Ene reductase variants

Abstract

The present invention lies in the technical field of enzyme technology and provides variants of ene reductases suitable for the production of dihydrochalcones. Furthermore, the present invention provides methods for the production of dihydrochalcones as well as uses of these variants for the production of hesperetin dihydrochalcone and hesperidin dihydrochalcone.

Description

[0001] Eisenfuhr Speiser

[0002] Munich, 5 February 2025

[0003] Our Ref.: SM 7072-01 WO SOE / HGR

[0004] Applicant: Symrise AG

[0005] Serial Number: New Application

[0006] Symrise AG

[0007] MiihlenfeldstraBe 1 , 37603 Holzminden, Germany

[0008] Ene reductase variants

[0009] The present invention lies in the technical field of enzyme technology and provides variants of ene reductases suitable for the production of dihydrochalcones. Furthermore, the present invention provides methods for the production of dihydrochalcones as well as uses of these variants for the production of hesperetin dihydrochalcone and hesperidin dihydrochalcone.

[0010] 5

[0011] There is a constant need of flavouring substances in the food technology area. Especially the class of dihydrochalcones is of interest, as these substances or mixtures of these substances exhibit superior properties compared to other flavouring substances. The natural source of dihydrochalcones are plants, an especially high content can be found in apple 0 leaves (Adamu et al., Investigations on the formation of dihydrochalcones in apple (Malus sp.) leaves. Acta Horticulturae 2019, 1242, 415-420). As the recovery and extraction of dihydrochalcones from plants is not favourable in terms of yield and process costs, there are several production methods for dihydrochalcones described in the literature.

[0012] 5 A desired product is hesperetin dihydrochalcone (3) as well as hesperidin dihydrochalcone (6).

[0013] *20250023988*

[0014]

[0015] 5 6

[0016] The aromatic effect of hesperetin dihydrochalcone (3) as flavouring substance is described for example in the international patent application WO 2017186299 A1. This property of hesperetin dihydrochalcone is also known from J. Agric. Food Chem., 25(4), 763-772 and J. Med., 1981, 24(4), 408-428. Mixtures of hesperetin dihydrochalcone (3) with corn syrup with increased fruit sugar content and other sweeteners are described in the international patent application WO 2019080990 A1.

[0017] It is described that the production of hesperetin chaicone (1) from hesperetin (2) can be achieved in a one pot reaction by reactingl ,8-Diazabicyclo[5.4.0]undec-7-ene, tert-Butyldimethylsilyl chloride and hydrochloric acid (Miles, Christopher O.; et al Australian Journal of Chemistry (1989), 42(7), 1103-13).

[0018] Further methods to generate hesperetin chaicone (1) comprise the aldol condensation of trihydroxyacetone with isovanillin by addition of potassium hydroxide (Wadher, S. J.; et al International Journal of Chemical Sciences (2006), 4(4), 761-766).In a further step, hesperetin chaicone (1) can be further reduced via hydrogenation using hydrogen or formic acid and palladium catalysts to form hesperetin dihydrochalcone (3) (Gan, Li-She; et al Bioorganic & Medicinal Chemistry Leters (2017), 27(6), 1441-1445, US 20180177758 A1).

[0019] It is also described in the state of the art that the production of hesperetin dihydrochalcone (3) can be achieved directly from hesperetin chaicone (2) by inorganic catalysts such as iron or platinum (CN111018684).

[0020] Hesperetin dihydrochalcone (3) can also be prepared by acidic hydrolysis of neohesperidine dihydrochalcon (WO 2019080990 A1). Furthermore, hesperetin dihydrochalcone (3) can be obtained from hesperetin chaicone (2) as described in DE 2148332 A1 or CN 111018684 by dissolving hesperetin chaicone (2) in 10% aqueous KOH solution and subsequent reduction by means of hydrogen (Pd / C catalyst). The use of protective groups, other bases or reducing agents and the possibility of an acid-catalysed aldol reaction are known to the skilled person.

[0021] However, all methods described in the state of the art cannot be declared as natural production methods according to EC 1334 / 2008 and are limited to producing a specific dihydrochalcone.

[0022] Labelling as natural is crucial for many consumers for purchase decision, so it is clear that there is a particular need for appropriate dihydrochalcones that are allowed to carry this label. Obtaining the dihydrochalcones from plant raw materials, is a timely and costly process and for some of the dihydrochalcones listed herein not possible, as they cannot be found in nature.

[0023] Concerning enzymatic or fermentative methods, the conversion of naringenin, eriodictyol and homoeriodictyol has been reported in the state of the art (EP 2963109A1 , Gall et. al Ange w. Chem. Int. Ed. 2013, 52, 1 - 5). It is furthermore mentioned that the single enzyme flavanonol-cleaving reductase is capable of converting naringenin and homoeriodictyol to the respective dihydrochalcones (Braune et. al. 2019 Appl Environ Microbiol 85:e01233-19). None of the 4-O-methylated derivatives were converted by the enzyme.

[0024] It is furthermore known that the gut bacterium Eubacterium ramulus is able to convert naringenin-7-O-glucoside via a pathway, which involves the production of the dihydroychalcone phloretin. Unfortunately, this is further degraded to phloroglucinol and dihydrocinnamic acid (H. Schneider, M. Blaut, Arch. Microbiol. 2000, 173, 71 - 75).Chaicone reduction activities to its respective dihydrochalcones are described for several organisms in literature, e.g. in Zyszka-Haberecht et al., 2018 and Stompor et al., 2016.

[0025] All of the reported methods and processes are either expensive, laborious or not available for commercial production.

[0026] The international application WO 2022184248 A1 discloses several double bond reductases, specifically the AtDBRI from Arabidopsis thaliana, were identified to reduce hesperetin chaicone to its dihydrochalcone form. This enzyme, the AtDBRI was further optimized via mutagenesis and several optimized variants are described in the international patent application WO 2022184248 A1. The enzymes were not described to be active with hesperidin chaicone (5) to produce hesperidin dihydrochalcone (6), nor do they show sufficient stability and activity fortheir use in large scale manufacturing.

[0027] It was thus the object of the present invention to provide suitable enzymes for the large scale production of dihydrochalcones. Especially for the production of hesperetin dihydrochalcone and hesperidin dihydrochalcone.

[0028] The primary object of the present invention was solved by providing an ene reductase variant having at least one mutation associated with enzyme stability selected from positions 19, 38, 84, 86, 90, 94, 155, 178, 198, 215, 224, 264, 265, 267, 269 and 315,

[0029] and

[0030] at least one mutation associated with enzyme activity at a position selected from positions 81 , 103, 275, 276, 285 and 290

[0031] of a parental polypeptide having a sequence according to SEQ ID NO.: 1 and, wherein the ene reductase variant has a sequence identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % to SEQ ID NO.: 1.

[0032] An ene reductase catalyzes the reduction of unsaturated double bonds. The variants according to the present invention are all derived from the enzyme AtDBRI from Arabidopsis thaliana, which is a double bond reductase. Such enzymes carrying one mutation have been described in WO 2022184248 A1.However, it was also found in terms of the present invention that these variants can be further optimized by including a mutation, which is associated either with enzyme stability or at least one mutation that is associated with enzyme activity. These mutations lead to a sufficient stability and activity of the enzyme in addition to the introduced disulphide bridge and can be used in economic large-scale production processes.

[0033] In terms of the present invention, the term “enzyme activity” relates to the ability and velocity of an enzyme to convert an educt to a product.

[0034] In terms of the present invention, the term “enzyme stability” relates to the ability of an enzyme to be still active on the conversion of a substrate at high temperatures and / or over a broad range of pH values. Preferably, the enzyme variants are stable at a temperature of more than 30 °C, more preferably of more than 40 °C and especially preferably at a temperature of more than 50 °C. Moreover, the enzyme variants are stable at such temperature for more than 30 Minutes, preferably more than one hour and especially preferably more than three hours.

[0035] In terms of the present invention at least two, preferably at least three mutations that are associated with activity of the enzyme, also have a beneficial effect on the stability of the enzyme. Such variants are also preferred in terms of the present invention.

[0036] Whenever the present disclosure refers to sequence homologies or sequence identity of amino acid sequences in terms of percentages, it refers to values, which can be calculated using EMBOSS Water Pairwise Sequence Alignments (Nucleotide) for nucleic acid sequences or EMBOSS Water Pairwise Sequence Alignments (Protein) for amino acid sequences. In the case of the local sequence alignment tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI), a modified Smith-Waterman algorithm is used (Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1):195-197). Furthermore, here, when performing the respective pairwise alignment of two sequences using the modified Smith-Waterman algorithm, reference is made to the default parameters currently given by EMBL-EBI. These are (i) for amino acid sequences: Matrix = BLOSUM62, Gap open penalty = 10 and Gap extend penalty = 0.5 and (ii) for nucleic acid sequences: Matrix = DNAfull, Gap open penalty = 10 and Gap extend penalty = 0.5.

[0037] The term "sequence homology" can be used interchangeably with "sequence identity" in the context of the present invention. Both terms always refer to the total length of an enzymeaccording to the invention compared to the total length of an enzyme to which the sequence identity or sequence homology is determined.

[0038] The enzyme variant according to the present invention is “genetically engineered”. “Genetically engineered” in terms of the present invention means that the enzyme according to the present invention is altered or modified in comparison to a naturally occurring enzyme or an enzyme known from the state of the art. Suitable modifications can be mutations in the amino acid sequence. Suitable mutagenesis methods, as well as the necessary conditions and reagents, are well known to those skilled in the art. Mutations occur at the gene level, for example, through the replacement (or substitution), removal (or deletion), or addition of bases. These mutations have different effects on the amino acid sequence of the resulting protein. In the case of substitution, so-called "nonsense" mutations can occur, causing protein biosynthesis to stop early and the resulting protein to remain dysfunctional. In the so-called "missense" mutation, only the encoded amino acid changes; these mutations result in a functional change in the resulting protein and, in the best case, may cause improved stability or activity of the resulting protein. In general nomenclature, amino acid substitution mutations are designated based on their position and the amino acid substituted, for example, as A143G. This notation means that at position 143 of the N- to C-terminal amino acid sequence, the amino acid alanine has been exchanged for guanine. This is called a “substitution mutation”. Preferably, the at least one mutation is a substitution.

[0039] Furthermore, it is preferred in terms of the present invention that the at least one mutation associated with enzyme stability is selected from T19K, T38S, I86V, G90K, Y94F, E155K, M178L, T198N, T215D, N224E, N264Q, Q265C, G267C, H269C and V315I.

[0040] It is also preferred in terms of the present invention that the at least one mutation associated with enzyme stability is selected from Y81F, I103F, I103M, I275G, V285Q, V285L, V285T, V285D, Y276A, Y276G, Y276F, Y290A, Y290F, Y290S and Y290G.

[0041] Furthermore, it is preferred in terms of the present invention that the ene reductase variant has at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen or at least eighteen mutations in total. Especially preferably, the ene reductase variant has at least five, moreover preferably at least ten mutations in total.Preferably, the ene reductase variants carries the substitution mutations Y276G, V258Q and Y290G (Act_10, SEQ ID NO.: 10).

[0042] Preferably, the ene reductase variants carries the substitution mutations Y276G, V258Q and Y290S (Stab_0, SEQ ID NO.: 11).

[0043] Preferably, the ene reductase variants carries the substitution mutations V285Q, Y290G and Y276F (Stab_8, SEQ ID NO.: 12).

[0044] Preferably, the ene reductase variants carries the substitution mutations V258Q and Y290G (Stab_9, SEQ ID NO.: 13).

[0045] Preferably, the ene reductase variants carries the substitution mutations V258Q, Y290G, I275G and Y276F (Stab_10, SEQ ID NO.: 14).

[0046] Preferably, the ene reductase variants carries the substitution mutations V258Q, Y290S and I275G (Stab_11 , SEQ ID NO.: 15).

[0047] Preferably, the ene reductase variants carries the substitution mutations Y276G, V258Q, Y290S and G267C (Stab_25, SEQ ID NO.: 16).

[0048] Preferably, the ene reductase variants carries the substitution mutations Y276G, V258Q, Y290S, Q265C and H269C (Stab_26, SEQ ID NO.: 17).

[0049] Preferably, the ene reductase variants carries the substitution mutations V258Q, Y290S, I275G, Y276F and I103M (Stab_42, SEQ ID NO.: 18).

[0050] Preferably, the ene reductase variants carries the substitution mutations V258Q, Y290S, I275G, Y276F and I103F (Stab_43, SEQ ID NO.: 19).

[0051] Preferably, the ene reductase variants carries the substitution mutations V258Q, Y290S, I275G, Y276F and I103W (Stab_44, SEQ ID NO.: 20).

[0052] Preferably, the ene reductase variants carries the substitution mutations V258Q, Y290S, I275G, Y276F and Y260F (Stab_45, SEQ ID NO.: 21).Preferably, the ene reductase variants carries the substitution mutations T38S, I86V, G90K, N264Q, M178L, E155K. T19K, I275G, Y276F, V285Q and Y290S (Stab_46, SEQ ID NO.: 22).

[0053] Preferably, the ene reductase variants carries the substitution mutations T38S, I86V, G90K, N264Q, M178L, E155K, T19K, Y94F, T215D, T198N, S84A, I275G, Y276F, V285Q, Y290S and G267C (Stab_47, SEQ ID NO.: 23).

[0054] Preferably, the ene reductase variants carries the substitution mutations T38S, I86V, G90K, N264Q, M178L, E155K, T19K, Y94F, T215D, T198N, S84A, I275G, Y276F, V285Q, Y290S, G267C and I103F (Stab_48, SEQ ID NO.: 24).

[0055] Preferably, the ene reductase variants carries the substitution mutations T38S, I86V, G90K, N264Q, M178L, E155K, T19K, Y94F, T215D, T198N, S84A, I275G, Y276F, V285Q, Y290S, G267C and I103M (Stab_49, SEQ ID NO.: 25).

[0056] Preferably, the ene reductase variants carries the substitution mutations T38S, I86V, G90K, N264Q, M178L, E155K, T19K, Y94F, T215D, T198N, S84A, I275G, Y276F, V285Q and Y290S (Stab_50, SEQ ID NO.: 26).

[0057] Preferably, the ene reductase variants carries the substitution mutations T38S, I86V, G90K, N264Q, M178L, E155K, T19K, Y94F, T215D, T198N, S84A, I275G, Y276F, V285Q, Y290S and I103F (Stab_51 , SEQ ID NO.: 27).

[0058] Preferably, the ene reductase variants carries the substitution mutations T38S, I86V, G90K, N264Q, M178L, E155K, T19K, Y94F, T215D, T198N, S84A, I275G, Y276F, V285Q, Y290S and I103M (Stab_52, SEQ ID NO.: 28).

[0059] Preferably, the ene reductase variants carries the substitution mutations G267C, I275G, Y276F, V285Q and Y290S (Stab_53, SEQ ID NO.: 29).

[0060] Preferably, the ene reductase variants carries the substitution mutations T38S, I86V, G90K, N264Q, M178L, E155K, T19K, Y94F, T215D, T198N, S84A, V315I, N224E, I275G, Y276F, V285Q and Y290S (Stab_54, SEQ ID NO.: 30).

[0061] It is also preferred in terms of the present invention that the ene reductase variant has an amino acid sequence selected from SEQ ID NO.:16 to SEQ ID NO.:30 ora sequence identityof at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % to an amino acid sequence according to any one of SEQ ID NO.: 16 to SEQ ID NO.: 30.

[0062] Especially preferably, the the ene reductase variant has an amino acid sequence selected from SEQ ID NO.:22 to SEQ ID NO.:30 or a sequence identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % to an amino acid sequence according to any one of SEQ ID NO.: 22 to SEQ ID NO.: 30.

[0063] Another aspect of the present invention relates to an ene reductase variant having at least one substitution to a cystein, which results in the introduction of a disulfide bridge, preferably at one, two or all positions selected from positions 265, 267 and 269 of a parental polypeptide having a sequence according to SEQ ID NO.: 1 and, wherein the ene reductase variant has a sequence identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % to SEQ ID NO.: 1.

[0064] It was surprisingly found in terms of the present invention that by introducing a disulfide bridge into the enzyme, several improvements not only in stability, but also in activity could be achieved.

[0065] A disulfide bridge (also known as a disulfide bond) is a covalent bond formed between the sulfur atoms of two cysteine amino acid residues within a protein. This bond is represented as -S-S- and is created through an oxidation reaction that links the sulfhydryl (-SH) groups of the cysteine side chains.

[0066] A disulfide bridge can be obtained by introducing a cysteine instead of another amino acid at a suitable position of the enzyme. It was found in terms of the present invention that such suitable position may be position 265, 267 and / or 269.

[0067] Another aspect of the present invention relates to a nucleic acid molecule encoding an ene reductase variant according to the invention.

[0068] It is preferred in terms of the nucleic acid molecule according to the present invention that the nucleic acid molecule has a sequence selected from the group consisting of SEQ ID NOs.: 46 to 60 or a nucleic acid sequence having at least 75 %, at least 80 %, at least 85 %,at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 46 to 60.

[0069] A further aspect of the present invention relates to a vector preferably a plasmid vector, comprising a sequence of a nucleic acid molecule according to the invention.

[0070] Yet another aspect of the present invention relates to a genetically modified cell comprising the vector according to the invention and / or the nucleic acid molecule according to the invention for expressing an ene reductase variant according to the invention.

[0071] A “genetically modified cell” in terms of the present invention is a cell of a microorganism, which was modified by introducing a nucleic acid into the cell, which expresses a protein of interest. Suitable methods for introducing a nucleic acid into a cell are known to the person skilled in the art. The introduction can be permanent, the organism is producing the protein of interest transgene, or non-permanent, the organism is transiently producing the protein of interest.

[0072] It is preferred in terms of the genetically modified cell according to the present invention that the cell is selected from the group consisting of Escherichia coli, Bacillus licheniformis, Bacillus subitilis, Bacillus megaterium, Bacillus amyloliquefaciens, Saccharomyces cerevesiae, Komagataella phaffii and Hansenula polymorpha, Yarrowia lipolytica, Kluyveromyces lactis, Corynebacterium glutamicum, Pseudomonas putida, Vibrio natringens, Aspergillus niger and Basfia succiniproducens.

[0073] Another aspect of the present invention relates to a method for biocatalytical manufacturing of dihydrochalcones, comprising or consisting of the steps:

[0074] i) providing at least one ene reductase according to the invention or an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or 100 % sequence homology to a sequence selected from the group consisting of SEQ ID NOs 16 to 30;

[0075] ii) providing at least one flavanone and / or at least one chaicone and / or at least one corresponding glycoside;iii) incubating the at least one ene reductase provided in step i) together with the at least one flavanone and / or at least one chaicone and / or the at least one corresponding glycoside provided in step ii);

[0076] iv) obtaining at least one dihydrochalcone;

[0077] v) optionally purifying the obtained dihydrochalcone.

[0078] Flavanones are the first flavonoid products of the flavonoid biosynthetic pathway. They are characterized by the presence of a chiral center at C2 and the absence of the C2-C3 bond. Flavanones are found at high concentrations in citrus fruits. They are preferably used as educts according to the present invention and are further specified below. Flavanones can also be used as educts in terms of the present invention as their natural hydrolysis to chaicone is causing the formation of chaicones, which can then be directly catalyzed by the ene reductase variants to dihydrochalcones. To enhance the conversion rate from flavanones to chaicones, also at least one chaicone isomerase can additionally be present, preferably a chaicone isomerase as disclosed in WO 2022184248 A1 , which content is herein incorporated by reference. Chaicone isomerases are enzymes, which catalyze the reaction from a chaicone to a flavanone and vice versa. Another name of chaicone isomerase is chalcone-flavanone isomerase.

[0079] The following reaction scheme depicts the enzymatic reaction with the ene reductase:

[0080]

[0081] Chaicones are a,p-unsaturated ketones, consisting of two aromatic rings (A and B) attached by a,p-unsaturated carbonyl system with different substituents. Chaicones preferably used as educts according to the present invention are further specified below. The term “corresponding glycosides” in connection with the used flavanones and / or chaicones refers to corresponding flavanones and / or chaicones having a sugar bound to another functional group via a glycosidic bond. Glycosides of flavanones and / or chaicones are especially present in natural sources of flavanones and / or chaicones.

[0082] The obtained at least one dihydrochalcone from the method according to the invention can be present as a mixture of different dihydrochalcones and / or in a mixture together with other compounds depending on the used flavanone and / or chaicone and / or their corresponding glycosides or, respectively the used starting material. They can be further purified by suitable methods known to the person skilled in the art. The obtained mixture of or purified dihydrochalcones are preferably incorporated as flavouring agents in preparations such as aroma compositions, preparations intended for nutrition or enjoyment.

[0083] Preferably, a preparation intended for nutrition or enjoyment may be selected from the group consisting of (reduced-calorie) baked goods (e.g. bread, dry biscuits, cakes, other baked articles), confectionery (e.g. muesli bar products, chocolates, chocolate bars, other productsin bar form, fruit gums, dragees, hard and soft caramels, chewing gum), nonalcoholic drinks (e.g. cocoa, coffee, green tea, black tea, (green, black) tea drinks enriched with (green, black) tea extracts, rooibos tea, other herbal teas, fruit-containing soft drinks, isotonic drinks, refreshing drinks, nectars, fruit and vegetable juices, fruit or vegetable juice preparations), instant drinks (e.g. instant cocoa drinks, instant tea drinks, instant coffee drinks), meat products (e.g. ham, fresh sausage or raw sausage preparations, spiced or marinated fresh or salt meat products), eggs or egg products (dried egg, egg white, egg yolk), cereal products (e.g. breakfast cereals, muesli bars, precooked ready-to-eat rice products), dairy products (e.g. full-fat or reduced-fat or fat-free milk drinks, rice pudding, yoghurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, ice-cream, partially or completely hydrolyzed milk-protein-containing products), products made from soy protein or other soybean fractions (e.g. soy milk and products produced therefrom, drinks containing isolated or enzymatically treated soy protein, drinks containing soy flour, preparations containing soy lecithin, fermented products such as tofu or tempeh or products produced therefrom and mixtures with fruit preparations and optionally flavors), dairy-like preparations (milk-type, yoghurt-type, dessert-type, ice cream) from protein rich plant materials (e.g. from seed materials of oat, almond, pea, lupine, lentils, faba beans, chickpea, rice, canola), plant protein-enriched non-dairy drinks, fruit preparations (e.g. jams, sorbets, fruit sauces, fruit fillings), vegetable preparations (e.g. ketchup, sauces, dried vegetables, frozen vegetables, precooked vegetables, boiled-down vegetables), snacks (e.g. baked or fried potato crisps or potato dough products, maize- or groundnut-based extrudates), fat- and oil-based products or emulsions thereof (e.g. mayonnaise, remoulade, dressings, in each case full-fat or reduced-fat), other readymade dishes and soups (e.g. dried soups, instant soups, precooked soups), spices, spice mixtures and in particular seasonings which are used, for example, in the snacks field, sweetener preparations, tablets or sachets, other preparations for sweetening or whitening drinks.

[0084] The preparation intended for nutrition or enjoyment within the meaning of the invention can also be present as dietary supplements in the form of capsules, tablets (uncoated and coated tablets, e.g. g astro- resista nt coatings), sugar-coated pills, granulates, pellets, solid mixtures, dispersions in liquid phases, as emulsions, as powders, as solutions, as pastes or as other formulations that can be swallowed or chewed.

[0085] The step of “incubation” the ene reductase variant with the at least one educt means contacting the educt and the enzyme for a specific time in order to achieve the catalytic conversion of the educt. Preferably, the incubation in step iv) is done for at least 5, 10, 15, 20, 25 minutes, preferably for at least 30 minutes.A preferred embodiment of the present invention relates to a method according to the invention, wherein the at least one ene reductase variant provided in step i) is purified or partially purified. A purified ene reductase refers to an enzyme which shows a purity of 90 % (w / w) or more when provided. Suitable methods for the purification of enzymes are known to the person skilled in the art. A partially purified enzyme refers to an enzyme, which has a purity of less than 90 % (w / w) and is not present in a living organism.

[0086] The reaction of the ene reductase variants according to the present invention is cofactor dependent on the availability of NADPH. It is thus preferred that besides the ene reductase variant, a suitable NADPH donor is available during the reaction. Suitable enzymes for combination are well known in the art. For example, the ene reductase variants according to the present invention may be combined with a glucose dehydrogenase for providing NADPH. Such suitable donor systems are described for example in Maier et al., “Formate Dehydrogenase: Recent Developments for NADH and NADPH Recycling in Biocatalysis" , ChemCatChem 2024, 16, e202401021 or Wang et al., “Cofactor NAD(P)H Regeneration Inspired by Heterogeneous Pathways", Chem, Volume 2, Issue 5, 2017, Pages 621-654, ISSN 2451-9294, especially table 2.

[0087] It is preferred in terms of the method of the present invention that the ene reductase is present in a whole-cell biocatalyst, partially purified or purified.

[0088] Furthermore, it is also preferred in terms of the method according to the present invention that the incubation in step ii) is done for at least 5, 10, 15, 20, 25 minutes, preferably for at least 30 minutes.

[0089] It is also preferred in terms of the method according to the present invention that wherein the at least one flavanone and / or at least one chaicone and / or at least one of the corresponding glycosides provided in step ii) is selected from the group consisting of homoeriodictyol, homoeriodictyol chaicone, hesperidin, hesperidin chaicone, hesperetin-7-glucosid, hesperetin monoglycoside, hesperetin monoglycoside chaicone, hesperetin chaicone, hesperetin, eriodictyol, eriodictyol chaicone, eriocitrin, eriocitrin chaicone, neohesperidin, neohesperidin chaicone, naringenin, naringin, naringenin chaicone, naringin chaicone, narirutin, butein, 4-o-methyl butein, liquiritigenin, pinocembrin, steppogenin, scuteamoenin, dihydroechiodinin, ponciretin, sakuranetin, isosakuranetin, 4,7-dihydroxy-flavanon, 4,7-dihydroxy-3’-methoxyflavanon, 3,7-dihydroxy-4’-methoxyflavanon, 3’4,7-trihydroxyflavanon, alpinentin, pinostrobin, 7-hydroxyflavanon, 4’-hydroxyflavanon, 3-hydroxyflavanon,tsugafolin, p-coumaric acid, caffeic acid ferulic acid, isoferulic acid, cinnamic acid and their respective -CoA derivatives, p-hydroxybenzalacetone, 3,4-dihydroxybenzylideneacetone, 4-(3-hydroxy-4-methoxyphenyl)but-3-en-2-one, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, resveratrol, 3-methoxy-4',5-dihydroxy-trans-stilbene, 5-(4-methoxystyryl)benzene-1 ,3-diol, hydrangeic acid, 2-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-6-hydroxybenzoic acid, 2-hydroxy-6-[(E)-2-(3-hydroxy-4-methoxyphenyl)ethenyl]benzoic acid, trans-deoxyrhapontigenin, stilbene-3,4'-diol, 3,3',4-trihydroxystilbene.

[0090] Preferably, the educt is selected from hesperetin chaicone and / or hesperetin dihydrochalcone.

[0091] Yet another aspect of the present invention relates to the use of an ene reductase variant according to the present invention for the manufacturing of hesperidin dihydrochalcone or hesperetin dihydrochalcone.

[0092] All of the embodiments described as preferred above, can be combined with each other interchangeably in terms of the present invention.

[0093] The present invention is further characterized by illustrative, non-limiting examples.Short description of figures

[0094] Figure 1 shows the fold improvement of enzyme activity towards hesperetin chaicone reduction. Fold improvement is calculated on the baseline activity of the AtDBRI -wildtype Act_0 variant. The wild type as well as the variants Act_0 to Act_9 were already described in WO2022184248A1.

[0095] Figure 2 shows the activity of AtDBRI -variants towards conversion of hesperidin chaicone. The activity is normalized on the baseline activity of Stab_0.

[0096] Figure 3 shows thermostability measurements of AtDBRI wildtype and variants at 30°C for 3 hours. Thermostability improvement is measured by measuring baseline enzyme activity directly after lysis and comparing it with enzyme activity after incubation without substrate for a certain time at different temperatures. An activity recovery of 100 % resembles a stable enzyme under the tested conditions.

[0097] Figure 4 shows thermostability measurements of AtDBRI wildtype and variants at 40 °C for 30 minutes. Thermostability improvement is measured by measuring baseline enzyme activity directly after lysis and comparing it with enzyme activity after incubation without substrate fora certain time at different temperatures. An activity recovery of 100 % resembles a stable enzyme under the tested conditions.

[0098] Figure 5 shows thermostability measurements of AtDBRI wildtype and variants at 50 °C for 30 minutes. Thermostability improvement is measured by measuring baseline enzyme activity directly after lysis and comparing it with enzyme activity after incubation without substrate fora certain time at different temperatures. An activity recovery of 100 % resembles a stable enzyme under the tested conditions.

[0099] Figure 6 shows a chromatogram of cultures with and without expression vector. Cells expressing Stab_48 were able to produce hesperidin dihydrochalcone from hesperidin.Short description of sequences

[0100] SEQ ID NO.: 1 is the amino acid sequence of the AtDBRI wild-type.

[0101] SEQ ID NO.: 2 to 9 are amino acid sequences carrying one mutation and known from WO 2022184248 A1.

[0102] SEQ ID NO.: 10 to 30 are amino acid sequences of inventive AtDBRI variants.

[0103] SEQ ID NO.: 31 is the nucleic acid sequence encoding the AtDBRI wild-type enzyme.

[0104] SEQ ID NOs.: 32 to 39 are nucleic acid sequences encoding ene reductase variants carrying one mutation and are known from WO 2022184248 A1.

[0105] SEQ ID NOs.: 40 to 60 are nucleic acid sequences encoding inventive AtDBRI variants.

[0106] The following table describes the names of the variants and the corresponding SEQ ID NO.

[0107] Table 1: Variant name and corresponding SEQ ID NO.

[0108]

[0109]

[0110] Examples

[0111] Example 1 - Generation of double bond reductase mutagenesis libraries

[0112] Expression vectors pET28a(+) were ordered (Twist Bioscience, San Francisco, USA) harboring DNA sequences which were codon-optimized for the expression in E. coli. The vectors comprise the nucleic acid according to SEQ ID NO.: 31 to 60 encoding the ene reductase wild type and variants according to SEQ ID NO.: 1 to 30 respectively. The obtained plasmids were transformed into chemically competent E. coli BL21(DE3) cells, selected using 50 pg mL-1neomycine.

[0113] Example 2 - Enzyme expression and determination of activity towards hesperetin chaicone

[0114] AtDBRI wildtype and variants were expressed using E. coli BL21(DE3) with a pET28a(+) expression system according to example 1 in 1 ml TB medium supplemented with 50 pg mL-1neomycine. Overnight precultures were used to inoculate the expression medium at OD600=0.5. After 90 min of cultivation at 37°C, shaking, 1 mM IPTG was added and the expression temperature lowered to 25 °C. Enzyme production was conducted for 16 hours. Cell cultures were centrifuged for 5 min, 5.000xg. The supernatant was discarded and the cell pellets resuspended in 400 pl B-PER buffer (ThermoFisher). After incubation at room temperature for 15 min, the suspension was centrifuged for 10 min, 5.000xg. Afterwards, the supernatant was used for production of hesperetin dihydrochalcone (1 mM hesperetin chaicone, 50 % (v / v) lysate, 50 mM citrate buffer pH 5.5, 1 mM NADPH, 30°C, 10 min). Reactions were stopped by dilution with methanol (1 :1). After centrifugation of cell debris, the supernatant was analyzed via LC-DAD measurements at 280 and 380 nm.

[0115] Figure 1 shows the fold improvement of enzyme activity towards hesperetin chaicone reduction. Fold improvement is calculated on the baseline activity of the AtDBRI -wildtype Act_0 variant (SEQ ID NO.: 1). The wild type as well as the variants Act_2 to Act_9 (SEQ ID NOs.: 2 to 9) were already described in WO 2022184248 A1.Example 3 - Enzyme expression and determination of activity towards hesperidin chaicone

[0116] AtDBRI variants were expressed as described in example 2. Cell cultures were centrifuged for 5 min, 5.000xg. The supernatant was discarded and the cell pellets resuspended in 400 pl B-PER buffer (ThermoFisher). After incubation at room temperature for 15 min, the suspension was centrifuged for 10 min, 5.000xg. Afterwards the supernatant was used for production of hesperidin dihydrochalcone (1 mM hesperidin chaicone, 50 % (v / v) lysate, 50 mM citrate buffer pH 5.5, 1 mM NADPH, 30°C, 10 min). Reactions were stopped by dilution with methanol (1 :1). After centrifugation of cell debris, the supernatant was analyzed via LC-DAD measurements at 280 and 380 nm.

[0117] Figure 2 depicts the activity of AtDBRI -variants towards conversion of hesperidin chaicone. The activity is normalized on the baseline activity of Stab_0 (SEQ ID NO.: 11).

[0118] Example 4 - Enzymes and stability testing

[0119] AtDBRI variants were expressed as described in example 2. Cell cultures were centrifuged for 5 min, 5.000xg. The supernatant was discarded and the cell pellets resuspended in 400 pl B-PER buffer (ThermoFisher). After incubation at room temperature for 15 min, the suspension was centrifuged for 10 min, 5.000xg. Afterwards the supernatant was used for stability testings. An aliquot of the supernatant was used for baseline activity measurement and a second aliquot was incubated at different temperatures (30°C, 40°C, 50°C) for a certain time period (30 min - 180 min). After this inactivation period, the activity was measured and compared to the baseline activity.

[0120] Figure 3, 4 and 5 show thermostability measurements of AtDBRI wildtype and variants. Figure 3 shows measurement at 30°C for 3 hours, Figure 4 at 40 °C for 30 minutes and Figure 5 at 50 °C for 30 minutes.

[0121] Thermostability improvement is measured by measuring baseline enzyme activity directly after lysis and comparing it with enzyme activity after incubation without substrate for a certain time at different temperatures. An activity recovery of 100 % resembles a stable enzyme under the tested conditions.Example 5 - Fermentation of Hesperidin

[0122] AtDBRI variant Stab_48 (SEQ ID NO.: 24) was expressed using E. coli BL21(DE3) with a pET28a(+) expression system in 1 ml TB medium supplemented with 50 pg mL-1 neomycine. Additionally, E. coli BL21 (DE3) cells without expression vector were cultivated as control. Overnight precultures were used to inoculate the expression medium at OD600=0.5. After 90 min of cultivation at 37°C, shaking, 1 mM IPTG was added and the expression temperature lowered to 25 °C. Additionally, 1 mM hesperidin was added to the culture. The cells were cultivated, shaking, for 16 hours. Reactions were stopped by dilution with methanol (1:1). After centrifugation of cell debris, the supernatant was analyzed via LC-DAD measurements at 280 nm and 380 nm.

[0123] Figure 6 shows a chromatogram of both cultures. Cells expressing Stab_48 were able to produce hesperidin dihydrochalcone from hesperidin.

Claims

Claims1. Ene reductase variant having at least one mutation associated with enzyme stability selected from positions 19, 38, 84, 86, 90, 94, 155, 178, 198, 215, 224, 264, 265, 267, 269 and 315,andat least one mutation associated with enzyme activity at a position selected from positions 81 , 103, 275, 276, 285 and 290 of a parental polypeptide having a sequence according to SEQ ID NO.: 1 and, wherein the ene reductase variant has a sequence identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % to SEQ ID NO.: 1.

2. Ene reductase variant according to claim 1, wherein the at least one mutation associated with enzyme stability is selected from T19K, T38S, I86V, G90K, Y94F, E155K, M178L, T198N, T215D, N224E, N264Q, Q265C, G267C, H269C and V315I.

3. Ene reductase variant according to any one of the preceding claims, wherein the at least one mutation associated with enzyme stability is selected from Y81F, I103F, I103M, I275G, V285Q, V285L, V285T, V285D, Y276A, Y276G, Y276F, Y290A, Y290F, Y290S and Y290G.

4. Ene reductase variant according to any one of the preceding claims, wherein the variant has at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen or at least eighteen mutations in total.

5. Ene reductase variant according to any one of the preceding claims, wherein the ene reductase variant has an amino acid sequence selected from SEQ ID NO.:16 to SEQ ID NQ.:30 or a sequence identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % to an amino acid sequence according to any one of SEQ ID NO.: 16 to SEQ ID NO.: 30.

6. Ene reductase variant having at least one substitution to a cystein, which results in the introduction of a disulfide bridge, preferably at one, two or all positions selected from positions 265, 267 and 269 of a parental polypeptide having a sequence according to SEQ ID NO.: 1 and, wherein the ene reductase variant has a sequence identity of at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % to SEQ ID NO.: 1.

7. Nucleic acid molecule encoding an ene reductase variant according to any one of claims 1 to 6.

8. Nucleic acid molecule according to claim 7, wherein the nucleic acid molecule has a sequence selected from the group consisting of SEQ ID NOs.: 46 to 60 or a nucleic acid sequence having at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 46 to 60.

9. Vector, preferably a plasmid vector, comprising a sequence of a nucleic acid molecule according to claims 7 or 8.

10. Genetically modified cell comprising the vector according to claim 9 and / or the nucleic acid molecule according to claims 7 or 8 for expressing an ene reductase variant according to any one of claims 1 to 6.

11. Genetically modified cell according to claim 10, wherein the cell is selected from the group consisting of Escherichia coli, Bacillus licheniformis, Bacillus subitilis, Bacillus amyloliquefaciens, Bacillus megaterium, Saccharomyces cerevesiae, Komagataella phaffii and Hansenula polymorpha, Yarrowia lipolytica, Kluyveromyces lactis, Corynebacterium glutamicum, Pseudomonas putida, Vibrio natringens, Aspergillus niger and Basfia succiniproducens.

12. Method for biocatalytical manufacturing of dihydrochalcones, comprising or consisting of the steps:i) providing at least one ene reductase according to any one of claims 1 to 6 or an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %,96 %, 97 %, 98 %, 99 % or 100 % sequence homology to a sequence selected from the group consisting of SEQ ID NOs 16 to 30;ii) providing at least one flavanone and / or at least one chaicone and / or at least one corresponding glycoside;iii) incubating the at least one ene reductase provided in step i) together with the at least one flavanone and / or at least one chaicone and / or the at least one corresponding glycoside provided in step ii);iv) obtaining at least one dihydrochalcone;v) optionally purifying the obtained dihydrochalcone.

13. Method according to claim 12, wherein the ene reductase is present in a whole-cell biocatalyst, partially purified or purifiedand / orwherein the incubation in step ii) is done for at least 5, 10, 15, 20, 25 minutes, preferably for at least 30 minutes.

14. Method according to any one of claims 12 or 13, wherein the at least one flavanone and / or at least one chaicone and / or at least one of the corresponding glycosides provided in step ii) is selected from the group consisting of homoeriodictyol, homoeriodictyol chaicone, hesperidin, hesperidin chaicone, hesperetin-7-glucosid, hesperetin monoglycoside, hesperetin monoglycoside chaicone, hesperetin chaicone, hesperetin, eriodictyol, eriodictyol chaicone, eriocitrin, eriocitrin chaicone, neohesperidin, neohesperidin chaicone, naringenin, naringin, naringenin chaicone, naringin chaicone, narirutin, butein, 4-o-methyl butein, liquiritigenin, pinocembrin, steppogenin, scuteamoenin, dihydroechiodinin, ponciretin, sakuranetin, isosakuranetin, 4,7-dihydroxy-flavanon, 4,7-dihydroxy-3’-methoxyflavanon, 3,7- dihydroxy-4’-methoxyflavanon, 3’4,7-trihydroxyflavanon, alpinentin, pinostrobin, 7- hydroxyflavanon, 4’-hydroxyflavanon, 3-hydroxyflavanon, tsugafolin, p-coumaric acid, caffeic acid ferulic acid, isoferulic acid, cinnamic acid and their respective -CoA derivatives, p-hydroxybenzalacetone, 3,4-dihydroxybenzylideneacetone, 4-(3- hydroxy-4-methoxyphenyl)but-3-en-2-one, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, resveratrol, 3-methoxy-4',5-dihydroxy-trans-stilbene, 5-(4- methoxystyryl)benzene-1 ,3-diol, hydrangeic acid, 2-[(E)-2-(3,4- dihydroxyphenyl)ethenyl]-6-hydroxybenzoic acid, 2-hydroxy-6-[(E)-2-(3-hydroxy-4- methoxyphenyl)ethenyl]benzoic acid, trans-deoxyrhapontigenin, stilbene-3,4'-diol, 3,3',4-trihydroxystilbene.

15. Use of an ene reductase variant according to any one of claims 1 to 6 for the manufacturing of hesperidin dihydrochalcone or hesperetin dihydrochalcone.

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