FIELD OF THE INVENTION

The invention relates to a method to provide a lactone by exposing a fatty acid derivative to an oxidoreductase. The invention also relates to use of the lactone obtainable by such method.

BACKGROUND OF THE INVENTION

Methods for providing lactones are known in the art. US2005130278A1, for instance, relates to a method for producing a lactone comprising culturing Candida sorbophila in a medium containing at least one selected from the group consisting of a hydroxy fatty acid, a hydroxy fatty acid derivative, and a hydrolysate of a hydroxy fatty acid derivative and recovering the produced lactone from the medium.

Dietrich, M. et al (2009), Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards y- and 6-positions; Journal of Biotechnology 139(1), 115-117 describes that using a rational site-directed mutagenesis approach, three elements were introduced into the substrate binding pocket of the cytochrome P450 BM-3 monooxygenase, which changed the product pattern of lauric acid hydroxylation.

Hammerer, L. et al (2018), Regioselective biocatalytic hydroxylation of fatty acids by cytochrome P450s; Catalysis Letters 148, 787-812 provides an overview of the regioselectivity of cytochrome P450 enzymes (CHs-terminal, in-chain, and carboxylate- terminal) and the optical purity of the hydroxylation products obtained from fatty acids.

Cirino, P. C. et al (2002), Regioselectivity and activity of cytochrome P450 BM- 3 and mutant F87A in reactions driven by hydrogen peroxide; Adv. Synth. Catal. 344(9), 932- 937 describes cytochrome P450 BM-3 (EC 1.14.14.1) as a monooxygenase that utilizes NADPH and dioxygen to hydroxylate fatty acids at subterminal positions.

W02020018729A1 describes methods for making gamma lactones comprising reacting a carboxylic acid substrate with a heterologous cytochrome P450 (CYP450) protein with carboxylic acid 4-hydroxylase activity.

SUMMARY OF THE INVENTION

Lactones are a group of organic molecules that comprise a cyclic ester. Naturally occurring lactones mainly comprise saturated and unsaturated y- and 6-lactones. y-Lactones and 6-lactones comprise a five-membered ring and a six-membered ring, respectively, which are the most stable structures for lactones. However, also other lactones exist. Naturally occurring lactones contribute to the aroma of foods such as fruits, cheese, and butter. Hence, lactones may be used as flavors and fragrances. In other applications, lactones may be used for the synthesis of polyester precursors.

Existing methods for lactone synthesis from (natural) fatty acids may e.g. comprise a four step aerobic-oxidation of ricinoleic acid, followed by a ring closing reaction. Alternative methods comprise fermentation by yeast or fungi. These methods may provide relatively low yields and titers.

The prior art may further describe complex industrial synthesis methods requiring a large number of steps, specialized equipment, harmful chemicals, and/or prohibited chemicals.

The prior art may further describe chemical production systems based on nonrenewable feedstocks.

The prior art may further describe enzymatic processes, such as based on a P450 monooxygenase, requiring one or more of cofactor regeneration systems, external aeration, and/or specific solvent conditions. Such specific requirements may lead to complex and expensive reaction processes. Further, such prior art processes may be relatively inefficient in terms of reaction rate and/or yield.

The prior art may further describe processes based on free fatty acids and/or natural oils, which may have solubility issues and may have surfactant activity leading to enzyme inactivation and foaming issues.

It appears desirable to produce lactones in a scalable and economic fashion in a simple and efficient process, and based on renewable feedstocks.

Hence, it is an aspect of the invention to provide an alternative method for providing a lactone, which preferably further at least partly obviates one or more of abovedescribed drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

According to a first aspect, the invention provides a method for providing a lactone. Especially, the method comprises one or more of a preparation stage, a first stage, and a second stage. The preparation stage may comprise providing a first compound according to formula (I):

In embodiments, X in the first compound may be selected from the group comprising O, NH, and S. Ri may in embodiments be selected from alkyl groups comprising 1-10 C atoms. In specific embodiments, R2 may comprise 5-25 C atoms. The first stage may in embodiments comprise exposing the first compound to an oxidoreductase, especially in the presence of a peroxide. The oxidoreductase may especially be selected from the group comprising oxygenases, peroxygenases, oxidases, and peroxidases. In embodiments, the oxidoreductase may comprise an unspecific peroxygenase. In embodiments, the oxidoreductase may be configured to catalyze a hydroxylation of the first compound at position 3, 4, 5, and/or 6 to provide a second compound, especially at position 4 or 5. The second stage may especially comprise providing the lactone from the second compound via a spontaneous or catalyzed ringformation reaction.

Hence, in specific embodiments, the invention provides a method for providing a lactone, wherein the method comprises: (i) a preparation stage comprising providing a first compound according to formula (I) wherein X is selected from the group comprising O, NH, and S, wherein Ri is selected from alkyl groups comprising 1-10 C atoms, and wherein R2 is selected from alkyl, alkene and alkyne groups comprising 5-25 C atoms; (ii) a first stage comprising exposing the first compound to an oxidoreductase in the presence of a peroxide, wherein the oxidoreductase is configured to catalyze hydroxylation of the first compound at position 3, 4, 5, and/or 6 to provide a second compound; and (iii) a second stage comprising providing the lactone from the second compound via a ring-formation reaction.

In other words, the method of the invention may use an oxidoreductase to hydroxylate a fatty acid derivative, and may subsequently form a lactone from the hydroxylated fatty acid derivative in a spontaneous or catalyzed ring-forming reaction.

A benefit of the invention may be that the invention may provide a simplified process for lactone synthesis. For instance, in embodiments, such as in embodiments wherein the oxidoreductase comprises an unspecific peroxygenase, the invention may not require using cofactors and/or regeneration systems and/or external aeration. Also, the use of fatty acid derivatives may have several advantages compared to the use of free fatty acids. Fatty acid derivatives may have a higher solubility in organic media, may have a reduced, such as no, surfactant activity, may have a higher reactivity in the lactonization process and may be easier in handling than free fatty acids. The method of the invention may further provide a scalable and economic production of lactones, such as y-decalactone and 6-decalactone having a higher yield compared to established methods such as fermentation methods. Especially, the method may in embodiments be performed as a one-step reaction, which may be much simpler compared to e.g. the four step aerobic-oxidation of ricinoleic acid, followed by a ring closing reaction. Compared to chemical synthesis, the reaction conditions may be more environmentally friendly.

In this way lactones may be provided in a more simple and efficient synthesis compared to established biocatalytic methods. Especially, additional cofactors and regeneration systems may be avoided. Further, the method of the invention may be independent from external aeration. In specific embodiments, the method may even be performed under nonaqueous conditions. Surprisingly, the inventors discovered that hydroxylation of a fatty acid derivative by an oxidoreductase provided similar conversion efficiencies as for fatty acids. Since the fatty acid derivatives may have a higher solubility in organic media and/or no surfactant activity and/or the corresponding second compounds may have a higher reactivity in the lactonization process, the use of a fatty acid derivative (such as a fatty acid ester, a fatty acid amide, and a fatty acid thioester) appears favorable over fatty acids.

Hence, the invention may provide a method for providing a lactone. The term lactone may herein refer to a cyclic carboxylic ester. In embodiments, the lactone may be formed from the first compound according to formula (lb):

Formula (lb) depicts a more detailed embodiment of the first compound according to Formula (I) wherein R2 essentially consists of -R4-C(Re)-R5. In embodiments, R4 comprises 1-4 C atoms. R4 may especially be selected from alkyl, alkene and alkyne groups comprising 1-4 C atoms. R5 may essentially comprise R2 (as defined below) minus R4-C-R5. In embodiments, R5 may be -H. In alternative embodiments, R5 may be selected from alkyl, alkene and alkyne groups. R5 may in embodiments comprise one or more heteroatoms such as a bromide, a iodide, and/or a hydroxy group. In further embodiments, Rs may be -H. In alternative embodiments, Rs may be selected from alkyl, alkene and alkyne groups. In yet further embodiments, Rs may comprise one or more heteroatoms such as a bromide, a iodide, and/or a hydroxy group. The first compound may be converted into the second compound according to formula (II):

The second compound may especially comprise a hydroxyl group on the carbon atom between R4 and R5.

The formed lactone may have a structure according to formula (III): wherein the oxygen of the hydroxyl of the second compound according to formula (II) now forms part of the ring structure. Especially, R4 may comprise 1-4 C atoms that may be part of the ring structure. Hence, the ring structure may comprise especially a four-membered ring, or especially a five-membered ring, or especially a six-membered ring, or especially a sevenmembered ring. In further embodiments, R4 may comprise (other) carbon atoms that may not be part of the ring structure. In embodiments, R5 may not form part of the ring structure. In further embodiments, Rs may not form part of the ring structure.

As indicated above, the method comprises one or more of a preparation stage, a first stage, and a second stage. Each stage will be described here in more detail.

The preparation stage may comprise providing a first compound according to formula (I): ^{( i )} especially wherein X is selected from the group comprising O, NH, and S, wherein Ri is selected from alkyl groups comprising 1-10 C atoms, and wherein R2 is selected from alkyl, alkene and alkyne groups comprising 5-25 C atoms.

In embodiments, X (in the first compound) may be selected from the group comprising O, NH, and S, especially O, or especially NH, or especially S. Hence, the first compound may especially comprise an ester, amide or thioester, especially an ester, or especially an amide, or especially a thioester. Such ester, amide, or thioester comprising compound may have improved solubility and/or no surfactant properties compared to the corresponding carboxylic acid compound. Therefore, such ester, amide, or thioester comprising compound may provide higher yields.

Ri may, in embodiments, be selected from alkyl groups comprising 1-25 C atoms, such as 1-10 C atoms, such as 1-6 C atoms, especially 1-4 C atoms. More especially, Ri may be selected from alkyl groups comprising 4-25 C atoms, such as 6-25 C atoms, especially 10-25 C atoms, more especially 15-25 C atoms. Furthermore, in embodiments, Ri may be selected from alkyl groups comprising at least 5 C atoms, such as at least 10 C atoms, especially at least 20 C atoms. Additionally or alternatively, Ri may be selected from alkyl groups comprising at most 20 C atoms, such as at most 10 C atoms, especially at most 5 C atoms. Especially, Ri X may act as a leaving group during the ring-formation reaction and hence both Ri and X may in embodiments not be part of the lactone obtained (in the second stage). Hence, selecting Ri may in embodiments be based on its ability to form a good leaving group RiX . Especially methyl-, ethyl-, propyl-, isopropyl-, butyl-, and glyceryl-, such as monoglyceride-, diglyceride- and triglyceride, groups may in embodiments form good leaving groups. Hence, in embodiments, Ri may comprise especially a methyl-, or especially an ethyl-, or especially a propyl-, or especially an isopropyl-, or especially a butyl-, or especially a monoglyceride-, or especially a diglyceride,- or especially a triglyceride group. It will be apparent to the skilled person that the leaving group in a chemical reaction refers to atoms or groups of atoms that detach from the primary part of the substrate (here especially the first compound).

R2 on the other hand may in embodiments become part of the lactone obtained. Especially, depending on the site of hydroxylation, one part of R2 may form the lactone ring, and another part of R2 may form a side chain of the lactone.

In embodiments, R2 may be selected from the group comprising alkyl, alkene and alkyne groups. Especially R2 may be an alkyl group, or especially an alkene group, or especially an alkyne group.

In embodiments, R2 may comprise 3-50 C atoms, such as 3-25 C atoms, especially 3-19 C atoms, such as 5-19 C atoms, especially 5-13 C atoms, more especially 7-9 C atoms. In other embodiments, R2 may comprise 4-25 C atoms, such as 10-25 C atoms, especially 15-25 C atoms. Yet further, in embodiments, R2 may comprise at least 3 C atoms, such as at least 5 C atoms, especially at least 7 C atoms, more especially at least 10 C atoms. Additionally or alternatively, R2may comprise at most 40 C atoms, such as at most 35 C atoms, especially at most 25 C atoms, more especially at most 17 C atoms. The first compound may thus comprise a hydrocarbon chain (-R2) with a terminal ester, amide or thioester group (-C(=O)XRi). Hence, the first compound may, in embodiments, comprise a fatty acid ester, a fatty acid amide, or a fatty acid thioester. Fatty acid esters, fatty acid amides, and fatty acid thioesters may herein collectively be referred to as “fatty acid derivatives”. Hence, the first compound may comprise a fatty acid derivative, especially a fatty acid ester, or especially a fatty acid amide, or especially a fatty acid thioester. Such fatty acid derivatives may have substantially less surfactant properties compared to fatty acids. Therefore fatty acid derivatives may react much more efficient.

Many embodiments for R2 are possible. Several embodiments will be discussed in the next paragraphs.

In embodiments, R2 may comprise a hydrocarbon chain. The hydrocarbon chain may comprise one or more of alkyl, alkene or alkyne groups, such as an alkyl group, or an alkene group, or an alkyne group. Especially, R2 may in embodiments be a branched hydrocarbon chain. Especially, in such embodiments the first compound may comprise a branched chain fatty acid (BCFA) derivative. In alternative embodiments, R2 may be an unbranched hydrocarbon chain. Especially, in such embodiments, the first compound may comprise a linear (unbranched) fatty acid (FA) derivative. In further embodiments, R2 may comprise a cyclic group.

Additionally or alternatively, R2 may in embodiments be an unsubstituted hydrocarbon chain. In such embodiments, R2 may consist of carbon atoms and hydrogen atoms. In other embodiments, R2 may be a substituted hydrocarbon chain. In such embodiments, one or more hydrogen atoms may be substituted with other groups such as hydroxy groups. Here, a hydroxy (or hydroxyl) group may be a functional group with the chemical formula -OH.

R2 may in embodiments be a saturated hydrocarbon chain, especially an alkyl group. In alternative embodiments, R2 may be an unsaturated hydrocarbon chain, especially one or more of alkene or alkyne groups.

In embodiments, R2 may be selected from unbranched, unsubstituted, saturated hydrocarbon chains. This embodiment will be described in more detail below (in relation to formula la). In other embodiments, R2 may be selected from unbranched, unsubstituted unsaturated hydrocarbon chains. In such embodiment, the first compound may essentially comprise a linear non-saturated fatty acid ester, a linear non-saturated fatty acid amide and/or a linear non-saturated fatty acid thioester. In other embodiments, R2 may be selected from unbranched, substituted, saturated hydrocarbon chains. In alternative embodiments, R2 may be selected from unbranched, substituted, unsaturated hydrocarbon chains. In other embodiments, R.2 may be selected from branched, unsubstituted, saturated hydrocarbon chains. In alternative embodiments, R2 may be selected from branched, unsubstituted, unsaturated hydrocarbon chains. In yet other embodiments, R2 may be selected from branched, substituted, saturated hydrocarbon chains. In alternative embodiments, R2 may be selected from branched, substituted, unsaturated hydrocarbon chains.

In embodiments, R2 may be selected from unbranched, unsubstituted, (unsaturated) alkene and alkyne groups. In more specific embodiments, R2 may especially comprise an unbranched (thus linear) alkene comprising 3-50 C atoms, such as 5-25 C atoms, especially 10-20 C atoms.

Hence, in embodiments, R2 may be unbranched. In further embodiments, R2 may be branched.

Similarly, in embodiments, R2 may be unsubstituted. In further embodiments, R2 may be substituted.

In further embodiments, R2 may be unsaturated. In alternative embodiments, R2 may be saturated.

In further embodiments, R2 may comprise at least one C=C bond, such as at least two C=C bonds, especially at least three C=C bonds.

In further embodiments, R2 may comprise at most 5 C=C bonds, such as at most three C=C bonds, especially at most two C=C bonds.

In further embodiments, R2 may comprise at least one C=C bond, such as at least two C=C bonds, especially at least three C=C bonds.

In further embodiments, R2 may comprise at most 5 C=C bonds, such as at most three C=C bonds, especially at most two C=C bonds.

It will be obvious to a person skilled in the art that many combinations of saturated/unsaturated, branched/unbranched and substituted/unsubstituted groups for R2 are possible. In specific embodiments, R2 may comprise dimers or trimers obtainable by dimerization or trimerization reactions of fatty acid (derivatives). In embodiments, R2 may comprise isostearic acid (derivatives) or ricinoleic acid (derivatives).

In embodiments, the method may comprise the first stage. The first stage may comprise exposing the first compound to an oxidoreductase, especially in the presence of a peroxide. The term “oxidoreductase” may herein refer to an enzyme that may be configured to catalyze a hydroxylation reaction. In particular, the oxidoreductase may be configured to catalyze hydroxylation of the first compound at position 3, 4, 5, and/or 6 to provide a second compound. Here, the term “at position 3, 4, 5, and/or 6” may refer to a specific carbon atom hydroxylated by the oxidoreductase. Especially, position 1 may refer to the carbon comprised by the -C(=O)X moiety, whereas subsequent positions (i.e., position 2, position 3, etc.) may refer to carbon atoms along R2. Hence, the term “position 2” may refer to the first carbon atom in R2, wherein said first carbon atom is connected to the carbon atom at position 1. Further, position 3 may refer to the second carbon atom in R2 (wherein said second carbon atom is connected to the carbon atom at position 2), position 4 may refer to the third carbon atom in R2 (wherein said third carbon atom is connected to the carbon atom at position 3), etc.. Such referring to carbon atoms by a numbered position is known to a person skilled in the art, and it will be apparent to a person skilled in the art how the carbon atoms in a specific structure according to formula I and/or formula lb should be labelled (with numbered positions) to execute the method of the invention.

Oxidoreductases are a large class of enzymes generally catalyzing reactions involving the transfer of electrons. Of specific interest herein are those oxidoreductases that catalyze reactions further involving the introduction of O into an organic molecule, such as reactions catalyzed by subclasses of oxidoreductases such as oxidases, peroxidases, oxygenases, and peroxygenases. Hence, in embodiments of the invention the oxidoreductase may be selected from the group consisting of oxidases, peroxidases, oxygenases and peroxygenases. It will be clear to one skilled in the art that the invention is not limited to the use of oxidoreductase (classified as) belonging to any one of the aforementioned subclasses. It will further be clear to one skilled in the art that not each enzyme belonging to the aforementioned subclasses will catalyze the hydroxylation of the first compound at position 3, 4, 5, and/or 6 to provide a second compound. Hence, it will be clear to one skilled in the art that the invention relates to any oxidoreductase configured to catalyze hydroxylation of the first compound at position 3, 4, 5, and/or 6 to provide the second compound.

Oxidases may catalyze oxidation-reduction reactions, especially oxidationreduction reactions involving O2. At least part of the oxidases may catalyze a reaction involving the introduction of an oxygen into an organic compound. For example, cytochrome P450 oxidase may catalyze a monooxygenase reaction of the form RH + O2 + NADPH + H ⁺ - ROH + H2O + NADP ⁺ . Similarly, a xanthine oxidase may catalyze a reaction of the form RH + 2 O2 introduction of an oxygen into an organic compound. For example, a peroxidase may catalyze a reaction selected from the group comprising Baeyer-Villiger oxidations, oxidations of styrene derivatives to corresponding ketones, and oxidations of sulfides to sulfoxides and sulfones.

Oxygenases may catalyze reactions involving the transfer of an oxygen atom from O2 to a substrate, especially to an organic compound. Oxygenases may be further classified into (i) monooxygenases, such as the aforementioned cytochrome P450 oxidase, which introduce one oxygen atom into a substrate, and (ii) dioxygenases which introduce both oxygen atoms from O2 into a substrate. In specific instances, the introduction of oxygen may occur via an epoxide intermediate.

Peroxygenases may catalyze reactions involving the transfer of an oxygen atom from a peroxide to a substrate, especially to an organic compound. Hence, peroxygenases may typically catalyze a reaction of the form R'H + R"00H R'OH + R"OH, especially wherein R" consists of H.

In embodiments, the oxidoreductase may comprise one or more of a cytochrome P450 enzyme, a heme-dependent and/or a vanadium-dependent oxygenase, a catalase, an unspecific peroxygenase (Enzyme Classification (EC) 1.11.2.1), a peroxidase (EC 1.11.1.7), a chloride peroxidase (EC 1.11.1.10) and a bromide peroxidase (EC 1.11.1.18), especially one or more of an unspecific peroxygenase, a peroxidase, a chloride peroxidase, and a bromide peroxidase, more especially an unspecific peroxygenase.

In embodiments wherein the oxidoreductase comprises an unspecific peroxygenase, the unspecific peroxygenase and/or a gene encoding the unspecific peroxygenase may be derived from one or more organisms selected from the group comprising Agrocybe aegerita, Agrocybe acericola, Agrocybe amara, Agrocybe aivalis, Agrocybe cylindracea, Agrocybe dura, Agrocybe erebia, Agrocybe farinacea, Agrocybe jinua, Agrocybe molesta, Agrocybe paludosa, Agrocybe parasitica, Agrocybe pediades, Agrocybe praecox, Agrocybe putaminum, Agrocybe re tiger a, Agrocybe semiorbiculcuis, Agrocybe sororia, Agrocybe vervacti, Coprinellus radians, Coprinellus amphithallus, Coprinellus angulatus, Coprinellus aureogranulatus, Coprinellus bipellis, Coprinellus bisporiger, Coprinellus bisporus, Coprinellus callinus, Coprinellus congregates, Coprinellus curtus, Coprinellus deliquescens, Coprinellus deminutus, Coprinellus dilectus, Coprinellus disseminates, Coprinellus domesticus, Coprinellus ellisii, Coprinellus ephemerus, Coprinellus flocculosus, Coprinellus heptemerus, Coprinellus heterosetulosus, Coprinellus hiascens, Coprinellus inipatiens, Coprinellus marculentus, Coprinellus mitrinodulisporus, Coprinellus pellucidus, Coprinellus plagioporus, Coprinellus pyrrhanthes, Coprinellus radians, Coprinellus sassii, Coprinellus sclerocystidiosus, Coprinellus subdisseminatus, Coprinellus subimpatiens, Coprinellus subpurpureus, Coprinellus truncoruni, Coprinellus velatopruinatus, Coprinellus verrucispei-imis, Coprinellus xcnithothrix, Coprinopsis cinerea, Marasmius rotula, and Sulfolobus tokodaii. Especially, the unspecific peroxygenases and/or gene encoding the enzyme may be derived from one or more organisms selected from the group consisting of Agrocybe aegerita, Coprinellus radians, Marasmius rotula and Sulfolobus tokodaii.

In embodiments wherein the oxidoreductase comprises a peroxidase, the peroxidase and/or a gene encoding the peroxidase may be derived from one or more organisms selected from the group comprising Acorns calamus, Aedes aegypti, Aggegatibacter actinomycetemcomitans, Allium sativum, Arabidopsis thalicnia, Arachis hypogaea, Armoracia rusticana, Arthromyces ramosus, Arundo donax, Beta vulgaris, Bjerkandera adusta, Bos taurus, Brassica napus, Brassica oleracea, Brassica rapa, Bubalus bubali, Butia capitata, Camellia sinensis, Capra hircus, Capsiam anmmm, Catharantus roseus, Chromolaena odorata, Cicer anetinmn, Coprinopsis cinerea, Cucumis melo, Cucumis melo var, inodorus, Cynara cardunculiis, Elaeis guineensis, Elizabethkingia meningoseptica, Escherichia coli, Euphorbia characias, Fagopyrtmi esculentum, Fragaria vesca, Fragaria x ananassa, Glycine max, Gossypiwv hirsutum, Helianthus annuus, Homo sapiens, Hordeum vulgare, Ipomoea batatas, Ipomoea caniea, Jubaea chilensis, Landoltia punctata, Leptogium saturninum, Malus x domestica, Mentha arvensis, Momordica charantia, Mus musculus, Mycobacterium tuberculosis, Neurospora crassa, Nicotiana sylvestris, Nicotiana tabacum, Oryza sativa, Ovis aries, a Pelargonium species, e.g., Pelargonium graveolens, Plasmodium falciparum, Pleurotus eryngii, Pleurotus ostreatus, Prunus persica, Pyrococcus furiosus, Raphanus sativus, Ratius norvegicus, Roystonea regia, Ruegeria pomeroyi DSS-3, Sabal minor, Sclerocarya birrea, Scutellaria baicalensis, Senecio squalidus, Sesbania rosfata, Solwmm lycopersicum, Solanum melongena, Sorghum bicolor, Sphagimm magellanicum, Streptomyces thermoviolaceus, Sulfolobus acidocaldarius, Sus scrofa, Trachycarpus fortunei, Triticum aestivum, Vitis vinifera, Washingtonia fdifera, and Yersinia pseudotuberculosis.

In embodiments wherein the oxidoreductase comprises a chloride peroxidase, the chloride peroxidase and/or a gene encoding the chloride peroxidase may be derived from one or more organisms selected from the group comprising Caldariomyces fumago, Aspergillus niger, Bazzania trilobata, Musa paradisiaca, and Streptomyces toyocaensis.

In embodiments wherein the oxidoreductase comprises a bromide peroxidase, the bromide peroxidase and/or a gene encoding the bromide peroxidase may be derived from one or more organisms selected from the group comprising Agrocybe aegerita, Ascophyllum nodosum, Corallina officinalis, Corallina pilulifera, Delisea pulchra, Ecklonia stolonifera, Fucus distichus, Gracilaria changii, Homo sapiens, Kappaphycus alvarezii, Laminaria hyperborea, Macrocystis pyrifera, Ochtodes secundiramea, Pseudomonas fluorescens, Pseudomonas putida, Saccharina latissima, Streptomyces aureofaciens, Streptomyces griseus, Streptomyces venezuelae, and Synechococcus sp.

It will be clear to one skilled in the art that the invention is not limited to the use of a native oxidoreductase of any one of the organisms specifically mentioned herein. Rather, the oxidoreductase may comprise an oxidoreductase of an organism not specifically mentioned herein, especially wherein the enzyme is homologous to an oxidoreductase of any one of the mentioned organisms. Alternatively or additionally, the oxidoreductase may comprise a mutant oxidoreductase, such as a mutant oxidoreductase comprising one or more amino acid substitutions, deletions and/or additions relative to a native oxidoreductase, especially wherein the mutant oxidoreductase is specifically designed through protein engineering.

In specific embodiments, the oxidoreductase may comprise a mutant unspecific peroxygenase. For example, the oxidoreductase may comprise one or more of the PaDa-I, the JaWa, and the Solo mutants of the unspecific peroxygenase of Agrocybe aegerita as described in Molina-Espeja et al. 2016 ChemBioChem and in WO2017081355A1. In further embodiments, the oxidoreductase may comprise the Jed-I mutant of the unspecific peroxygenase of Agrocybe aegerita as described in Ramirez-Escudero et al. 2018 ACS chemical biology. Hence, in embodiments, the oxidoreductase may comprise a wildtype and/or mutant unspecific peroxygenase, especially a wildtype and/or mutant unspecific peroxygenase of Agrocybe aegerita, more especially one or more of the PaDa-I, the JaWa, the Solo, and the Jed-I mutants of the unspecific peroxygenase of Agrocybe aegerita.

In embodiments, the oxidoreductase may be provided via one or more microorganisms producing the oxidoreductase, or via an addition of isolated oxidoreductase. Herein the term “isolated oxidoreductase” refers to biologically, especially microbially, produced oxidoreductase that has been isolated from the production organism. The isolated oxidoreductase may essentially comprise purified oxidoreductase. In general, embodiments of the invention involve the use of isolated oxidoreductase.

In embodiments, the oxidoreductase may be produced by an organism naturally producing the oxidoreductase. Alternatively or additionally, the oxidoreductase may be produced by a genetically modified organism. For example, in an embodiment the unspecific peroxygenase apol gene of Agrocybe aegerita is heterologously expressed in Pichia pastoris X-33, which exports the Apol protein (the unspecific peroxygenase) into a medium, for example into a liquid (growth) medium. In alternative embodiments, the oxidoreductase may be produced by an organism naturally producing the oxidoreductase, especially wherein the organism exports the oxidoreductase into a medium. In further embodiments, the oxidoreductase may be isolated from the medium to obtain an isolated oxidoreductase. For example, the media comprising (microbial) cells and oxidoreductase may be centrifuged such that the cells precipitate while the oxidoreductase remains in the supernatant. The supernatant comprising the oxidoreductase may be used as a crude enzyme preparation in the first stage, i.e., in embodiments the oxidoreductase may be provided as crude enzyme preparation. The oxidoreductase may also be further purified from the crude enzyme preparation, i.e., in embodiments the oxidoreductase may be provided in purified form. Methods for heterologous gene expression, protein production, protein isolation, and protein purification will be known by a person skilled in the art.

It will be clear to a person skilled in the art that an enzyme may require a cofactor. Hence in embodiments, the first mixture may comprise a cofactor that is suitable for the oxidoreductase. In embodiments, the first mixture may comprise one or more of heme, NAD ⁺ and NADH.

The oxidoreductase may, in embodiments, especially comprise an unspecific peroxygenase. Unspecific peroxygenases may be particularly suitable for the method of the invention as they may have fewer requirements than other oxidoreductases. For instance, unspecific peroxygenases may not require additional cofactors and corresponding regeneration systems, may function independently from external aeration, and may be stable and active under non-aqueous reaction conditions. The unspecific peroxygenase may, in embodiments, be an enzyme from a fungal protein family, identified by EC no 1.11.2.1. Hence, the unspecific peroxygenase may, in embodiments, belong to enzyme class EC 1.11.2.1. Especially, the unspecific peroxygenase may be an isolated protein, such as isolated from their natural biological source. Alternatively, in embodiments, the unspecific peroxygenase may be a recombinant protein which may be synthesized in vivo or in vitro.

In embodiments, the oxidoreductase may be configured to catalyze a hydroxylation of the first compound at position 3, 4, 5, and/or 6 to provide a second compound. The oxidoreductase may be configured to catalyze hydroxylation of the first compound especially at position 3, and/or especially at position 4, and/or especially at position 5, and/or especially at position 6. In further embodiments, the oxidoreductase may be configured to catalyze the hydroxylation of the first compound at position 4 or 5. Especially, the position of hydroxylation may determine a ring-size of the lactone obtained by the method. For instance, hydroxylation at a carbon at a position x may result in a x+1 membered ring, i.e., hydroxylation at position 4 may result in a lactone with 5 atoms in its ring structure.

In embodiments, in the first stage, the peroxide may especially provide a hydroxy group for hydroxylation of the first compound by the oxidoreductase. In embodiments, the peroxide may be selected from the group of hydroperoxides, such as hydrogen peroxide, tert-butyl peroxide, urea hydrogen peroxide, and cumene hydroperoxide. The method of the invention has, for instance, been performed successfully with hydrogen peroxide and tert-butyl peroxide as peroxide. Hence, in embodiments, the peroxide may comprise hydrogen peroxide. Additionally or alternatively, the peroxide may in embodiments comprise tert-butyl peroxide. Hence, in specific embodiments, the peroxide comprises one or more of hydrogen peroxide and tert-butyl peroxide.

In embodiments, the method may comprise the second stage. The second stage may especially comprise providing the lactone from the second compound. In embodiments, the lactone formation may be a catalyzed ring-formation reaction. Embodiments of the catalyzation are described below. However, in particular, the second stage may comprise a spontaneous ring-formation reaction, especially in embodiments wherein the oxidoreductase hydroxylates the first compound at position 4 or 5, no second catalyst may be required as the second compound may spontaneously provide a stable five- or six-membered ring, respectively.

In embodiments, the first stage and the second stage may coincide i.e. a part of the first stage and a part of the second stage may be carried out concurrently. Especially, the first stage and the second stage may be performed simultaneously in one reaction vessel. In alternative embodiments, the preparation stage, first stage and second stage may be performed sequentially. More especially, the first stage and second stage may be performed sequentially in one reaction vessel. Alternatively, in embodiments, the first stage may be performed in a first reaction vessel and the second stage may be performed (subsequently) in a second reaction vessel.

As mentioned before, the preparation stage may in embodiments comprise providing the first compound. Further embodiments of the first compound will be described here. As indicated above, in specific embodiments, R2 may be selected from unbranched, unsubstituted, saturated alkyl groups. Formula la depicts the structural formula of such embodiment of the first compound.

Especially, in embodiments of the first compound, n (as depicted in formula la) may be selected from the range of 0-30, such as 0-22, like 0-18, especially 0-12. More especially, n may be selected from the range of 0-10, like 2-8, such as 2-6, especially 2-4. Hence, in embodiments, the first compound has a structural formula according to formula la, wherein n is selected from the range of 0-18. Such embodiments of the first compound may be naturally available, especially from renewable feedstocks. In further embodiments, n is selected from the range of 2-6. With such embodiments of the first compound particularly good experimental results have been obtained (see below).

As indicated above, in embodiments, Ri may be selected from alkyl groups. Especially, Ri may be branched or unbranched (linear). Although Ri may generally not become part of the final lactone product, various properties of Ri may be relevant for the method of the invention. These properties may include, for instance, solubility (in the solvent; see below), reactivity, (bio)-availability (as a renewable feedstock), melting temperature, as well as handleability. Especially, first compounds comprising -CH3 (methyl) and -CH2CH3 (ethyl) groups for Ri may be relatively easy to handle and/or affordable. Therefore, the first compound may especially comprise one or more of a fatty acid methyl ester, fatty acid methyl amide, fatty acid methyl thioester, fatty acid ethyl ester, fatty acid ethyl amide and fatty acid ethyl thioester. Hence, in specific embodiments Ri comprises -CH3 or -CH2CH3.

In specific embodiments, the first compound may be selected from the group comprising monoglyceride esters, diglyceride esters, triglyceride esters, methyl esters, and ethyl esters. Esters may be prepared from natural fatty acid (derivatives) as described in Khan et al., Current developments in esterification reaction: A review on process and parameters, Journal of Industrial and Engineering Chemistry, Volume 103, 2021, 80-101.

In embodiments wherein the oxidoreductase hydroxylates the first compound at position 4 or 5, a stable 5 or 6-membered ring may be obtained respectively. Due to the stability of such a 5- or 6-membered ring, the ring forming reaction may especially occur spontaneously and no (second) catalyst may be required.

In embodiments wherein the oxidoreductase hydroxylates the first compound at position 3 or 6, the corresponding lactone may comprise a 4- or 7-membered ring, respectively. Such a lactone may be less stable and in embodiments, a second catalyst may be required to catalyze the ring forming reaction. Hence, the method may especially comprise providing a second catalyst to the second compound. More especially, the second catalyst may be provided during and/or preceding the second stage. Further, in embodiments, the second catalyst may comprise a hydrolase. Especially, the hydrolase may comprise one or more of a lipase, an esterase, a transacylase and a protease. In alternative embodiments, the second catalyst may comprise an acid, such as a Lewis acid and/or a Bronsted acid. Especially, the acid may comprise one or more of simple mineral acids, and metal salts (free in solution or embedded in heterogeneous caries (e.g. zeolites). Hence, in specific embodiments, the method comprises providing a second catalyst to the second compound during and/or preceding the second stage, wherein the second catalyst comprises one or more of a hydrolase and an acid.

The first stage may in embodiments comprise applying a (first) reaction temperature. The oxidoreductase may perform especially well at a temperature selected from the range of 10-60°C. Especially, the first stage may comprise exposing the first compound to the oxidoreductase at the (first) reaction temperature. In embodiments, the (first) reaction temperature may be selected from the range of 5-150°C, such as from the range of 20°C - 100°C, especially from the range of 25°C - 80°C, more especially from the range of 30-60°C, such as from the range of 40-60°C. Hence, in specific embodiments, the first stage comprises exposing the first compound to the oxidoreductase at a reaction temperature, wherein the reaction temperature is selected from the range of 20°C - 100°C.

Further, in embodiments, the first compound may be a liquid at the reaction temperature. Especially, the first compound may in embodiments form a liquid phase. More especially, the liquid phase (of the first compound) may be a solvent for the first stage. Especially the liquid phase (of the first compound) may be the only solvent for the first stage. Especially, in embodiments the solvent comprises a water content wherein the water content is less than 5% such as less than 2%, like less than 1%, especially less than 0.1%. Hence, in this way, compared to conventional methods, the first stage (and/or the second stage) may be carried out under non-aqueous conditions. Especially, since the first compound may be a (liquid phase) solvent, this may provide the advantage of not requiring additional (aqueous) solvents. Water may in embodiments contribute to denaturation of proteins, such as the oxidoreductase. Therefore, the oxidoreductase, especially the unspecific peroxygenase, may be more stable under non-aqueous conditions. Hence, performing the method under non-aqueous conditions may improve the efficiency of the method.

Especially, in such non-aqueous conditions, the peroxide may comprise tertbutyl peroxide. Tert-butyl peroxide may be better soluble in organic solvents than other peroxides such as hydrogen peroxide. Better solubility of the peroxide may improve a reaction rate of the method and hence may be more efficient. On the other hand, in aqueous conditions, the peroxide may in embodiments comprise hydrogen peroxide as hydrogen peroxide may have a higher solubility in water compared to tert-butyl peroxide.

Additionally or alternatively, the first stage may in embodiments be performed under dehydrating conditions. In such embodiments, water may evaporate, e.g. at 100°C at ambient pressure or lower temperatures at lower pressures. As indicated above, the oxidoreductase, especially the unspecific peroxygenase, may be more stable under nonaqueous conditions. Hence, performing the method under dehydrating conditions may also improve the yield of the method.

In alternative embodiments, the first stage may also comprise one or more other solvent (in addition to the first compound). Furthermore, the other solvent may especially comprise an organic solvent, such as acetonitrile, or dimethyl sulfoxide. In further embodiments, the other solvent may comprise a hydrophobic organic solvent, such as hexane, heptane, pentane, butanol, octanol, benzene, ethyl acetate (EtOAc), or tert-butylmethylether (MtBE).

In embodiments, the reaction temperature may be selected such that the first compound may be in a liquid phase. In other words, in embodiments, the reaction temperature may be selected such that the first compound may be melted. Hence, in embodiments, the reaction temperature may be selected above a melting temperature of the first compound. The melting temperature of the first compound may especially depend on Ri and R2, which will be known to a person skilled in the art. In specific embodiments, the reaction temperature may be selected from the range of 10 - 150°C, such as from the range of 20°C - 100°C, especially from the range of 25°C - 80°C. In further embodiments, the reaction temperature may be at least 5°C, such as at least 10°C, like at least 15°C. Additionally or alternatively, the reaction temperature may be below 180°C, especially below 150°C, such as below 100°C, especially below 90°C.

Further, in embodiments, the first stage may comprise providing a first mixture. In embodiments, the first mixture may comprise one or more of the first compound, the oxidoreductase, especially an (unspecific) peroxygenase, and the peroxide. Hence, in specific embodiments, the first stage comprises providing a first mixture comprising the first compound, the oxidoreductase, and the peroxide, wherein the reaction temperature is selected from the range of 20-100°C, and wherein the first compound forms a liquid phase.

In embodiments, the first stage may in embodiments comprise applying a (first) reaction pressure. The (first) reaction pressure may, over a temperature range of 5-150 °C, especially be higher than the vapor pressure of the first compound. That is, at every temperature in the range of 5-150 °C, the (first) reaction pressure may be higher than the vapor pressure of the first compound (at that temperature). In embodiments, the first stage may comprise exposing the first compound to the oxidoreductase at the (first) reaction pressure. In embodiments, the (first) reaction pressure may be selected from the range of 0.5-15 bar, such as from the range of 1-10 bar, especially from the range of 1-8 bar. Hence, in specific embodiments, the first stage comprises exposing the first compound to the oxidoreductase at a reaction pressure, wherein the reaction pressure is selected from the range of 0.5-15 bar. Exposing especially the first compound to a pressure selected from the range of 0.5-15 bar may facilitate maintaining the first compound in a liquid state, thereby preventing evaporation and possible loss of the first compound. Hence, availability of the first compound for the oxidoreductase (present in solution) may be improved at pressures of 0.5-15 bar, especially at pressures above the vapor pressure of the first compound. Improved availability of the first compound may facilitate improving the yield of the reaction (i.e. the amount of second compound formed in the first stage). Hence, in specific embodiments, the first stage comprises exposing the first compound to the oxidoreductase at a reaction pressure, wherein the reaction pressure (at the reaction temperature) is higher than a vapor pressure of the first compound (at the reaction temperature).

Likewise, the second stage may in embodiments comprise selecting a second reaction temperature. Especially, the second stage may comprise exposing the second compound to the second reaction temperature. As indicated above, the first stage and the second stage may in embodiments overlap. That is, (a part of) the first stage may in embodiments be carried out concurrently (or simultaneously) with the second stage. Therefore, in such embodiments, the second reaction temperature may be equal to the (first) reaction temperature. However, in embodiments wherein the first stage and the second stage are (at least partly) sequential, the second stage may in embodiments comprise applying a second reaction temperature that differs from the (first) reaction temperature. However, in alternative embodiments, the second reaction temperature may in embodiments be selected equal to the (first) reaction temperature (even when the first stage and the second stage are fully sequential).

In embodiments, the second reaction temperature may be selected such that the second compound may be in a liquid phase. In other words, in embodiments, the reaction temperature may be selected such that the second compound may be melted. Hence, in embodiments, the reaction temperature may be selected above a melting temperature of the second compound. The melting temperature of the second compound may especially depend on Ri and R2, which will be known to a person skilled in the art. In specific embodiments, the second reaction temperature may be selected from the range of 10 - 150°C, such as from the range of 20°C - 100°C, especially from the range of 25°C - 80°C. In further embodiments, the second reaction temperature may be at least 5°C, such as at least 10°C, like at least 15°C. Additionally or alternatively, the reaction temperature may be below 250°C, such as below 200°C, like below 150°C. In embodiments, the second reaction temperature may be selected from the range of 15°C - 200°C, like from the range of 20°C - 100°C, especially from the range of 30-60°C, such as from the range of 20°C - 80°C. Hence, in specific embodiments, the second stage comprises exposing the second compound to a second reaction temperature, wherein the second reaction temperature is selected from the range of 20°C - 100°C.

Further, in embodiments, the second stage may in embodiments comprise applying a second reaction pressure. The second reaction pressure may, over a temperature range of 5-150 °C, especially be higher than the vapor pressure of the second compound. That is, at every temperature in the range of 5-150 °C, the second reaction pressure may be higher than the vapor pressure of the second compound (at that temperature). In embodiments, the second stage may comprise providing the lactone from the second compound at the second reaction pressure. In embodiments, the second reaction pressure may be selected from the range of 0.5-15 bar, such as from the range of 1-10 bar, especially from the range of 1-8 bar. Hence, in specific embodiments, the second stage comprises providing the lactone from the second compound at a second reaction pressure, wherein the second reaction pressure is selected from the range of 0.5-15 bar. Exposing the second compound to a pressure selected from the range of 0.5-15 bar may facilitate maintaining the second compound in a liquid state, thereby preventing evaporation and possible loss of the second compound. Hence, the yield of the lactone (i.e. the amount of lactone formed) may be improved. In embodiments, the second reaction pressure may be equal to the (first) reaction pressure. However, in embodiments wherein the first stage and the second stage are (at least partly) sequential, the second stage may in embodiments comprise applying a second reaction pressure that differs from the (first) reaction pressure. However, in alternative embodiments, the second reaction pressure may in embodiments be selected equal to the (first) reaction pressure (even when the first stage and the second stage are fully sequential).

As mentioned above, the first compound may in embodiments form a liquid phase (e.g. the first compound may melt at the first reaction temperature). Further, in embodiments, the first compound may be provided in an aqueous solution. Thus, the first stage may in embodiments comprise providing a first mixture in an aqueous solution. Especially, the first mixture may comprise one or more of the first compound, the oxidoreductase, especially the (unspecific) peroxygenase, and the peroxide. In specific embodiments, the aqueous solution may have a pH selected from the range of 2.0-14.0, such as from the range of 4.0-11.0, especially from the range of 5.0-8.0, such as from the range of 6.0-7.0. Hence, in specific embodiments, the first stage comprises providing a first mixture in an aqueous solution, wherein the first mixture comprises the first compound, the oxidoreductase, especially the (unspecific) peroxygenase, and the peroxide wherein the aqueous solution has a pH selected from the range of 5.0-8.0. In further embodiments, the first mixture may further comprise an organic solvent, such as acetonitrile and/or dimethyl sulfoxide. The organic solvent may in embodiments make up 0-99 vol.% such as 0-50 vol.%, especially 2-20 vol.% of the total solvent. Further, in embodiments, the organic solvents may make up 10-90 vol.%, such as 40- 80 vol.%, especially 50-70 vol.%.

As indicated above, the method may be independent from external aeration. Hence, in embodiments the first mixture may have an oxygen level below 15 vol.%, such as below 10 vol. %, especially below 7 vol.%, such as below 4 vol.%. In alternative embodiments, the first mixture may have an oxygen level above 4 vol.%, such as above 7 vol.%, especially above 10 vol.%.

The method of the invention may especially comprise using an oxidoreductase. In further embodiments, the oxidoreductase may comprise an unspecific peroxygenase. The term “unspecific peroxygenase” may herein refer to an enzyme that may be configured to catalyze a variety (hence “unspecific”) of hydroxylation reactions. The unspecific peroxygenase is an enzyme, hence a protein and thus has an amino acid sequence. In specific embodiments, unspecific peroxygenase may have an amino acid sequence having at least 30%, like at least 50% such as at least 70%, like at least 80%, especially at least 90%, such as at least 95%, including 100% sequence identity to a reference amino acid sequence with respect to a sequence alignment between the amino acid sequence and the reference amino acid sequence. In embodiments, the sequence alignment may have a length of at least 40%, such as at least 65%, like at least 70%, such as at least 80%, especially at least 90%, such as at least 99%, including 100% of a sequence length of the reference amino acid sequence. In embodiments, the reference amino acid sequence may be one or more of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3. Especially, the reference amino acid sequence may in embodiments be selected from the group comprising SEQ ID NO: 1-3, especially SEQ ID NO:1, or especially SEQ ID NO:2, or especially SEQ ID NO:3. Hence, in embodiments, unspecific peroxygenase has an amino acid sequence having at least 80% sequence identity to a reference amino acid sequence with respect to a sequence alignment between the amino acid sequence and the reference amino acid sequence, wherein the sequence alignment has a length of at least 70% of a sequence length of the reference amino acid sequence, wherein the reference amino acid sequence is selected from the group comprising SEQ ID NO: 1-3. In further embodiments, a peroxygenase having at least 80% sequence identity to SEQ ID NO:2 or to SEQ ID NO:3 may be more active and/or selective to a reaction of interest. Hence, in embodiments, the reference amino acid sequence is selected from the group comprising SEQ ID NO:2-3. Also, such peroxygenase may be smaller (having a shorter amino acid sequence length) compared to SEQ ID NO:1 and hence may be easier and/or cheaper to produce.

In general, if two proteins consist of (highly) similar amino acid sequences, these two proteins may be likely to perform the same biological function. This relation between amino acid sequence and protein function may, for example, be used to predict the function of a protein based on its sequence identity with proteins of known function (annotation by sequence homology based inference). The term “sequence identity” herein refers to the percentage of the characters (such as amino acids in an amino acid sequence) in the shorter of two sequences matching an identical character in the longer of the two sequences in a sequence alignment (also see below). The higher the sequence identity between two proteins, the higher the chance may be that these two proteins have the same or a similar function. Although there may not be a hard rule for inferring functional identity or similarity based on a threshold value for sequence identity, especially as the threshold value may need to be adjusted based on (relative) sequence length and/or protein function, proteins may have been successfully annotated based on a common rule-of-thumb threshold of at least 30-40% sequence identity. Hence, proteins similar to SEQ ID NO: 1-3 in both length (including both shorter and longer) and amino acid sequence may have a similar activity as SEQ ID NO: 1-3.

Amino acid sequence alignments may especially be obtained using BLASTp at the website of the National Center for Biotechnology Information (NCBI). Two sequences may be aligned via BLASTp, especially using default algorithm parameters, such as using a BLOSUM62 matrix with a gap cost of 11 : 1 (existence:extension).

Hence, in embodiments, the sequence alignment of the unspecific peroxygenase amino acid sequence and one or more of SEQ ID NO: 1-3 may be a BLASTP pairwise sequence alignment obtained with a BLOSUM62 matrix with an existence gap cost of 11 and an extension gap cost of 1.

In embodiments, the unspecific peroxygenase amino acid sequence may be shorter or longer than one or more of SEQ ID NO: 1-3. Hence, in embodiments, the unspecific peroxygenase amino acid sequence of the unspecific peroxygenase protein may have a sequence length > 50% of the sequence length of one or more of SEQ ID NO: 1-3, such as > 60%, especially > 70%, such as > 80, especially > 90, such as > 100%, especially > 120%. Similarly, in further embodiments, the unspecific peroxygenase amino acid sequence of the unspecific peroxygenase protein may have a sequence length < 200% of the sequence length of one or more of SEQ ID NO: 1-3, such as < 180%, especially < 160%, such as < 140%, especially < 130%, such as < 120%, especially < 110%, such as < 100%.

Specifically, SEQ ID NO:1 corresponds to the amino acid sequence of the unspecific peroxygenase protein of Agrocybe aegerita unspecific peroxygenase ( /cUPO). Further, SEQ ID NO:2 corresponds to the amino acid sequence of the unspecific peroxygenase protein of Daldinia caldariorum unspecific peroxygenase (/Jcz/UPO). Likewise, SEQ ID NO:3 corresponds to the amino acid sequence of the unspecific peroxygenase protein of Collariella virescens unspecific peroxygenase (Cw'UPO).

In specific embodiments, the oxidoreductase may comprise an unspecific peroxygenase. Especially, the unspecific peroxygenase may comprise an Agrocybe aegerita unspecific peroxygenase ( /cUPO) protein, i.e., an unspecific peroxygenase (derived) from Agrocybe aegerita. Additionally or alternatively, the unspecific peroxygenase may in embodiments comprise a Daldinia caldariorum unspecific peroxygenase (/Jcz/UPO) protein. In further embodiments, the unspecific peroxygenase may comprise a Collariella virescens unspecific peroxygenase (CvzUPO) protein. In specific embodiments, the unspecific peroxygenase may comprise one or more of /cUPO, DcaUPO, and CvzUPO.

In further embodiments, the oxidoreductase amino acid sequence may comprise at least one difference to the reference sequence, especially to one or more of SEQ ID NO: 1-3, or especially to (each of) the reference sequences. Hence, in embodiments, the oxidoreductase protein may be a recombinant unspecific peroxygenase protein, wherein an oxidoreductase amino acid sequence of the recombinant unspecific peroxygenase protein is engineered to have at least one difference with respect to (each of) the reference sequences. The difference may especially be an amino acid deletion, addition, and/or substitution. Hence, in embodiments, the oxidoreductase protein may be a non-naturally occurring protein.

In embodiments, the oxidoreductase protein may comprise 100 - 800 amino acids, such as 200 - 600 AA.

The method of the invention may be used to provide one or more of y- decalactone and 6-decalactone. In embodiments of the method, the first compound may be according to formula la, wherein X=O, i.e., the first compound comprises an ester. Especially, in embodiments, n=4. In such embodiments, the method may provide one or more of a y- decalactone and a 6-decalactone. In further embodiments, the peroxide may comprise hydrogen peroxide. Hence, in specific embodiments, X=O, wherein n=4, wherein the peroxide comprises hydrogen peroxide, and wherein the method provides one or more of a y-decalactone and a 6- decalactone. In this way, the industrially relevant lactones y-decalactone and 6-decalactone may be provided via a relatively scalable and more cost-effective method.

In yet further embodiments, the first compound may be according to formula la, wherein X=NH or X=S , i.e., the first compound comprises an amide or thioester. Especially, in embodiments, n=4. In such embodiments, the method may provide one or more of a y- decalactone and a 6-decalactone. Hence, in specific embodiments, X=NH or X=S. In further embodiments, the peroxide may comprise hydrogen peroxide. In more specific embodiments, X= NH or X=S, wherein n=4, wherein the peroxide comprises hydrogen peroxide, and wherein the method provides one or more of a y-decalactone and a 6-decalactone. Also in this way, the industrially relevant lactones y-decalactone and 6-decalactone may be provided via a relatively scalable and more cost-effective method.

In embodiments, the first compound may comprise a fatty acid derivative. The term “fatty acid derivative” may especially refer to a one or more of a fatty acid ester, a fatty acid amide and a fatty acid thioester. Also, in embodiments the fatty acid derivative may comprise a natural fatty acid derivative. Especially in such embodiments, the first compound may originate from a biological sample. However, the natural fatty acid derivative may in embodiments be chemically synthesized. Alternatively, in embodiments the fatty acid derivative may comprise a non-natural fatty acid derivative. Although one or more of the first compound and the oxidoreductase may originate from a biological sample, the method of the invention may in embodiments especially comprise exposing the first compound to the oxidoreductase in vitro. The term “/// vitro" may herein refer to “outside of their natural (biological) context”. Hence the first compound may in embodiments comprise an isolated fatty acid derivative. Likewise, the oxidoreductase may in embodiments comprise an isolated enzyme. Especially, the first stage may in embodiments be in vitro, such as in a (first) reaction vessel, reaction tube etc.

In a further aspect, the invention may provide a use of the lactone obtainable by the method of the invention. In embodiments, the use of the lactone may comprise use as a flavor ingredient. Additionally or alternatively, in embodiments, the use of the lactone may comprise use as a fragrance ingredient. In summary, the invention may in embodiments comprise use of the lactone in the flavor and fragrance industry. This embodiment may (also) comprise the use of the lactone as a food ingredient and hence, use of the lactone in food industry. Further embodiments may comprise the use of the lactone as a polymer building block, for e.g. as a monomer for ring opening polymerization of polycaprolactone. Hence, in specific embodiments the invention provides a use of the lactone obtainable by the method of the invention as one or more of a flavor and fragrance ingredient or a polymer building block.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig. 1 schematically depicts an embodiment of the method of the invention; Fig. 2 schematically depicts further embodiments of the method of the invention; Fig. 3 schematically depicts an embodiment of the method of the invention; and Figs. 4 and 5 depict experimental data. The schematic drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Fig. 1 schematically depicts embodiments of a method for providing a lactone 10, wherein the method comprises: a preparation stage, a first stage and a second stage. In the depicted embodiment, these stages coincide. In embodiments, the preparation stage may comprise providing a first compound 1 according to formula (I):

Especially, X may be selected from the group comprising O, NH, and S, especially O, or especially NH, or especially S. Ri may in embodiments be selected from alkyl groups comprising 1-10 C atoms. In specific embodiments, Ri may comprise -CH3 or -CH2CH3. In further embodiments, R2 may be selected from branched or unbranched, substituted or unsubstituted hydrocarbon chains, especially one or more of alkyl, alkene, and alkyne groups. Especially, R2 may comprise 5-25 C atoms. The first stage may in embodiments comprise exposing the first compound 1 to an oxidoreductase 3, especially an unspecific peroxygenase, in the presence of a peroxide 4. The oxidoreductase 3 may especially be configured to catalyze hydroxylation of the first compound 1 at position 3, 4, 5, and/or 6, especially at position 3, and/or at position 4, and/or at position 5, and/or at position 6. In embodiments, the second stage may comprise providing the lactone 10 from the second compound 2 via a (spontaneous) ringformation reaction. In further embodiments, the first stage may comprise exposing the first compound 1 to the oxidoreductase 3 at a reaction temperature. Especially, the reaction temperature may be selected from the range of 20°C - 100°C. In further embodiments, the first stage may comprise providing a first mixture comprising the first compound 1, the oxidoreductase 3, and the peroxide 4 wherein the reaction temperature may be selected from the range of 20-100°C, wherein the first compound 1 forms a liquid phase.

In alternative embodiments, the first stage may comprise providing a first mixture in an aqueous solution. The first mixture may especially comprise the first compound 1, the oxidoreductase 3, and the peroxide 4. In embodiments, the aqueous solution may have a pH selected from the range of 5.0-8.0.

In embodiments of the method of the invention, the oxidoreductase 3 may have an amino acid sequence having at least 80% sequence identity to a reference amino acid sequence with respect to a sequence alignment between the amino acid sequence and the reference amino acid sequence. Especially, the sequence alignment may have a length of at least 70% of a sequence length of the reference amino acid sequence. In specific embodiments the reference amino acid sequence may be selected from the group comprising SEQ ID NO:1- 3. In embodiments, the reference amino acid sequence may be SEQ ID NO: 1. In alternative embodiments, the reference amino acid sequence may be selected from the group comprising SEQ ID NO:2-3, especially SEQ ID NO:2, or especially SEQ ID NO:3.

In embodiments, the peroxide 4 may comprise one or more of hydrogen peroxide and tert-butyl peroxide. In specific embodiments, the method may comprise exposing the first compound 1 to the oxidoreductase 3 in vitro.

The invention may further provide use of the lactone 10 obtainable by the method of the invention as one or more of a flavor and fragrance ingredient or a polymer building block.

Fig. 2a-b schematically depict embodiments wherein the method may comprise providing a second catalyst 5 to the second compound 2 during and/or preceding the second stage. Fig. 2a schematically depicts embodiments wherein the first stage and the second stage overlap. In embodiments, the method may comprise providing the second catalyst 5 to the second compound 2 during and/or preceding the first stage. The second catalyst 5 may in embodiments comprise one or more of a hydrolase and an acid.

Fig. 2b schematically depicts embodiments wherein the first stage precedes the second stage. In such embodiments, the second catalyst 5 may be provided after the first stage. Hence, in embodiments the method may comprise providing the second catalyst 5 to the second compound 2 during and/or preceding the second stage.

In further embodiments, the second stage may comprise exposing the second compound 2 to a second reaction temperature. Especially, the second reaction temperature may be selected from the range of 20°C - 100°C.

Fig. 3 schematically depicts specific embodiments of the method of the invention. Especially, the first compound 1 may in embodiments have a structural formula as depicted in Fig. 3. In specific embodiments, n may be selected from the range of 0-18. In more specific embodiments n may be selected from the range of 2-6. In specific embodiments n=4. In further embodiments, X=O, i.e., the first compound may comprise an ester. Especially, the peroxide may in embodiments comprise hydrogen peroxide. In the depicted embodiment, the oxidoreductase 3 may be configured to hydroxylate the first compound 1 at position 4 and/or 5. Hence, as depicted, two distinct second compounds 2 may be formed. In such embodiments, the method may provide one or more of a y-decalactone and a 6-decalactone. In alternative embodiments, X=NH or X=S, i.e., the first compound may comprise an amide or thioester.

Experiments

Unless described otherwise, the experiments described herein are performed using the materials and methods described hereinafter.

Example with methyl hexadecanoate and Agrocybe aegerita unspecific peroxygenase (AaeUPO)

The reaction was (separately) performed in 1 mL with two different first compounds 1 : methyl hexanoate and methyldecanoate, with 1 pM r /cUPO mutant, 5 mM/h H2O2 (continuously) and buffer (50 mM KPi, pH 7.0), and was under 600 rpm shaking speed at 25 °C for 24 h. Samples were withdrawn and extracted with ethyl acetate (containing 5 mM cyclooctane as internal standard 9). The organic layer was dried over magnesium sulfate (MgSCU) for gas chromatography analysis.

In these conditions, methyl 4-hydroxyhexanoate (obtained with a regioselectivity co- 1/ co-2 ratio of 99: 1) appeared as the main product. The second compound 2, the lactonization product, was also observed in these conditions at 8.8 minutes as depicted in Fig. 4a. Fig 4a schematically depicts a GC chromatogram of samples taken at multiple time points of hydroxylation reaction from first compound 1 methyl hexanoate to second compound 2 methyl 5-hydroxyhexanoate. Peaks for the first compound 1, the second compound 2, an internal standard 9, and the lactone 10 can be observed. The reaction was followed over time with samples taken at tl= 1 h, t2=2h, t4=4h, t6=6h, and t24=24h. An intensity I (pV) is plotted against a retention time t (min).

In order to enlarge the proportion of the lactone 10, the product was isolated in ethyl acetate. The organic layer was dried over MgSC and then evaporated with N2 flow. After removing the solvent, liquid was dissolved in CH2Q2 (5 mL/g lactone) and TFA (0.04 mL/g lactone). The lactonization reaction was performed under 300 rpm at 25°C for 18 h. And the reaction was diluted with ethyl acetate and worked up with 100 mM NaHCOs solution. The organic layer was then washed with water and dried with MgSC . The solvent was evaporated to obtain 6-hexalactone, which was confirmed with ^X H NMR (400 MHz) and ¹³ C NMR (100 MHz) spectra (obtained in CDCh). Fig. 4b schematically depicts a GC-MS analysis of the obtained 6-hexalactone in amount A (in arbitrary units) against mass m (in u).

Example with octanoic acid and the unspecific peroxygenases from Daldinia caldariorum (Dca PO).

In 250 pL of sodium phosphate buffer (100 mM, pH6), 1 pM of /Jcz/UPO was added (as a cell free extract from heterologous production in Escherichia colt) as well as 5 mM of octanoic acid from an acetonitrile solution (final concentration of acetonitrile: 10 v/v %). 2 mM of hydrogen peroxide (H2O2) was added to start the reaction. 2 mM of H2O2 was supplemented every hour until 4h.

The reaction was quenched by the addition of 5 pL IM HC1 solution and extracted two times with 250 pL of a solution of 5 mM 1 -octanol in ethyl acetate. The organic phase was dried on magnesium sulfate and injected in gas chromatography. Fig. 5 schematically depicts a GC chromatogram of the dried organic phase. An intensity I (in pV) is plotted against a retention time t (in min). Samples were measured at tO=Oh, 11 = 1 h, t2=2h and tl 5= 15h. The gamma hydroxylation product (second compound 2) and corresponding lactone 10 were detected.

Example with methyl decanoate and the unspecific peroxygenases from Daldinia caldariorum (DcaU PO) and Agrocybe aegerita (AaeU PO)

In 200 pL of sodium phosphate buffer (100 mM, pH6), 1 pM of /Jcz/UPO or /cUPO was added (as a cell free extract from heterologous production in Escherichia colt) as well as 5 mM of methyl decanoate from an acetonitrile solution (final concentration of acetonitrile: 10 v/v %). 2 mM of hydrogen peroxide (H2O2) was added to start the reaction. 2 mM of H2O2 was supplemented every hour until 4h.

The reaction was quenched by the addition of 5 pL IM HC1 solution and extracted two times with 200 pL of a solution of 5 mM n-dodecane in ethyl acetate. The organic phase was dried on magnesium sulfate and injected in gas chromatography. Fig. 6A schematically depicts a GC chromatogram of the dried organic phase. An intensity I (in pV) is plotted against a retention time t (in min). Samples were measured after 15h. The gamma hydroxylation product (second compound 2) and corresponding lactone 10 were detected.

Example with methyl octanoate and the unspecific peroxygenases from Daldinia caldariorum (DcaU PO) and Agrocybe aegerita (AaeV PO)

In 200 pL of sodium phosphate buffer (100 mM, pH6), 1 pM of DcaUPO or AaeUPO was added (as a cell free extract from heterologous production in Escherichia colt) as well as 5 mM of methyl octanoate from an acetonitrile solution (final concentration of acetonitrile: 10 v/v %). 2 mM of hydrogen peroxide (H2O2) was added to start the reaction. 2 mM of H2O2 was supplemented every hour until 4h.

The reaction was quenched by the addition of 5 pL IM HC1 solution and extracted two times with 200 pL of a solution of 5 mM n-dodecane in ethyl acetate. The organic phase was dried on magnesium sulfate and injected in gas chromatography. Fig. 6B schematically depicts a GC chromatogram of the dried organic phase. An intensity I (in pV) is plotted against a retention time t (in min). Samples were measured after 15h. The gamma hydroxylation product (second compound 2) and corresponding lactone 10 were detected.

These examples show that the method of the invention may provide lactones starting from fatty acid derivatives and using an unspecific peroxygenase.

The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’. The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

The project leading to this application has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 966788).