WO2025017042A1

WO2025017042A1 - New enzymes for the manufacturing of 4'-o-methylated phenolic substances

Info

Publication number: WO2025017042A1
Application number: PCT/EP2024/070199
Authority: WO
Inventors: Uwe Bornscheuer; Andreas KUNZENDORF; Jakob Peter Ley; Lars MILKE; Bastian ZIRPEL
Original assignee: Symrise Ag
Priority date: 2023-07-17
Filing date: 2024-07-17
Publication date: 2025-01-23
Also published as: WO2025016533A1

Abstract

The present invention relates to new enzymes for the manufacturing of 4'-O-methylated phenolic substances from the corresponding educt and a method for producing a mixture with 3'-O- and 4'-O-methylated substances as well as the use of the enzymes for producing such a mixture and the use of a manufactured mixture as taste modifier in compositions.

Description

New enzymes for the manufacturing of 4’-O-methylated phenolic substances

The present invention relates to new enzymes for the manufacturing of 4’-O-methylated phenolic substances from the corresponding educt and a method for producing a mixture with 3’-O- and 4’-O-methylated substances as well as the use of the enzymes for producing such a mixture and the use of a manufactured mixture as taste modifier in compositions.

(Poly)phenolic substances are frequently present in plants and are thus also found in human nutrition. A specific group of phenolic substances are flavonoids. The group of flavonoids comprise compounds exhibiting sweet taste or sweet taste optimization. Those compounds are frequently used in various applications to either increase the sweet impression or to mask bittering substances of foodstuffs, pharmaceuticals, beverages or similar finished goods. There is thus a constant need to provide flavonoids as safe food additive and consequently methods to provide said substances in a reliable manner.

The manufacturing of homoeriodictyol dihydrochalcone (1) as well as its sweetness enhancing properties are described in W02007107596A1 . Furthermore, mixtures of homoeriodictyol dihydrochalcone (1) with salivation increasing agents in flavouring compositions are described in US20080227867. Also, a masking with homoeriodictyol dihydrochalcone (1) of the bitter taste impression of caffeine was described in US20080227867. The manufacturing of (1) was described in W02007107596A1 as a catalysed aldol reaction with piperidine of 1 ,4-di-O-benzyolacetophenone with vanillin. In this chemical reaction, the double bond of the obtained chaicone is hydrated with the aid of a Pd/C catalyst. Further methods comprise the usage of protective groups, other bases or reducing agents. All known methods require organic solvents and can therefore also not be classified as natural manufacturing methods according to EC 1334/2008.

3 4

The use and effect of hesperetin dihydrochalcone (2) for modifying unpleasant taste impressions is described in WO 2017186299A1. These characteristics are also described in J. Agric. Food Chem. 1977, 25(4), 763-772 as well as in J. Med. Chem. 1981 , 24(4), 408-428. Mixtures of (2) and corn syrup with an increased content of fructose as well as other sweeteners are described in W02019080990A1 . Homoeriodictyol dihydrochalcone (1) comprises a 3’-0-methylation, wherein hesperetin dihydrochalcone (2) comprises a 4’- O-methylation.

Homoeriodictyol dihydrochalcone (1) and hesperetin dihydrochalcone (2) can be manufactured via enzymatic hydroxylation of phloretin (3) to eriodictyol dihydrochalcone (4) and subsequent enzymatic methylation of (4) which is described in WO 2021/058115 A1 . This application also discloses enzyme variants for varying the specificity of an O- methyltransferase from Myxococcus xanthus regarding 3’-0-methylation and 4’-O- methylation.

Catechols are difunctional phenolic substances, which can be methylated. There are published results regarding altering the regioselectivity of catechol O-methyltransferases for the methylation of different catechols (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).

The paper of Wils et al., 2013 “A single amino acid determines position specificity of an Arabidopsis thaliana CCoAOMT-like O-methyltransferase” describes a mutation study to identify amino acid residues relevant for position specificity in the plant Arabidopsis thaliana. A position specificity towards a 3’-0-methylation of the substrate was achieved.

The paper of 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. Herein, the regioselectivity of a human catechol-O-methyltransferase heterologous expressed in E.coli was investigated.

As there is a steadily increasing awareness of consumers towards natural products over the last few years, the labelling as a natural or ecological product is a strong purchasing argument. It is therefore clear, that a need for naturally manufactured dihydrochalcones which have the same properties as their chemically manufactured pendants is present and rapidly increasing. Notably, an extraction from natural raw materials is not possible due to the unavailability of dihydrochalcones in natural compounds. Therefore, the most promising natural manufacturing method is a biocatalytic approach. However, so far no enzyme is described which has a high regioselectivity for conveying a 4’-0-methylation of specific phenolic substances, in particular for 4’-0-methylation of eriodictyol dihydrochalcone (4) to increase the yield of hesperetin dihydrochalcone (2) and decrease the by-product yields of 3’-O-methylated homoeriodictyol dihydrochalcone (1).

It was thus a primary object of the present invention to provide enzymes having an increased regioselectivity towards the 4’-O-methylated as well as methods for producing 4’-O-methylated phenolic substances.

This primary object was solved by providing an O-methyltransferase variant having a substitution at one, two or all amino acid positions) selected from positions 41 , 42, 43, 173 and 174 of a parental polypeptide, wherein the parental polypeptide is SEQ ID NO.: 1 and, wherein the at least one O-methyltransferase has a sequence identity of 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 % to SEQ ID NO.: 1 .

The term “variant” in terms of the present invention relates to an enzyme, which is altered or modified in comparison to the enzyme, which is occurring naturally. This naturally occurring enzyme is called “wild-type” enzyme.

The term “parental polypeptide” in terms of the present invention relates to the O- methyltransferase according to SEQ ID NO.: 1 to which modifications are made to obtain the O-methyltransferase variant according to the invention.

The numbering of the amino acid positions at which substitutions are present is preferably according to the numbering of the amino acid residues of the parental polypeptide. The term "numbering is according to" as used herein, refers to the way each of the amino acid residues in a polypeptide of the present invention is numbered. The skilled person knows that when, e.g. position 202 is numbered according to SEQ ID NO.: 1 , he would know that by alignment of any other polypeptide with SEQ ID NO.: 1 , he will be able to determine the corresponding amino acid residue in the other polypeptide.

A “substitution” in terms of the present invention relates to the change of an amino acid residue in comparison 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 called a substitution. Such a substitution is either described by the 3-letter amino acid code as Asn41 Gly or by the one letter code N41 G.

Whenever the present disclosure relates to the percentage of 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 Alignments (nucleotide) program or the EMBOSS Water Pairwise Sequence Alignments (polypeptide) program for amino acid sequences. Alignments or sequence comparisons as used herein refer to an alignment over the whole length of two sequences compared to each other. Those tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1):195-197). When conducting an alignment, the default parameters defined by the EMBL-EBI are used. Those parameters are (i) for amino acid sequences: Matrix = BLOSUM62, gap open penalty = 10 and gap extend penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = DNAfull, gap open penalty = 10 and gap extend penalty = 0.5. The skilled person is well aware of the fact that, for example, a nucleic acid sequence encoding a polypeptide can be "codon-optimized" if the respective sequence is to be used in another organism in comparison to the original organism a molecule originates from.

A methyltransferase is an enzyme, which transfers a methyl group from a donor to an accepting molecule, substrate or educt. In particular, it catalyses the selective methylation of C-, N-, and O-centered nucleophiles via a methyl transfer from the sulfonium functional group of the cofactor S-adenosyl methionine (SAM). The O-methylation of eriodictyol dihydrochalcone by the O-methyltransferase from Zooshikella ganghwensis is depicted in Figure 1 .

This figure shows that the educt eriodictyol dihydrochalcone (1 b) is converted to the 3’-O- methylated homoeriodictyol dihydrochalcone (2b) and the 4’-O-methylated hesperetin dihydrochalcone (3b). It was surprisingly discovered that the provided O-methyltransferase variants comprising very specific amino acid substitutions are able to increase the product yields of the 4’-O- methylated phenolic substance and thus increase the regioisomeric ratio of 4’-O- methylated to 3’-O-methylated phenolic substance.

It is preferred that the O-methyltransferase variant according to the invention comprises a substitution at one amino acid position selected from positions 41 , 42, 43, 173 and 174 of a parental polypeptide, wherein the parental polypeptide is SEQ ID NO.: 1 and, wherein the at least one O-methyltransferase has a sequence identity of 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 % to SEQ ID NO.: 1. Moreover preferably, the substitution is selected from positions 41 , 42 and 174.

Preferably, the O-methyltransferase variant according to the invention comprises a substitution at two amino acid position selected from positions 41 , 42, 43, 173 and 174, especially preferably at positions 41 and 174 or 42 and 174, of a parental polypeptide, wherein the parental polypeptide is SEQ ID NO.: 1 and, wherein the at least one O- methyltransferase has a sequence identity of 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 % to SEQ ID NO.: 1 .

Moreover preferably, the O-methyltransferase variant according to the invention comprises three substitutions at the amino acid positions 41 , 41 and 174, wherein the parental polypeptide is SEQ ID NO.: 1 and, wherein the at least one O-methyltransferase has a sequence identity of 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 % to SEQ ID NO.: 1 .

It is preferred that 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. Especially preferably, the substitution is an amino acid selected from the group consisting of phenylalanine, lysine, glutamine, leucine, threonine, valine, proline, asparagine and histidine.

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. Moreover preferably, the O-methyltransferase variant comprises the substitutions M42T and S174R, N41 L and S174R, N41 K and S174R or N41 K and S174K.

It is preferred that the O-methyltransferase variant according the invention comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs.: 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35 and 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 11 , 13, 15, 17, 19, 21 , 23,

25, 27, 29, 31 , 33, 35 and 37.

Another aspect of the present invention relates to a vector system, preferably a plasmid vector system, consisting of a vector comprising at least one nucleic acid section (a) comprising a gene encoding an O-methyltransferase having a nucleic acid sequence selected from the group consisting of SEQ ID NOs.: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 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 NOs.: 12, 14, 16, 18, 20, 22, 24,

26, 28, 30, 32, 34, 36 and 38, and preferably comprising at least one nucleic acid section (b) comprising a gene encoding a 4-coumarate 3-hydroxylase and a gene encoding a 4-hydroxyphenylacetate 3- monooxygenase reductase, and/or preferably at least one nucleic acid section (c) comprising a gene encoding a S- Adenosylmethionine synthetase, wherein the nucleic acid sections (a), (b) and/or (c), if (b) and/or (c) are present, are preferably provided on the same vector, or in two or three different vectors, each comprising one nucleic acid section (a) or (b) or (c).

The vector system according to the invention comprises the O-methyltransferase variant itself as well as preferably further accessory enzymes.

The first preferred accessory enzymes are the combination of a 4-coumarate 3-hydroxylase and a 4-hydroxyphenylacetate 3-monooxygenase reductase, which provides the hydroxylation of the phenolic substances for being used as substrate for the O- methyltransferase.

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 a nucleic acid according to SEQ ID NO.: 4. 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 a nucleic acid according to SEQ ID NO.: 6.

The compatibility of both enzymes 4-coumarate 3-hydroxylase and 4- hydroxyphenylacetate 3-monooxygenase reductase is beneficial for a complete and efficient reaction. It was surprisingly found that these two accessory enzymes could be combined, even though they are not derived from the same organism.

The third preferred accessory enzyme is an 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 required as co-substrate for the reaction of the O-methyltransferase.

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

Preferably, the S-Adenosylmethionine synthetase has a nucleic acid sequence selected from SEQ ID NOs.: 8 and 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 SEQ ID NOs.: 8 and 10.

Another aspect of the present invention relates to a genetically modified cell comprising a vector system according to the invention.

Preferably, the genes encoding the O-methyltransferase and/or the genes encoding the 4- coumarate 3-hydroxylase and 4-hydroxyphenylacetate 3-monooxygenase and/or S- Adenosylmethionine synthetase are stably integrated into the genome of the cell. Preferably one, two, three or all of the genes are under the control of an inducible promoter.

Preferably, the cell is selected from the group consisting of Escherichia coli spp., such as E. coli BL21, E. coli MG1655, preferably E. co// W3110, Bacillus spp., such as Bacillus licheniformis, Bacillus subitilis, or Bacillus amyloliquefaciens, Saccharomyces spp., preferably S. cerevesiae, Hansenula or Komagataella spp., such as. K. phaffii and H. polymorpha, preferably K. phaffii, Yarrowia spp. such as Y. lipolytica, Kluyveromyces spp, such as Kluyveromyces. lactis, Corynebacterium glutamicum and Pseudomonas putida. Also preferably, the O-methyltransferase variant as described herein as well as the accessory enzymes 4-coumarate 3-hydroxylase and 4-hydroxyphenylacetate 3- monooxygenase reductase and/or S-Adenosylmethionine synthetase, if one or all of these accessory enzymes is/are present, are expressed in a cell-free expression system. Cell- free expression is also called in vitro protein expression and is the production of recombinant proteins in solution using the biomolecular translation machinery extracted from cells.

Yet another aspect of the present invention relates to a biocatalytic method for the manufacturing of 4'-O- and 3'-O-methylated phenolic substances, preferably hesperetin dihydrochalcone, comprising the step of providing an O-methyltransferase variant according to the invention.

Preferably, the term “4'-O- and 3'-O-methylated phenolic substance”, as used herein, refers to a compound selected from the group consisting of ferulic acid, dihydroferulic acid, homoeriodictyol dihydrochalcone, homobutein, homoeriodictyol, 2,3-dihydro-7-hydroxy-2- (4-hydroxy-3-methoxyphenyl)-4H-1-benzopyran-4-one, isorhapontigenin, 3'-O-Methyl-(-)- epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1 H-2-benzopyran-1- one, isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2- yl]oxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)propan-1-one, 5-hydroxy-2-(4-hydroxy-3- methoxyphenyl)-7-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyloxan- 2yl)oxymethyl]oxan-2-yl]oxy-2,3-dihydrochromen-4-one, narcissin, p-methoxycinnamic acid, isoferulic acid, dihydroisoferulic acid, isosakuranetin dihydrochalcone, hesperetin dihydrochalcone, 2',4'-dihydroxy-4-methoxychalcone, (2E)-1 -(2,4-dihydroxyphenyl)-3-(3- hydroxy-4-methoxyphenyl)-2-propen-1-one, isosakuranetin, hesperetin, 2,3-dihydro-7- hydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one, 2,3-dihydro-7-hydroxy-2-(3- hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, desoxyrhapontigenin, rhapontigenin, (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)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, phyllodulcin, tamarixetin, kaempferid, hesperetin dihydrochalcone glucoside, hesperidin, 1-[4-(p-D-glucopyranosyloxy)-2,6- dihydroxyphenyl]-3-(4-methoxyphenyl)-1 -propanone, 3-[[6-0-(6-deoxy-a-L- mannopyranosyl)-p-D-glucopyranosyl]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)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-dimethoxyphenyl)-3,4- dihydro-2H-1-benzopyran-3,5,7-triol, thunberginol G, dillenetin, calomelanone, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, their glycosides, and mixtures thereof. It is preferred that the method according to the invention is a fermentative method, comprising the steps of: i. Providing at least one recombinant microorganism comprising a nucleic acid, which encodes at least one O-methyltransferase variant according to the invention, preferably a genetically engineered cell according to the invention; ii. Cultivating the at least one recombinant microorganism under conditions allowing the expression of the at least one O-methyltransferase;

Hi. Adding at least one phenolic substance selected from the group consisting of benzoic acids, phenylacetic acids, mandelic acids, cinnamic acids, dihydrocinnamic acids, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3- hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, aglycones, glycosides , particularly O- glycosides, of the aforementioned and mixtures, preferably mixtures of aglycones and glycosides, thereof, preferably selected from the group consisting of flavanones, chaicones, dihydrochalcones, phenylpropanoids, flavanols, dihydroisocoumarines, stilbenecarboxylates, stilbenes, their glycosides, and mixtures thereof to the recombinant microorganism; iv. Obtaining a mixture of 4'-O- 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, moreover preferably 99.9:0.1 to 80:20, especially preferably 99.9:0.1 to 90:10, even more preferable 99.9:0.1 to 95:5.

A “fermentative” method according to the invention is to be understood as a method involving the cultivation of a recombinant microorganism. Preferably, a fermentative method according to the invention is a method, wherein no purified or partially purified enzymes or cell lysates are present.

The term “phenolic substance”, as used herein, preferably refers to a compound, which possesses one or more, preferably at least one, preferably at least two, preferably two, hydroxyl groups, preferably wherein the hydroxyl group(s) is/are bound to a phenyl group or a derivative thereof. Preferably, the term refers to a compound, which possesses a catechol group. Particularly preferably, the term relates to a compound selected from the group consisting of benzoic acids, phenylacetic acids, mandelic acids, cinnamic acids, dihydrocinnamic acids, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3-hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, aglycons, glycosides , particularly O-glycosides, of the aforementioned and mixtures, preferably mixtures of aglycones and glycosides, thereof, preferably selected from the group consisting of flavanones, chaicones, dihydrochalcones, phenylpropanoids, flavanols, dihydroisocoumarines, stilbenecarboxylates, stilbenes, their glycosides, and mixtures thereof.

It is preferred that the phenolic substance is selected from naringenin, eriodictyol, phloretin, eriodictyol dihydrochalcone, hydrangenol, hydrangeic acid, thunberginol G, thunberginol G acid, their glycosides, preferably O-glycosides of the aforementioned and mixtures, preferably mixtures of aglycones and glycosides, thereof.

Furthermore, when a “phenolic substance”, as described herein, is methylated at the or one of the hydroxyl groups, an “O-methylated phenolic substance”, as described herein, is obtained. Thus, the term “the corresponding phenolic substance” refers to a phenolic substance, which can be reacted to the respective O-methylated phenolic substance by methylation O-methylation. The 4’-0-methylation is preferred over the 3’-0-methylation in terms of the present invention.

Suitable reaction conditions for cultivating the recombinant microorganism such as buffers, additives, temperature and pH conditions, suitable co-factors, and optionally further proteins can easily be determined by a person skilled in the art.

Especially preferably, the method relates to the manufacturing of hesperetin dihydrochalcone by 4’-0-methylation of the phenolic substance 3-hydroxyphloretin (eriodictyol dihydrochalcone).

Moreover preferably, the method relates to the manufacturing of hesperetin dihydrochalcone from the phenolic substance phloretin, which is initially hydroxylated by the enzymes 4-coumarate 3-hydroxylase and 4-hydroxyphenylacetate 3-monooxygenase reductase to 3-hydroxyphloretin (eriodictyol dihydrochalcone) and subsequently 4’-O- methylated by an O-methyltransferase variant according to the invention to hesperetin dihydrochalcone.

Moreover preferably, the method relates to the manufacturing of hesperetin from the phenolic substance naringenin, which is initially hydroxylated by the enzymes 4-coumarate 3-hydroxylase and 4-hydroxyphenylacetate 3-monooxygenase reductase to eriodictyol and subsequently 4’-O-methylated by an O-methyltransferase variant according to the invention to hesperetin.

Moreover preferably, the method relates to the manufacturing of phyllodulcin and/or phyllodulcinic acid from the phenolic substance hydrangenol and/or hydrangeic acid, which is initially hydroxylated by the enzymes 4-coumarate 3-hydroxylase and 4- hydroxyphenylacetate 3-monooxygenase reductase to thunberginol G and/or thunberginol G acid subsequently 4’-O-methylated by an O-methyltransferase variant according to the invention to phyllodulcin and or phyllodulcinic acid.

It is preferred in terms of the present invention that the method according to the invention is an enzymatic method comprising the steps of: i. Providing at least one O-methyltransferase variant according to the invention; ii. Adding at least one phenolic substance selected from the group consisting of benzoic acids, phenylacetic acids, mandelic acids, cinnamic acids, dihydrocinnamic acids, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3- hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, aglycons, glycosides , particularly O- glycosides, of the aforementioned and mixtures, preferably mixtures of aglycones and glycosides, thereof, preferably selected from the group consisting of flavanones, chaicones, dihydrochalcones, phenylpropanoids, flavanols, dihydroisocoumarines, stilbenecarboxylates, stilbenes, their glycosides, and mixtures thereof to the recombinant microorganism;

Hi. Obtaining a mixture of 4'-O- 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, moreover preferably 99.9:0.1 to 80:20, especially preferably 99.9:0.1 to 90:10, even more preferable 99.9:0.1 to 95:5.

An “enzymatic method” in terms of the present invention is to be understood as a biocatalytic method, wherein the biocatalyst is provided as a purified or partially purified enzyme.

In context of the present invention, a purified enzyme or partially purified enzyme means the processing of a biotechnological manufactured enzyme to decrease the by-products. This can be done with different separation methods well-known in the art, e.g. chromatography, including affinity chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and the like, precipitation, membrane filtration, centrifugation, crystallization or sedimentation. A purified enzyme hereby relates to a total content of at least 90 % (w/v) enzyme in relation to the complete mixture, wherein a partially purified enzyme relates to a total content of maximum 90 % (w/v) enzyme in relation to the complete mixture. The skilled person can easily determine the content of and the degree of purity of at least one enzyme of interest in a cell culture lysate and/or supernatant of interest and he can easily combine at least one, two, or at least three or several steps of purification to obtain a higher degree of purity, if desired.

It is preferred that the method according to the invention additionally comprises the steps of ii.a-1 Providing at least one 4-coumarate 3-hydroxylase and a 4-hydroxyphenylacetate 3- monooxygenase reductase; and/or ii.a-2 Providing at least one methyl group donor, wherein the at least one methyl group donor is selected from S-adenosylmethionine and/or the combination of methionine and a S-adenosylmethionine synthase.

The additional provision of accessory enzymes 4-coumarate 3-hydroxylase and 4- hydroxyphenylacetate 3-monooxygenase reductase (step ii.a-1) provides for the hydroxylation of the corresponding phenolic substances for use as the substrate for the O- methyltransferase variant according to the invention.

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

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 an amino acid sequence according to SEQ ID NO.: 3, or a nucleic acid sequence encoding the respective amino acid sequence and/or that the 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 to an amino acid sequence according to SEQ ID NO.: 5, or a nucleic acid sequence encoding the respective amino acid sequence, and/or that the S-adenosylmethionine synthase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs.: 7 and 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 7 and 9, or a nucleic acid sequence encoding the respective amino acid sequence.

Another aspect of the present invention relates to the use of an O-methyltransferase variant according to the invention for producing at least one 4'-O-methylated phenolic substance, preferably for producing hesperetin dihydrochalcone, hesperetin and/or phyllodulcin

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

Preferably, the mixture obtained or obtainable by a method according to the invention is a mixture comprising hesperetin dihydrochalcone and homoeriodictiol dihydrochalcone, wherein the ration of hesperetin dihydrochalcone to homoeriodictiol dihydrochalcone is at least 70 : 30, preferably at least 80 : 20, moreover preferably at least 90 to 10 and especially preferably at least 99.9 : 0.1 .

Preferably, the mixture according to the invention is used in finished goods intended for nutrition or enjoyment, this can be particularly products such as bakery products (e.g. bread, dry biscuits, cake, other pastries), confectionery (e.g. chocolates, chocolate bar products, other bar products, fruit gum, hard and soft caramel, chewing gum), alcoholic or non-alcoholic drinks (e.g. coffee, tea, wine, drinks containing wine, beer, drinks containing beer, liqueurs, schnapps, brandies, lemonades containing fruit, isotonic drinks, refreshing drinks, nectars, fruit and vegetable juices, fruit and 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, flavoured or marinated fresh or salt meat products), eggs or egg products (dry egg, protein, yolk), cereal products (e.g. breakfast cereals, muesli bars, precooked instant rice products), milk products (e.g. milk drinks, milk ice cream, yoghurt, kefir, cream cheese, soft cheese, hard cheese, dry milk powder, whey, butter, buttermilk, partly or completely hydrolysed products containing milk protein), products made of soy protein or other soy bean fractions (e.g. soy milk and products obtained therefrom, compositions containing soy lecithin, fermented products as tofu or tempeh or products made therewith), fruit preparations (e.g. jams, fruit ice cream, fruit sauces, fruit fillings), vegetable preparations (e.g. ketchup, sauces, dry vegetables, frozen vegetables, precooked vegetables, boiled down vegetables), snacks (e.g. baked or fried potato chips or potato dough products, extrudates based on corn or peanut), products based on fat and oil or emulsions thereof (e.g. mayonnaise, remoulade, dressings), other finished products and soups (e.g. dry soups, instant soups, precooked soups).

Another aspect of the present invention relates to a mixture obtained or obtainable by a method according to the present invention, 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, moreover preferably 99.9:0.1 to 80:20, especially preferably 99.9:0.1 to 90:10, even more preferable 99.9:0.1 to 95:5.

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

What was said herein with regard to the method and/or mixture according to the invention applies accordingly to the uses according to the invention, where applicable. What was said herein with regard to the uses according to the invention applies accordingly to the method and/or mixture according to the invention, where applicable.

Short description of the sequences

SEQ ID NOs.: 1 and 2: Amino acid and nucleic acid sequences of the parental O- methyltransferase from Zooshikella ganghwensis.

SEQ ID NOs.: 3 and 4: Amino acid and nucleic acid sequences of 4-coumarate 3- hydroxylase from Saccharothrix espanaensis.

SEQ ID NOs.: 5 and 6: Amino acid and nucleic acid sequences of 4-hydroxyphenylacetate 3-monooxygenase reductase from Pseudomonas aeruginosa.

SEQ ID NOs.: 7 to 10: Amino acid and nucleic acid sequences of S-adenosylmethionine synthases from Bacillus subtilis and Saccharomyces cerevisiae. SEQ ID NOs.: 11 to 38: Amino acid and nucleic acid sequences of O-methyltransferase variants.

SEQ ID NOs.: 39 to 67: Nucleic acid sequences of amplification primers.

Short description of

Figure 1 : Reaction scheme of the methylation reaction by the parental O- methyltransferase from Zooshikella ganghwensis (ZgOMT). The substrate eriodictyol dihydrochalcone (1 b) is methylated under presence of S- adenosylmethionine (SAM) to the 3’-O-methylated product homoeriodictyol dihydrochalcone (2b) and the 4’-O-methylated product hesperetin dihydrochalcone (3b).

Figure 2: Conversion and regioisomeric ratio of 3’-0 to 4’-O-methylated product of the reaction of different ZgOMT variants. Eriodictyol dihydrochalcone is the substrate and products are homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone.

Figure 3: Regioisomeric ratio and substrate conversion of ZgOMT wildtype and variants with different substrates. Figure 3A shows the schematic methylation of different phenolic substances to the 3’-O- and 4’-O-methylated product. Figure 3B shows the dihydrochalcone educts phloretin, eriodictyol dihydrochalcone and sieboldin and the corresponding conversion rates and isomeric ratios of 3’-O- to 4’-O-methylated product when either the wild-type ZgOMT (A) or an inventive ZgOMT variant (B) is used. Figure 3C shows the flavanone educts naringenin and eriodictyol and the corresponding conversion rates and isomeric ratios of 3’-O- to 4’-O-methylated product when either the wild-type ZgOMT (A) or an inventive ZgOMT variant (B) is used. Figure 3D shows the catechol educts caffeic acid and 3,4-dihydroxybenzaldehyde.

Figure 4: Regioisomeric ratio of methoxylated dihydrochalcones produced from phloretin by different E. coli strains expressing SeSAM5 and ZgOMT wildtype or variants, but also BsSAMS and PaHpaC. The ratio is the amount of hesperetin dihydrochalcone divided by the total amount of methoxylated dihydrochalcone products in percent.

Figure 5: Chromatogram of hydroxylated and methoxylated compounds produced from hydrangenol by E. coli W3110 (DE3) SeSAM5_ZgOMT-N41 K-S174R, Sampling 1 h after hydrangenol supplementation. Cells were cultivated with hydrangenol for 16 h at 25 °C in total.

Figure 6: Chromatogram of hydroxylated and methoxylated compounds produced from hydrangenol by E. coli W3110 (DE3) SeSAM5_ZgOMT-N41 K-S174R, Sampling 16 h after hydrangenol supplementation. Cells were cultivated with hydrangenol for 16 h at 25 °C in total. Figure 7: Chromatogram of hydroxylated and methoxylated compounds produced from naringenin by E. coli W3110 (DE3) SeSAM5_ZgOMT-N41 K-S174R. Cells were cultivated with naringenin for 24 h at 25 °C

The present invention is further explained by means of the following examples, which are not intended to limit the scope of the present invention, but serve as illustration only.

Examples

Example 1 - Generation of methyltransferase mutagenesis libraries

SEQ ID NO.: 2 was synthesized (Twist Bioscience, San Francisco, USA) and cloned into a pET28a(+) vector via restriction-ligation using enzymes Ncol and Xhol to obtain the vector pET28a_ZgOMT. Site-saturation mutagenesis of active site residues was performed via PCR using pET28a_ZgOMT template and degenerated primers containing NNK. The following PCR program was used with Q5 high-fidelity DNA polymerase (New England Biolabs, Frankfurt am Main, Germany) and one of the primer pairs of 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, 30 s (initial denaturation), followed by 18 cycles with 98 °C, 10 s (denaturation), 60 °C, 20 s (annealing), 72 °C, 3 min (elongation) and a final elongation at 72 °C, 2 min. Subsequently, the PCR mixture was column purified and digested with 1 pL Dpn\ at 37 °C for 1 h. The obtained DNA product was directly used to transform chemical competent E. coli TOP10 cells. After outgrowth overnight on LB agar plates supplemented with 50 pg/ml kanamycin, the bacterial cells were resuspended in LB medium, the plasmid DNA was isolated and used to transform chemical competent E. coli BL21 (DE3).

Example 2 - Site-directed mutagenesis of the methyltransferase gene

Site-directed mutagenesis of active site residues was performed via PCR using pET28a_ZgOMT template and Q5 site-directed mutagenesis kit (New England Biolabs, 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 one of SEQ ID NO.: 56 to SEQ ID NO.: 67: 98 °C, 30 s (initial denaturation), followed by 25 cycles with 98 °C, 10 s (denaturation), 60 °C, 20 s (annealing), 72 °C, 3 min (elongation) and a final elongation at 72 °C, 2 min. Subsequently, 1 pL of the PCR product was treated with KLD enzyme mix (total volume: 10 pL) at room temperature for 1 h. The obtained DNA product was directly used to transform chemical competent E. coli TOP10 cells. After outgrowth overnight on LB agar plates supplemented with 50 pg mL-1 kanamycin, the bacterial cells were resuspended in LB medium, the plasmid DNA was isolated and used to transform chemical competent E. coli BL21 (DE3). Example 3 - Protein expression, purification and determination of regioisomeric ratio

Wild-type O-methyltransferase of Zooshikella gagnhwensis (ZgOMT) and ZgOMT variants were expressed using E. coli BL21 (DE3) with a pET28a(+) expression system in 100 mL TB medium supplemented with 50 pg mL-1 kanamycin, 0.2 % (w/v) lactose and 0.05 % (w/v) glucose at 37 °C, 180 rpm for 3 h followed by 25 °C overnight. Afterwards, the cells were collected by centrifugation (10 min, 4500 g), the cell pellet frozen at - 80 °C for 1 h, the pellet resuspended in 15 mL lysis buffer containing 50 mM Tris-HCI pH 7.5, 1 mg mL- 1 lysozyme and 5 pg mL-1 DNAse I from bovine (Sig ma-Ald rich, Steinheim, Germany) and incubated at 37 °C for 1 h. The insoluble cell fragments were removed by centrifugation (15 min, 5000 g). Either cell-free extract (CFE) was used directly for the activity assay or CFE was used for protein purification.

For protein purification the buffer was adjusted to 10 mM imidazole and the supernatant was incubated for 30 min at 4 °C with Ni-NTA beads (Sigma-Aldrich, Steinheim, Germany) before loading on a gravity-flow column. The column was washed three times with 5 mL 50 mM Tris-HCI pH 7.5 supplemented with 50 mM imidazole and eluted with 3 mL 50 mM Tris- HCI pH 7.5 supplemented with 250 mM imidazole. The eluted fractions containing protein were collected and rebuffered on a PD-10 desalting column (Cytiva, Freiburg im Breisgau, Germany) in 50 mM Tris-HCI pH 7.5.

Regioisomeric ratio of the enzyme variants was determined with either 20 pL CFE or 5 pl purified enzyme, 100 pM substrate (various phenolic substances), 2 mM MgCL and 0.5 mM SAM in 25 mM Tris-HCI pH 7.5 (total volume: 100 pL) for 1 h at room temperature. The reactions were quenched with 75 pL acetonitrile, incubated for 20 min at room temperature and insoluble proteins were removed by centrifugation (15 min, 600 g). 90 pL of the reaction mixture were transferred into a new 96-well plate for reverse-phase HPLC analysis. Enzymatic reaction products were analyzed by reverse-phase HPLC using an Inertsil ODS- 3 5 pm, 4 x 100 mm C-18 column (GL Sciences, Tokyo, Japan) with a flow rate of 1.2 mL min-1 , 30 % acetonitrile/ 70 % H2O containing 0.1 % (v/v) formic acid at 50 °C. Table 1 : Regioisomeric ratio of ZgOMT variants with eriodictyol dihydrochalcone as substrate

The conversion rate and regioisomeric ratio of different O-methyltransferase with eriodictyol dihydrochalcone as substrate are further depicted in Figure 2.

Figure 3 shows the conversion rate as well as the regioisomeric ratio of 4’-0-methylation to 3’-0-methylation. It can be observed that phenolic substances with one hydroxy group cannot be methylated, wherein phenolic substances with two hydroxygroups can be methylated by the O-methyltransferase. For substrates with only one hydroxy group, a prior methylation must be performed, such as e.g. by the accessory enzymes 4-coumarate 3- hydroxylase and 4-hydroxyphenylacetate 3-monooxygenase reductase as described in terms of the present invention. The regioisomeric ratio as well as the conversion rate could be increased using the O-methyltransferase variant according to SEQ ID NO.: 35. Example 4 - Generation of E. coli strain for the hydroxylation and methylation of phenolic substances

SEQ ID NOs.: 2, 4, 6 and 8 were synthesized (Twist Bioscience, San Francisco, USA). SEQ ID NO.: 4 and one of the DNA sequences of SEQ ID NOs.:2 or 12 - 38 were cloned into pCDFDuet-1 (Merck, Darmstadt, Germany) via restriction-ligation using restriction enzymes Ncol, EcoRI and Ndel, Kpnl, respectively, to obtain vectors for expression of SeSAM5 together with ZgOMT wildtype or its variants. SEQ ID NOs.: 6 and 8 were cloned into pRSFDuet-1 (Merck, Darmstadt, Germany) via restriction ligation using restriction enzymes Ncol, EcoRI and Ndel, Kpnl, respectively, to obtain vector pRSFDuet_PaHpaC_BsSAMS. The obtained pCDFDuet vectors and pRSFDuet_PaHpaC_BsSAMS were transformed into chemically competent E. coli Z 10 (DE3) cells to obtain the expression cells shown in table 2:

Table 2: Generated E. coli strains for the production of methoxylated dihydrochalcone from phloretin (PaHpaC: 4-hydroxyphenylacetate 3-monooxygenase reductase; BsSAMS: S- Adenosylmethionine Synthetase; SeSAM5: 4-coumarate 3-hydroxylase; ZgOMT: O- Methyltransferase)

Example 5 - Production of methoxylated dihydrochalcone mixture from phloretin using SeSAM5 and ZgOMT wildtype or variants

The strains generated in example 4 were cultivated in 10 mL TB medium (yeast extract 24 g/L, tryptone 20 g/L, glycerol 4 ml/L, 0.017 M KH2PO4, 0.072 M K2HPO4) supplemented with 50 pg mL-1 neomycin, 50 pg mL-1 streptomycin at 37 °C, 180 rpm for 1 h. Afterwards 0.1 mM IPTG was added and the temperature decreased to 25 °C. Cells were incubated for 7 h at 180 rpm. Phloretin was added at a concentration of 200 mg L-1 and cells were incubated for another 16 h, 180 rpm, 25 °C. For HPLC analysis, 10 ml acetonitrile was added to the cultures and supernatant used for analysis after incubation on ice for 1 h and subsequent centrifugation of cell debris. Determined regioisomeric ratio of methoxylated dihydrochalcones are depicted in figure 4.

Example 6 - Production of methoxylated dihydroisocoumarine mixture from hydrangenol using SeSAM5 and ZgOMT wildtype or variants

The strain E. coli W3110 (DE3) SeSAM5_ZgOMT-N41 K-S174R comprising the ZgOMT variant according to SEQ ID NO.: 35 generated in example 4 was cultivated in 10 mL TB medium (yeast extract 24 g/L, tryptone 20 g/L, glycerol 4 ml/L, 0.017 M KH2PO4, 0.072 M K2HPO4) supplemented with 50 pg mL^-1 neomycin, 50 pg mL^-1 streptomycin at 37 °C, 180 rpm for 1 h. Afterwards 0.1 mM IPTG was added and the temperature was decreased to 25 °C. Cells were incubated for 7 h at 180 rpm. Hydrangenol was added at a concentration of 200 mg L^-1 and cells were incubated for another 16 h, 180 rpm, 25 °C. For HPLC-MS analysis, 10 ml acetonitrile was added to the cultures and supernatant used for analysis after incubation on ice for 1 h and subsequent centrifugation of cell debris. Figures 5 and 6 depict the reaction results after incubation time with hydrangenol of 1 hour and 16 hours. The results as depicted in the chromatograms of Figures 5 and 6 are:

Figure 5:

Figure 6:

Example 7 - Production of methoxylated flavonoid mixture from naringenin using SeSAM5 and ZGOMT wildtype or variants

The strain E. coli W3110 (DE3) SeSAM5_ZgOMT-N41 K-S174R comprising the ZgOMT variant according to SEQ ID NO.: 35 generated in example 4 was cultivated in 10 mL TB medium (yeast extract 24 g/L, tryptone 20 g/L, glycerol 4 ml/L, 0.017 M KH2PO4, 0.072 M K2HPO4) supplemented with 50 pg mL^-1 neomycin, 50 pg mL^-1 streptomycin at 37 °C, 180 rpm for 1 h. Afterwards 0.1 mM IPTG was added and the temperature was decreased to 25 °C. Cells were incubated for 7 h at 180 rpm. Naringenin was added at a concentration of 200 mg L^-1 and cells were incubated for another 16 h, 180 rpm, 25 °C. For HPLC-MS analysis, 10 ml acetonitrile was added to the cultures and supernatant used for analysis after incubation on ice for 1 h and subsequent centrifugation of cell debris. Figures 7 depict the reaction results after incubation time with naringenin of 16 hours. The results as depicted in the chromatograms of Figure 7 are:

Figure 7:

Claims

1. An O-methyltransferase variant having a substitution at one, two or all amino acid positions) selected from positions 41 , 42, 43, 173 and 174 of a parental polypeptide, wherein the parental polypeptide is SEQ ID NO.: 1 and, wherein the at least one O- methyltransferase has a sequence identity of 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 % to 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 an amino acid sequence selected from the group consisting of SEQ ID NOs.: 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35 and 37 or an amino acid sequence 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35 and 37.

4. A vector system, preferably a plasmid vector system, consisting of a vector comprising at least one nucleic acid section (a) comprising a gene encoding an O- methyltransferase variant having a nucleic acid sequence selected from the group consisting of SEQ ID NOs.: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 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 NOs.: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38, and preferably comprising at least one nucleic acid section (b) comprising a gene encoding a 4-coumarate 3-hydroxylase and a gene encoding a 4- hydroxyphenylacetate 3-monooxygenase reductase, and/or preferably at least one nucleic acid section (c) comprising a gene encoding a S-Adenosylmethionine synthetase, wherein the nucleic acid sections (a) ,(b) and/or (c), if (b) and/or (c) are present, are preferably provided on the same vector, or in two or three different vectors, each comprising one nucleic acid section (a) or (b) or (c).

5. A genetically modified cell comprising a vector system according to claim 4.

6. The genetically modified cell according to claim 5, wherein the cell is selected from the group consisting of Escherichia coli spp., such as E. coli BL21, E. coli MG1655, preferably E. coli W31 10, Bacillus spp., such as Bacillus licheniformis, Bacillus subitilis, or Bacillus amyloliquefaciens, Saccharomyces spp., preferably S. cerevesiae, Hansenula 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, Pseudomonas putida.

7. A biocatalytic method for the manufacturing of 4'-O- and 3'-O-methylated phenolic substances, preferably hesperetin dihydrochalcone, 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 is a fermentative method comprising the steps of: i. Providing at least one recombinant microorganism comprising a nucleic acid, which encodes at least one O-methyltransferase variant according to any one of claims 1 to 3, preferably a genetically engineered cell according to claims 5 or 6; ii. Cultivating the at least one recombinant microorganism under conditions allowing the expression of the at least one O-methyltransferase;

Hi. Adding at least one phenolic substance selected from the group consisting of benzoic acids, phenylacetic acids, mandelic acids, cinnamic acids, dihydrocinnamic acids, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3-hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, aglycons, glycosides, particularly O-glycosides, of the aforementioned and mixtures, preferably mixtures of aglycones and glycosides, thereof, preferably selected from the group consisting of flavanones, chaicones, dihydrochalcones, phenylpropanoids, flavanols, dihydroisocoumarines, stilbenecarboxylates, stilbenes, their glycosides, and mixtures thereof to the recombinant microorganism; iv. Obtaining a mixture of 4'-O- 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, moreover preferably 99.9:0.1 to 80:20, especially preferably 99.9:0.1 to 90:10, even more preferable 99.9:0.1 to 95:5.

9. The method according to claim 7, wherein the method is an enzymatic method comprising the steps of: i. Providing at least one O-methyltransferase according to any one of claims 1 to 3; ii. Adding at least one phenolic substance selected from the group consisting of benzoic acids, phenylacetic acids, mandelic acids, cinnamic acids, dihydrocinnamic acids, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3-hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, aglycons, glycosides, particularly O-glycosides, of the aforementioned and mixtures, preferably mixtures of aglycones and glycosides, thereof, preferably selected from the group consisting of flavanones, chaicones, dihydrochalcones, phenylpropanoids, flavanols, dihydroisocoumarines, stilbenecarboxylates, stilbenes, their glycosides, and mixtures thereof to the recombinant microorganism;

Hi. Obtaining a mixture of 4'-O- 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, moreover preferably 99.9:0.1 to 80:20, especially preferably 99.9:0.1 to 90:10, even more preferable 99.9:0.1 to 95:5.

10. The method according to claims 8 or 9, wherein the phenolic substance is selected from the group consisting of phloretin and 3-hydroxyphloretin, hydrangenol hydrangeic acid, thunberginol G and thunberginol G acid, naringenin and eriodictyolor mixtures thereof.

11 . The method according to claims 8 to 10, wherein the method additionally comprises the steps of ii.a-1 Providing at least one 4-coumarate 3-hydroxylase and a 4- hydroxyphenylacetate 3-monooxygenase reductase; and/or ii.a-2 Providing at least one methyl group donor, wherein the at least one methyl group donor is selected from S-adenosylmethionine and/or the combination of methionine and a S-adenosylmethionine synthase.

12. The method according to claim 11 , wherein 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 an amino acid sequence according to SEQ ID NO.: 3, or a nucleic acid sequence encoding the respective amino acid sequence and/or wherein the 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 to an amino acid sequence according to SEQ ID NO. 5, or a nucleic acid sequence encoding the respective amino acid sequence, and/or wherein the S-adenosylmethionine synthase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs.: 7 and 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs.:7 and 9, or a nucleic acid sequence encoding the respective amino acid sequence.

13. A use of an O-methyltransferase variant according to any one of claims 1 to 3 for producing at least one 4'-O-methylated phenolic substance, preferably for producing hesperetin dihydrochalcone.

14. A use of a mixture obtained or obtainable by a method according to claims 7 to 12 as a sweetness enhancer and/or sweetness modulator and/or flavouring agent, preferably wherein the sweetness enhancer and/or flavouring agent is used in finished goods selected from the group consisting of goods intended for nutrition or enjoyment.

15. Mixture obtained or obtainable by a 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, moreover preferably 99.9:0.1 to 80:20, especially preferably 99.9:0.1 to 90:10, even more preferable 99.9:0.1 to 95:5.