WO2017207484A1

WO2017207484A1 - Mutant sucrose synthases and their uses

Info

Publication number: WO2017207484A1
Application number: PCT/EP2017/062885
Authority: WO
Inventors: Tom Desmet; Griet DEWITTE; Margo DIRICKS
Original assignee: Universiteit Gent
Priority date: 2016-05-31
Filing date: 2017-05-29
Publication date: 2017-12-07

Abstract

The present invention relates to the field of enzymatic glycosylation reactions. More specifically, the present invention provides mutant sucrose synthases which efficiently catalyze the conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose. The latter UDP-glucose can then be used as a donor to glycosylate various substrates via the usage of a glycosyltransferase. A specific glucosyltransferase capable of glycosylating a specific set of acceptors is further provided.

Description

Mutant sucrose synthases and their uses

Technical field of invention

Background art

Sucrose (Sue) is a major photosynthetic end-product in plants and plays an important role in their development, growth, carbon storage, stress protection, and signal transduction (Winter & Huber 2000). One of the enzymes involved in its metabolism is Sucrose Synthase (SuSy, EC 2.4.1.13), which catalyzes the reversible conversion of a nucleoside diphosphate (NDP) and Sue into NDP-glucose and fructose. The first report of this enzyme dates back to 1955 and subsequent research was mainly focused on plant SuSys (Cardini et al. 1955). Forty-four years later, in 1999, the first prokaryotic SuSy was purified from the cyanobacterial Anabaena and recently also SuSys from non-photosynthetic bacteria were characterized (Porchia, Curatti, & Salerno, 1999; Diricks, De Bruyn, Van Daele, Walmagh, & Desmet, 2015). The sugar nucleotides produced by SuSy are mainly directed toward cellulose or starch biosynthesis in plants, whereas they are linked to the synthesis of glycogen and other structural polysaccharides in bacteria (Haigler et al. 2001; Baroja-Fernandez et al. 2003; Koch 2004; Curatti et al. 2008).

Besides its important physiological role, SuSy also has a lot of potential in industrial context. Indeed, plant and cyanobacterial SuSys have been extensively used in coupled processes together with glycosyltransferases (GTs) to create valuable (pharmaceutical) glycosides in a cost-effective way (Brinkmann et al. 2001; Masada et al. 2007; Son et al. 2009; Terasaka et al. 2012; Gutmann et al. 2014; Bungaruang et al. 2013; Schmolzer et al. 2015). In such a one-pot reaction, SuSy provides and regenerates the expensive UDP-GIc in situ, which is subsequently used as donor substrate by a GT that attaches the sugar moiety to an acceptor. Glycosylation of small molecules, secondary metabolites in particular, also occurs in vivo and has a profound impact on their solubility, stability or bioactivity (Desmet et al. 2012). Although several nucleoside diphosphates (UDP, CDP, GDP, ADP, TDP) have been shown to act as acceptor nucleotides for SuSy, biochemical characterization has revealed that plant enzymes preferentially use UDP whereas the small amount of data available for bacterial SuSys points towards a preference for ADP (Delmer 1972; Tsai 1974; Morell & Copeland 1985; Tanase & Yamaki 2000; Baroja- Fernandez et al. 2003; Baroja-Fernandez et al. 2012; Murata 1971; Nomura & Akazawa 1973; Moriguchi & Yamaki 1988; Ross & Davies 1992; Figueroa et al. 2013; Wu et al. 2015; Diricks et al. 2015). For example, SuSy from Acidothiobacillus caldus (SuSyAc) has a K_m value for UDP (7.8 mM) which is 25 times higher compared to ADP (0.3 mM) (Diricks et al. 2015). The latter authors further replaced amino acids of SuSyAc which were thought to be responsible for the nucleotide (ADP) preference by those occurring in plant SuSys'. However, the results couldn't confirm the hypothesis that specific amino acid positions are responsible for the difference in nucleotide preference between bacteria and plants.

As plant Susy's suffer from low stability and poor expression levels, there is a need to provide bacterial SuSys's having UDP as a preferred nucleotide which can then be used as a donor to glycosylate substrates via a glycosyltransferase.

The present invention provides mutant SuSyAc's with excellent properties for use in such coupled glycosylation reactions and provides a particular glycosyltransferase -previously disclosed in US 2016/0010133- which is particularly useful in the latter reactions. Brief Description of figures

Figure 1: Amino acid distribution of plant (upper part) and bacterial (lower part) SuSys at positions around the nucleotide substrate including those constituting the QN motif. Residues within 4A of the uracil moiety of UDP (trapped within the crystal structure of SuSyAtl) are marked with an asterisk. The sequences of SuSyAtl and SuSyAc were chosen as plant and bacterial representative, respectively and the QN motif is highlighted in bold.

Figure 2: QN motif of SuSyAtl (A) and SuSyAc (B) using a crystal structure (PDB ID 3S27) and a homology model, respectively. Possible hydrogen bonds are represented by dashed lines.

Figure 3: Kinetic parameters for UDP of SuSyAc WT and QN mutants. 1M of Sue was used as co- substrate. Km values are reported in mM (A), V_max values in U-mg ¹ (B). Figure 4: Schematic representation of coupled reaction between SuSy and GT as an approach for the glycosylation of target acceptors with UDP-glucose (re)generation

Figure 5: Acceptor promiscuity of UGT-76GlSr. Qualitative survey of enzymatic activity towards 58 diverse acceptors. Reaction conditions: 0.25 mg mL ¹ UGT-76GlSr, 0.5 mM acceptor, 0.5 mM UDP- glucose. Reactions were buffered at pH 7.0, 37 ⁵C Figure 6: Glycosylation of polydatin by direct action of UGT-76GlSr (full line) and coupled reaction between SuSyAc double mutant LMDKVVA and UGT-76GlSr (dashed line). The production of the O- glucoside of polydatin is presented in function of time. Reaction conditions: 1 mg mL ¹ UGT-76GlSr, 1 mM polydatin, 2 mM UDP-glucose (full line); 50 μg mL ¹ SuSy, 1 mg mL ¹ UGT-76GlSr, 1 mM polydatin, 1 mM UDP, 100 mM Sue. Both reactions were buffered at pH 7.5, 37°C

Description of invention

Sucrose Synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate (NDP) into NDP-glucose and fructose. Biochemical characterization of several plant and bacterial SuSys has revealed that the eukaryotic enzymes preferentially use UDP whereas prokaryotic SuSys prefer ADP as acceptor. In the present invention, SuSy from the bacterium Acidithiobacillus caldus, which has a higher affinity for ADP as reflected by the 25-fold lower K_m value compared to UDP, was used to scrutinize the effect of introducing plant residues at positions in a putative nucleotide binding motif surrounding the nucleobase ring of NDP. Hence, the present invention discloses mutants having similar activities as the wild type enzyme but having significantly reduced K_m values for UDP (up to 60 times). Furthermore, the present invention also led to the establishment of bacterial SuSy mutants that are suitable for the recycling of UDP during glycosylation reactions. The latter has been demonstrated -as a non-limiting example- by combining one of the mutants with a glycosyltransferase in a one-pot reaction for the glucosylation of polydatin, a derivative of the polyphenolic anti-oxidant resveratrol. Moreover, the present invention discloses a glycosyltransferase that was found to have a surprisingly broad specificity, which can be exploited for the glucosylation of a wide range of acceptor molecules. Hence, the present invention in first instance relates to mutated sucrose synthase characterized by:

-its sequence comprising the amino acid sequence of SEQ ID N° 1,

-its sequence containing at least one of the following mutations in SEQ ID N° 1: K639R, L637M, T640V, L637M and T640V, L636Q and K639R and V641R and A642N, L637M and K639R and T640V, or, L636Q and L637M and K639R and T640V and V641R and A642N, and

- it has a higher affinity with the substrate UDP when compared to the wild type enzyme.

In other words, the present invention relates to mutated sucrose synthase characterized by:

-its sequence comprising the amino acid sequence of SEQ ID N° 1 which contains at least one of the following mutations: K639R, L637M, T640V, L637M and T640V, L636Q and K639R and V641R and A642N, L637M and K639R and T640V, or, L636Q and L637M and K639R and T640V and V641R and A642N, and

- it has a higher affinity with the substrate UDP when compared to the wild type enzyme. The term 'sequence comprising the amino acid sequence of SEQ ID N°l refers to the following amino acid sequence from Acidithiobacillus caldus having UniProt ID A0A059ZV61 (and wherein the amino acid positions 636, 637, 639, 640, 641 and 642 are underlined):

The term 'sequence containing at least one of the following mutations in SEQ ID N° 1: K639R, L637M, T640V, L637M and T640V, L636Q and K639R and V641R and A642N, L637M and K639R and T640V, or, L636Q and L637M and K639R and T640V and V641R and A642N' refers to the fact that specific amino acids at specific positions, or combinations thereof, of wild type SEQ ID N° 1 (i.e. at amino acid positions 636, 637, 639, 640, 641 and/or 642) have been substituted by other specific amino acids such as L636Q, L637M, K639R, T640V, V641R and A642N. The latter substitutions can be obtained by any method known in the art such as site-directed mutagenesis. The term 'at least one of the following mutations' specifically refers to the single mutants K639R, L637M, T640V, or, to the double mutant L637M and T640V, or, to the triple mutant L637M and K639R and T640V, or, to the quadruple mutant L636Q and K639R and V641R and A642N, or, to the sextuple mutant L636Q and L637M and K639R and T640V and V641R and A642N.

The term 'a higher affinity with the substrate UDP when compared to the wild type enzyme' relates to the fact that the mutants of the present invention show a significantly lower (i.e. 2x lower, 3x lower, 4 x lower, ... ,60 x lower,...) Km value vis-a-vis UDP when compared to the Km value of the wild type enzyme Km value vis-a-vis UDP.

The present invention also relates to a mutated sucrose synthase characterized by:

-its sequence having at least 90% sequence identity with SEQ ID N° 1, -its sequence containing at least one of the following mutations in SEQ ID N° 1: K639 , L637M, T640V, L637M and T640V, L636Q and K639R and V641R and A642N, L637M and K639R and T640V, or, L636Q and L637M and K639R and T640V and V641R and A642N, and

- it has a higher affinity with the substrate UDP when compared to the wild type enzyme. The term 'sequence having at least 90% sequence identity with SEQ ID N° refers to variants of the enzyme comprising the amino acid sequence of SEQ ID N° 1 which have at least 90% sequence identity (i.e. having 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity with SEQ ID N° 1). Hence, orthologues, or genes in other genera and species (than the species Acidithiobacillus caldus from which SEQ ID N° 1 is derived which encode for a polypeptide with at least 90% identity at amino acid level are part of the present invention. The percentage of amino acid sequence identity is determined by alignment of the two sequences and identification of the number of positions with identical amino acids divided by the number of amino acids in the shorter of the sequences x 100. The latter 'variant' may also differ -besides that its sequence contains at least one of the following mutations in SEQ ID N° 1: K639R, L637M, T640V, L637M and T640V, L636Q and K639R and V641R and A642N, L637M and K639R and T640V, or, L636Q and L637M and K639R and T640V and V641R and A642N- from the protein as depicted by SEQ ID N° 1 only in further conservative substitutions and/or modifications, such that the ability of the protein to have enzyme activity is retained. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of protein chemistry would expect the nature of the protein to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also (or alternatively) be proteins as described herein modified by, for example, the deletion or addition of amino acids that have minimal influence on the enzyme activity as defined above, secondary structure and hydropathic nature of the enzyme.

The present invention thus further relates to an isolated nucleic acid encoding for a mutant sucrose synthase as defined above. An example of a nucleic acid encoding for a mutant sucrose synthase of the present invention (= SEQ ID N° 2) is as follows (the codons of which at least one should be mutated according to the present invention are underlined):

The present invention also relates to a vector comprising a nucleic acid as described above.

The present invention further relates to a host cell comprising a vector as described above.

The present invention further relates to the usage of the mutated sucrose synthases as defined above to produce UDP-glucose. In other words, the present invention discloses mutant sucrose synthases that efficiently catalyze the conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose.

The present invention also relates to the usage of mutated sucrose synthase to produce UDP-glucose wherein said mutated sucrose synthase is characterized by: -its sequence comprising the amino acid sequence of SEQ ID N° 1,

-its sequence containing at least the following mutations in SEQ ID N° 1: L636Q and A642N, and -it has a higher affinity with the substrate UDP when compared to the wildtype enzyme.

In other words, the present invention also relates to the usage of mutated sucrose synthase to produce UDP-glucose wherein said mutated sucrose synthase is characterized by: -its sequence comprising the amino acid sequence of SEQ ID N° 1 which contains at least the following mutations: L636Q and A642N, and

-it has a higher affinity with the substrate UDP when compared to the wildtype enzyme.

The latter usage takes preferably place when the concentration of sucrose is higher than 200 mM (i.e. 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 mM...). More specifically, the latter usage takes place when the concentration of sucrose is about 1M.

Moreover, the present invention specifically relates to mutated sucrose synthase characterized by: -its sequence comprising the amino acid sequence of SEQ ID N° 1,

-its sequence containing at least one of the following mutations in SEQ ID N° 1: K639 , L637M, or, L637M and T640V, and

- it has a both a higher affinity and a higher activity with the substrate UDP when compared to the substrate ADP.

In other words, the present invention specifically relates to mutated sucrose synthase characterized by: -its sequence comprising the amino acid sequence of SEQ ID N° 1 which contains at least one of the following mutations: K639 , L637M, or, L637M and T640V, and

The term 'both a higher affinity and a higher activity with the substrate UDP when compared to the substrate ADP' indicates that for these 3 particular mutants the Km values for UDP are lower than for ADP, and that the Vmax values for UDP are higher than for ADP.

The present invention further relates to a process to glycosylate a substrate comprising: -using a mutated sucrose synthase as indicated above to produce UDP-glucose,

-using a glycosyltransferase and said UDP-glucose to covalently attach a glucose moiety to said substrate.

The latter refers to the fact that present invention also led to the establishment of bacterial SuSy mutants that are suitable for the recycling of UDP during glycosylation reactions. With the term 'a glycosyltransferase' is meant any enzyme that uses NDP-glucose as a donor to glycosylate any substrate or acceptor (where NDP stands for a nucleoside diphosphate, for example UDP).

More specifically, the present invention relates to a process as described above wherein said glycosyltransferase is a glycosyltransferase comprising the amino acid sequence corresponding to UniProt ID Q6VAB4 -previously described by US 2016/0010133- and the following amino acid sequence (SEQ ID N° 3):

The present invention further relates to a process as described above wherein the substrate is chosen from the list of: vanillin, para-nitrophenol, anisyl alcohol, catechin, epicatechin, hesperetin, resveratrol, phloretin, curcumin, polydatin, salicin, p-nitrophenol a-glc, p-nitrophenol a-gal, esculin, ethanol, t-cyclohexanediol, cinnamyl alcohol, phenol, orcinol, methylgallate, ethylgallate and propylgallate. Indeed, the present invention discloses a particular glycosyltransferase (corresponding to SEQ ID N° 3 and previously described by US 2016/0010133) which was found to have a surprisingly broad specificity and which can thus be exploited for the glucosylation of a wide range of acceptor molecules. More specifically, the present invention relates to a process as described above wherein said substrate is polydatin.

Moreover, the present invention relates to the usage of a glycosyltransferase comprising the amino acid sequence of SEQ ID N° 3, or functional variants thereof, to glycosylate a substrate chosen from the list of: vanillin, para-nitrophenol, anisyl alcohol, catechin, epicatechin, hesperetin, resveratrol, phloretin, curcumin, polydatin, salicin, p-nitrophenol alpha-glc, p-nitrophenol alpha-gal, esculin, ethanol, t-cyclohexanediol, cinnamyl alcohol, phenol, orcinol, methylgallate, ethylgallate and propylgallate.

It should be noted that the usage of variants or fragments of said glycosyltransferase comprising the amino acid sequence of SEQ ID N° 3 are also part of the present invention. Variants or fragments are proteins/enzymes as described herein modified by, for example, the deletion, substitution or addition of amino acids that have no or minimal influence on the enzyme activity as defined above. The latter variants or fragments can thus be defined as "functional variants/fragments" of said glycosyltransferase comprising the amino acid sequence of SEQ ID N° 3.

The present invention will further be illustrated by the following non-limiting examples. Examples

Materials and methods

'Materials'

Unless otherwise stated, all chemicals were bought from Sigma-Aldrich, Merck or Carbosynth and were of the highest purity. 'Amino acid distribution'

All amino acid sequences annotated as Sucrose Synthase were retrieved from the UniProtKB database. Sequences that were not unique, did not start with a methionine, were too long (>2000 amino acids), too short (<600 amino acids) or contained undefined amino acids were removed. In total, 68 prokaryotic sequences and 110 plant sequences were retained and aligned separately with Clustal Omega (default parameters) (Sievers et al. 2011). Per species, only one isoform was considered. The amino acid distribution of the positions in the QN domain were calculated using a simple python script. 'Homology modeling'

To model the structure of SuSyAc, the l-TASSER server for protein and structure prediction was used with the crystal structure of SuSyAtl (PDB ID 3S27, chain A) as template. With a C-score of 2, the homology model can be considered of high quality. To evaluate the interactions of SuSyAc with the nucleotide substrate, the homology model was superposed with the crystal structure of SuSyAtl, which includes UDP.

'Site-directed mutagenesis'

The SuSy sequence from Acidithiobacillus caldus (SuSyAc, UniProt ID A0A059ZV61), which is codon optimised for Escherichia coli (E. coli), provided with a C-terminal His₆-tag and cloned into a pXCP34h expression vector was used as template DNA to construct all QN mutants (Aerts et al. 2011; Diricks et al. 2015). Site-directed mutations were introduced with a modified two-stage megaprimer based whole plasmid PCR method (Sanchis et al. 2008). In each case, oMEM0351_RV_5'rrnB T2 (5'- AAAGGGAATAAGGGCGACAC-3') was used as reverse primer and forward primers are described in Table 1.

Table 1. Primers used for site-directed mutagenesis of Sucrose Synthase from Acidithiobacillus caldus (SuSyAc). Codons and amino acids subjected to mutagenesis are underlined

Mutant Template SEQ ID N° Fw Primer Sequence (5'→ 3')

The PCR mix contained Q5 reaction buffer, 0.02 (U-μΙ^"1) Q5 High-Fidelity DNA Polymerase (Bioke), 0.2 mM dNTP mix, 0.002-0.02 ng-μΙ ¹ template plasmid DNA, 0.5 μΜ forward and reverse primer in a total volume of 50 μΙ. The amplification program started with an initial denaturation (30 s at 98°C), followed by 5 cycles of denaturation for 10 s at 98°C, annealing for 20 s at 66°C and extension for 30 s-kb ¹ (size megaprimer) at 72°C. The second stage consisted of 25 cycles of 10 s at 98°C and extension for 1 min-kb ¹ (size whole plasmid) at 72°C and one final extension of 2 min at 72°C. After digestion of the template DNA by Dpn\ (Westburg), mutant plasmids were transformed in E. coli BL21 (DE3) (Novagen). All constructs were subjected to nucleotide sequencing (LGC genomics sequence service, Berlin).

'Enzyme production and purification'

His₆-tagged SuSyAc WT and SuSyAc mutants were constitutively expressed in E. coli BL21 (DE3) and purified by Ni-NTA chromatography according to the protocol previously described by Diricks et al. (Diricks et al. 2015).

The gene coding for UGT-76GlSr (UniProt ID Q6VAB4) was chemically synthesized with a codon usage that is optimal for expression in E. coli. It was subsequently inserted into a pET21a vector via Gibson Assembly (Gibson et al. 2009), using the primers listed in Table 2. As a result, a C-terminal His₆-tag (underlined) is added to the protein sequence of SEQ ID N° 3:

Ampicillin (100 μg/mΙ) was used for selection of clones, and correct inserts were revealed via sequencing (LGC Genomics). UGT-76GlSr inoculum was routinely grown at 37 °C in 5 mL LB medium supplemented with 100 μg/mL ampicillin. After overnight incubation, 1% (v/v) of inoculum was added to 1 L shake flasks containing 250 mL LB medium, supplemented with ampicillin (100 μg/mL). After approximately 2 hours of incubation at 37 °C (ΟD₆₀₀≈ 0.6), expression was induced by adding 1 mM IPTG to the inoculum. After 18h incubation at 16°C, the culture was centrifuged and the obtained cell pellets were frozen at -20 °C. Afterwards, the pellets were redissolved in lysis buffer (50 mM NaPB pH 7.4 and 500 mM NaCI, 10 mM imidazole, 100 μΜ PMSF and 1 mg/mL lysozyme) and exposed to 2 times 3 min of sonication (Branson sonifier 250, level 3, 50% duty cycle). Then, cell debris was removed by centrifugation, yielding crude cell extract containing the soluble protein fraction. The His6-tagged proteins were purified by Ni-NTA affinity chromatography using gravity-flow columns as described by the supplier (MC Lab). Finally, buffer was exchanged to 100 mM MOPS pH 7.0 in 30 K Amicon Ultra centrifugal filters (Merck).

Table 2. Primers used for subcloning of UGT-76GlSr

'Characterization of mutant SuSys'

The bicinchoninic acid (BCA) method was used to detect fructose, which is released by SuSy during the cleavage of Sue. The color reagent is prepared by combining 23 parts of a solution containing 1.5 g-L ¹ 4,4'-dicarboxy-2,2'-biquinoline dipotassium salt and 62.3 g-L ¹ anhydrous Na2CQ3, 1 part of a solution composed of 23 g-L^"1 aspartic acid, 33 g-L^"1 anhydrous Na2CC>3 and 7.3 g-L^"1 CuS0₄ and 6 parts ethanol. Sample (25 μL) is added to 150 μL of assay solution. Afterwards the microtiter plate is covered by a plastic foil and incubated for 30 min at 70°C. After cooling to room temperature, the absorbance is measured at 540 nm. One unit of SuSy activity is defined as the amount of enzyme that released 1 μιτιοΙ of fructose min ¹ under the specified conditions. Kinetic parameters for ADP and UDP (apparent Km and Vmax values) were determined with 1 M Sue at 60°C in 100 mM MOPS pH 7 and values were calculated by non-linear regression of the Michaelis-Menten equation using Sigma Plot 11.0. Alternatively, substrate inhibition was fitted according to the equation (V_max*S)/(S+K_m+(S²/Ki)) with Vmax = maximal reaction velocity (U-mg ^_1); S = substrate concentration (mM); K, = inhibition dissociation constant; K_m = Michaelis-Menten constant (Copeland 2000).

'Glycosylation reactions'

Glucosylation of 1 mM polydatin by 1 mg mL ¹ UGT-767GlSr was achieved directly from 2 mM UDP- glucose. Reactions were also coupled to UDP-glucose (re)generation from 1 mM UDP and 100 mM Sue by 50 μg mL^"1 SuSyAc or SuSyAc LMDKVVA. Reactions were buffered at pH 7.5 with 50 mM HEPES containing 50 mM KCI, 12 mM MgC and 0.13% BSA. Conversions were performed on a scale of 200 μL in 1.5 mL reaction tubes at 30 °C and started by adding enzymes to the preheated reaction solutions. To monitor the conversion, aliquots of 30 μL were withdrawn and enzymes were inactivated by mixing with 30 μL acetonitrile. By centrifugation at 14000 rpm for 15 min precipitated proteins were removed. The concentrations of polydatin and its glucoside were determined by analyzing 5 μL of the supernatant with ion-pairing reversed-phase HPLC. A Kinetex™ C18 column (5μιτι, 100 A, 50 x 4.6 mm) was used for HPLC analysis at 35°C. 20 mM potassium phosphate, pH 5.9 containing 40 mM TBAB were used as mobile phase A and acetonitrile was used as mobile phase B. Separation was achieved using following method at a constant flow rate of 2 mL min^"1: 5% B (1 min), 4-40% B (3.20 min), 40-90% B (0.01 min), 90% B (0.79 min), 90-5% B (0.01 min), 5% B (0.99 min), with detection at 320 nm. Results

Nucleotide preference and its relation to the QN domain

Plant SuSys are known to prefer UDP, although they are also able to use other nucleotides such as ADP, CDP and GDP (Delmer 1972; Tsai 1974; Morell & Copeland 1985; Tanase & Yamaki 2000; Baroja- Fernandez et al. 2003; Baroja-Fernandez et al. 2012; Murata 1971; Nomura & Akazawa 1973; Moriguchi & Yamaki 1988; Ross & Davies 1992). In contrast, most of the bacterial SuSys prefer ADP (Figueroa et al. 2013; Wu et al. 2015; Diricks et al. 2015).. One of the most clear examples of ADP preference is provided by the bacterial SuSyAc. Indeed, this enzyme has a K_m value for UDP (7.8 mM) that is 25 times higher compared to ADP (0.3 mM). To unravel the determinants underlying the difference in nucleotide specificity between prokaryotic and eukaryotic SuSys, residues surrounding the nucleobase ring of the nucleotide acceptor were targeted. To localize these residues, the crystal structure of SuSy from Arabidopsis thaliana (SuSyAtl), a plant enzyme which is 48% identical to SuSyAc, was used (PDB 3S27) (Zheng et al. 2011). In the active site of this closed structure, both UDP and Fru are trapped and all positions within 4A of the uracil unit of UDP are presented in Fig. 1, together with the distribution of amino acids in plant and bacterial SuSy sequences.

The first five residues are identical between SuSyAtl and SuSyAc. Position 596 and 635 in SuSyAc do differ from those in SuSyAtl but in 51% of the other bacterial SuSys, the former position is occupied by the same residue as in SuSyAtl and the amino acid from SuSyAc occurring at the other position can be found in 54% of the plant SuSys. Consequently, these six positions were not included in the mutagenesis strategy. According to the crystal structure of SuSyAtl, the main chain of Gln-648 (Q) and the side chain of Asn-654 (N), residues which are highly conserved in plants, make hydrogen bonds with the uracil unit of UDP (Fig. 2). These two amino acids flank a motif of seven residues in total, hereinafter referred to as the 'QN motif, and are situated in the catalytic GT-Bc domain of the SuSy enzyme (Fig. 1 and Fig. SI) (Zheng et al. 2011). In SuSyAc, it seems that the corresponding residues of Gln-648 (Leu-636) and Asn-654 (Ala-642) do not participate in hydrogen bonding with UDP, which was to be expected particularly for Ala-642 because of its hydrophobic side chain (Fig. 2).

Interestingly, the distribution of amino acids in the QN motif also differs significantly between plants and bacteria (Fig 1). In general, the residues in plants are highly conserved and the range of possible amino acids at less conserved positions is small while in bacteria they show no or little conservation (except for Leu-637 and Lys-639) and a lot of different amino acids can be found. Furthermore, the most prevalent amino acids observed in plant sequences, rarely occur in bacterial ones.

'Mutational analysis of the QN motif To determine which residues have an effect on the nucleotide preference, several amino acids in the QN motif of SuSyAc (LLDKTVA) were replaced by those occurring in SuSyAtl, which can be regarded as a representative sequence for plant SuSys. In total, eight mutants were constructed: three single, two double, one triple, one quadruple and one sextuple mutant. Two of these mutants, QLD T N and QLDKTVN, contain plant residues that are highly conserved and include the two residues making hydrogen bonds with UDP. LLDRTVA has a highly conserved residue of plants that does not participate in hydrogen bonding. LMDKVVA, LLDKVVA and LMDKTVA contain mutations that are less conserved in plants and mutants LMD VVA and QMDRVRN (complete plant QN motif) have a combination of conserved and non-conserved residues (Fig. 1). Positions in the QN motif that are mutated are underlined.

Next, the kinetic parameters for both UDP and ADP were determined for each mutant and results can be found in Fig 3 and Table 3. Table 3. Kinetic parameters of QN mutants. V_max values are expressed in U-mg ¹ and K_m/Ki values in mM. If no inhibition was observed below 20 mM UDP, '/' is used

Compared to the wild-type (WT) enzyme, all mutants had a considerable higher affinity for UDP in the presence of 1 M Sue and half of them also showed a higher activity. This strongly supports the hypothesis that the residues within the QN motif are important for the difference in nucleotide specificity between eukaryotes and prokaryotes. Double mutant LMDKVVA exhibited the highest (60- fold) improvement in K_m. Although the affinity for ADP also increased for all mutants, except for QMDRVRN, the improvement is much smaller compared to UDP. Only three out of eight mutants displayed a higher V with ADP. The K_m value for UDP of the QLDKTVN_^ double mutant, was found to be 25 times higher with 200 mM Sue compared to 1M Sue (Diricks et al. 2015).

Although the eight mutants all showed a significantly improved affinity for UDP compared to the WT, only three of them showed a true switch in nucleotide preference. Indeed, only LLDRTVA, LMDKVVA, and LMDKTVA had both a higher affinity and a higher activity with UDP compared to ADP. Interestingly, none of these enzymes had mutations at the two positions that are highly conserved in plant SuSys and are involved in hydrogen bonding with U DP. Moreover, the side chain of their mutated residues (L637M, K639 and T640V) point away from the substrate which may indicate that their effect is rather secondary. One could argue for example that introducing larger residues (as is the case for L637M and K639R) moved the QN motif loop towards the nucleotide, resulting in a tighter binding pocket, which could intensify the interactions with the substrate.

Next, the best performing mutant LMDKVVA, exhibiting the lowest K_m for UDP and one of the highest associated maximal velocities was used as test case to study the effect on CDP and GDP. Results are summarized in Table 4.

SuSyAc WT has a higher affinity for GDP, which is just like ADP a purine derivative, but the maximum velocity is higher with CDP. Neither SuSyAc WT nor the mutant showed substrate inhibition below 25 mM CDP/GDP but the mutant had a two-fold improved affinity for both CDP and GDP. This clearly demonstrates the general regulatory role of these residues in nucleotide binding.

'Coupling reactions'

If SuSy is used in a coupled process together with a GT (Fig 5), laborious isolation of nucleotide sugars can be bypassed and only catalytic amounts of the expensive nucleotide has to be supplied. Furthermore, conversion efficiencies are increased by this strategy as reverse glycosylation and inhibition of GT by high concentrations of UDP is suppressed (lchikawa et al. 1994; Owens & Mcintosh 2009; Terasaka et al. 2012; Zhang et al. 2006; Schmolzer et al. 2015). To create an efficient and cost- effective SuSy/GT coupled process, it is thus of utmost importance that only a low amount of UDP has to be supplied, requiring a SuSy enzyme with high affinity for UDP. To demonstrate this, the mutant LMDKVVA, was evaluated in a cascade reaction together with the glycosyltransferase UGT-76GlSr from Stevia rebaudiana. This enzyme is involved in the biosynthetic pathway for the natural sweetener stevia, where it converts stevioside into rebaudioside A or steviolbioside into rebaudioside B through the addition of a -l,3-linked glucose moiety (Richman et al. 2005; Mohamed et al. 2011). However, the enzyme was found here to have a remarkably broad specificity, which can be exploited for the glucosylation of a wide range of acceptor molecules (Fig 6).

As proof-of-concept, the glucosylation of polydatin was examined in more detail, both with and without coupling to SuSy (Fig 7). If UGT-76GlSr was used without SuSy, the acceptor yield was only 50% after 24h whereas a yield of 90% could be obtained in the coupled process. This indicates that UGT-76GlSr is inhibited by UDP, a problem that is efficiently solved in the coupled process. The glucoside in the coupled system using the SuSy LMDKVVA mutant was formed at a rate of 138 μΜ-h^"1, which is about 9 times faster compared to the coupled reaction using SuSyAc WT. These results clearly demonstrate that the increased affinity of SuSyAc mutants for UDP can be translated to improved performance in coupled reactions. Furthermore, taking into account the excellent features of the bacterial SuSyAc such as high stability, high activity and high expression yields (Diricks et al. 2015), the mutants of the present invention, such as the LMDKVVA mutant, provide promising alternatives to the commonly used plant enzymes for cascade reactions.

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Claims

1. A mutated sucrose synthase characterized by:

-its sequence comprising the amino acid sequence of SEQ ID N° 1 which

contains at least one of the following mutations: K639R, L637M, T640V, L637M and T640V, L636Q and K639R and V641R and A642N, L637M and K639R and T640V, or, L636Q and L637M and K639R and T640V and V641R and A642N, and

2. A mutated sucrose synthase characterized by:

-its sequence having at least 90% sequence identity with SEQ ID N° 1,

3. A mutated sucrose synthase characterized by:

-its sequence comprising the amino acid sequence of SEQ ID N° 1 which

contains at least one of the following mutations : K639R, L637M, or, L637M and T640V, and

4. An isolated nucleic acid encoding for a mutant sucrose synthase according to claims 1-3.

5. Use of a mutated sucrose synthase according to claims 1-3 to produce UDP-glucose.

6. Use of mutated sucrose synthase to produce UDP-glucose wherein said mutated sucrose synthase is characterized by:

-its sequence comprising the amino acid sequence of SEQ ID N° 1 which

contains at least the following mutations : L636Q and A642N, and

-it has a higher affinity with the substrate UDP when compared to the wild type enzyme.

7. A process to glycosylate a substrate comprising:

-using a mutated sucrose synthase according to claims 5-6 to produce UDP-glucose, -using a glycosyltransferase and said UDP-glucose to covalently attach a glucose moiety to said substrate.

8. A process according to claim 7 wherein said glycosyltransferase is a glycosyltransferase comprising the amino acid sequence of SEQ ID N° 3.

9. A process according to claim 8 wherein the substrate is chosen from the list of: vanillin, para- nitrophenol, anisyl alcohol, catechin, epicatechin, hesperetin, resveratrol, phloretin, curcumin, polydatin, salicin, p-nitrophenol a-glc, p-nitrophenol a-gal, esculin, ethanol, t-cyclohexanediol, cinnamyl alcohol, phenol, orcinol, methylgallate, ethylgallate and propylgallate.

10. A process according to claim 9 wherein said substrate is polydatin.