Novel enzymes for manufacture of 4 '-O-methylated phenolic substances

Abstract

The invention relates to a novel enzyme for producing 4 '-O-methylated phenolic substances from a corresponding extract and to a method for producing a mixture with 3'-O-methylated substances and 4 '-O-methylated substances, and to the use of said enzyme for producing such a mixture and to a mixture produced for use as a taste modifier in a composition and applications thereof.

Description

TECHNICAL FIELD

[0001] The present invention relates to a novel enzyme for the manufacture of 4'-O-methylated phenolic substances from the corresponding leachate and a process for the production of a mixture with 3'-O-methylated substances and 4'-O-methylated substances and the use of said enzyme for the production of such a mixture and the use of the manufactured mixture as a taste modifier in a composition. BACKGROUND

[0002] (Poly)phenolic substances are generally present in plants and thus also in human nutrition. A specific group of phenolic substances are the flavonoids. The group of flavonoids comprises compounds which exhibit a sweet taste or sweet taste optimization. These compounds are generally used in various applications to increase the sweet taste impression or to mask bitter substances of food, pharmaceutical, beverage or similar end products. Thus, there is a constant need to provide flavonoids as safe food additives and thus a need for a process to provide the substances in a reliable way.

[0003] The manufacture of homoeriodictyol dihydrochalcone (1) and its sweetening properties are described in WO2007107596A1. Furthermore, a mixture of homoeriodictyol dihydrochalcone (1) with a saliva secretion increasing agent in a flavoring composition is described in US20080227867. Furthermore, the masking of the bitter taste impression of caffeine with homoeriodictyol dihydrochalcone (1) is described in US20080227867. The manufacture of (1) described in WO2007107596A1 is a catalytic aldol reaction of 1,4-di-O-benzoylacetophenone with vanillin with piperidine. In this chemical reaction the double bond of the obtained chalcone is hydrated with the help of a Pd / C catalyst. Additional methods include the use of protecting groups, other bases or reducing agents. All known methods require organic solvents and thus cannot be classified as natural manufacturing methods according to EC 1334 / 2008.

[0004]

[0005] The use and effect of hesperetin dihydrochalcone (2) for the modification of unpleasant taste impressions is described in WO2017186299A1. These features are also described in J. Agric. Food Chem. 1977, 25(4), 763-772 and J. Med. Chem. 1981, 24(4), 408-428. A mixture of (2) and high fructose corn syrup and other sweeteners is described in WO2019080990A1. Homoeriodictyol dihydrochalcone (1) comprises a 3'-O-methylation, wherein hesperetin dihydrochalcone (2) comprises a 4'-O-methylation.

[0006] As described in WO 2021 / 058115 Al, high-eriodictyol dihydrochalcone (1) and hesperetin dihydrochalcone (2) can be manufactured via the enzymatic hydroxylation of phloretin (3) to eriodictyol dihydrochalcone (4), followed by the enzymatic methylation of (4). The present application also discloses enzyme variants for altering the specificity of O-methyltransferases from Myxococcus xanthus with respect to 3'-O-methylation and 4'-O-methylation.

[0007] Catechols are bifunctional phenolic substances that can be methylated. Results on altering the regioselectivity of catechol O-methyltransferases for the methylation of different catechols were published (Dippe et al., Altering the Regioselectivity of a Catechol O-methyltransferase through Rational Design: Vanilloid vs. Isovanilloid Motifs in the B-ring of Flavonoids, 2022; Su et al., Regioselectivity Inversion of an O-Methyltransferase via Semi-rational Mutagenesis Combined with Metal Ion Substitution, 2022).

[0008] The paper by Wils et al. 2013, A single amino acid determines position specificity of an Arabidopsis thaliana CCoAOMT-like O-methyltransferase, describes a mutational study for identifying amino acid residues in the plant Arabidopsis thaliana that are relevant for the position specificity. A position specificity for 3'-O-methylation of the substrate was achieved.

[0009] Law et al. 2016, Effects of Active-Site Modification and Quaternary Structure on the Regioselectivity of Catechol-O-Methyltransferase, also deals with the regioselectivity of catechol-O-methyltransferases. This paper investigates the regioselectivity of human catechol-O-methyltransferase heterologously expressed in E. coli.

[0010] With the steadily increasing awareness of natural products by consumers over the past years, the label natural or ecological product is a strong buying argument. Therefore, it is obvious that there is a demand for dihydrochalcones manufactured in a natural way, which have the same properties as their chemically manufactured counterparts, and which is rapidly growing. Notably, dihydrochalcones are not available in natural compounds and therefore extraction from natural raw materials is not possible. Therefore, the most promising natural manufacturing method is a biocatalytic pathway. However, so far no enzyme has been described which has a high regioselectivity for the 4'-O-methylation of specific phenolic substances, in particular the 4'-O-methylation of eriodictyol dihydrochalcone (4) to increase the yield of hesperetin dihydrochalcone (2) and to decrease the yield of the 3'-O-methylation of homoeriodictyol dihydrochalcone (1) as a by-product. SUMMARY

[0011] Therefore, it is a main object of the present application to provide an enzyme having increased regioselectivity for 4'-O-methylated phenolic substances and a method for producing 4'-O-methylated phenolic substances.

[0012] This main object is achieved by providing an O-methyltransferase variant having a substitution at one, two or all of the amino acid positions selected from positions 41, 42, 43, 173 and 174 of a parent polypeptide, wherein the parent polypeptide is SEQ ID NO.: 1, and wherein the at least one O-methyltransferase has 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 SEQ ID NO.: 1.

[0013] For the purposes of the present application, the term "variant" relates to an enzyme which is altered or modified compared to a naturally occurring enzyme. Such naturally occurring enzyme is referred to as "wild-type" enzyme.

[0014] For the present invention, the term "parental polypeptide" relates to the O-methyltransferase according to SEQ ID NO.: 1 which is modified to obtain the O-methyltransferase variant according to the present invention.

[0015] The numbering of the amino acid positions at which substitutions exist is preferably according to the numbering of the amino acid residues of the parental polypeptide. The term "numbering according to" as used herein refers to the way in which each amino acid residue in the polypeptide of the present invention is numbered. The skilled person is aware that when, for example, position 202 is numbered according to SEQ ID NO.: 1, by aligning any other polypeptide with SEQ ID NO.: 1, the corresponding amino acid residue in the other polypeptide will be able to be determined.

[0016] For the present invention, "substitution" relates to a change of an amino acid residue compared to the parental polypeptide. For example, if an asparagine is present at position 41 of the parental polypeptide according to SEQ ID NO.: 1 and a glycine is present in the O-methyltransferase variant, this is referred to as a substitution. Such a substitution is described by the 3-letter amino acid code as Asn41Gly or by the one-letter code as N41G.

[0017] Whenever the present disclosure refers to the percentage identity of nucleic acid or amino acid sequences to each other, these values define those values as obtained by using the EMBOSS Water pairwise sequence alignment (nucleotides) program or the EMBOSS Water pairwise sequence alignment (polypeptides) program for amino acid sequences. Alignment or sequence comparison as used herein refers to an alignment over the full length of the two sequences compared to each other. Those tools for local sequence alignment provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) use a modified Smith-Waterman algorithm (see Smith, T.F. and Waterman, M.S. Identification of common molecular subsequences. Journal of Molecular Biology, 1981 147 (1 ): 195-197). When performing the alignment, the default parameters as defined by EMBL-EBI are used. For amino acid sequences, those parameters are (i): Matrix = BLOSUM62, Gap Open Penalty = 10 and Gap Extension Penalty = 0.5; or for nucleic acid sequences, (ii): Matrix = DNAfull, Gap Open Penalty = 10 and Gap Extension Penalty = 0.5. The skilled person is well aware of the fact that a nucleic acid sequence encoding a polypeptide can be "codon-optimized" if, for example, the respective sequence is used in another organism compared to the original organism from which the molecule originates.

[0018] Methyltransferases are enzymes that transfer a methyl group from a donor to an acceptor molecule, substrate or leachate. In particular, they catalyze the selective methylation of C-centered, N-centered and O-centered nucleophiles via methyl transfer from the sulfonium function of the co-factor S-adenosylmethionine (SAM). The O-methylation of scillipiden dihydrochalcone by an O-methyltransferase from Zooshikella ganghwensis is depicted in Figure 1 .

[0019] This figure shows the conversion of the leachate scillipiden dihydrochalcone (1b) to the 3'-O-methylated homo-scillipiden dihydrochalcone (2b) and the 4'-O-methylated hesperetin dihydrochalcone (3b).

[0020] Surprisingly, it was found that the provided O-methyltransferase variants comprising very specific amino acid substitutions are able to increase the product yield of 4'-O-methylated phenolic substances, thus increasing the regioisomer ratio of 4'-O-methylated phenolic substances to 3'-O-methylated phenolic substances.

[0021] It is preferred that the O-methyltransferase variant according to the present application comprises a substitution at one amino acid position selected from positions 41, 42, 43, 173 and 174 of a parent polypeptide, wherein the parent polypeptide is SEQ ID NO.: 1, and wherein the at least one O-methyltransferase has 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 SEQ ID NO.: 1. It is further preferred that the substitution is selected from positions 41, 42 and 174.

[0022] It is preferred that the O-methyltransferase variant according to the present application comprises a substitution at two amino acid positions selected from positions 41, 42, 43, 173 and 174 of a parent polypeptide, in particular at positions 41 and 174 or 42 and 174, wherein the parent polypeptide is SEQ ID NO.: 1, and wherein the at least one O-methyltransferase has 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 SEQ ID NO.: 1.

[0023] Even further preferably, the O-methyltransferase variant according to the application comprises three substitutions at said amino acid positions 41, 41 and 174, wherein said parent polypeptide is SEQ ID NO.: 1 and wherein said at least one O-methyltransferase has 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 SEQ ID NO.: 1.

[0024] Preferably, said substitution is an amino acid selected from the group consisting of aliphatic amino acids such as leucine, isoleucine and valine and / or positively charged amino acids such as lysine, arginine and histidine and / or threonine. Especially preferably, said substitution is an amino acid selected from the group consisting of phenylalanine, lysine, glutamine, leucine, threonine, valine, proline, asparagine and histidine.

[0025] Especially preferably, the O-methyltransferase variant comprises a substitution selected from N41 P, N41 K, N41 Q, N41 L, M42T, M42V, M42F, S174R, S174H or S174K. Even further preferably, the O-methyltransferase variant comprises the substitutions M42T and S174R, N41 L and S174R, N41 K and S174R or N41 K and S174K.

[0026] Preferably, the O-methyltransferase variant according to the application comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO.: 11, SEQ ID NO.: 13, SEQ ID NO.: 15, SEQ ID NO.: 17, SEQ ID NO.: 19, SEQ ID NO.: 21, SEQ ID NO.: 23, SEQ ID NO.: 25, SEQ ID NO.: 27, SEQ ID NO.: 29, SEQ ID NO.: 31, SEQ ID NO.: 33, SEQ ID NO.: 35, and SEQ ID NO.: 37 or an amino acid sequence having 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 with an amino acid sequence selected from the group consisting of SEQ ID NO.: 11, SEQ ID NO.: 13, SEQ ID NO.: 15, SEQ ID NO.: 17, SEQ ID NO.: 19, SEQ ID NO.: 21, SEQ ID NO.: 23, SEQ ID NO.: 25, SEQ ID NO.: 27, SEQ ID NO.: 29, SEQ ID NO.: 31, SEQ ID NO.: 33, SEQ ID NO.: 35, and SEQ ID NO.: 37.

[0027] Another aspect of the present application relates to a vector system, preferably a plasmid vector system, consisting of a vector comprising at least one nucleic acid segment (a) comprising a gene encoding an O-methyltransferase having a nucleic acid sequence selected from the group consisting of SEQ ID NO.: 12, SEQ ID NO.: 14, SEQ ID NO.: 16, SEQ ID NO.: 18, SEQ ID NO.: 20, SEQ ID NO.: 22, SEQ ID NO.: 24, SEQ ID NO.: 26, SEQ ID NO.: 28, SEQ ID NO.: 30, SEQ ID NO.: 32, SEQ ID NO.: 34, SEQ ID NO.: 36, and SEQ ID NO.: 38 or a nucleic acid having at least 70%, 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 a nucleic acid selected from the group consisting of SEQ ID NO.: 12, SEQ ID NO.: 14, SEQ ID NO.: 16, SEQ ID NO.: 18, SEQ ID NO.: 20, SEQ ID NO.: 22, SEQ ID NO.: 24, SEQ ID NO.: 26, SEQ ID NO.: 28, SEQ ID NO.: 30, SEQ ID NO.: 32, SEQ ID NO.: 34, SEQ ID NO.: 36, and SEQ ID NO.: 38; and

[0028] comprising preferably at least one nucleic acid segment (b) comprising a gene encoding a 4-coumarate 3-hydroxylase and a gene encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase;

[0029] and / or preferably at least one nucleic acid segment (c) comprising a gene encoding an S-adenosylmethionine synthetase,

[0030] wherein the nucleic acid segments (a), (b) and / or (c), if (b) and / or (c), are preferably provided on the same vector or in two or three different vectors, each vector comprising one nucleic acid segment (a) or (b) or (c).

[0031] The vector system according to the present application comprises the O-methyltransferase variant itself as well as preferably further auxiliary enzymes.

[0032] The first preferred co-enzyme is a combination of 4-coumarate 3-hydroxylase and 4-hydroxyphenylacetate 3-monooxygenase reductase, which provides hydroxylation of phenolic substances for use as a substrate for O-methyltransferases.

[0033] Preferably, the 4-coumarate 3-hydroxylase has a nucleic acid sequence according to SEQ ID NO.: 4 or a nucleic acid having at least 70%, 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 the nucleic acid according to SEQ ID NO.: 4.

[0034] Preferably, the 4-hydroxyphenylacetate 3-monooxygenase reductase has a nucleic acid sequence according to SEQ ID NO.: 6 or a nucleic acid having at least 70%, 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 the nucleic acid according to SEQ ID NO.: 6.

[0035] The compatibility of both enzymes, 4-coumarate 3-hydroxylase and 4-hydroxyphenylacetate 3-monooxygenase reductase, is beneficial for a complete and efficient reaction. Surprisingly, it was found that these two co-enzymes can be combined, even though they are not derived from the same organism.

[0036] The third preferred co-enzyme is S-adenosylmethionine synthetase, which is able to catalyze the conversion of ATP and methionine to S-adenosylmethionine. S-adenosylmethionine is a methyl group donor and is required as a co-substrate of the reaction of O-methyltransferases.

[0037] The term “methyl donor” as used herein refers to a chemical structure that donates a methyl group to another substance in a chemical or enzymatic reaction. The methyl donor as described herein can also be a mixture of several chemical structures in such a chemical structure.

[0038] Preferably, the S-adenosylmethionine synthetase has a nucleic acid sequence selected from the group consisting of SEQ ID NO.: 8 and SEQ ID NO.: 10 or a nucleic acid having at least 70%, 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 a nucleic acid selected from the group consisting of SEQ ID NO.: 8 and SEQ ID NO.: 10.

[0039] Another aspect of the present application relates to a genetically modified cell comprising a vector system according to the present application.

[0040] Preferably, the gene encoding the O-methyltransferase and / or the gene encoding the 4-coumaric acid 3-hydroxylase and 4-hydroxyphenylacetic acid 3-monooxygenase and / or the S-adenosylmethionine synthetase is stably integrated into the genome of the cell. Preferably, one, two, three or all genes are under control of an inducible promoter.

[0041] Preferably, the cell is selected from the group consisting of Escherichia coli spp., such as E. coli BL21, E. coli MG1655, preferably E. coli W3110; Bacillus spp., such as Bacillus licheniformis, Bacillus subtilis or Bacillus amyloliquefaciens; Saccharomyces spp., preferably S. cerevisiae; Hansenula spp. or Komagataella spp., such as K. phaffii and H. polymorpha, preferably K. phaffii; Yarrowia spp., such as Y. lipolytica; Kluyveromyces spp, such as K. lactis; Corynebacterium glutamicum; and Pseudomonas putida.

[0042] Also preferably, the O-methyltransferase variant as described herein and the auxiliary enzymes 4-coumaric acid 3-hydroxylase and 4-hydroxyphenylacetic acid 3-monooxygenase reductase and / or S-adenosylmethionine synthetase, if one or all of these auxiliary enzymes are present, are expressed in a cell-free expression system. Cell-free expression is also known as in vitro protein expression and is the production of recombinant proteins in solution using the translation machinery of biomolecules extracted from cells.

[0043] Yet another aspect of the present application relates to a biocatalytic process for the production of 4'-O-methylated and 3'-O-methylated phenolic substances, preferably hesperetin dihydrochalcones, said process comprising the step of providing the O-methyltransferase variant according to the present application.

[0044] Preferably, as used herein, the terms "4'-O-methylated phenolic substances and 3'-O-methylated phenolic substances" refer to compounds selected from the group consisting of: ferulic acid, dihydroferulic acid, high-senna dihydrochalcone, high-purple chalcone, high-senna, 2,3-dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopyran-4-one, isopantherin, 3'-O-methyl-(-)-epicatechin, 3,4-dihydro-8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1H-2-benzopyran-1-one, isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5-trihydroxy-6-(hydroxymethyl)oxane-2-yl]oxyphenyl]-3-( 4-Hydroxy-3-methoxyphenyl)prop-1-one, 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyloxane-2-yl)oxymethyl]oxane-2-yl]oxy-2,3-dihydrochromogen-4-one, narcissin, p-methoxycinnamic acid, isoflavonic acid, dihydroisoferulic acid, isopyrosine dihydrochalcone, hesperidin dihydrochalcone, 2',4'-dihydroxy-4-methoxychalcone, (2E)-1-(2,4-dihydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-2-propen-1-one, isopyrosine, hesperidin, 2,3-dihydro-7-hydroxy-2-(4 2,3-Dihydro-7-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, deoxyrhein, emodin, (2R,3R)-3,4-dihydro-2-(4-methoxyphenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-methyl-(-)-epicatechin, 3,4-dihydro-8-hydroxy-3-(4-methoxyphenyl)-1H-2-benzopyran-1-one, citronella derivatives, dihydrocitronella derivatives, phytosalicylate, tamarindin, kaempferol, hesperidin dihydrochalcone glucoside, hesperidin, 1-[4-(β-D-pyranoglycoloxy)-2,6-dihydroxyphenyl] -3-(4-methoxyphenyl)-1-propanone, 3-[[6-O-(6-deoxy-α-L-pyranomannosyl)-β-D-pyranoglucopyranosyl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, 3',4'-dimethoxycinnamic acid, 3-(3,4-dimethoxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone, (2E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1-one, 2-(3,4-dimethoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4-dimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-dimethoxyphenyl)vinyl]-1,3-benzendiol, (2R,3R)-2-(3,4-dimethoxyphenyl)-3,4-dihydro-2H-1 -benzopyran-3,5,7-triol, thunberginol G, pentaceroiin, calomelanone, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillo-mandelic acid, isovanillo-mandelic acid, sinapinic acid, their glycosides and mixtures thereof.

[0045] It is preferred that the process according to the present application is a fermentation process, said fermentation process comprising the following steps:

[0046] i. providing at least one recombinant microorganism comprising a nucleic acid encoding at least one O-methyltransferase variant according to the present application, preferably a genetically engineered cell according to the present application;

[0047] ii. cultivating said at least one recombinant microorganism under conditions allowing the expression of said at least one O-methyltransferase;

[0048] iii. adding at least one phenolic substance to said recombinant microorganism, said at least one phenolic substance being selected from the group consisting of benzoic acid, phenylacetic acid, mandelic acid, cinnamic acid, dihydrocinnamic acid, chalcone, dihydrochalcone, flavan, catechin, flavanone, flavone, 3-hydroxyflavone, anthocyane, stilbene, dihydrostilbene, dihydroisocoumarin, isocoumarin, phenylpropanoid, flavanol, aglycone, glycosides of the aforementioned substances, in particular O-glycosides, and mixtures thereof, preferably aglycones and glycosides mixtures thereof, preferably selected from the group consisting of flavanone, chalcone, dihydrochalcone, phenylpropanoid, flavanol, dihydroisocoumarin, carboxylic acid stilbene, stilbene, glycosides thereof and mixtures thereof;

[0049] iv. obtaining a mixture of 4’-O-methylated phenolic substances and 3’-O-methylated phenolic substances, preferably wherein the ratio of 4’-O-methylated phenolic substances to 3’-O-methylated phenolic substances is 99.9:0.1 to 60:40, preferably 99.9:0.1 to 70:30, more preferably 99.9:0.1 to 80:20, especially preferably 99.9:0.1 to 90:10, even more preferably 99.9:0.1 to 95:5.

[0050] A “fermentation” process according to the present application is to be understood as referring to a process involving the cultivation of a recombinant microorganism. Preferably, the fermentation process according to the present application is a process wherein no purified or partially purified enzymes or cell lysates are present.

[0051] The term "phenolic substance" as used herein preferably refers to a compound having one or more, preferably at least one, preferably at least two, preferably two hydroxyl groups, preferably wherein the hydroxyl groups are bound to a phenyl group or a derivative thereof. Preferably, the term refers to a compound having a catechol group. Particularly preferred, the term relates to a compound selected from the group consisting of benzoic acid, phenylacetic acid, mandelic acid, cinnamic acid, dihydrocinnamic acid, chalcone, dihydrochalcone, flavan, catechin, flavanone, flavone, 3-hydroxyflavone, anthocyanidin, stilbene, dihydrostilbene, dihydroisocoumarin, isocoumarin, phenylpropanoid, flavanol, aglycone, glycosides of the aforementioned, in particular O-glycosides, and mixtures thereof, preferably aglycones and glycoside mixtures thereof, preferably selected from the group consisting of flavanone, chalcone, dihydrochalcone, phenylpropanoid, flavanol, dihydroisocoumarin, carboxylic acid stilbene, stilbene, glycosides thereof and mixtures thereof.

[0052] It is preferred that the phenolic substance is selected from the group consisting of naringenin, eriodictyol, phloretin, eriodictyol dihydrochalcone, rhodoviolanthin, rhodoviolaxanthin, thunberginol G, thunberginol G acid, glycosides thereof, preferably O-glycosides of the foregoing, and mixtures thereof, preferably mixtures of aglycones and glycosides thereof.

[0053] Further, when a "phenolic substance" as described herein is methylated at the hydroxyl group or one of the hydroxyl groups, an "O-methylated phenolic substance" as described herein is obtained. Accordingly, the term "corresponding phenolic substance" refers to a phenolic substance which can be reacted with the corresponding O-methylated phenolic substance by methylation O-methylation. In terms of the present application, 4'-O-methylation is preferred over 3'-O-methylation.

[0054] The skilled person can readily determine suitable reaction conditions for cultivating the recombinant microorganism, such as buffers, additives, temperature and pH conditions, suitable co-factors and optionally additional proteins.

[0055] Especially preferred, the method involves manufacturing hesperetin dihydrochalcone by 4'-O-methylation of the phenolic substance 3-hydroxyphloretin (eriodictyol dihydrochalcone).

[0056] Further preferred, the method involves manufacturing hesperetin dihydrochalcone from the phenolic substance phloretin which is initially hydroxylated to 3-hydroxyphloretin (eriodictyol dihydrochalcone) by the enzymes 4-coumarate 3-hydroxylase and 4-hydroxyphenylacetic acid 3-monooxygenase reductase, followed by 4'-O-methylation to hesperetin dihydrochalcone by the O-methyltransferase variant according to the present application.

[0057] More preferably, the method involves the production of hesperidin from the phenolic substance naringenin, which is initially hydroxylated to sennaol by enzymes 4-coumaric acid 3-hydroxylase and 4-hydroxyphenylacetic acid 3-monooxygenase reductase, and subsequently 4'-O-methylated to hesperidin by the O-methyltransferase variant according to the invention.

[0058] More preferably, the method involves the production of phyllodulcinic acid and / or phyllodulcinic acid from phenolic substances physolic acid and / or physolic acid, wherein the phenolic substances physolic acid and / or physolic acid are initially hydroxylated to thunberginol G and / or thunberginol G acid by enzymes 4-coumaric acid 3-hydroxylase and 4-hydroxyphenylacetic acid 3-monooxygenase reductase, and subsequently 4'-O-methylated to phyllodulcinic acid and / or phyllodulcinic acid by O-methyltransferase variant according to the invention.

[0059] Preferably, the method according to the present invention is an enzymatic method, which includes the following steps:

[0060] i. Provide at least one O-methyltransferase variant according to the invention;

[0061] ii. Adding at least one phenolic substance to the recombinant microorganism, said at least one phenolic substance being selected from the group consisting of: benzoic acid, phenylacetic acid, mandelic acid, cinnamic acid, dihydrocinnamic acid, chalcone, dihydrochalcone, flavan, catechin, flavanone, flavonoid, 3-hydroxyflavone, anthocyanin, arbutin, dihydroarbutin, dihydroisocoumarin, isocoumarin, phenylpropanoid, flavanol, aglycone, glycosides of the above substances, especially O-glycosides, and mixtures thereof, preferably mixtures of their aglycones and glycosides, preferably selected from the group consisting of: flavanone, chalcone, dihydrochalcone, phenylpropanoid, flavanol, dihydroisocoumarin, arbutin carboxylate, arbutin, their glycosides, and mixtures thereof;

[0062] iii. Obtaining a mixture of 4'-O-methylated phenolic substances and 3'-O-methylated phenolic substances, preferably wherein the ratio of 4'-O-methylated phenolic substances to 3'-O-methylated phenolic substances is 99.9:0.1 to 60:40, preferably 99.9:0.1 to 70:30, more preferably 99.9:0.1 to 80:20, particularly preferably 99.9:0.1 to 90:10, and even more preferably 99.9:0.1 to 95:5.

[0063] For the purposes of this invention, "enzymatic method" should be understood as a biocatalytic method in which the biocatalyst is provided in the form of a purified or partially purified enzyme.

[0064] In the context of the present application, a purified enzyme or a partially purified enzyme means that the biotechnologically produced enzyme is processed to reduce side products. This can be done with different separation methods well known in the art, such as chromatography (including affinity chromatography, hydrophobic interaction chromatography, size exclusion chromatography, etc.), precipitation, membrane filtration, centrifugation, crystallization or sedimentation. A purified enzyme refers hereby to an enzyme with a total content of at least 90% (w / v) of the enzyme as compared to the complete mixture, wherein a partially purified enzyme refers to an enzyme with a total content of maximally 90% (w / v) of the enzyme as compared to the complete mixture. The skilled person can easily determine the content and purity of at least one enzyme of interest in a cell culture lysate and / or a supernatant of interest and he can easily combine at least one, two or at least three or several purification steps to obtain a higher purity, if desired.

[0065] It is preferred that the method according to the present application additionally comprises the following steps:

[0066] ii. a-1 providing at least one 4-coumarate 3-hydroxylase and a 4-hydroxyphenylacetic acid 3-monooxygenase reductase;

[0067] and / or

[0068] ii. a-2 providing at least one methyl group donor, wherein the at least one methyl group donor is selected from the group consisting of S-adenosylmethionine and / or a combination of methionine and S-adenosylmethionine synthase.

[0069] The additional provision of the helper enzymes 4-coumarate 3-hydroxylase and 4-hydroxyphenylacetic acid 3-monooxygenase reductase (step ii. a-1) provides for the hydroxylation of the corresponding phenolics to be used as a substrate for the O-methyltransferase variant according to the present application.

[0070] The additional provision of a methyl group donor provides for a co-substrate of the O-methyltransferase as described herein.

[0071] It is preferred that the 4-coumarate 3-hydroxylase comprises or consists of an amino acid sequence according to SEQ ID NO.: 3 or an amino acid having 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 the amino acid sequence according to SEQ ID NO.: 3 or a nucleic acid sequence encoding the respective said amino acid sequence,

[0072] and / or

[0073] 4-hydroxyphenylacetate 3-monooxygenase reductase comprises or consists of an amino acid sequence according to SEQ ID NO.: 5 or an amino acid sequence having 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 with the amino acid sequence according to SEQ ID NO.: 5 or a nucleic acid sequence encoding the respective said amino acid sequence, and / or

[0074] S-adenosylmethionine synthetase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO.: 7 and SEQ ID NO.: 9 or an amino acid sequence having 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 with the amino acid sequence selected from the group consisting of SEQ ID NO.: 7 and SEQ ID NO.: 9 or a nucleic acid sequence encoding the respective said amino acid sequence.

[0075] Another aspect of the present application relates to the use of the O-methyltransferase variant according to the present application for the production of at least one 4'-O-methylated phenolic substance, preferably for the production of hesperetin dihydrochalcone, hesperetin and / or phloridzin

[0076] Yet another aspect of the present application relates to the use of the mixture obtained or obtainable by the method according to the present application as a sweetness enhancer and / or a sweetness modulator and / or a flavoring agent, preferably wherein the sweetness enhancer and / or the flavoring agent is used in a finished product selected from the group consisting of goods intended to obtain nutrition or enjoyment.

[0077] Preferably, the mixture obtained or obtainable by the method according to the present application is a mixture comprising hesperetin dihydrochalcone and phloridzin dihydrochalcone, wherein the ratio of hesperetin dihydrochalcone to phloridzin dihydrochalcone is at least 70:30, preferably at least 80:20, more preferably at least 90:10 and especially preferably at least 99.9:0.1.

[0078] Preferably, the mixture according to the invention is used in a finished product intended to obtain nutrition or enjoyment, which may in particular include products such as: baked goods (e.g., bread, biscuits, cakes, other pastries), sweets (e.g., chocolate, chocolate bar products, other bar products, fruit gummies, hard caramel and soft caramel, chewing gum), alcoholic or non-alcoholic beverages (e.g., coffee, tea, wine, wine-containing beverages, beer, beer-containing beverages, liqueurs, spirits, brandy, fruit-containing lemon beverages, isotonic beverages, soft drinks, nectar, fruit and vegetable juices, fruit and vegetable juice preparations), instant beverages (e.g., instant cocoa beverages, instant tea beverages, instant coffee beverages), meat products (e.g., ham, fresh sausage or raw sausage products, seasoned or cured fresh or salted meat products), eggs or egg products (dried eggs, egg whites, egg yolks), and grain products (e.g., Breakfast cereals, cereal bars, pre-cooked instant rice products), dairy products (e.g., dairy beverages, dairy ice cream, yogurt, kefir, cream cheese, soft cheese, hard cheese, milk powder, whey, buttermilk, partially or fully hydrolyzed products containing milk proteins), products made from soy protein or other soy fractions (e.g., soy milk and products derived therefrom, compositions containing soy lecithin, fermented products in the form of tofu or tempeh, or products made therefrom), fruit products (e.g., jams, fruit ice cream, fruit sauces, fruit fillings), vegetable products (e.g., tomato sauce, sauces, dried vegetables, frozen vegetables, pre-cooked vegetables, boiled dried vegetables), snacks (e.g., baked or fried potato chips or potato ball products, corn or peanut-based extrusions), fat and oil-based products or their emulsions (e.g., mayonnaise, remora, seasonings), other finished products and soups (e.g., dry soups, instant soups, pre-cooked soups).

[0079] Another aspect of the invention relates to mixtures obtained or available by the method according to the invention, wherein the ratio of 4'-O-methylated phenolic substance to 3'-O-methylated phenolic substance is 99.9:0.1 to 60:40, preferably 99.9:0.1 to 70:30, more preferably 99.9:0.1 to 80:20, particularly preferably 99.9:0.1 to 90:10, and even more preferably 99.9:0.1 to 95:5.

[0080] Preferably, the mixture obtained or obtainable by the method according to the invention has a ratio of 99.9:0.1 to 60:40, more preferably 99.9:0.1 to 70:30, more preferably 99.9:0.1 to 80:20, particularly preferably 99.9:0.1 to 90:10, and even more preferably 99.9:0.1 to 95:5 of 4'-O-methylated phenolic substances to other O-methylated phenolic substances.

[0081] Preferably, the mixture obtained or obtainable by the method according to the invention is a mixture comprising hesperidin dihydrochalcone and succinylcholine dihydrochalcone, wherein the ratio of hesperidin dihydrochalcone to succinylcholine dihydrochalcone is at least 70:30, preferably at least 80:20, more preferably at least 90:10, and particularly preferably at least 99.9:0.1.

[0082] Where applicable, the contents described herein with respect to the methods and / or mixtures according to the invention are accordingly applicable to the uses according to the invention.

[0083] Sequence brief description

[0084] SEQ ID NO.: 1 and SEQ ID NO.: 2 Amino acid and nucleic acid sequences of the parental O-methyltransferase from *Rhodotorula rubrum* from Ganghwa Island.

[0085] SEQ ID NO.: 3 and SEQ ID NO.: 4 Amino acid and nucleic acid sequences of 4-coumaric acid 3-hydroxylase from Saccharothrix espanaensis.

[0086] SEQ ID NO.: 5 and SEQ ID NO.: 6 Amino acid and nucleic acid sequences of 4-hydroxyphenylacetic acid 3-monooxygenase reductase from Pseudomonas aeruginosa.

[0087] SEQ ID NO.: 7 to SEQ ID NO.: 10 Amino acid and nucleic acid sequences of S-adenosylmethionine synthases from Bacillus subtilis and Saccharomyces cerevisiae.

[0088] SEQ ID NO.: 11 to SEQ ID NO.: 38 : Amino acid and nucleic acid sequences of the O-methyltransferase variant.

[0089] SEQ ID NO.: 39 to SEQ ID NO.: 67 : The nucleic acid sequence of the amplification primer. Attached Figure Description

[0090] Figure 1 : A reaction protocol for methylation via parental O-methyltransferase (ZgOMT) from *Rhodotorula oryzae* from Ganghwa Island. The substrate senna dihydrochalcone (1b) was methylated in the presence of S-adenosylmethionine (SAM) to the 3'-O-methylated product hypersenna dihydrochalcone (2b) and the 4'-O-methylated product hesperidin dihydrochalcone (3b).

[0091] Figure 2 The conversion and regioisomerism ratio of the 3'-O-methylated and 4'-O-methylated products of different ZgOMT variants were studied. Senna dihydrochalcone was the substrate, and the products were high-senna dihydrochalcone and hesperidin dihydrochalcone.

[0092] Figure 3 Regional isomerism ratios and substrate conversion rates of ZgOMT wild-type and variants with different substrates. Figure 3 A shows a schematic diagram of the methylation of different phenolic substances into 3'-O-methylated and 4'-O-methylated products. Figure 3 B shows the conversion rates and isomerism ratios of 3'-O-methylated and 4'-O-methylated products of dihydrochalcone extract, senna dihydrochalcone, and blue sieve phlotoxin, as well as the conversion rates and isomerism ratios of the 3'-O-methylated products to the 4'-O-methylated products when using wild-type ZgOMT (A) or the ZgOMT variant of the present invention (B). Figure 3 C shows the conversion and isomerism ratio of the flavanone extracts naringenin and sennaol, as well as the corresponding conversion and isomerism ratio of the 3'-O-methylated product to the 4'-O-methylated product when using wild-type ZgOMT (A) or the ZgOMT variant of the present invention (B). Figure 3 D shows the catechol extract of caffeic acid and 3,4-dihydroxybenzaldehyde.

[0093] Figure 4 The regioisomerism ratio of methoxylated dihydrochalcone produced by rhodopsin from different Escherichia coli strains expressing SeSAM5 and ZgOMT wild-type or variants, as well as BsSAMS and PaHpaC. The ratio is the amount of hesperidin dihydrochalcone divided by the total amount of methoxylated dihydrochalcone product (in percentage).

[0094] Figure 5 Chromatolated and methoxylated compounds produced by hydrazine from *Escherichia coli* W3110(DE3)SeSAM5_ZgOMT-N41K-S174R were obtained by sampling 1 hour after hydrazine supplementation. Cells were incubated with hydrazine at 25°C for a total of 16 hours.

[0095] Figure 6 Chromatolated and methoxylated compounds produced by hydrazine from *Escherichia coli* W3110(DE3)SeSAM5_ZgOMT-N41K-S174R were obtained by sampling 16 hours after hydrazine supplementation. Cells were incubated with hydrazine at 25°C for a total of 16 hours.

[0096] Figure 7Chromatograms of hydroxylated and methoxylated compounds produced from naringenin by *Escherichia coli* W3110(DE3)SeSAM5_ZgOMT-N41K-S174R. Cells were incubated with naringenin at 25°C for 24 hours.

[0097] The invention is further explained by the following examples, which are not intended to limit the scope of the invention but are merely illustrative. Detailed Implementation

[0098] Example

[0099] Example 1 - Generation of a methyltransferase mutagenesis library

[0100] SEQ ID NO.: 2 (Twist Bioscience, San Francisco, USA) was synthesized and cloned into the pET28a(+) vector via restriction ligation using the enzymes NcoI and XhoI to obtain the vector pET28a_ZgOMT. Site saturation mutagenesis of the active site residues was performed by PCR using the pET28a_ZgOMT template and degenerate primers containing NNK. The following PCR procedure was used with Q5 high-fidelity DNA polymerase (New England Biolabs, Frankfurt am Main, Germany) and one of the following primer pairs: SEQ ID NO.: 39 and SEQ ID NO.: 40, or SEQ ID NO.: 41 and SEQ ID NO.: 42, or SEQ ID NO.: 43 and SEQ ID NO.: 44, or SEQ ID NO.: 45 and SEQ ID NO.: 46, or SEQ ID NO.: 47 and SEQ ID NO.: 48: 98 °C for 30 seconds (initial denaturation), followed by 18 cycles: 98 °C for 10 seconds (denaturation); 60 °C for 20 seconds (annealing); 72 °C for 3 minutes (extension); and finally, 72 °C for 2 minutes (extension). The PCR mixture was then column purified and digested with 1 μL of DpnI at 37 °C for 1 hour. The obtained DNA product was used directly to transform chemically competent Escherichia coli TOP10 cells. After growing overnight on LB agar plates supplemented with 50 μg / ml kanamycin, the bacterial cells were resuspended in LB medium, and plasmid DNA was isolated and used to transform chemically competent Escherichia coli BL21(DE3).

[0101] Example 2: Site-directed mutagenesis of the methyltransferase gene

[0102] Site-directed mutagenesis of active site residues was performed via PCR using the pET28a_ZgOMT template and the Q5 site-directed mutagenesis kit (New England BioSciences, Frankfurt am Main, Germany). The following PCR program was used with Q5 high-fidelity DNA polymerase and one of the primer pairs of SEQ ID No.: 48 and SEQ ID No.: 49, or SEQ ID No.: 50 and SEQ ID No.: 51, or SEQ ID No.: 52 and SEQ ID No.: 53, or SEQ ID No.: 54 and SEQ ID No.: 56 to SEQ ID No.: 67: 98°C for 30 seconds (initial denaturation), followed by 25 cycles: 98°C for 10 seconds (denaturation); 60°C for 20 seconds (annealing); 72°C for 3 minutes (extension); and finally, 72°C for 2 minutes (extension). Subsequently, 1 μL of the PCR product was treated with a KLD enzyme mixture (total volume: 10 μL) for 1 hour at room temperature. The obtained DNA product was used directly to transform chemically competent Escherichia coli TOP10 cells. After growing overnight on LB agar plates supplemented with 50 μg mL⁻¹ kanamycin, the bacterial cells were resuspended in LB medium, and plasmid DNA was isolated and used to transform chemically competent Escherichia coli BL21(DE3).

[0103] Example 3 - Protein Expression, Purification, and Regioisomeric Ratio Determination

[0104] Wild-type O-methyltransferase and ZgOMT variant of *Rhizopus oryzae* (ZgOMT) were expressed using the pET28a(+) expression system in 100 mL TB medium supplemented with 50 μg mL⁻¹ kanamycin, 0.2% (w / v) lactose and 0.05% (w / v) glucose for 3 hours at 37 °C and 180 rpm, followed by overnight expression at 25 °C. Cells were then collected by centrifugation (10 min, 4500 g), and the cell clumps were frozen at -80°C for 1 hour. The clumps were then resuspended in 15 mL of lysis buffer (Sigma-Aldrich, Steinheim, Germany) containing 50 mM Tris-HCl pH 7.5, 1 mg mL⁻¹ lysozyme, and 5 μg mL⁻¹ bovine DNase, and incubated at 37°C for 1 hour. Insoluble cell fragments were removed by centrifugation (15 min, 5000 g). Activity was determined directly using cell-free extract (CFE), or protein purification was performed using CFE.

[0105] For protein purification, the buffer was adjusted to 10 mM imidazole, and the supernatant was incubated with Ni-NTA beads (Sigma-Aldrich, Steinham, Germany) for 30 minutes at 4°C, then loaded onto a gravity flow column. The column was washed three times with 5 mL of 50 mM Tris-HCl pH 7.5 supplemented with 50 mM imidazole, and eluted with 3 mL of 50 mM Tris-HCl pH 7.5 supplemented with 250 mM imidazole. The elution fraction containing protein was collected and reburied in 50 mM Tris-HCl pH 7.5 on a PD-10 desalting column (Cytiva, Freiburg im Breisgau, Germany).

[0106] The regioisomerism ratio of enzyme variants was determined at room temperature using 20 μL CFE or 5 μL purified enzyme in 25 mM Tris-HCl pH 7.5, 100 μM substrate (various phenolic substances), 2 mM MgCl2, and 0.5 mM SAM (total volume: 100 μL) for 1 hour. The reaction mixture was quenched with 75 μL acetonitrile, incubated at room temperature for 20 min, and insoluble proteins were removed by centrifugation (15 min, 600 g). 90 μL of the reaction mixture was transferred to a new 96-well plate for reversed-phase HPLC analysis. The enzymatic reaction products were analyzed by reversed-phase HPLC using an Inertsil ODS-3 5 μm, 4 × 100 mm C-18 column (GL Sciences, Tokyo, Japan) at 50 °C, at a flow rate of 1.2 mL min⁻¹, with 30% acetonitrile / 70% H₂O containing 0.1% (v / v) formic acid.

[0107] Table 1: Regioisomer ratios of ZgOMT variants based on sennal dihydrochalcone

[0108]

[0109] Figure 2 The conversion rates and regioisomerism ratios of different O-methyltransferases based on senna dihydrochalcone were further described.

[0110] Figure 3The conversion rates and regioisomerism ratios for 4'-O-methylation and 3'-O-methylation are shown. It can be observed that phenolic substances with one hydroxyl group cannot be methylated, while phenolic substances with two hydroxyl groups can be methylated by O-methyltransferases. As described for the purposes of this invention, for substrates with only one hydroxyl group, pre-methylation must be performed, for example, by coenzymes such as 4-coumaric acid 3-hydroxylase and 4-hydroxyphenylacetic acid 3-monooxygenase reductase. The regioisomerism ratio and conversion rate can be increased using an O-methyltransferase variant according to SEQ ID NO.: 35.

[0111] Example 4 - Generation of Escherichia coli strains for hydroxylation and methylation of phenolic substances

[0112] Synthesized SEQ ID NO.: 2, SEQ ID NO.: 4, SEQ ID NO.: 6, and SEQ ID NO.: 8 (TopVest Biotech, San Francisco, USA). One of the DNA sequences of SEQ ID NO.: 4 and SEQ ID NO.: 2 or SEQ ID NO.: 12-38 was cloned into pCDFDuet-1 (Merck, Darmstadt, Germany) via restriction ligation using restriction enzymes NcoI, EcoRI, and NdeI, respectively, to obtain a vector for expressing SeSAM5 and ZgOMT wild-type or variants thereof. SEQ ID NO.: 6 and SEQ ID NO.: 8 were cloned into pRSFDuet-1 (Merck, Darmstadt, Germany) via restriction ligation using restriction enzymes NcoI, EcoRI, and NdeI, respectively, to obtain the vector pRSFDuet_PaHpaC_BsSAMS. The obtained pCDFDuet vector and pRSFDuet_PaHpaC_BsSAMS were transformed into chemically competent Escherichia coli W3110(DE3) cells to obtain the expression cells shown in Table 2:

[0113] Table 2: Escherichia coli strains used for the production of methoxylated dihydrochalcone from phloretin (PaHpaC: 4-hydroxyphenylacetic acid 3-monooxygenase reductase; BsSAMS: S-adenosylmethionine synthase; SeSAM5: 4-coumaric acid 3-hydroxylase; ZgOMT: O-methyltransferase)

[0114]

[0115] Example 5 - Production of a mixture of methoxylated dihydrochalcones from phlorizin using SeSAM5 and ZgOMT wild-type or variants

[0116] The strain generated in Example 4 was cultured for 1 hour at 37°C and 180 rpm in 10 mL TB medium (24 g / L yeast extract, 20 g / L tryptone, 4 mL / L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) supplemented with 50 µg mL⁻¹ neomycin and 50 µg mL⁻¹ streptomycin. Then, 0.1 mM IPTG was added, and the temperature was lowered to 25°C. The cells were incubated at 180 rpm for 7 hours. Phloretin at a concentration of 200 mg L⁻¹ was added, and the cells were incubated for another 16 hours at 180 rpm and 25°C. For HPLC analysis, the cells were incubated on ice for 1 hour, followed by centrifugation of cell debris. 10 mL of acetonitrile was added to the culture, and the supernatant was used for analysis. Figure 4 The regioisomer ratio of the determination of methoxylated dihydrochalcone is described in the figure.

[0117] Example 6 - Production of a mixture of methoxylated dihydroisocoumarins from hyacinthol using SeSAM5 and ZgOMT wild-type or variants

[0118] The strain of *Escherichia coli* W3110(DE3)SeSAM5_ZgOMT-N41K-S174R, containing the ZgOMT variant according to SEQ ID NO.: 35, generated in Example 4, was subjected to a reaction at 37°C and 180 rpm with 50 µg mL of water. -1 Neomycin, 50 µg / mL -1 Cells were cultured in 10 mL TB medium (24 g / L yeast extract, 20 g / L tryptone, 4 mL / L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) for 1 hour. Then, 0.1 mM IPTG was added, and the temperature was lowered to 25°C. Cells were incubated at 180 rpm for 7 hours. The added concentration was 200 mg / L. -1 The cells were incubated with acetonitrile and then incubated at 180 rpm and 25°C for 16 hours. For HPLC-MS analysis, the cells were incubated on ice for 1 hour, followed by centrifugation of cell debris. 10 ml of acetonitrile was added to the culture, and the supernatant was used for analysis. Figure 5 and Figure 6 The reaction results after incubation with hyacinthol for 1 hour and 16 hours were described. Figure 5 and Figure 6 The results depicted in the chromatogram are:

[0119] Figure 5 :

[0120]

[0121] Figure 6 :

[0122]

[0123] Example 7 - Production of a mixture of methoxylated flavonoids from naringenin using SeSAM5 and ZGOMT wild-type or variants

[0124] The strain of *Escherichia coli* W3110(DE3)SeSAM5_ZgOMT-N41K-S174R, containing the ZgOMT variant according to SEQ ID NO.: 35, generated in Example 4, was subjected to a reaction at 37°C and 180 rpm with 50 µg mL of water. -1 Neomycin, 50 µg / mL -1 Cells were cultured in 10 mL TB medium (24 g / L yeast extract, 20 g / L tryptone, 4 mL / L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) for 1 hour. Then, 0.1 mM IPTG was added, and the temperature was lowered to 25°C. Cells were incubated at 180 rpm for 7 hours. The added concentration was 200 mg / L. -1 The cells were incubated with naringenin at 180 rpm and 25°C for 16 hours. For HPLC-MS analysis, the cells were incubated on ice for 1 hour, followed by centrifugation of cell debris. 10 ml of acetonitrile was added to the culture, and the supernatant was used for analysis. Figure 7 The results of the reaction after incubation with naringenin for 16 hours were described. Figure 7 The results depicted in the chromatogram are:

[0125] Figure 7 :

[0126]

Claims

1. An O-methyltransferase variant, said O-methyltransferase variant having substitutions at one, two, or all amino acid positions selected from positions 41, 42, 43, 173, and 174 of the parent polypeptide, wherein, The parent polypeptide is SEQ ID NO.:1, and wherein the at least one O-methyltransferase has 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 with SEQ ID NO.:

1.

2. The O-methyltransferase variant according to claim 1, wherein, The substitution is an amino acid selected from the group consisting of aliphatic amino acids such as leucine, isoleucine and valine and / or positively charged amino acids such as lysine, arginine and histidine and / or threonine.

3. The O-methyltransferase variant according to any one of claims 1 or 2, wherein, The O-methyltransferase comprises or consists of the following amino acid sequences selected from the group consisting of SEQ ID NO.: 11, SEQ ID NO.: 13, SEQ ID NO.: 15, SEQ ID NO.: 17, SEQ ID NO.: 19, SEQ ID NO.: 21, SEQ ID NO.: 23, SEQ ID NO.: 25, SEQ ID NO.: 27, SEQ ID NO.: 29, SEQ ID NO.: 31, SEQ ID NO.: 33, SEQ ID NO.: 35 and SEQ ID NO.: 37, or the amino acid sequence selected from the group consisting of SEQ ID NO.: 11, SEQ ID NO.: 13, SEQ ID NO.: 15, SEQ ID NO.: 17, SEQ ID NO.: 19, SEQ ID NO.: 21, SEQ ID NO.: 23, SEQ ID NO.: 25, SEQ ID NO.: 27, SEQ ID NO.: 29, ...3, SEQ ID NO.: 14, SEQ ID NO.: 15, SEQ ID NO.: 1 31, the amino acid sequences comprising SEQ ID NO.: 33, SEQ ID NO.: 35 and SEQ ID NO.: 37 have amino acid sequences having 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.

4. A vector system, preferably a plasmid vector system, said vector system comprising a vector containing at least one nucleic acid segment (a), said at least one nucleic acid segment (a) containing a gene encoding an O-methyltransferase variant, said O-methyltransferase variant having a nucleic acid sequence selected from the group consisting of SEQ ID NO.: 12, SEQ ID NO.: 14, SEQ ID NO.: 16, SEQ ID NO.: 18, SEQ ID NO.: 20, SEQ ID NO.: 22, SEQ ID NO.: 24, SEQ ID NO.: 26, SEQ ID NO.: 28, SEQ ID NO.: 30, SEQ ID NO.: 32, SEQ ID NO.: 34, SEQ ID NO.: 36 and SEQ ID NO.: 38 or a nucleic acid sequence selected from SEQ ID NO.: 12, SEQ ID NO.: 14, SEQ ID NO.: 16, SEQ ID NO.: 18, SEQ ID NO.: 20, SEQ ID NO.: 22, SEQ ID NO.:

38. The nucleic acids of the group consisting of SEQ ID NO.: 24, SEQ ID NO.: 26, SEQ ID NO.: 28, SEQ ID NO.: 30, SEQ ID NO.: 32, SEQ ID NO.: 34, SEQ ID NO.: 36, and SEQ ID NO.: 38 have at least 70%, 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; and Preferably, it includes at least one nucleic acid segment (b), said at least one nucleic acid segment (b) including a gene encoding 4-coumaric acid 3-hydroxylase and a gene encoding 4-hydroxyphenylacetic acid 3-monooxygenase reductase; And / or preferably at least one nucleic acid segment (c), said at least one nucleic acid segment (c) comprising a gene encoding S-adenosylmethionine synthase, in, The nucleic acid segments (a), (b) and / or (c), if present, are preferably provided on the same vector or in two or three different vectors, each containing one nucleic acid segment (a) or (b) or (c).

5. A genetically modified cell, said genetically modified cell comprising the vector system according to claim 4.

6. The genetically modified cell according to claim 5, wherein, The cells are selected from the group consisting of: certain species of Escherichia coli, such as Escherichia coli BL21, Escherichia coli MG1655, preferably Escherichia coli W3110; certain species of Bacillus, such as Bacillus licheniformis, Bacillus subtilis or Bacillus amyloliquefaciens; certain species of Yeast, preferably Saccharomyces cerevisiae; certain species of Hansenula or certain species of Cotyledon, such as Cotyledon phaf. phaf. phaf. phaf. phaf. phaf. phaf. phaf.; certain species of Yersinia, such as Yersinia lipophila; certain species of Kluyveromyces, such as Kluyveromyces lactis; Corynebacterium glutamicum; and Pseudomonas putida.

7. A biocatalytic method for producing 4'-O-methylated phenolic substances and 3'-O-methylated phenolic substances, preferably hesperidin dihydrochalcone, said method comprising the step of providing an O-methyltransferase variant according to any one of claims 1 to 3.

8. The method according to claim 7, wherein, The method includes the following fermentation steps: i. Providing at least one recombinant microorganism containing nucleic acid, preferably a genetically engineered cell according to claim 5 or 6, wherein the nucleic acid encodes at least one O-methyltransferase variant according to any one of claims 1 to 3; ii. Culture the at least one recombinant microorganism under conditions that allow expression of the at least one O-methyltransferase; iii. Adding at least one phenolic substance to the recombinant microorganism, said at least one phenolic substance being selected from the group consisting of: benzoic acid, phenylacetic acid, mandelic acid, cinnamic acid, dihydrocinnamic acid, chalcone, dihydrochalcone, flavan, catechin, flavanone, flavonoid, 3-hydroxyflavone, anthocyanin, piracetam, dihydropiracetam, dihydroisocoumarin, isocoumarin, phenylpropanoid, flavanol, aglycone, glycosides of the above substances, especially O-glycosides, and mixtures thereof, preferably mixtures of their aglycones and glycosides, preferably selected from the group consisting of: flavanone, chalcone, dihydrochalcone, phenylpropanoid, flavanol, dihydroisocoumarin, piracetam carboxylate, piracetam, their glycosides, and mixtures thereof; iv. Obtain a mixture of 4'-O-methylated phenolic substances and 3'-O-methylated phenolic substances, preferably wherein, The ratio of 4'-O-methylated phenolic substances to 3'-O-methylated phenolic substances is 99.9:0.1 to 60:40, preferably 99.9:0.1 to 70:30, more preferably 99.9:0.1 to 80:20, particularly preferably 99.9:0.1 to 90:10, and even more preferably 99.9:0.1 to 95:

5.

9. The method according to claim 7, wherein, The method is an enzymatic method comprising the following steps: i. To provide at least one O-methyltransferase according to any one of claims 1 to 3; ii. Adding at least one phenolic substance to the recombinant microorganism, said at least one phenolic substance selected from the group consisting of: benzoic acid, phenylacetic acid, mandelic acid, cinnamic acid, dihydrocinnamic acid, chalcone, dihydrochalcone, flavan, catechin, flavanone, flavonoid, 3-hydroxyflavone, anthocyanin, arbutin, dihydroarbutin, dihydroisocoumarin, isocoumarin, phenylpropanoid, flavanol, aglycone, glycosides of the above substances, especially O-glycosides, and mixtures thereof, preferably mixtures of their aglycones and glycosides, preferably selected from the group consisting of: flavanone, chalcone, dihydrochalcone, phenylpropanoid, flavanol, dihydroisocoumarin, arbutin carboxylate, arbutin, their glycosides, and mixtures thereof; iii. Obtaining a mixture of 4'-O-methylated phenolic substances and 3'-O-methylated phenolic substances, preferably wherein, The ratio of 4'-O-methylated phenolic substances to 3'-O-methylated phenolic substances is 99.9:0.1 to 60:40, preferably 99.9:0.1 to 70:30, more preferably 99.9:0.1 to 80:20, particularly preferably 99.9:0.1 to 90:10, and even more preferably 99.9:0.1 to 95:

5.

10. The method according to claim 8 or 9, wherein, The phenolic substance is selected from the group consisting of: phloretin and 3-hydroxyphloretin, physalisol, physalis acid, thunberginol G and thunberginol G acid, naringenin and sennaol or mixtures thereof.

11. The method according to claims 8 to 10, wherein, The method further includes the following steps: ii. a-1 provides at least one 4-coumaric acid 3-hydroxylase and 4-hydroxyphenylacetic acid 3-monooxygenase reductase; and / or ii. a-2 provides at least one methyl group donor, wherein the at least one methyl group donor is selected from S-adenosylmethionine and / or a combination of methionine and S-adenosylmethionine synthase.

12. The method according to claim 11, wherein, The 4-coumaric acid 3-hydroxylase comprises or consists of the following: an amino acid sequence according to SEQ ID NO.: 3 or an amino acid sequence having 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 with the amino acid sequence according to SEQ ID NO.: 3, or a nucleic acid sequence encoding the corresponding amino acid sequence, and / or The 4-hydroxyphenylacetic acid 3-monooxygenase reductase comprises or consists of the following: an amino acid sequence according to SEQ ID NO.:5 or an amino acid sequence having 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 with the amino acid sequence according to SEQ ID NO.:5, or a nucleic acid sequence encoding the corresponding amino acid sequence, and / or The S-adenosylmethionine synthase comprises or consists of the following: an amino acid sequence selected from the group consisting of SEQ ID NO.: 7 and SEQ ID NO.: 9, or an amino acid sequence having 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 with the amino acid sequence selected from the group consisting of SEQ ID NO.: 7 and SEQ ID NO.: 9, or a nucleic acid sequence encoding the corresponding amino acid sequence.

13. Use of an O-methyltransferase variant according to any one of claims 1 to 3, said use being for the production of at least one 4'-O-methylated phenolic substance, preferably for the production of hesperidin dihydrochalcone.

14. Use of a mixture obtained or available by the method according to claims 7 to 12, the use being as a sweetness enhancer and / or sweetness modifier and / or flavoring agent, preferably wherein the sweetness enhancer and / or the flavoring agent is used in a finished product selected from the group consisting of goods intended to provide nutrition or enjoyment.

15. A mixture obtained or obtainable by the method according to any one of claims 7 to 12, wherein, The ratio of 4'-O-methylated phenolic substances to 3'-O-methylated phenolic substances is 99.9:0.1 to 60:40, preferably 99.9:0.1 to 70:30, more preferably 99.9:0.1 to 80:20, particularly preferably 99.9:0.1 to 90:10, and even more preferably 99.9:0.1 to 95:5.

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