Enzymatic synthesis of glycosylated anthranilate derivatives

Abstract

The present invention concerns enzymatic synthesis of glycosides of anthranilate derivatives, such as methyl anthranilate N-glucoside, ethyl anthranilate N-glucoside, and butyl anthranilate N-glucoside. These anthranilate derivatives may preferably be used as insect and / or bird repellent.

Description

[0001] TITLE: Enzymatic synthesis of glycosylated anthranilate derivatives

[0002] FIELD OF THE INVENTION

[0003] The present invention concerns enzymatic synthesis of glycosides of anthranilate derivatives, such as methylanthranilate (MANT) / V-glucoside, ethylanthranilate (EANT) / V-glucoside, and butylanthranilate (BANT) / V-glucoside.

[0004] These anthranilate derivatives may preferably be used as insect and / or bird repellent.

[0005] BACKGROUND OF THE INVENTION

[0006] Anthranilate derivatives, such as methylanthranilate (MANT), ethylanthranilate (EANT), and butylanthranilate (BANT), are valuable compounds, which are currently synthesized petrochemically. More sustainable solutions are in high demand. Microbial production of MANT was recently published (Luo et al 2019), but yields were low due to the MANT compound being toxic to the cells.

[0007] Methyl anthranilate acts as a bird repellent. It is food-grade and can be used to protect corn, sunflowers, rice, fruit, and golf courses. Further, methyl anthranilate both as a component of various natural essential oils and as a synthesised aroma-chemical is used extensively in modern perfumery.

[0008] Further, anthranilate-based insect repellents (such as repellent of mosquito and fly Drosophila Suzuki, which is a plague in fruit plantations) include methyl anthranilate (MANT), N,N-dimethylanthranilic acid (DMA), ethyl anthranilate (EANT), and butyl anthranilate (BANT). Chemically, they are esters of anthranilic acid.

[0009] Anthranilate derivatives, such as MANT, EANT, and BANT are volatile and poorly water- soluble.

[0010] SUMMARY OF THE INVENTION

[0011] In a first aspect, the present invention provides an in vitro method for glycosylation of an anthranilate derivative, comprising the steps of a. providing an isolated UDP-glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40. b. mixing said enzyme with a UDP-sugar and an anthranilate derivative having the structure of formula I, wherein R. is an alkyl: formula I c. letting the mixture react for a selected period of time, d. optionally purifying the glycosylated anthranilate derivative.

[0012] Preferably, the amino group of the anthranilate derivatives is glycosylated.

[0013] Preferably, the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consist of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14.

[0014] Preferably, the amino acid sequence of the polypeptide comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0015] In one preferred embodiment of the first aspect, R is CnH2n+i, n is 1-4, and the UDP- sugar is UDP-glucose.

[0016] In a second aspect, the present invention provides a composition comprising

[0017] (I) an anthranilate derivative having the structure of formula I, wherein R is an alkyl,

[0018] (II) an isolated UGT-glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40, and

[0019] (III) a UDP-sugar.

[0020] Preferably, the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consist of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14.

[0021] Preferably, the amino acid sequence of the polypeptide comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14. In one preferred embodiment of the second aspect, R is CnH2n+i, n is 1-4, and the UDP- sugar is UDP-glucose. Preferably R-group is selected from methyl, ethyl, propyl, butyl, isopropyl, and isobutyl, more preferably the R-group is selected from methyl, ethyl, propyl, and butyl, most preferably methyl.

[0022] In a third aspect, the present invention concerns the use of an isolated UGT- glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40, in the process of glycosylating anthranilate derivatives having the structure of formula I, wherein R is alkyl.

[0023] Preferably, the amino group of the anthranilate derivatives is glycosylated.

[0024] Preferably, the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consist of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14.

[0025] Preferably, the amino acid sequence of the polypeptide comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0026] In one preferred embodiment of the third aspect, R is CnH2n+i, n is 1-4, and the UDP- sugar is UDP-glucose.

[0027] In a fourth aspect, the present invention provides an in vivo method for producing a glycosylated anthranilate derivative, comprising the steps of a. providing a microbial cell comprising

[0028] (I) a nucleic acid sequence encoding a UDP-glycosyltransferase enzyme, wherein said enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40, and

[0029] (II) one or more nucleic acid sequences encoding one or more enzymes for synthesizing an anthranilate derivative having the structure of formula I, wherein R is an alkyl; b. growing said microbial cell in a growth medium, and expressing said UDP- glycosyltransferase enzyme and said one or more enzymes for synthesizing the anthranilate derivative, wherein a UDP-sugar is provided (i) as a component of the growth medium or (ii) by said microbial cell further comprising one or more nucleic acid sequences encoding one or more enzymes for synthesis of the UDP-sugar and said microbial cell expressing said enzymes for synthesis of the UDP-sugar; and c. optionally purifying the glycosylated anthranilate derivative.

[0030] Preferably, the amino group of the anthranilate derivatives is glycosylated.

[0031] Preferably, the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consist of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14.

[0032] Preferably, the amino acid sequence of the polypeptide comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0033] In one preferred embodiment of the fourth aspect, R is CnH2n+i, n is 1-4, and the UDP- sugar is UDP-glucose. Preferably R-group is selected from methyl, ethyl, propyl, butyl, isopropyl, and isobutyl, more preferably the R-group is selected from methyl, ethyl, propyl, and butyl, most preferably methyl.

[0034] In a further embodiment of the fourth aspect, the microbial cell is selected from E. coli, C. glutamicum, S. cerevisiae, and P. putida.

[0035] In a further embodiment of the fourth aspect, the one or more enzymes for synthesizing the anthranilate derivative comprise (i) a methyltransferase or (ii) an anthranilate-CoA ligase and an anthraniloyl-coenzyme A (CoA): methanol acyltransferase.

[0036] In a fifth aspect, the present invention provides a method for producing an insect and / or bird repellent composition, comprising the steps of a. providing a UDP-glycosyltransferase enzyme, a UDP-sugar, and an anthranilate derivative having the structure of formula I, wherein R is an alkyl, b. mixing said enzyme, said UDP-sugar, and said anthranilate derivative, c. letting the enzyme catalyze glycosylation of the anthranilate derivative for a selected period of time to produce a glycosylated anthranilate derivative having insect and / or bird repelling properties, and d. optionally purifying the glycosylated anthranilate derivative, and e. optionally further formulating the glycosylated anthranilate derivative by addition of one or more additives.

[0037] Preferably, the amino group of the anthranilate derivatives is glycosylated. Preferably, the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consist of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14.

[0038] Preferably, the amino acid sequence of the polypeptide comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0039] In one preferred embodiment of the fifth aspect, R is CnH2n+i, n is 1-4, and the UDP- sugar is UDP-glucose. Preferably R-group is selected from methyl, ethyl, propyl, butyl, isopropyl, and isobutyl, more preferably the R-group is selected from methyl, ethyl, propyl, and butyl, most preferably methyl.

[0040] In a further embodiment of the fifth aspect, the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40.

[0041] In a sixth aspect, the present invention concerns the use of a glycosylated anthranilate derivative as an insect and / or bird repellent, wherein the glycosylated anthranilate derivate has the structure of formula II, wherein R is CnH2n+i and n is 1-4; and X is glucose: formula II.

[0042] In a seventh aspect, the present invention provides a polypeptide having glycosyltransferase activity (enzyme classification EC:2.4.1.-), wherein the amino acid sequence of said polypeptide has at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO. 14, and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14.

[0043] Preferably, the amino acid sequence of the polypeptide comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14. DESCRIPTION OF THE INVENTION

[0044] Brief description of the figures:

[0045] Figure 1: Structure model of enzyme UGT16 predicted by alphafold2.

[0046] Figure 2: Structure model of enzyme UGT43 predicted by alphafold2.

[0047] Figure 3: Structure model of enzyme UGT53 predicted by alphafold2.

[0048] Figure 4: Identification of pH optimum of UGT16, UGT43, and UGT53. Each reaction contained 0.40 mM UDP-glucose, 0.11 mM MANT from a 50 mM DMSO stock, and UGT (0.7 pM for UGT16 and 1.1 pM for UGT43 and UGT53). The reaction was incubated at room temperature and quenched at 5 time points (2 min, 5 min, 10 min, 15 min, and 30 min) by transferring 50 pL of the reaction mixture to 70 pL of cold methanol. The reaction mixtures were analyzed via HPLC. The following buffering agents were utilized for the respective pH ranges: pH value: pH 4-7, 50 mM citrate-phosphate; pH 7-8, 50 mM phosphate; pH 8-9, 50 mM Trizma; pH 9-11, 50 mM glycine. The data was analyzed as means ± standard deviations of duplicate experiments.

[0049] Figure 5: Identification of temperature optimum of UGT16, UGT43, and UGT53. Each reaction contained 0.40 mM UDP-glucose, 0.11 mM MANT from a 50 mM DMSO stock, and UGT (0.7 pM for UGT16 and 1.1 pM for UGT43 and UGT53), 50 mM phosphate buffer pH 8.0 was used and the reaction was carried out at temperatures ranging from 30 °C to 54 °C for UGT16 and UGT43 and 15 °C to 37 °C UGT53 on a thermocycler. The reaction was stopped at four different time points (5 min, 10 min, 15 min, and 30 min) by thermal denaturation at 95 °C for 20 seconds. The reaction mixtures were analyzed via HPLC. The data was analyzed as means ± standard deviations of duplicate experiments.

[0050] Figure 6: UGT16 variants tested on the AEAA substrate panel: MANT, EANT, BANT, IBANT, and CANT. Reaction conditions: 2.5 pM of UGT16 WT or variants, 2.0 mM UDP- glucose, and 1 mM of MANT, EANT, BANT, IBANT, and CANT, respectively, from a 10 mM DMSO stock in 50 mM phosphate buffer pH 8.0 at 35 °C for 15 minutes. Reactions were stopped by thermal denaturation at 95 °C for 20 seconds. The reaction mixtures were analyzed via HPLC as means ± standard deviations of two independent experiments. The activity was quantified via the product peak area and subsequently normalized to the variant with the largest peak area for each substrate, respectively.

[0051] Figure 7: Chemo-tolerance of UGT16. (A) 0.25-1.5 pM UGT16, 1.0 mM UDP-glucose, and 0.12-10.0 mM MANT from a 100 mM DMSO stock in 50 mM phosphate buffer pH 8.0 at 35 °C for 2, 5, 10, and 15 minutes. (B) 0.75 pM UGT16, 1.0 mM UDP-glucose, and 0.23-20.0 mM MANT from a 100 mM DMSO stock in 50 mM phosphate buffer pH 8.0 at 35 °C for 2, 5, 10, and 15 minutes. The reaction mixtures were analyzed via HPLC.

[0052] Figure 8: The effect of MANT on microbial growth (A) E. coli and, (B) P. putida. The effect of MANT-N-glucoside on microbial growth (C) E. coli and (D) P. putida. A control of 5% DMSO (the maximum, added in the 7.5 mM samples) is included.

[0053] Figure 9: Molecular docking of MANT, EANT, BANT, and IBANT into an AlphaFold32 model of UGT16.

[0054] Abbreviations, terms, and definitions:

[0055] Amino acid sequence identity: The term "sequence identity" as used herein, indicates a quantitative measure of the degree of similarity between two amino acid sequences of essentially equal length. The two sequences to be compared must be aligned to give a best possible fit, by means of the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as ((Nref- Ndif)100) / (Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Sequence identity calculations are preferably automated using the BLAST program e.g. the BLASTP program (Pearson W.R and D.J. Lipman (1988)) (www.ncbi.nlm.nih.gov / cgi-bin / BLAST). Sequence alignment may be performed using program MAFFT24 (Multiple Alignment using Fast Fourier Transform; Katoh et al 2019) using default parameters (SCORING MATRIX: blosum62, gap opening penalty: 1.53, gap extension penalty 0.123).

[0056] Preferably, the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide as compared to its comparator polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions. Preferably the substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1: Glycine, Alanine, Valine, Leucine, Isoleucine; group 2: Serine, Cysteine, Selenocysteine, Threonine, Methionine; group 3: Proline; group 4: Phenylalanine, Tyrosine, Tryptophan; Group 5: Aspartate, Glutamate, Asparagine, Glutamine; Group 6: Histidine. Lysine, Arginine.

[0057] In vitro, In vivo: The term "in vitro" as used herein refers to processes, reactions, methods, procedures, experiments, tests, and the like that are conducted outside of a living organism or biological system. In contrast, the term "in vivo" as used herein refers to processes, reactions, methods, procedures, experiments, tests, and the like that are conducted within a living organism.

[0058] Isolated polypeptide: The term "isolated polypeptide" as used herein refers to a polypeptide that is isolated from a source. In a preferred aspect, the polypeptide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE.

[0059] Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation.

[0060] The polypeptides of the present invention are preferably in a substantially pure form, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

[0061] Mature polypeptide: The term "mature polypeptide" is defined herein as a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

[0062] Glycosylation: The term glycosylation is defined herein as the reaction in which a carbohydrate (e.g. glucose) is transferred from a glycosyl donor (e.g. UDP-glucose) to a functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. Specifically, herein the term refers to an enzyme-catalyzed reaction. The carbohydrate is preferably a simple sugar, such as glucose, xylose, galactose, rhamnose, / V-acetyl glucosamine, glucuronic acid, / V-acetyl galactosamine, mannose, arabinose, galacturonic acid, or fucose; most preferably glucose.

[0063] Glycoside: The term glycoside refers herein to a broad class of compounds where a carbohydrate sugar molecule is bonded to a non-sugar molecule through a glycosidic bond. The carbohydrate sugar is preferably a simple sugar, such as glucose, xylose, galactose, rhamnose, / V-acetyl glucosamine, glucuronic acid, / V-acetyl galactosamine, mannose, arabinose, galacturonic acid, or fucose; most preferably glucose. The term glucoside refers herein to a glycoside, wherein the carbohydrate sugar is glucose.

[0064] Alkyl: An alkyl is a functional group of an organic compound that contains only carbon and hydrogen atoms, which are arranged in a linear chain or branched structure. They have general formula CnH2n+i.

[0065] Glycosyltransferases (GTs): Glycosyltransferases are enzymes (EC 2.4) that establish glycosidic linkages. They catalyze the transfer of saccharide moieties from an activated nucleotide sugar (also known as the "glycosyl donor") to a nucleophilic glycosyl acceptor molecule. UDP-glycosyltransferases (UGTs) transfer the saccharide moiety of a UDP- sugar to an acceptor molecule. The UDP-sugar may be selected from UDP-glucose, UDP- xylose, UDP-galactose, UDP-rhamnose, UDP- / V-acetyl glucosamine, UDP-glucuronic acid, UDP- / V-acetyl galactosamine, UDP-mannose, UDP-arabinose, UDP-galacturonic acid, and UDP-fucose; most prefereably UDP-glucose.

[0066] Bird: The term "bird" refers herein to any type of warm-blooded, feathered vertebrate belonging to the class Aves in the animal kingdom, including geese and seagulls.

[0067] Insect: The term "insect" refers herein to any type of insect belonging to the class Insecta in the animal kingdom, and of particular relevance for the present invention the term in preferred embodiments refer to mosquitos and fruit flies (e.g. Drosophila suzuki).The term "comprising" means that the invention encompasses the listed elements, but may also include additional elements not specifically mentioned (i.e. permits the presence of additional components or steps). The term "consisting of" on the other hand signifies that the invention consists solely of the elements listed (i.e. it excludes any additional elements).

[0068] ANT = Anthranilic acid; MANT = Methyl anthranilate; EANT = Ethyl anthranilate; BANT = Butyl anthranilate; IBANT = Isobutyl anthranilate; CANT = Cyclohexyl anthranilate; AEAAs = alkyl esters of anthranilic acid.

[0069] Detailed description of the invention:

[0070] The present invention provides a sustainable route of synthesizing glycosylated anthranilate derivatives, such as glycosylated alkyl-anthranilates, e.g. glycosylated methyl anthranilate, glycosylated ethyl anthranilate, and glycosylated butyl anthranilate. Glycosylation of such small compounds is considered a means of improving solubility as well as stability and making them less volatile. The anthranilate derivatives are preferably glycosylated using simple sugars, such as sugar monomers, e.g. glucose. For example, glycosylating MANT to form MANT- / V-glucoside increases its water solubility and makes it less volatile. Increased water solubility may be advantageous with regards to its use as a bird or insect repellant; and especially the reduction of its volatility may allow for reducing the frequency of application on the solution when used as a bird or insect repellant. Hence, the glycosylated anthranilate derivatives of the present invention provide an improved alternative to currently used anthranilates.

[0071] The enzymatic glycosylation of anthranilate derivatives according to the invention is mediated by an enzyme having glycosyltransferase activity. Specifically, the present invention provides a solution, wherein the amino group of anthranilate derivatives is glycosylated; preferably glycosylation of the amino group of alkyl anthranilates, i.e. yielding alkyl anthranile N-glucosides.

[0072] UDP-glycosyltransgerases (UGTs) are used in a variety of biotech applications to attach a sugar moiety to different chemical compounds. They utilize a nucleotide-activated sugar donor to transfer a sugar moiety to an acceptor substrate, most often uridine diphosphate (UDP) glucose. The present inventors have screened multiple UGT enzymes and identified the best performing enzymes for specific anthranilate derivatives, and have further identified a versatile UGT enzyme capable of synthesizing significant amounts of different N-glycosylated anthranilate derivatives, such as MANT-, EANT-, and BANT- / V-glucoside. The solution provided by the present invention is therefore to use the best performing enzymes and / or the versatile UGT for glycosylation of at least MANT, EANT, and BANT and other related compounds, such as IGT16 (SEQ ID NO. 14), IGT43 (SEQ ID NO 32), and IGT53 (SEQ ID NO 40) (see Example 2).

[0073] Biochemical characterization with MANT revealed UGT16 from Solanum lycopersicum to be the most catalytically efficient UGT, leading to further biochemical characterization with additional AEAAs, where it displayed the highest catalytic efficiency with BANT (see Example 3).

[0074] A structure-function analysis of UGT16 revealed critical residues for AEAA activity, strongly indicating a critical role of both Ile80 and Ile84 in the AEAA glycosylating activity of UGT16, and further showing that other critical residues included Phel35 and Glyl41. In addition, variant Phel45Met exhibited improved activity with bulkier AEAAs compared to WT UGT16 and enabled activity with cyclohexyl anthranilate (CANT) (see Example 5).

[0075] UGT16 exhibited remarkably high chemo stability towards MANT and DMSO, enabling gram-scale production of MANT-N-glucoside achieving a 74 % yield of 99 % pure (HPLC) MANT-N-glucoside (see Example 3 and Example 6, respectively).

[0076] The purified MANT- / V-glucoside was employed in a bird repellence study where it displayed an improved efficacy (100%) compared to previous studies of MANT (70%) (see Example 7). Additionally, the growth of Escherichia coli and Pseudomonas putida remained unaffected by MANT- / V-glucoside, indicating glycosylation to be a promising strategy to alleviate MANT cell factory toxicity and that MANT-N-glucoside is suitable for microbial production (see Example 9).

[0077] I. An in vitro method of glycosylating anthranilate derivatives

[0078] In a first aspect, the present invention concerns in vitro glycosylation of anthranilate derivatives using glycosyltransferase enzymes.

[0079] Specifically, the present invention provides a method for glycosylating an anthranilate derivative, comprising the steps of a. providing a UDP-glycosyltransferase enzyme, b. mixing said enzyme with said anthranilate derivative and a UDP-sugar, c. letting the mixture react for a selected period of time, and d. optionally purifying the glycosylated anthranilate derivative.

[0080] Specifically, the present invention provides a method, wherein the amino group of the anthranilate derivative is glycosylated.

[0081] In one aspect, the present invention provides enzymes and methods for glycosylation of specific anthranilate derivatives.

[0082] In one preferred embodiment, the anthranilate derivative is methyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 2, 14, 24, 26, 28, 32, 34, 36, 38, 40, 68, and 70 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 2, 14, 24, 26, 28, 32, 34, 36, 38, 40, 68, or 70 . The resulting product is methyl anthranilate N-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is methyl anthranilate / V-glucoside.

[0083] In one more preferred embodiment, the anthranilate derivative is methyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 24, 26, 32, 34, 36, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 24, 26, 32, 34, 36, or 40. The resulting product is methyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is methyl anthranilate N-glucoside. In one most preferred embodiment, the anthranilate derivative is methyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40. The resulting product is methyl anthranilate / V-glycoside. Preferably the UDP- sugar is UDP-glucose, and the resulting product is methyl anthranilate / V-glucoside.

[0084] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is methyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14. The resulting product is methyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is methyl anthranilate / V-glucoside.

[0085] In one most preferred embodiment, the anthranilate derivative is methyl anthranilate and the UDP-glycosyltransferase enzyme is selected from SEQ ID NO. 14, 32, and 40, the resulting product is methyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is methyl anthranilate N-glucoside.

[0086] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is methyl anthranilate and the UDP-glycosyltransferase enzyme is SEQ ID NO. 14, the resulting product is methyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is methyl anthranilate / V-glucoside.

[0087] In one preferred embodiment, the anthranilate derivative is ethyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 8, 14, 16, 28, 32, 38, 40, and 68 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 8, 14, 16, 28, 32, 38, 40, or 68. The resulting product is ethyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is ethyl anthranilate / V-glucoside.

[0088] In one more preferred embodiment, the anthranilate derivative is ethyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 8, 14, 16, 32, 38, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 8, 14, 16, 32, 38, or 40. The resulting product is ethylanthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is ethyl anthranilate N- glucoside.

[0089] In one most preferred embodiment, the anthranilate derivative is ethyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40. The resulting product is ethyl anthranilate N-glycoside. Preferably the UDP-sugar is UDP- glucose, and the resulting product is ethyl anthranilate / V-glucoside.

[0090] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is ethyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14. The resulting product is ethyl anthranilate N-glycoside. Preferably the UDP-sugar is UDP- glucose, and the resulting product is ethyl anthranilate / V-glucoside.

[0091] In one most preferred embodiment, the anthranilate derivative is ethyl anthranilate and the UDP-glycosyltransferase enzyme is selected from SEQ ID NO. 14, 32, and 40, the resulting product is ethyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP- glucose, and the resulting product is ethyl anthranilate / V-glucoside.

[0092] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is ethyl anthranilate and the UDP-glycosyltransferase enzyme is SEQ ID NO. 14, the resulting product is ethyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is ethyl anthranilate / V-glucoside.

[0093] In one most preferred embodiment, the anthranilate derivative is propyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40. The resulting product is propyl anthranilate / V-glycoside. Preferably the UDP- sugar is UDP-glucose, and the resulting product is propyl anthranilate / V-glucoside.

[0094] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is propyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14. The resulting product is propyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is propyl anthranilate / V-glucoside.

[0095] In one most preferred embodiment, the anthranilate derivative is propyl anthranilate and the UDP-glycosyltransferase enzyme is selected from SEQ ID NO. 14, 32, and 40, the resulting product is propyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is propyl anthranilate / V-glucoside.

[0096] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is propyl anthranilate and the UDP-glycosyltransferase enzyme is SEQ ID NO. 14, the resulting product is propyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is propyl anthranilate / V-glucoside.

[0097] In one most preferred embodiment, the anthranilate derivative is butyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40. The resulting product is butyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP- glucose, and the resulting product is butyl anthranilate / V-glucoside.

[0098] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is butyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14. The resulting product is butyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP- glucose, and the resulting product is butyl anthranilate / V-glucoside.

[0099] In one most preferred embodiment, the anthranilate derivative is butyl anthranilate and the UDP-glycosyltransferase enzyme is selected from SEQ ID NO. 14, 32, and 40, the resulting product is butyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP- glucose, and the resulting product is butyl anthranilate / V-glucoside.

[0100] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is butyl anthranilate and the UDP-glycosyltransferase enzyme is SEQ ID NO. 14, the resulting product is butyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is butyl anthranilate / V-glucoside. In one most preferred embodiment, the anthranilate derivative is isobutyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40. The resulting product is isobutyl anthranilate / V-glycoside. Preferably the UDP- sugar is UDP-glucose, and the resulting product is isobutyl anthranilate / V-glucoside.

[0101] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is isobutyl anthranilate and the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40. The resulting product is isobutyl anthranilate / V-glycoside. Preferably the UDP- sugar is UDP-glucose, and the resulting product is isobutyl anthranilate / V-glucoside.

[0102] In one most preferred embodiment, the anthranilate derivative is isobutyl anthranilate and the UDP-glycosyltransferase enzyme is selected from SEQ ID NO. 14, 32, and 40, the resulting product is isobutyl anthranilate / V-glycoside. Preferably the UDP-sugar is UDP-glucose, and the resulting product is isobutyl anthranilate / V-glucoside.

[0103] In one most preferred embodiment of the first aspect of the invention, the anthranilate derivative is isobutyl anthranilate and the UDP-glycosyltransferase enzyme is SEQ ID NO. 14, the resulting product is isobutyl anthranilate / V-glycoside. Preferably the UDP- sugar is UDP-glucose, and the resulting product is isobutyl anthranilate / V-glucoside.

[0104] The UDP-glycosyltransferase enzyme may be provided by expressing said enzyme in a suitable host, as recognized by a person skilled in the art. Such suitable host may preferably secrete said enzyme, or the enzyme may be released from the host cell by other means such as breaking open the cell. Example 1 provides an example of expression of UGT enzymes using E. coli.

[0105] The UDP-glycosyltransferase enzyme may be purified by standard protein purification methods, as recognized by a person skilled in the art, to obtain an isolated enzyme, such as a substantially pure enzyme. In one embodiment, such isolated UDP- glycosyltransferase enzyme is provided, such as a substantially pure enzyme.

[0106] In another aspect, the present invention provides versatile UDP-glycosyltransferase (UGT) enzymes and methods for glycosylation of various anthranilate derivatives. Preferably, the present invention provides a method of using versatile UGT enzymes for glycosylating the amine group of an anthranilate derivative, wherein said anthranilate derivative has the structure of formula I: formula I wherein R is an alkyl.

[0107] Preferably, the R-group is an alkyl having have the general formula -CnH2n+i.

[0108] In one embodiment, the R-group is an alkyl, wherein the n is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or n is 1. Preferably, R is CnH2n+i and n is selected from 1, 2, 3, or 4.

[0109] In one embodiment, the R-group is a straight chain alkyl, such as a straight chain alkyl selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

[0110] In one preferred embodiment, the R-group is selected from methyl, ethyl, propyl, butyl, isopropyl, and isobutyl, more preferably the R-group is selected from methyl, ethyl, propyl, and butyl, most preferably methyl.

[0111] Preferably the UDP-glycosyltransferase enzyme for glycosylating the anthranilate derivate of formula I (as disclosed above) is selected from SEQ ID NO. 14, 32, and 40.

[0112] Most preferably the UDP-glycosyltransferase enzyme for glycosylating the anthranilate derivate of formula I (as disclosed above) is SEQ ID NO. 14.

[0113] In one preferred embodiment, the present invention provides a method of glycosylating the amine group of an anthranilate derivative having the structure of formula I: formula I wherein R is an alkyl, said method comprising the steps: a. providing a UDP-glycosyltransferase enzyme selected from SEQ ID NO. 14, 32, and 40, preferably SEQ ID NO. 14, b. mixing said enzyme with said anthranilate derivative and a UDP-sugar, c. letting the mixture react for a selected period of time, and d. optionally purifying the glycosylated anthranilate derivative.

[0114] The sugar moiety of the UDP-sugar is preferably a simple sugar, such as a monomer or dimer, such as selected from glucose, xylose, galactose, rhamnose, / V-acetyl glucosamine, glucuronic acid, / V-acetyl galactosamine, mannose, arabinose, galacturonic acid, fucose; preferably the UDP-sugar is UDP-glucose.

[0115] In one embodiment, the present invention concerns the use of an UDP- glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40, preferably consists of or comprises amino acid sequence SEQ ID NO. 14, or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40, preferably consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, in the process of glycosylating anthranilate derivatives having the structure of formula I. The UDP-glycosyltransferase enzyme may be an isolated enzyme, such as a substantially pure enzyme.

[0116] In one preferred embodiment, the present invention concerns the use of an UDP- glycosyltransferase enzyme selected from SEQ ID NO. 14, 32, and 40, preferably SEQ ID NO. 14, in the process of glycosylating anthranilate derivatives having the structure of formula I.

[0117] In one most preferred embodiment, a method of glycosylating the amine group of an anthranilate derivative having the structure of formula I is provided as disclosed herein, wherein the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0118] In one most preferred embodiment, a method of glycosylating the amine group of an anthranilate derivative having the structure of formula I is provided as disclosed herein, wherein the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0119] In one most preferred embodiment, a method of glycosylating MANT, EANT, BANT, or IBANT is provided, wherein the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0120] In one most preferred embodiment, a method of glycosylating MANT, EANT, BANT, or IBANT is provide, wherein the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

[0121] 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequences comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0122] It is further preferred that in a method of glycosylating EANT, BANT, or IBANT, the amino acid sequence of the UDP-glycosyltransferase enzyme comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14.

[0123] In one embodiment, a method of glycosylating the amine group of an anthranilate derivative having the structure of formula I is provided, wherein R. in an akyl CnH2n+i larger than methyl - i.e. wherein n is >1, such as 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2- 3, or 2; preferably n is selected from 2, 3, and 4, such as ethyl, propyl, butyl, or isobutyl; and wherein the UDP-glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

[0124] 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, and wherein the amino acid sequence of the UDP-glycosyltransferase enzyme comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14. Preferably further wherein the amino acid sequence of the UDP-glycosyltransferase enzyme comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14. In another aspect, the present invention provides UDP-glycosyltransferase (UGT) enzyme variants and methods for glycosylation of more bulky anthranilate derivatives, such as a method of glycosylating the amine group of an anthranilate derivative having the structure of formula III: wherein -R2comprises a cyclic hydrocarbon, such as comprises a phenyl or a cycloalkyl. In one embodiment, the R2-group is a cycloalkyl, most preferably cyclohexyl. In one embodiment, the R2-group is a cycloalkyl -CnH2n-i, wherein the n is 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, or 3, preferably n is selected from 3, 4, 5, and 6, most preferably n is 6. In one embodiment, the R2-group is selected from phenyl, phenethyl, and benzyl.

[0125] In one such embodiment, the method for glycosylating an anthranilate derivative having the structure of formula III, comprising the steps: a. providing a UDP-glycosyltransferase consisting of or comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14, b. mixing said enzyme with said anthranilate derivative and a UDP-sugar, c. letting the mixture react for a selected period of time, and d. optionally purifying the glycosylated anthranilate derivative.

[0126] In one most preferred embodiment, a method of glycosylating is provided as disclosed herein, wherein the anthranilate derivative is CANT, and wherein the UDP- glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14.

[0127] It is further preferred that in the method of glycosylating an anthranilate derivative having the structure of formula III, such as CANT, that the amino acid sequence of the UDP-glycosyltransferase enzyme comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

[0128] In the method of the present invention, according to any of the embodiments above, the anthranilate derivative is preferably incubated with the glycosyltransferase enzyme at temperature and pH conditions optimal for the enzyme. In one embodiment, the incubation temperature applied should be in the range 20-65°C, such as 20-60°C, such as 25-50°C, such as 25-45°C, preferably in the range 30-40°C. In one embodiment, the incubation pH applied should be in the range pH 5-9, such as pH 5.5-8.5, such as preferably pH 6-8. In one preferred embodiment, the incubation pH is pH 7-9, more preferably pH 7.5-8.5, most preferably around pH 8.

[0129] In one embodiment, an anthranilate derivative according to formula I is incubated with an UGT-glycosyltransferase enzyme consisting of or comprising an amino acid sequence according to SEQ ID NO. 14 or an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14; at an incubation temperature in the range 20- 65°C, such as 20-60°C, such as 25-55°C, such as 25-45°C, preferably in the range 30- 40°C, such as preferably around 35°C.

[0130] In one embodiment, an anthranilate derivative according to formula I is incubated with an UGT-glycosyltransferase enzyme consisting of or comprising an amino acid sequence according to SEQ ID NO. 32 or an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 32; at an incubation temperature in the range 20- 65°C, such as 20-60°C, such as 25-55°C, such as 30-50°C, preferably in the range 35- 45°C, such as preferably around 39°C.

[0131] In one embodiment, an anthranilate derivative according to formula I is incubated with an UGT-glycosyltransferase enzyme consisting of or comprising an amino acid sequence according to SEQ ID NO. 40 or an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 40; at an incubation temperature in the range 20- 65°C, such as 20-55°C, such as 20-45°C, such as 25-40°C, preferably in the range 25- 35°C, such as preferably around 30°C.

[0132] The enzymatic reaction may take place in buffered solution for stabilizing the enzymes, as a person skilled in the art would recognize and routinely optimize. The glycosylated compounds produced by the method of the present invention may be detected by HLPC-UV, LC-MS, NMR, or similar equipment as recognized by a person skilled in the art.

[0133] II. Composition comprising an anthranilate derivative and UGT enzyme

[0134] In a second aspect, the present invention provides a composition comprising (I) an anthranilate derivative as defined in formula I, (II) an UDP-glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40, and (III) a UDP-sugar. The UDP-glycosyltransferase enzyme may be an isolated enzyme, such as a substantially pure enzyme.

[0135] In one preferred embodiment, a composition is provided comprising (I) an anthranilate derivative as defined in formula I, (II) an glycosyltransferase enzyme selected from SEQ ID NO. 14, 32, and 40, and (III) a UDP-sugar.

[0136] In one preferred embodiment, the present invention provides a composition comprising (I) an anthranilate derivative as defined in formula I, such as MANT, EANT, BANT, or IBANT, (II) an UDP-glycosyltransferase enzyme (i) consisting of or comprising amino acid sequence SEQ ID NO. 14 or (ii) consisting of or comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, and (III) a UDP- sugar. The UDP-glycosyltransferase enzyme may be an isolated enzyme, such as a substantially pure enzyme.

[0137] In one preferred embodiment, a composition is provided comprising (I) an anthranilate derivative as defined in formula I, such as MANT, EANT, BANT, or IBANT, (II) an glycosyltransferase enzyme SEQ ID NO. 14, and (III) a UDP-sugar.

[0138] In one preferred embodiment, the present invention provides a composition comprising (I) an anthranilate derivative as defined in formula I or formula III, such as MANT, EANT, BANT, IBANT, or CANT, (II) an UDP-glycosyltransferase enzyme consisting of or comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14, and (III) a UDP sugar. In one most preferred embodiment, a composition is provided comprising (I) an anthranilate derivative as defined in formula I wherein n is at least 2 or formula III, such as EANT, BANT, IBANT, or CANT, (II) an UDP-glycosyltransferase enzyme consisting of or comprising an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14, and (III) a UDP sugar.

[0139] The composition of the present invention comprises a UDP-sugar, which is required for the glycosylation reaction catalyzed by the glycosyltransferase. In one embodiment, the UDP-sugar, is selected from UDP-glucose, UDP-xylose, UDP-galactose, UDP-rhamnose, UDP- / V-acetyl glucosamine, UDP-glucuronic acid, UDP- / V-acetyl galactosamine, UDP- mannose, UDP-arabinose, UDP-galacturonic acid, UDP-fucose. In a preferred embodiment, the UDP-sugar is UDP-glucose. UDP-glucose may be provided directly as UDP-glucose or indirectly in the form of other sugars or sugar-containing molecules which are then converted into UDP-glucose. One example of such indirect provision of UDP-glucose is by providing sucrose along with UDP, which then by enzymatic catalysis (such as using Sucrose synthase (SuSy) EC 2.4.1.13) is converted to UDP-glucose. Another example of indirect providing of UDP-glucose is by using sucrose phosphorylase (converts sucrose and phosphate into glucose-l-P and fructose), glucose-l-phosphate uridylyltransferase (converts UTP and glucose-l-P into UDP-glucose and PPi); and further to regenerate the UTP from UDP, using acetate kinase which requires acetyl-P as substrate in equimolar amounts (converts Acetyl-P + UDP to UTP + acetate) (Lee et al 2004, Bruyn et al 2015). in. An in vivo method of glycosylating anthranilate derivatives

[0140] As mentioned, previous attempts have been made to prepare MANT microbially, but yields were low due to toxicity of MANT to the host cell. Adding a glycosylation step will alleviate this toxicity and pave the way for economically bio-based MANT production (see Example 9). As one example, the present invention thereby provides an improved microbial cell factory for use in a method of producing of MANT : The cell factory produces MANT- / V-glycoside, and the glucose unit is then chemically or enzymatically cut off to yield the final product MANT.In a third aspect, the present invention concerns in vivo glycosylation of anthranilate derivatives using glycosyltransferase enzymes.

[0141] In one embodiment, the UGT enzymes disclosed herein are expressed in a selected host along with the metabolic pathway for producing a desired anthranilate derivative, preferably an anthranilate derivative having the structure of formula I, as disclosed herein.

[0142] Specifically, the present invention provides an in vivo method for producing a glycosylated anthranilate derivative, comprising the steps of a. providing a microbial cell comprising (i) a nucleic acid sequence encoding a UDP-glycosyltransferase enzyme, and (ii) one or more nucleic acid sequences encoding one or more enzymes for synthesizing an anthranilate derivative, b. growing said microbial cell in a growth medium, and expressing said UDP- glycosyltransferase enzyme and said one or more enzymes for synthesizing the anthranilate derivative and c. optionally purifying the glycosylated anthranilate derivative.

[0143] Specifically, the present invention provides a method, wherein the amino group of the anthranilate derivative is glycosylated. In one preferred embodiment, the anthranilate derivative has the structure of formula I, as disclosed herein; and in a further preferred embodiment, the UDP-glycosyltransferase enzyme is a versatile enzyme, such as selected from SEQ ID NO. 14, 32, and 40, preferably SEQ ID NO. 14.

[0144] In one embodiment, the microbial cell for in vivo production of glycosylation of anthranilate derivatives is capable of producing anthranilic acid. The microbial cell may be capable of producing anthranilic acid naturally or because it has been genetically engineered. In one embodiment, the microbial cell is selected from a strain of E. coli, C. glutamicum, S. cerevisiae, and P. putida. In a preferred embodiment, the microbial cell for in vivo production of glycosylated anthranilate derivatives is a strain of E. coli.

[0145] Anthranillic acid is naturally produced by E. coli and other microbes as a precursor of the amino acid tryptophan. This may be over-produced by tuning the tryptophan biosynthesis pathway using metabolic engineering approaches. Anthranilate derivatives may be produced enzymatically from anthranillic acid. For example, methyl anthranilate may be produced from anthranilic acid by a methyltransferase enzyme.

[0146] UDP-sugar is needed for the glycosylation reaction by the UDP-glycosyltransferase enzyme. The UDP-sugar may either be present in the growth medium, or may be synthesized by the microbial cell. Hence, in one embodiment, the microbial cell further comprises one or more nucleic acid sequences encoding one or more enzymes for synthesizing a UDP-sugar, preferably UDP-glucose (example of such enzyme is disclosed herein as SEQ ID NO. 82 (sucrose synthase from Glycine max)). In one embodiment, the in vivo method for producing a glycosylated anthranilate derivative, comprising the steps of a. providing a microbial cell comprising

[0147] (I) a nucleic acid sequence encoding a UDP-glycosyltransferase enzyme, preferably said enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14, 32, or 40, and

[0148] (II) one or more nucleic acid sequences encoding one or more enzymes for synthesizing an anthranilate derivative having the structure of formula I, wherein R. is an alkyl, b. growing said microbial cell in a growth medium, and expressing said UDP- glycosyltransferase enzyme and said one or more enzymes for synthesizing the anthranilate derivative, wherein a UDP-sugar is provided (i) as a component of the growth medium or (ii) by said microbial cell further comprising one or more nucleic acid sequences encoding one or more enzymes for synthesis of the UDP-sugar and said microbial cell expressing said enzymes for synthesis of the UDP-sugar; and c. optionally purifying the glycosylated anthranilate derivative.

[0149] Most preferably, the UDP-glycosyltransferase enzyme (i) consists of or comprises amino acid sequence SEQ ID NO. 14 or (ii) consist of or comprises an amino acid sequence having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 14.

[0150] In one embodiment, an in vivo method of glycosylating MANT is provided; wherein the UDP-glycosyltransferase enzyme is preferably selected from SEQ ID NO. 2, 14, 24, 26, 28, 32, 34, 36, 38, 40, 68, and 70, more preferably SEQ ID NO. 14, 24, 26, 32, 34, 36, and 40, most preferably SEQ ID NO. 14, 32, and 40, most preferably SEQ ID NO. 14, or an enzyme having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to any of these preferred SEQ ID NOs.; and wherein the enzyme for synthesizing MANT is a methyltransferase (example of such enzyme is disclosed herein as SEQ ID NO. 76 (Aamtl from Zea mays)). The resulting product is methyl anthranilate / V-glycoside (MANT- / V-glycoside). Preferably the UDP-sugar is UDP-glucose, and the resulting product is methyl anthranilate N-glucoside. Another route for the synthesis of alkyl anthranilates, such as methyl anthranilate and ethyl anthranilate, from anthranillic acid may be catalyzed by two enzymes. Anthranilate is converted into anthraniloyl-CoA by anthranilate-CoA ligase and then it is converted into alkyl anthranilate by AMAT (encoding anthraniloyl-coenzyme A (CoA): methanol acyltransferase). Which alkyl group is attached to anthranilate depends on the alcohol used, such as disclosed in Lee et al 2019. AMAT uses diverse alcohols as an alkyl group donor and anthraniloyl-CoA as an alkyl group acceptor.

[0151] In one embodiment, an in vivo method of glycosylating MANT is provided; wherein the UDP-glycosyltransferase enzyme is preferably selected from SEQ ID NO. 2, 14, 24, 26, 28, 32, 34, 36, 38, 40, 68, and 70, more preferably SEQ ID NO. 14, 24, 26, 32, 34, 36, and 40, most preferably SEQ ID NO. 14, 32, and 40, most preferably SEQ ID NO. 14, or an enzyme having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to any of the preferred SEQ ID NOs.; and wherein the enzymes for synthesizing MANT include an anthranilate-CoA ligase (examples of such enzyme is disclosed herein as SEQ ID NO. 80 (PqsA from Pseudomonas aeruginosa)) and an anthraniloyl-coenzyme A (CoA): methanol acyltransferase (examples of such enzyme is disclosed herein as SEQ ID NO. 78 (AMAT from Vitis labrusca)). The microbial cell is grown in the presence of methanol. The resulting product is methyl anthranilate / V-glycoside (MANT- / V-glycoside). Preferably the UDP-sugar is UDP-glucose, and the resulting product is methyl anthranilate N-glucoside.

[0152] In one embodiment, an in vivo method of glycosylating EANT is provided; wherein the UDP-glycosyltransferase enzyme is preferably selected from SEQ ID NO. 8, 14, 16, 28, 32, 38, 40, and 68, more preferably SEQ ID NO. 8, 14, 16, 32, 38, and 40, most preferably SEQ ID NO. 14, 32, and 40, most preferably SEQ ID NO. 14, or an enzyme having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to any of the preferred SEQ ID NOs; and wherein the enzymes for synthesizing EANT include an anthranilate- CoA ligase (examples of such enzyme is disclosed herein as SEQ ID NO. 80 (PqsA from Pseudomonas aeruginosa)) and an anthraniloyl-coenzyme A (CoA): methanol acyltransferase (examples of such enzyme is disclosed herein as SEQ ID NO. 78 (AMAT from Vitis labrusca')'). The microbial cell is grown in the presence of ethanol. The resulting product is ethyl anthranilate / V-glycoside (EANT- / V-glycoside). Preferably the UDP-sugar is UDP-glucose, and the resulting product is ethyl anthranilate N-glucoside, wherein the glucoside is a glucose moiety.

[0153] In one embodiment, the anthranilate derivative is BANT, and the UDP- glycosyltransferase enzyme is preferably selected from SEQ ID NO. 14, 32, and 40, most preferably SEQ ID NO. 14, or an enzyme having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to any of the preferred SEQ ID NOs.; and wherein the enzymes for synthesizing BANT include an anthranilate-CoA ligase (examples of such enzyme is disclosed herein as SEQ ID NO. 80 (PqsA from Pseudomonas aeruginosa)) and an anthraniloyl-coenzyme A (CoA): methanol acyltransferase (examples of such enzyme is disclosed herein as SEQ ID NO. 78 (AMAT from Vitis labrusca')'). The microbial cell is grown in the presence of butanol. The resulting product is butyl anthranilate / V-glycoside (BANT- / V-glycoside). Preferably the UDP-sugar is UDP-glucose, and the resulting product is butyl anthranilate / V-glucoside, wherein the glucoside is a glucose moiety.

[0154] In one embodiment, the anthranilate derivative is iso-BANT, and the UDP- glycosyltransferase enzyme is preferably selected from SEQ ID NO. 14, 32, and 40, most preferably SEQ ID NO. 14, or an enzyme having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to any of the preferred SEQ ID NOs.; and wherein the enzymes for synthesizing iso-BANT include an anthranilate-CoA ligase (examples of such enzyme is disclosed herein as SEQ ID NO. 80 (PqsA from Pseudomonas aeruginosa)) and an anthraniloyl-coenzyme A (CoA): methanol acyltransferase (examples of such enzyme is disclosed herein as SEQ ID NO. 78 (AMAT from Vitis labrusca)). The microbial cell is grown in the presence of iso-butanol. The resulting product is iso-butyl anthranilate N- glycoside (iso-BANT- / V-glycoside). Preferably the UDP-sugar is UDP-glucose, and the resulting product is iso-butyl anthranilate / V-glucoside, wherein the glucoside is a glucose moiety.

[0155] The growth medium for the in vivo production of a glycosylated anthranilate derivative may further comprise one or more components selected from the list glucose, urea, KH2PO4, (NH4)2HPO4, MgSO4-7H2O, citric acid, 3-morpholinopropanesulfonic acid (MOPS), and different trace metals (e.g. CaCI2-2H2O, FeSO4-7H2O, MnSO4-5H2O, ZnSO4'7H2O, CuSO4-5H2O, and NiCI2-6H2O). Preferably, such media conditions are applied, if the microbial cell used is a prokaryotic strain, such as E. coll or C. glutamicum. Growth medium for S. cerevisiae may comprise YPD medium (10 g 1-1 yeast extract, 20 g 1-1 peptone and 20 g 1-1 D-glucose).

[0156] In one aspect, the present invention provides a method for producing an anthranilate derivative, comprising producing a glycosylated anthranilate derivative in vivo in a microbial cell as disclosed herein, wherein said glycosylated anthranilate derivative is secreted by or extracted from said microbial cell, followed by chemically or enzymatically breaking the glycosidic bond of the glycosylated anthranilate derivative in vitro, thereby obtaining the anthranilate derivative. In one embodiment, the glycosidic bond is chemically hydrolyzed by use of an acid, such as HCI (see Example 10). IV. Glycosylated anthranilate derivatives as an insect and / or bird repellant

[0157] In a fourth aspect, the present invention concerns the use of glycosylated anthranilate derivatives as an insect and / or bird repellant.

[0158] In one embodiment, the present invention concerns the use of glycosylated anthranilate derivatives as an insect repellant. In one embodiment, the present invention concerns the use of glycosylated anthranilate derivatives as a bird repellant.

[0159] In one embodiment, the glycosylated anthranilate derivative used as an insect and / or bird repellent has a structure of formula II: formula II wherein R is an alkyl or alkyl substitute, and wherein X is a sugar moiety.

[0160] Preferably, the R-group is an alkyl having have the general formula -CnH2n+i.

[0161] In one embodiment, the R-group is an alkyl, wherein the n is between 1-10, between 1- 9, between 1-8, between 1-7, between 1-6, between 1-5, between 1-4, between 1-3, between 1-2, or n is 1.

[0162] In one embodiment, the R-group is a straight chain alkyl, such as a straight chain alkyl selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

[0163] In one preferred embodiment, the R-group is selected from methyl, ethyl, propyl, butyl, isopropyl, and isobutyl, more preferably the R-group is selected from methyl, ethyl, propyl, and butyl, most preferably methyl.

[0164] In one embodiment, the X-group is selected from glucose, xylose, galactose, rhamnose, / V-acetyl glucosamine, glucuronic acid, / V-acetyl galactosamine, mannose, arabinose, galacturonic acid, fucose. Preferably the X-group is glucose.

[0165] In a preferred embodiment, the invention concerns the use of a glycosylated anthranilate derivative as an insect and / or bird repellent, wherein the glycosylated anthranilate derivate has the structure of formula II, wherein R is CnH2n+i and n is 1-4; and X is glucose.

[0166] In a preferred embodiment, the invention concerns the use of methyl anthranilate N- glucoside as an insect and / or bird repellant. In a preferred embodiment, the invention concerns the use of ethyl anthranilate N- glucoside as an insect and / or bird repellant. In a preferred embodiment, the invention concerns the use of propyl anthranilate / V-glucoside as an insect and / or bird repellant. In a preferred embodiment, the invention concerns the use of butyl anthranilate N- glucoside as an insect and / or bird repellant. In a preferred embodiment, the invention concerns the use of iso-butyl anthranilate / V-glucoside as an insect and / or bird repellant.

[0167] In one embodiment, the present invention provides a method for repelling insects and / or birds from an item, using / V-glycosylated anthranilate derivatives. In one embodiment, the method for repelling insects and / or birds from an item comprises applying an aqueous solution comprising a / V-glycosylated anthranilate derivative to the item, such as spraying it onto the item. The aqueous solution comprising the / V-glycosylated anthranilate derivative may be applied to the surface of any such item where the presence of birds is undesired. Examples include applying the solution to areas of vegetation, such as grass fields, including recreational areas and sports fields such as golf courses and football / soccer field, or agricultural fields. Other examples include applying the solution to buildings and roads, such as airports and airfields, or food processing facilities and surrounding areas.

[0168] In one preferred embodiment, the present invention provides a method of repelling birds using methyl anthranilate / V-glucoside.

[0169] V. A method of producing an insect and / or bird repellent

[0170] In a fifth aspect, the present invention provides a method for producing an insect and / or bird repellent composition, comprising the steps of a. providing a UDP-glycosyltransferase enzyme, a UDP-sugar, and an anthranilate derivative, b. mixing said enzyme, said UDP-sugar, and said anthranilate derivative, c. letting the enzyme catalyze glycosylation of the anthranilate derivative for a selected period of time to produce a glycosylated anthranilate derivative having insect and / or bird repelling properties, and d. optionally purifying the glycosylated anthranilate derivative, and e. optionally further formulating the glycosylated anthranilate derivative by addition of additives.

[0171] In one preferred embodiment, in the method for producing an insect and / or bird repellent, the anthranilate derivative has the structure of formula I, preferably wherein R is methyl, ethyl, propyl, or butyl, most preferably methyl; the UDP-glycosyltransferase enzyme is selected from SEQ ID NO. 14, 32, and 40, or an enzyme having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to SEQ ID NO. 14, 32, or 40; and the UDP-sugar is a sugar monomer, most preferably UDP-glucose, resulting in the glycosylated anthranilate derivative having the structure of formula II, most preferably the structure of formula II wherein X is glucose.

[0172] In one most preferred embodiment, in the method for producing an insect and / or bird repellent, the anthranilate derivative has the structure of formula I, preferably wherein R is methyl, ethyl, propyl, or butyl, most preferably methyl; the UDP-glycosyltransferase enzyme is SEQ ID NO. 14, or an enzyme having at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to SEQ ID NO. 14; and the UDP-sugar is a sugar monomer, most preferably UDP-glucose, resulting in the glycosylated anthranilate derivative having the structure of formula II, most preferably the structure of formula IV wherein X is glucose.

[0173] In one embodiment, one or more additive(s) selected from fatty acid, antioxidant, buffering solution, surfactant (i.e. a substance that decreases the surface tension between two substances that do not readily mix), and cosolvent (i.e. a substance added to a primary solvent to increase solubility) is added to the bird and / or insect repellent solution.

[0174] Insect and / or bird repellant properties may be measured as disclosed in Example 7 and Example 8. For example, bird-repellent properties of a glycosylated anthranilate may be tested on live birds through an aqueous formulation comprising the glycosylated anthranilate, by offering the birds untreated seeds as well as seeds treated with the aqueous formulation, and measuring and comparing the consumption of each type of seed.

[0175] VI. Improved UGT variant

[0176] In one aspect, the present invention provides an improved UGT variant. By amino acid residue substation Phel45Met in UGT16, the inventors discovered more than 10-fold increase in glycosylation of CANT, more than 3-fold increase in glycosylation of IBANT, more than 1.5-fold increase in glycosylation of BANT, and more than 1.3-fold increase in glycosylation of EANT, compared to WT UGT16 (see Example 5). Hence, compared to SEQ ID NO 14, such enzyme comprising amino acid residue substitution Phel45Met has improved efficiency especially in glycosylating CANT. Hence, compared to SEQ ID NO 14, such enzyme comprising amino acid residue substitution Phel45Met has improved enzyme activity in regards to glycosylating CANT.

[0177] In one preferred embodiment, the present invention provides a polypeptide having glycosyltransferase activity (enzyme classification EC:2.4.1.-), wherein the amino acid sequence of said polypeptide has at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO. 14, and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14. Compared to SEQ ID NO 14, such enzyme comprising amino acid residue substitution Phel45Met has improved efficiency in glycosylating (I) anthranilate derivatives according to formula I, wherein R in an akyl CnH2n+i larger than methyl - i.e. wherein n is >1, such as 2-10, 2-9, 2-8, 2- 7, 2-6, 2-5, 2-4, 2-3, or 2; preferably n is selected from 2, 3, and 4, such as ethyl, propyl, butyl, or isobutyl; and / or (II) anthranilate derivatives according to formula III, wherein R2in a cycloakyl -CnH2n-i, wherein n is at least 3, such as 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, or 3, preferably n is selected from 3, 4, 5, and 6, most preferably n is 6.

[0178] In one preferred embodiment, the present invention provides a polypeptide having glycosyltransferase activity (enzyme classification EC:2.4.1.-), wherein the amino acid sequence of said polypeptide has at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO. 14, and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14, and wherein the efficiency of said glycosyltransferase activity in glycosylating CANT, IBANT, BANT and / or EANT is improved, compared to SEQ ID NO. 14.

[0179] The inventors further identified amino acid residues Ile80, Ile84, Phel35 and Glyl41 of UGT16 as essential for maintaining glycosylation activity (see Example 5). A mutation of one or more of these may cause loss of glycosylation activity. Hence, in one further preferred embodiment, the present invention provides a polypeptide having glycosyltransferase activity (enzyme classification EC:2.4.1.-), wherein the amino acid sequence of said polypeptide has at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with SEQ ID NO. 14, and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14, and wherein said amino acid sequences comprises amino acid residues Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14. In a further aspect, the present invention provides a composition comprising the improved UGT variant disclosed herein.

[0180] In a further aspect, the present invention provides an in vivo method for producing a glycosylated anthranilate derivative using the improved UGT variant disclosed herein.

[0181] VII. Advantages and commercial application

[0182] As discussed previously, and further evidenced in the below examples, the preferred glycosyltransferase enzymes selected for use in the present invention of glycosylating anthranilate derivatives are versatile in their activity towards and tolerance of several different R-groups in the structure of formula I, while most other enzymes tested showed lower activity in general or only good activity on a single type of anthranilate derivative. Such versatile activity is highly relevant commercially.

[0183] Further, the in vivo method of the present invention is particularly suited as a greener alternative to current petrochemical processes - i.e. in vivo glycosylation of anthranilate derivatives is a feasible way of avoiding the toxicity associated with anthranilate derivatives.

[0184] Finally, the glycosylated anthranilate derivatives are preferably used as bird repellents, and compared to non-glycosylated anthranilate derivatives they are less volatile which means they can be applied less frequently while still maintaining their bird repelling effect. Further, the glycosylated anthranilate derivatives have improved water solubility, leading to easier formulation.

[0185] NUMBERED EMBODIMENTS

[0186] Numbered embodiment 1. An in vitro method for glycosylation of an anthranilate derivative, comprising the steps of a. providing an UDP-glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40. b. mixing said enzyme with a UDP-sugar and an anthranilate derivative having the structure of formula I, wherein R is an alkyl: formula I c. letting the mixture react for a selected period of time, d. optionally purifying the glycosylated anthranilate derivative.

[0187] Numbered embodiment 2. The in vitro method according to Numbered embodiment 1, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

[0188] Numbered embodiment 3. A composition comprising

[0189] (I) an anthranilate derivative having the structure of formula I, wherein R is an alkyl,

[0190] (II) an UDP-glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40, and

[0191] (III) a UDP-sugar.

[0192] Numbered embodiment 4. The composition according to Numbered embodiment 3, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

[0193] Numbered embodiment 5. Use of an UDP-glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40, in the process of glycosylating anthranilate derivatives having the structure of formula I, wherein R is alkyl.

[0194] Numbered embodiment 6. The use according to Numbered embodiment 5, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

[0195] Numbered embodiment 7. An in vivo method for producing a glycosylated anthranilate derivative, comprising the steps of a. providing a microbial cell comprising (I) a nucleic acid sequence encoding a UDP-glycosyltransferase enzyme, wherein said enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40, and

[0196] (II) one or more nucleic acid sequences encoding one or more enzymes for synthesizing an anthranilate derivative having the structure of formula I, wherein R is an alkyl; b. growing said microbial cell in a growth medium, and expressing said UDP- glycosyltransferase enzyme and said one or more enzymes for synthesizing the anthranilate derivative, wherein a UDP-sugar is provided (i) as a component of the growth medium or (ii) by said microbial cell further comprising one or more nucleic acid sequences encoding one or more enzymes for synthesis of the UDP-sugar and said microbial cell expressing said enzymes for synthesis of the UDP-sugar; and c. optionally purifying the glycosylated anthranilate derivative.

[0197] Numbered embodiment 8. The in vivo method according to Numbered embodiment 7, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

[0198] Numbered embodiment 9. The in vivo method according to Numbered embodiment 7 or 8, wherein the microbial cell is selected from a strain of E. coli, C. glutamicum, S. cerevisiae, and P. putida.

[0199] Numbered embodiment 10. The in vivo method according to any one of Numbered embodiment 7-9, wherein the one or more enzymes for synthesizing the anthranilate derivative comprise (i) a methyltransferase or (ii) an anthranilate-CoA ligase and an anthraniloyl-coenzyme A (CoA): methanol acyltransferase.

[0200] Numbered embodiment 11. A method for producing an insect and / or bird repellent composition, comprising the steps of a. providing a UDP-glycosyltransferase enzyme, a UDP-sugar, and an anthranilate derivative having the structure of formula I, wherein R is an alkyl, b. mixing said enzyme, said UDP-sugar, and said anthranilate derivative, c. letting the enzyme catalyze glycosylation of the anthranilate derivative for a selected period of time to produce a glycosylated anthranilate derivative having bird repelling properties, and d. optionally purifying the glycosylated anthranilate derivative, and e. optionally further formulating the glycosylated anthranilate derivative by addition of one or more additives.

[0201] Numbered embodiment 12. The method according to Numbered embodiment 11, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

[0202] Numbered embodiment 13. The method according to Numbered embodiment 11 or 12, wherein the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40.

[0203] Numbered embodiment 14. The method according to any one of Numbered embodiment 11-13, wherein the one or more additives is selected from a fatty acid, an antioxidant, a buffering solution, a surfactant, and a cosolvent.

[0204] Numbered embodiment 15. Use of a glycosylated anthranilate derivative as an insect and / or bird repellent, wherein the glycosylated anthranilate derivate has the structure of formula II, wherein R is CnH2n+i and n is 1-4; and X is glucose formula II.

[0205] EXAMPLES

[0206] Example 1: Enzyme expression and purification

[0207] 1.1 E. coli expressing UGT enzymes

[0208] The full-length histidine-tagged DNA sequence of the UGTs was synthesized and cloned into a pET-28a(+) vector. The protein was expressed in a 1 L culture of One Shot BL21 Star (DE3) E. coli cells in 2xYT media. The culture was started at OD600 = 0.05 and incubated at 37 °C until OD600 reaches 0.6-0.8. The culture was then left to reach room temperature followed by induction with 0.2 mM IPTG. The culture was incubated overnight at 20 °C where it was then harvested via centrifugation.

[0209] Table 1 provides an overview of the different UGTs tested.

[0210] *The DNA sequences provided in the sequence listing encoding these UGT enzymes are synthetic constructs prepared based on the amino acid sequences of the UGT enzymes originating from the organisms as listed here in table 1.

[0211] 1.2 Protein production

[0212] The pellet from 1 L culture was resuspended in 85 mL of lysis buffer (20 mM imidazole, 50 mM HEPES, 300 mM NaCI, 1.5 tablets complete EDTA-free protease inhibitor cocktail tablets (Roche), 10 pg / mL DNase, 0.1 mg / mL lysozyme, 0.05 % TRITON X). Sonication was performed for 4 min. at 70% amplitude for 30 sec. on / 30 sec. off, keeping the sample in ice. Centrifugation at 14.000 x g for 20 min at 4° C was used to remove cell debris. The protein was purified using the AKTA FPLC system (kept at 10 °C) after the cleared extracts were loaded into 1 mL nickel affinity columns (HisTrap FF, Cytiva). Elution was carried out using buffer A (20 mM imidazole, 50 mM HEPES, 300 mM NaCI, pH 7.5) and buffer B (500 mM imidazole, 50 mM HEPES, 300 mM NaCI, pH 7.5). The peak fraction was analysed by SDS-PAGE. Selected fractions were combined and concentrated to 2.5 ml using a 30 KDa Amicon Ultra-15 Centrifugal Filter Unit (Merck). The buffer was exchanged with a PD-10 desalting column, previously equilibrated with 10 resin bed volumes of storage buffer (50 mM HEPES, 150 mM NaCI, pH 7.4), eluting with 3.5 mL of storage buffer. The protein concentration (mg / mL) was quantified using a NanoDroplOOO spectrophotometer (Thermo Fisher Scientific)

[0213] Example 2: Enzyme screening

[0214] 2.1 Screening reaction

[0215] The enzymes were screened for their activity on different anthranilate derivatives.

[0216] To determine whether the enzymes were active on methyl anthranilate and ethyl anthranilate, screening was carried out on a pre-made microplate containing 50 pL of enzyme in 50 mM HEPES pH 7 supplemented with 2 mM UDP-glucose. In each well of the microplate, the substrate of interest previously dissolved in DMSO was added to a final concentration of 0.2 mM. After an overnight reaction at room temperature, product formation was measured by HPLC. The best hits were subsequently screened for their activity on butyl anthranilate, and isobutyl anthranilate making an overnight reaction at room temperature using 1 mM of UDP-glucose, 2.5 pM of enzyme and 0.5 mM of the substrate of interest.

[0217] A control without enzyme was included in all experiments. 2.2 Analytical procedure

[0218] Product formation was followed by HPLC, using an Ultimate 3000 Series apparatus (Thermo Fisher) and a Kinetex 2.6 pm C18 100 A 100x4.6 mm analytical column (Phenomenex). 0.1 % formic acid in water (A) and acetonitrile (B) were used as mobile phases following the gradient in table 2. The flow rate was set to 1 mL / min and the column temperature was set to 40° C. The products in the eluate were revealed using their absorbance at 330 nm. The peaks were manually integrated using the Chromeleon software (Thermo Fisher Scientific). The conversion was determined by analyzing the ratio between the substrate in the reaction with enzyme and the control reaction without enzyme.

[0219] 2.3 Results

[0220] Table 3 provides the data from the screening on methyl-, ethyl- and butyl anthranilate.

[0221] Grey shaded boxes with no numeric value or comment means not tested.

[0222] UGT1, UGT41, UGT50, UGT151, and UGT152 showed good activity on methyl anthranilate; while UGT16, UGT36, UGT38, UGT43, UGT46, UGT47, and UGT53 showed very good activity on methyl anthranilate. UGT9, UGT16, UGT17, UGT43, UGT50, and UGT53 showed >90% conversion of ethyl anthranilate; while UGT41 and UGT151 showed >60% conversion of ethyl anthranilate.

[0223] UGT16, UGT43, and UGT53 showed >80% conversion of butyl anthranilate.

[0224] UGT16, UGT43, and UGT53 were found to be versilate in the sense that they perform very well on all three substrates methyl-, ethyl-, and butyl anthranilate. These three versatile enzymes UGT16, UGT43, and UGT53 were additionally tested in isobutyl anthranilate (Table 4) and were found to also be able to glycosylate this substrate; with UGT53 showing >60% conversion. UGT16, UGT43, and UGT53 were also tested with cyclohexyl anthranilate (CANT). Here, UGT16 did not display activity with CANT. Meanwhile, UGT43 and UGT53 displayed activity, with UGT53 displaying the highest activity with CANT.

[0225] Finally, the three versatile enzymes UGT16, UGT43, and UGT53 were also tested on N- methyl anthranilate, where the methyl group is placed one the amine group, therefore blocking the possibility of glycosylating this site. No or close to none conversion was seen for this substrate, indicating that at the tested conditions, the UGT16, UGT43, and UGT53 are specific for glycosylating the amine group of anthranilate derivatives. Further experiments have shown that the reaction equilibrium is significantly shifted towards the non-glycosylated N-methyl anthranilate. Using sucrose synthase (SuSy) in combination with sucrose, for regeneration of UDP-glucose, helped push the reaction equilibrium towards the production of significant amounts of glycosylated N-methyl anthranilate.

[0226] Example 3: Properties of UGT16, UGT43, and UGT53

[0227] 3.1 Analytical procedure

[0228] The samples were analysed as described in example 2, section 2.2. However, the formation of the product was determined by analysing the ratio between the product peak and acceptor peak on the HPLC chromatograms, assuming the absorbance at 330 nm.

[0229] 3.2 Biochemical characterization with MANT

[0230] To assess the kinetic properties of UGT16, UGT43, and UGT53 with MANT, the optimal reaction temperature and pH were determined pH optimum: Reactions were performed at a pH range to find the optimum pH of UGT16, UGT43, and UGT53. The following buffering agents were utilized for the respective pH ranges: pH value: pH 4-7, 50 mM citrate-phosphate; pH 7-8, 50 mM phosphate; pH 8- 9, 50 mM Trizma; pH 9-11, 50 mM glycine. Each reaction contained 0.40 mM UDP- glucose, 0.11 mM MANT from a 50 mM DMSO stock, and UGT (0.7 pM for UGT16 and 1.1 pM for UGT43 and UGT53). The reaction was incubated at room temperature and quenched at 5 time points (2 min, 5 min, 10 min, 15 min, and 30 min) by transferring 50 pL of the reaction mixture to 70 pL of cold methanol. The reaction mixtures were analyzed via HPLC. The data was analyzed as means ± standard deviations of duplicate experiments.

[0231] Temperature optimum: Reactions were carried out at a temperature range to find the optimum temperature of UGT16, UGT43, and UGT53. Each reaction contained 0.40 mM UDP-glucose, 0.11 mM MANT from a 50 mM DMSO stock, and UGT (0.7 pM for UGT16 and 1.1 pM for UGT43 and UGT53). 50 mM phosphate buffer at the previously determined optimal pH was used and the reaction was carried out at temperatures ranging from 30 °C to 54 °C for UGT16 and UGT43 and 15 °C to 37 °C UGT53 on a thermocycler. The reaction was stopped at four different time points (5 min, 10 min, 15 min, and 30 min) by thermal denaturation at 95 °C for 20 seconds. The reaction mixtures were analyzed via HPLC. The data was analyzed as means ± standard deviations of duplicate experiments.

[0232] It was found that all UGTs displayed optimum reaction conditions in phosphate buffer pH 8.0, see Figure 4. The optimum temperature was 35 °C for UGT16, 39 °C for UGT43, and 30 °C for UGT53, see Figure 5.

[0233] 3.3 Kinetic characterization of UGT16, UGT43, and UGT53 with MANT

[0234] The above found option reaction conditions were used to establish the kinetic properties of the UGTs.

[0235] Kinetic parameters of UGT16, UGT43, and UGT53 with MANT were determined in 50 mM phosphate buffer at the optimal pH and the optimal temperature for the respective UGTs. Reaction mixtures consisted of 0.8 mM UDP-glucose, an appropriate UGT concentration (0.7 pM for UGT16, 1.1 pM for UGT43, and 0.5 pM for UGT53), and a MANT concentration ranging from 1000 pM to 11.56 pM from a 50 mM stock in DMSO. Reactions were performed on a thermocycler. The reaction was stopped at four different time points (2 min, 5 min, 10 min, and 15 min) by thermal denaturation at 95 °C for 20 seconds. The reaction mixtures were analyzed via HPLC. The data was analyzed as means ± standard deviations of duplicate experiments. Michaelis-Menten plots were generated and analyzed in R (https: / / www.R-project.org / ) using RStudio (https: / / www.RStudio.com).

[0236] These reaction conditions were used to establish the kinetic properties of the UGTs (Table 1). All UGTs demonstrated Michaelis-Menten-like kinetics, with UGT16 having the highest catalytic efficiency.

[0237] 3.4 Kinetic characterization of UGT16 with EANT, BANT, and IBANT

[0238] UGT16 was selected for further biochemical characterization with EANT, BANT, and IBANT.

[0239] Kinetic parameters of UGT16 with EANT, BANT, and IBANT were determined using the optimal pH and temperature achieved with MANT (50 mM phosphate buffer pH 8.0 at 35 °C). For EANT, reaction mixtures consisted of 0.40 mM UDP-glucose, 0.40 pM UGT16, and an EANT concentration ranging from 1000 pM to 11.56 pM from a 50 mM stock in DMSO. The reaction was stopped at four different time points (2 min, 5 min, 10 min, and 15 min). For BANT, reaction mixtures consisted of 0.4 mM UDP-glucose, 0.25 pM UGT16, and a BANT concentration ranging from 1000 pM to 11.56 pM from a 10 mM stock in DMSO (lower water solubility compared to MANT). The reaction was stopped at four different time points (1 min, 2 min, 3 min, and 5 min). For IBANT, reaction mixtures consisted of 0.40 mM UDP-glucose, 0.25 pM UGT16, and an IBANT concentration ranging from 1000 pM to 11.56 pM. The reaction was stopped at four different time points (1 min, 2 min, 3 min, and 5 min). Reactions were stopped by thermal denaturation at 95 °C for 20 seconds. The reaction mixtures were analyzed via HPLC. The data was analyzed as means ± standard deviations of duplicate experiments. Michaelis-Menten plots were generated and analyzed in R (https: / / www.R-project.org / ) using RStudio (https: / / www.RStudio.com).

[0240] UGT16 displayed a higher catalytic efficiency with EANT, BANT, and IBANT compared to MANT (Table 1).

[0241] Table 1. Kinetic properties of UGTs with MANT, EANT, BANT, and IBANT

[0242] Km Vmax kcat kcat / Km

[0243] UGT Acceptor

[0244] (pM) (pM min1) (s1) (s’1M1)

[0245] UGT16 MANT 347.1 ± 45.7 12.1 ± 1.2 0.287 ± 0.027 840 ± 190

[0246] UGT43 MANT 212.2 ± 19.1 2.8 ± 0.9 0.042 ± 0.013 203 ± 82

[0247] UGT53 MANT 164.5 ± 22.0 2.4 ± 0.7 0.079 ± 0.022 473 ± 69

[0248] UGT16 EANT 879.6 ± 89.8 35.8 ± 1.9 1.490 ± 0.081 1698 ± 81

[0249] UGT16 BANT 226.8 ± 17.7 15.6 ± 0.8 1.038 ± 0.055 4602 ± 602

[0250] UGT16 IBANT 259.9 ± 47.6 4.0 ± 0.1 0.267 ± 0.008 1048 ± 223

[0251] 3.5 Chemo tolerance of UGT16 towards MANT

[0252] The chemo tolerance of UGT16 was tested by reacting various concentrations of UGT16 with 1.0 mM UDP-glucose, and various concentrations of MANT from a 100 mM DMSO stock in 50 mM phosphate buffer pH 8.0 at 35 °C for 2, 5, 10, and 15 minutes. The reaction mixtures were analyzed via HPLC. The data was analyzed as means ± standard deviations of duplicate experiments.

[0253] This probing of UGT16 with increasing MANT concentrations revealed that UGT16 tolerated up to 20 mM MANT (Figure 7A&B), which also means that it tolerated up to 20% (v / v) DMSO, the solvent used for the MANT stock. This indicates that UGT16 is remarkably chemotolerant, making it an excellent candidate catalyst for industrial production of MANT-N-glucoside.

[0254] 3.6 Testing UGT43 with different UDP-sugars

[0255] The activity of UGT43 was additionally tested on MANT using UDP-xylose.

[0256] Specifically, to determine whether the enzyme could glycosylate methyl anthranilate using UDP-xylose, the following reaction was set up: 50 mM HEPES pH 7, 0.35 mM of UDP-xylose, 5 pM of UGT43 enzyme and 0.25 mM of the MANT substrate; incubation overnight at room temperature for 16 hours; and product formation was measured by HPLC. It was found that UGT43 could reach a conversion percentage of 99 %.

[0257] Example 4: UGT structural predictions

[0258] The crystal structure, PDB 5V2K (Protein data bank, Crystal structure of UDP- glucosyltransferase, UGT74F2 (T15A) (SEQ ID NO. 84), with UDP and 2-bromobenzoic acid; PDB DOI: https: / / doi.org / 10.2210 / pdb5v2k / pdb), was used to model the substrate, 2-bromobenzoic acid, into the AlphaFold models of enzymes UGT16, UGT43, and UGT53.

[0259] From this, residues that line the substrate binding site of the three enzymes were identified, see below. Here, the residues within 10 angstroms of the substrate are listed.

[0260] UGT16 5V2K 10 A residues:

[0261] Serl2, Prol3, Glyl4, Metl5, Glyl6, Leul8, Pro20, Val78, Lys79, Ile80, Glu81, Thr82, Arg83, Ile84, Ser85, Thr87, Aspll4, Leull5, Phel35, Prol36, Serl37, Thrl38, Glyl41, Phel45, Leul79, Leul80, Aspl81, Vall83, Glnl84, Serl90, Tyrl91, Ser272, Gly273, Gly274, Asn308, Phe311, Phe312, His360, Cys361, Gly362, Trp363, Asn364, Leu381, Tyr382, Ala383, Gln385, Lys386, Met387, and Asn388 of SEQ ID NO. 84.

[0262] UGT43 5V2K 10 A residues:

[0263] Ser29, Pro30, Gly31, Met32, Gly33, His34, Leu35, Pro37, Ala93, Arg94, Ile95, Glu96, Thr97, Leu98, Met99, SerlOO, Aspl26, Leul27, Phel28, Phel47, Prol48, Serl49, Thrl50, Glyl53, Phel57, Leul91, Leul92, Aspl93, Prol94, Vall95, Ala202, Tyr203, Val206, Ser279, Gly280, Asn315, Thr317, Tyr318, Phe319, His367, Cys368, Gly369, Trp370, Asn371, Leu388, Tyr389, Ala390, Glu391, Gln392, Lys393, Met394, and Asn395 of SEQ ID NO. 84.

[0264] UGT53 5V2K 10 A residues:

[0265] Phel4, Prol5, Phel6, Serl7, Glyl8, Hisl9, Ile20, Ala22, Trp46, Leu48, Phe50, Ile51, Phe87, Phe90, Ala91, Glu92, Ser93, Tyr94, Ile95, Leu96, Tyr98, Val99, Aspl31, Phel32, Phel33, Cysl34, Phel51, Leul52, Thrl53, Cysl54, Serl55, Phel58, Metl61, Metl62,

[0266] Pro200, Ser201, Gly202, Leu203, Thr208, Pro211, Trp212, Leu215, Arg263, Ser293,

[0267] Leu294, Lys295, His369, Cys370, Gly371, Trp372, Asn373, Tyr391, Ala392, Glu393,

[0268] Gln394, Gln395, Leu396, and Asn397 of SEQ ID NO. 84.

[0269] Figures 1, 2, and 3 shows illustrations of the crystal structures of enzymes UGT16,

[0270] UGT43, and UGT53, respectively.

[0271] Example 5: UGT16 variants

[0272] To investigate the structure-function relationship underlying the substrate preferences of UGT16, the active site was probed through site-directed mutagenesis.

[0273] Two strategies were employed to investigate the structure-function of UGT16. The first strategy aimed to explore the substrate preference of UGT16 observed in Table 5. Initially, MANT, EANT, BANT, and IBANT were docked into an AlphaFold32 model of UGT16 with UDP-Glucose from PDB entry 6SU6 (SEQ ID NO. 85) superimposed into the model.

[0274] Molecular docking of MANT, EANT, BANT, IBANT, and CANT into an AlphaFold32 model of UGT16 was performed in AutoDock Vina vl.1.2 using standard settings. To help define the grid space, binary complexes of protein and sugar donor were obtained by structurally aligning the UGT16 AlphaFold model on the crystal structure of PtUGTl from Polygonum tinctorium, which has a bound UDP-glucose molecule in its active site (PDB: 6SU6). The grid space was placed near the glucose moiety of UDP-glucose and docked poses were in-spected in PyMOL (v2.5.2). Poses, where the aniline was not pointing towards the anomeric carbon of UDP-glucose, were not considered.

[0275] For all docked substrates, a similar binding mode was found with the ester alkyl chain pointing in the same direction (Figure 9). Based on this, Ile80 and Ile84 were identified as targets for site-directed mutagenesis to understand the role of this residue pair. The two residues were swapped with bulkier residues, Trp / Phe, or a smaller residue, Ala, leading to the synthesis of variants, Ile80Phe / Trp / Ala, Ile84Phe / Trp / Ala, and Ile80 / 84Ala. Swapping the residues with bulkier residues could potentially improve catalytic efficiency with MANT, while replacing them with Ala would help elucidate the importance of the residue pair in AEAA glycosylation activity.

[0276] The second strategy utilized the active site architecture of another UGT known for its methyl salicylate activity, UGT71C3 (SEQ ID NO 86), as a template to alter the active site of UGT16. Residues within 5 A of docked MANT were swapped with the equivalent residue in UGT71C3 which was identified via structural alignment, yielding nine UGT16 variants: Lys79Ile, Ile80Leu, Leull5Phe, Phel35Leu, Prol36Thr, Serl37Cys, Glyl41Phe, Phel45Met, and Leul94Trp.

[0277] From the two strategies, a total of 16 variants were tested on the AEAA substrate panel: MANT, EANT, BANT, IBANT, and CANT (Figure 6). Conversion yields were determined by reacting 2.5 pM of UGT16 WT and variants with 2.0 mM UDP-glucose and 1 mM of MANT, EANT, BANT, IBANT, and CANT, respectively, from a 10 mM DMSO stock in 50 mM phosphate buffer pH 8.0 at 35 °C for 15 minutes. Reactions were stopped by thermal denaturation at 95 °C for 20 seconds. The reaction mixtures were analyzed via HPLC as means ± standard deviations of two independent experiments. The samples were analysed as described in example 2, section 2.2. However, the activity was quantified via the product peak area and subsequently normalized to the variant with the largest peak area for each substrate, respectively.

[0278] It was observed (see Figure 6) that any mutation on residues Ile80 and Ile84 led to a significant decrease in activity with all AEAAs with Ile84 being the most sensitive residue. Interestingly, the less invasive swap of Ile80 with Leu (the corresponding residue in UGT71C3) also led to a significant decrease in activity compared to WT UGT16. These results strongly indicate a critical role of both Ile80 and Ile84 in the AEAA glycosylating activity of UGT16. Other critical residues included Phel35 and Glyl41. Lastly, the Phel45Met variant demonstrated improved activity with the bulkier AEAAs while still retaining activity with MANT comparable to that of the WT UGT16. This included activity with CANT. Interestingly, the corresponding position in UGT53, which was active with CANT, is occupied by a Met.

[0279] Example 6: Gram-scale production and purification of MANT-N-glucoside

[0280] UGT16 was purified from the lysate of 22 L E. coli culture. This yielded 221 mg purified UGT16, which was utilised in a 2 L reaction consisting of 20 mM MANT and 30 mM UDP- glucose. After overnight incubation, 90 % of MANT was converted to MANT-N-glucoside.

[0281] Separation of the production from the remaining constituents of the reaction mixture via flash chromatography and subsequent evaporation in vacuo and lyophilisation yielded 9.3 g of an off-white powder at 99 % purity (HPLC). ID and 2D NMR confirmed the powder to be MANT-N-glucoside. The resulting product yield was 74.4 % from the reaction, with a recovery rate from the purification process of 82.3 %.

[0282] Example 7: Glycosylated anthranilate derivatives as bird repellent

[0283] Anthranilates, including MANT, are widely implemented as benign bird control agents, acting by deterring the birds from an area without harming them. Since glycosylation changes the physicochemical properties of a compound, including its volatility and solubility, the bird deterrence of MANT-N-glucoside could be drastically changed compared to MANT.

[0284] The bird-repellent properties of the glycosylated anthranilates are tested on live birds through a water-based formulation. Birds of the species, European starlings, are captured and undergo a two-week adaptation period in their respective pens. The now experimentally naive starlings are randomly assigned to one of three to six groups of 8- 10 birds where water consumption is now monitored to ensure consumption within the normal range. Each group are then assigned to receive one of two to five concentrations of the anthranilate-glucoside (dependent on the number of groups) where one group is used as a control which will receive tap water. Water with added anthranilate-glucoside is then provided for the respective test groups and the fluid intakes are monitored every hour. At the end of the test, unlimited food and water are provided for the birds to understand if any carry-over effect is observed after treatment.

[0285] The bird repellency of MANT-N-glucoside was tested in a one-day two-choice test with MANT-N-glucoside-treated and untreated sunflower achenes on red-winged blackbirds in captivity (Table 2). The feeding experiment was conducted at the United States Department of Agriculture, National Wildlife Research Center's (NWRC) outdoor animal research facility in Fort Collins, Colorado (USA). 11 male red-winged blackbirds (Agelaius phoeniceus) were live-captured for the experiment. The capture, care, and use of all birds associated with the feeding experiment were approved by the NWRC Animal Care and Use Committee.

[0286] Blackbirds were maintained in a 4.9 x 2.4 x 2.4-m cage within a wire mesh-sided building for at least two weeks before the experiment (i.e., quarantine, holding; Table 2). Free access to grit (sand) and a maintenance diet was provided to all birds during quarantine and holding. The maintenance diet included two parts millet : one part cracked corn : one part milo : one part safflower. The experiment was conducted in visually-isolated individual cages (0.9 x 1.8 x 0.9 m) within a wire mesh-sided building. Water was provided ad libitum to all birds throughout the experiment (quarantine, holding, preference experiment).

[0287] Seed treatments offered during the experiment were formulated by applying aqueous suspensions (60 mL / kg) to whole oilseed sunflower (Northern Colorado Feeders Supply, Fort Collins, CO, USA) using a rotating mixer and household spray equipment. All blackbirds were offered untreated sunflower seed ad libitum in two food bowls for five days of acclimation in individual cages (Table 2). Each blackbird was subsequently offered one bowl of untreated sunflower and one bowl of sunflower treated with 1% of MANT-N-glucoside (targeted concentration, wt / wt) at 08:00 h during the test (i.e., Monday; Table 2). Daily sunflower consumption was measured during the one-day preference experiment. Unconsumed sunflower seeds (remaining in each food bowl) and spillage were collected (at 08:00 h on day subsequent to test; Tuesday) and weighed (± 0.1 g). Weight change (e.g., desiccation) of sunflower seeds was measured daily by weighing seeds offered within a vacant cage throughout the preference experiment.

[0288] Table 6. Experimental timeline depicting the days or weeks for each experimental phase

[0289] Experimental phase Timeline

[0290] Quarantine / Holding 2+ weeks

[0291] Acclimation Wednesday-Sunday (5 days)

[0292] Test Monday (1 day)

[0293] The dependent measure for the preference experiment was the average (i.e., daily) test consumption of treated and untreated sunflower seeds. Descriptive statistics (± SEM) and a paired t-test were used to summarize and analyze the consumption of treated and untreated seeds throughout the experiment.

[0294] It was found that relative to average consumption of treated sunflower, blackbirds consumed significantly more untreated sunflower during the preference experiment (P = 0.0005). Blackbirds consumed an average of 0 ± 0.7 g of treated sunflower and 6.9 ± 0.8 g of untreated sunflower during the experiment. We therefore conclude that blackbirds preferred untreated sunflower when offered a choice between untreated sunflower and sunflower treated with 1% anthranilate glucoside (w / w).

[0295] In a similar study using the same experimental setup (Avery et al 1995), it was previously showed that MANT had a 70% bird deterrency (70 % decrease in consumption when applied to the feed at a concentration of 1.0 % (w / w)). In this study, a 100 % decrease in consumption was achieved when applying MANT-N-glucoside to the feed at 1.0 % (w / w), so MANT-N-glucoside seems to be even more efficient as bird repellent compared to the non-glycosylated parent compound.

[0296] Example 8: Glycosylated anthranilate derivatives as insect repellent

[0297] The insect-repellent properties of the glycosylated anthranilates are tested on live insects through a water-based formulation. Insects of the species, Drosophila suzukii, are used in the Two-Choice Trap Assay. Ten female flies are placed in a Petri dish containing two traps. Traps are made with 1.5 ml microcentrifuge tubes with an opening cut in the bottom of the tube. Both traps contain the fly's normal laboratory food at the base. The neck of one trap has a filter paper with the test compound while the other trap acts as a control containing only the solvent (water in this case). Five microliters of water (negative control) and five microliters of 10% DEET in hexane (positive control) or test compounds in water are applied to the stem part of filter paper inserted into the upper part of the pipette tip near the entrance of the trap to allow flies to walk across the treated surface. Traps are placed in a fume hood for 5 minutes to allow the hexane to volatilize before being placed in the 1% agarose-treated Petri dish chamber.

[0298] Example 9: Potential of glycosylated anthranilate derivatives in cell factories

[0299] As mentioned, previously developed cell factories for MANT to replace petroleum-based synthesis are limited by MANT's toxicity towards the microbial host. To investigate if this effect is alleviated by glycosylation, the growth of two common cell factories, E. coli and Pseudomonas putida, was monitored upon exposure to a range of MANT and MANT-N- glucoside concentrations (Figure 8).

[0300] Both organisms exhibited a dose-dependent growth-inhibition by MANT (Figure 8A&B), with P. putida seemingly being more sensitive than E. coli. However, no response to MANT-N-glucoside was found in any of the organisms (Figure 8C&D), indicating that glycosylation could be a strategy to enhance cell factory titers of MANT and other AEAAs.

[0301] Example 10: Degradation of MANT-N-glucoside

[0302] Given how the glycosylation of MANT can be used to remediate its antimicrobial effects, it can be used as a strategy to increase the titers in microbial production. However, the compound of interest could potentially still be the aglycon. Therefore, the possibility of retrieving the aglycon from the corresponding N-glucoside was investigated. This was first examined via chemical degradation using acid to catalyze the hydrolytic reaction. Here, it was observed that using 100 mM HCI cleaves the glycosidic bond of the entire 5 mM MANT-N-glucoside mixture within an hour. This could also be achieved with 10 mM HCI but only within approximately 5 hours.

[0303] References

[0304] Avery et al 1995. Methyl anthranilate as a rice seed treatment to deter birds. The Journal of Wildlife Management, Vol. 59, No. 1 (Jan 1995), pp. 50-56. De Bruyn et al. 2015. Development of an in vivo glucosylation platform by coupling production to growth: Production of phenolic glucosides by a glycosyltransferase of Vitis vinifera. Biotechnol. Bioeng (2015) 112, 1594-1603, doi.org / 10.1002 / bit.25570.

[0305] Lee et al 2004. One-pot enzymatic synthesis of UDP-D-glucose from UMP and Glucose- 1-Phosphase using ATP regeneration system. Journal of Biochemistry and Molecular Biology, Vol. 37, No. 4, July 2004, pp. 503-506.

[0306] Lee et al 2019. Synthesis of Methylated Anthranilate Derivatives Using Engineered Strains of Escherichia coli. J. Microbiol. Biotechnol. 2019; 29(6): 839-844. https: / / doi.org / 10.4014 / jmb.1904.04022 Luo et al 2019. Microbial production of methyl anthranilate, a grape flavor compound. pnas.org / cgi / doi / 10.1073 / pnas.1903875116.

Claims

CLAIMS1. An in vitro method for glycosylation of an anthranilate derivative, comprising the steps of a. providing an UDP-glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40, and having the ability of glycosylating the anthranilate derivative, b. mixing said enzyme with a UDP-sugar and an anthranilate derivative having the structure of formula I, wherein R is an alkyl:formula I c. letting the mixture react for a selected period of time, and d. optionally purifying the glycosylated anthranilate derivative, wherein glycosylation takes place at the amine moiety of the anthranilate derivative having the structure of formula I.

2. The in vitro method according to claim 1, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

3. The in vitro method according to claim 1 or 2, wherein the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14.

4. The in vitro method according to claim 1, wherein R is CnH2n+i and n is >1, preferably n is 2-4; wherein the UDP-glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14; and wherein the UDP-sugar is UDP-glucose.

5. The in vitro method according to any one of claims 1-4, wherein the UDP- glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

6. A composition comprising(I) an anthranilate derivative having the structure of formula I, wherein R is an alkyl,(II) an UDP-glycosyltransferase enzyme (i) consisting of or comprising an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consisting of or comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40, and having the ability of glycosylating an anthranilate derivative, and(III) a UDP-sugar.

7. The composition according to claim 6, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

8. The composition according to claim 6 or 7, wherein the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14.

9. The composition according to claim 6, wherein R is CnH2n+i and n is >1, preferably n is 2-4; wherein the UDP-glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14; and wherein the UDP-sugar is UDP-glucose.

10. The composition according to any one of claims 6-9, wherein the UDP- glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

11. Use of an UDP-glycosyltransferase enzyme in a process of glycosylating an anthranilate derivatives having the structure of formula I, wherein R is alkyl, and wherein said UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40, and has the ability of glycosylating the anthranilate derivative.

12. The use according to claimll, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

13. The use according to claim 11 or 12, wherein the UDP-glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO.14.

14. The use according to claim 11, wherein R is CnH2n+i and n is >1, preferably n is 2- 4; wherein the UDP-glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14; and wherein the UDP-sugar is UDP-glucose.

15. The use according to any one of claims 11-14, wherein the UDP-glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

16. An in vivo method for producing a / V-glycosylated anthranilate derivative, comprising the steps of a. providing a microbial cell comprising(I) a nucleic acid sequence encoding a UDP-glycosyltransferase enzyme, wherein said enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40, and has the ability of glycosylating an anthranilate derivative having the structure of formula I, wherein R is an alkyl, and(II) one or more nucleic acid sequences encoding one or more enzymes for synthesizing the anthranilate derivative having the structure of formula I, wherein R is an alkyl; b. growing said microbial cell in a growth medium, and expressing said UDP- glycosyltransferase enzyme and said one or more enzymes for synthesizing the anthranilate derivative, wherein a UDP-sugar is provided (i) as a component of the growth medium or (ii) by said microbial cell further comprising one or more nucleic acid sequences encoding one or more enzymes for synthesis of the UDP-sugar and said microbial cell expressing said one or more enzymes for synthesis of the UDP-sugar; andc. optionally purifying the / V-glycosylated anthranilate derivative.

17. The in vivo method according to claiml6, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

18. The in vivo method according to claim 16 or 17, wherein R is methyl, and wherein the UDP-sugar is UDP-glucose.

19. The in vivo method according to any one of claims 16-18, wherein the microbial cell is selected from a strain of E. coli, C. glutamicum, S. cerevisiae, and P. putida, preferably selected from a strain of E. coli and P. putida.

20. The in vivo method according to any one of claims 16-19, wherein the one or more enzymes for synthesizing the anthranilate derivative comprise (i) a methyltransferase or (ii) an anthranilate-CoA ligase and an anthraniloyl-coenzyme A (CoA): methanol acyltransferase.

21. The in vivo method according to any one of claims 16-20, wherein the one or more enzymes for synthesis of the UDP-sugar comprises a sucrose synthase.

22. The in vivo method according to any one of claims 16-21, wherein the UDP- glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14.

23. The in vivo method according to any one of claims 16-22, wherein the UDP- glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

24. The in vivo method according to any one of claims 16-23, wherein R is CnH2n+i and n is >1, preferably n is 2-4; wherein the UDP-glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14; and wherein the UDP-sugar is UDP-glucose.

25. A method for producing an anthranilate derivative, comprising producing a N- glycosylated anthranilate derivative in vivo according to any one of claims 16-24, wherein said / V-glycosylated anthranilate derivative is secreted by or extracted from said microbial cell, followed by chemically or enzymatically breaking the glycosidic bond of the / V-glycosylated anthranilate derivative in vitro, thereby obtaining the anthranilate derivative.

26. A method for producing an insect and / or bird repellent composition, comprising the steps of a. providing a UDP-glycosyltransferase enzyme, a UDP-sugar, and an anthranilate derivative having the structure of formula I, wherein R is an alkyl, b. mixing said enzyme, said UDP-sugar, and said anthranilate derivative, c. letting the enzyme catalyze glycosylation of the anthranilate derivative for a selected period of time to produce the insect and / or bird repellent composition comprising a / V-glycosylated anthranilate derivative, and e. optionally further formulating the insect and / or bird repellent composition comprising the / V-glycosylated anthranilate derivative by addition of one or more additives.

27. The method according to claim 26, wherein R is CnH2n+i and n is 1-4; and wherein the UDP-sugar is UDP-glucose.

28. The method according to claim 26 or 27 , wherein R is methyl, and wherein the UDP- sugar is UDP-glucose.

29. The method according to any one of claims 26-28, wherein the UDP- glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence selected from SEQ ID NO. 14, 32, and 40 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14, 32, or 40, and has the ability of glycosylating the anthranilate derivative.

30. The method according to any one of claims 26-29, wherein the UDP- glycosyltransferase enzyme (i) consists of or comprises an amino acid sequence SEQ ID NO. 14 or (ii) consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14.

31. The method according to claim 26, wherein R is CnH2n+i and n is >1, preferably n is 2-4; wherein the UDP-glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14; and wherein the UDP-sugar is UDP-glucose.

32. The method according to any one of claims 26-31, wherein the UDP- glycosyltransferase enzyme consists of or comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO. 14 and wherein said amino acid sequence comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

33. The method according to any one of claims26-32, wherein the one or more additives is selected from a fatty acid, an antioxidant, a buffering solution, a surfactant, and a cosolvent.

34. Use of a glycosylated anthranilate derivative as an insect and / or bird repellent, wherein the glycosylated anthranilate derivate has the structure of formula II, wherein R is CnH2n+i and n is 1-4; and X is glucoseformula II.

35. The use according to claim 32, wherein R is methyl.

36. A method for repelling birds from an item or area, comprising applying an aqueous solution comprising a glycosylated anthranilate derivative to the item or area, wherein the glycosylated anthranilate derivate has the structure of formula II, wherein R is CnH2n+i and n is 1-4, and X is glucose.

37. The method according to claim 36, wherein the glycosylated anthranilate derivate is methyl anthranilate / V-glucoside.

38. The method according to claim 36 or 37, wherein the item or aera is selected from areas of vegetation, such as grass fields, including recreational areas and sports fields such as golf courses and football / soccer field, or agricultural fields; buildings and roads, such as airports and airfields; or food processing facilities and surrounding areas.

39. A polypeptide having glycosyltransferase activity (enzyme classification EC: 2.4.

1. - ), wherein the amino acid sequence of said polypeptide has at least 75% sequence identity with SEQ ID NO. 14, and wherein said amino acid sequence comprises amino acid residue substitution Phel45Met with respect to SEQ ID NO. 14.

40. The polypeptide according to claim 39, wherein the polypeptide, compared to SEQ ID NO 14, has improved efficiency in glycosylating (I) anthranilate derivatives according to formula I, wherein R is an alkyl -CnH2n+i and n is >1, preferably R is ethyl, propyl, butyl, or isobutyl; and / or (II) anthranilate derivatives according to formula III, wherein R2is a cycloakyl -CnH2n-i and n is at least 3, preferably R2is cyclohexyl.

41. The polypeptide according to claim 39 or 40, wherein the polypeptide, compared to SEQ ID NO 14, has improved efficiency in glycosylating cyclohexyl anthranilate.

42. The polypeptide according to any one of claims 39-41, wherein the amino acid sequence of the UDP-glycosyltransferase enzyme comprises one or more of amino acid residues selected from Ile80, Ile84, Phel35 and Glyl41 of SEQ ID NO. 14.

43. A composition comprising a polypeptide according to any one of claim 39-42.

44. An in vitro method for / V-glycosylation of an anthranilate derivative, comprising the steps of a. providing an UDP-glycosyltransferase enzyme according to any one of claims 38-42, b. mixing said enzyme with a UDP-sugar and an anthranilate derivative having the structure of formula III, wherein R2is a cycloalkyl:formula III c. letting the mixture react for a selected period of time, d. optionally purifying the / V-glycosylated anthranilate derivative.

45. The in vitro method according to claim 44, wherein R2is cyclohexyl; and wherein the UDP-sugar is UDP-glucose.

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