[00155] The enantioselectivity of EPA hydroxylation to 18-HEPE was determined by HPLC on a column with chiral stationary phase:

Column: Chiralpak AD-3 250 mm x 4.6 mm (3 pm)

Eluent: w-Heptane/2-propanol/formic acid 95/5/0.1% (v/v)

Flow: 0.8 mL/min

Temperature: 20°C

Injection volume: 5 pL

Detection: UV 237 nm

[00156] Racemic 18-HEPE (0.04 mg/ml, Cayman Chemicals) was base-line separated under these conditions (FIG. 14).

[00157] The enantioselectivity of the hydroxylations catalyzed by P450-BM3 variants B4 (F87A/A328I) and C4 (F87A/A328V) was examined using the conditions described above. Both enzymes have preference for making 18-HEPE peak #2, which is in analogy to the elution patterns of 17-hydroxy-DHA enantiomers, the CS')-enantiomer.

[00158] The enantiomeric excess was estimated to be 69% (S) for variant B4 (F87A/A328I) and 67% (5) for variant C4 (F87A/A328V), respectively.

Claims

CLAIMS What is claimed is:

1. A method for the production of one or more hydroxylated polyunsaturated fatty acid derivatives comprising reacting a polyunsaturated fatty acid (PUFA) with a P450 enzyme or an enzyme that is at least 70% homologous thereto, whereby one or more hydroxylated PUFAs are produced.

2. The method of claim 1, wherein the PUFA is eicosapentaenoic acid (EP A).

3. The method of claim 1 or 2, wherein the P450 enzyme has an amino acid sequence comprising the sequence of SEQ ID NO: 1, 2, 3 or 4.

4. The method of claim 1 or 2, wherein the P450 enzyme has an amino acid sequence consisting of the sequence of SEQ ID NO: 1, 2, 3 or 4.

5. The method of claim 1 or 2, wherein the P450 enzyme has an amino acid sequence comprising the sequence of SEQ ID NO:2.

6. The method of claim 1 or 2, wherein the P450 enzyme has an amino acid sequence consisting of the sequence of SEQ ID NO:2.

7. The method of claim 1 or 2, wherein the P450 enzyme has an amino acid sequence comprising the sequence of SEQ ID NO:3.

8. The method of claim 1 or 2, wherein the P450 enzyme has an amino acid sequence consisting of the sequence of SEQ ID NO:3.

9. The method of claim 1 or 2, wherein the P450 enzyme has an amino acid sequence comprising the sequence of SEQ ID NO:4

10. The method of claim 1 or 2, wherein the P450 enzyme has an amino acid sequence consisting of the sequence of SEQ ID NO:4.

11. The method of claim 1, wherein the one or more hydroxylated PUFA derivative is a monohydroxylated EPA having a single hydroxy group at carbons 5, 12, or 18 from the carboxyl group end.

12. The method of claim 11, wherein the one or more hydroxylated PUFA derivative is 18- hydroxy eicosapentaenoic acid (18-HEPE).

13. The method of any of claims 1-12, wherein the method further comprises one or more of:

24 a) decolorization; b) filtration and optionally, washing of the solids filtrated; c) extraction with a water-immiscible organic solvent; and/or d) drying. The method of claim 13, wherein the drying comprises evaporation. The method of claim 13, wherein the drying comprises freeze drying. The method of claim 13, wherein the drying comprises drying under vacuum. The method of any of claims 1-16, wherein the method further comprises: e) a chromatography step. The method of claim 17, wherein the chromatography step comprises HPLC chromatography. The method of claim 17, wherein the chromatography step comprises reversed-phase chromatography. The method of any of claims 1-19, wherein said PUFA and said P450 enzyme are reacted in the presence of molecular oxygen. The method of any of claims 1-20, wherein steps a) through e) are performed in any order. The method of any of claims 1-20, wherein the steps a) through e) are performed in the order provided in claims 13 and 17. The hydroxylated PUFA derivative obtained according to the method of any of claims 1- 22. The hydroxylated PUFA derivative of claim 23, wherein the hydroxylated PUFA derivative is a monohydroxylated EPA having a single hydroxy group at carbons 5, 12, or 18 from the carboxyl group end. The hydroxylated PUFA derivative of claim 24, wherein the hydroxylated PUFA derivative is 18-HEPE. Use of the hydroxylated PUFA derivative obtained according to the method of any of claims 1-22 in a food preparation. The use according to claim 26, wherein the food is a human food. The use according to claim 26, wherein the food is an infant food. The use according to claim 26, wherein the food is an infant formula or an infant supplement. Use of the hydroxylated PUFA derivative obtained according to the method of any of claims 1-22 in a dietary supplement. Use of P450 enzyme or an enzyme that is at least 70% homologous thereto to obtain a hydroxylated PUFA derivative.