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Document Type and Number:
WIPO Patent Application WO/2007/034190
Kind Code:
We describe a screening method for the identification of glycosyltransferase polypeptides that regioselectively modify aglycones and the use of said glycosyltransferase polypeptides to modify aglycones.

Application Number:
Publication Date:
August 16, 2007
Filing Date:
September 21, 2006
Export Citation:
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International Classes:
C12Q1/48; C12N9/10; C12N15/54; C12P19/18
Domestic Patent References:
Other References:
LIM ENG-KIAT ET AL: "Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides", BIOTECHNOLOGY AND BIOENGINEERING, vol. 87, no. 5, 5 September 2004 (2004-09-05), pages 623 - 631, XP008077945, ISSN: 0006-3592
BOWLES D ET AL: "Glycosyltransferases: managers of small molecules", CURRENT OPINION IN PLANT BIOLOGY, QUADRANT SUBSCRIPTION SERVICES, GB, vol. 8, no. 3, June 2005 (2005-06-01), pages 254 - 263, XP004869338, ISSN: 1369-5266
NAGASHIMA SHIGEYUKI ET AL: "cDNA cloning and expression of isoflavonoid-specific glucosyltransferase from Glycyrrhiza echinata cell-suspension cultures.", PLANTA (BERLIN), vol. 218, no. 3, January 2004 (2004-01-01), pages 456 - 459, XP008077944, ISSN: 0032-0935
Attorney, Agent or Firm:
GILHOLM HARRISON (Westminster Place York Business Par, Nether Poppleton York YO26 6RW, GB)
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galatGsyltransferases; glucuronσsylixansferases; thamnosyltransferases; and mannosyltransferases.

In a preferred method of the invention said glycosyltransferase is a plant glucosyltransferase.

Ih & further preferred method of the invention said nucleic acid molecule encodes a glucosyltransfer&se selected from the group consisting of: i) nucleic add molecules consisting of a nucleic acid sequence as represented in Table 1;

U) nucleic acid molecules tiaat hybridise under stringent hybridisation conditions to the nucleic acid molecules in (i) and which encode a polypeptide with glucosyltransferase activity; iii) a nucleic acid molecule ϋhat is degenerate as a result of the genetic code to the sequences as defined in (i) and (ii) above.

Ih a preferred method of the invention said nucleic acid molecule consists of a nucleic acid sequence as represented in Table 1.

In an alternative preferred -method of the invention said glycosyltransferase is a mammalian glycosyltransferase. Preferably said mammalian glycosyltransferase is human.

In a preferred method of the invention said cell is a prokgryotϊc cell. Preferably said prokaryotic cell is Eschercheria coli.

In an alternative preferred method of the invention said cell is a eukaryotic cell.

In a preferred method of the invention said eukaryotϊc cell is selected from the group consisting of: a yeast cell- an insect cell; a mammalian cell or a plant cell.

Ih a preferred method of the invention said nucleic acid molecule is part of a vector adapted for the expression of said glycosyltransferase.

Typically said adaptationincludes, by example and not by way of limitation, the provision, of transcription control sequences (promoter sequences) that mediate cell specific expression. These promoter sequences may be cell specific, inducible or constitutive.

Promoter is an art recognised term and, for the sake of clarity, includes the following features which are provided by example only, Enhancer elements are cϊs acting nucleic acid sequences often found 5' to the transcription initiation, site of a gene (enhancers can also be found 3' to a gene sequence or even located in intronic sequences and is therefore position, independent). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors that have been shown to bind specifically to enhancer elements. The binding/activily of transcription factors ' (please see Eukaryofic Transcription Factors, by

David S Latchman, Academic Press Ltd , San Diego) is responsive to a number of environmental cues that include, by example and not by way of limitation , intermediary metabolites (e.g. sugars), environmental effectors (e,g. light heat). Promoter elements also include so called TATA box and RNA polymerase initiation selection (RIS) sequences that function to select a site of transcription initiation. These sequences also bind polypeptides that function, inter alia , to facilitate transcription initiation selection by RNA polymerase.

Adaptations also include the provision of selectable markers and autonomous replication sequences that both facilitate the maintenance of said vector in either the eukaryoticcell or prokaryotic host Vectors that are maintained autonomously are referred to as episomal vectors. Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-50kb DNA). Episomal vectors of this type are described in WO9S/07876.

Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) that function to maximise expression of vector encoded genes arranged in bicistronic or multi-cistronic expression cassettes-

These adaptations are well known in the art. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbow, NY and references therein; Marston, F (19S7) DNA Cloning Techniques: A Practical Approach VoI 3H IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, ϊnc (1994).

The invention, features polypeptide sequences having at least 75% identity -with the polypeptide sequences as herein disclosed, or fragments and fimctϊonaily equivalent polypeptides thereof, in one embodiment, the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the amino acid sequences illustrated herein and which retain or has enhanced glycosyltransferase activity.

In a preferred method of the invention said test substrate is selected from the group consisting of; other sugars, proteins, peptides, lipids and other organic substrates, for example intermediate metabolites (e.g. phenylpropanoid der_vatives 3 coumarins, fjavonoids, isofiavones., for example diadzein j stilbenes, for example tratis-resveratrol).

Bi a preferred method of the invention said cell is further transformed or transfeoted with a nucleic acid molecule that encodes a polypeptide or peptide substrate for said glycosyltransferase.

In a preferred method of the invention said preparation further includes a test agent wherein said agent is a potential modulator of said glycosyltransferase.

In a preferred method of the invention said agent is an antagonist of said glycosyltransferase.

The screening of large numbers of aglycones and/or agents requires preparing arrays of cells for the handling and the administration of substrates/agents. Standard muMwell micro titre plates with formats such as 6, 12, 48, 96 and 384 wells are typically used for compatibility with automated loading and robotic handling systems. Typically, high -throughput screens use homogeneous mixtures of agents with an indicator compound that is either converted or modified resulting in the production of a signal The signal is measured by suitable means (for example detection of fluorescence emission, optical density; or radioactivity) followed by integration of the signals from each well containing the cells, substrate/agent and indicator compound. The present invention utilises the detection of a sugar in cell culture medium and this detection may be the result of the direct detection of the sugar or an indirect measure of the concentration of cleaved sugar irom a modified substrate.

Am embodiment of the invention will now be described by example only and with reference to the following figures :

Figure 1: Design of the rapid screening method. This method consists of three stages: agiycone biotransformation (stage I), cleavage of the glucoside (stage 2), and detection of the released D-giucose ia a coupled enzymatic assay (stage 3);

Figure 2: Screening of a GT-library against the agiycone scopoletin. a) The readings at λ 40 5 s m for D-glucose detection are presented in a colored code format b) The correlation of the colorimetric detection at Anosm a and the HPLC analysis. HPLC quantifications of glycosides are normalized on the strongest peak and annotated in percentage, c) Examples of RP-HPLC chromatographs of active and non-active GTs in whole-cell biocatalysϊs are illustrated;

Figure 3: Screening of a GT-library against the agiycone daidzein. a) The readings at •^40 5 nm for D-glucose detection are presented is, a colored code format b) Examples of RP-HPLC trace of active and non-active GTs in whole-cell biocatalysϊs are illustrated, c) The regioεelectivily of the active GTs towards daidzein. defined by the percentage of a regiospedfϊc gluooside in the total amount of monoglucosides formed;

Figure 4: Screening of a GT-libraiy against the aglycone frans-xesve&txol a) The readings . at Am n m for D-glucose detection are presented in a colored code format b) Examples of RP HPLC trace of active and non active GTs in -whole cell biocatalysxs are illustrated, c) The regioselectivity of the active GTs towards ^rrtrø-resveratrol, defined by the percentage of a regiospecϋϊc glucoside in the total amount of monoglueosides formed;

Figure 5: Jhvestigatάσn of ecsulin hydrolysis. Neither a) autohydrolysis in MES buffer nor b) hydrolysis in bacterial culture of esculin (12) was detected. Samples at 24 h s 44 h incubation and additionally a standard of fbs aglycone esculetin (11) axe illustrated;

Figuxe 6: Cleavage of esculin by Q-glucosidase. Samples of the cleavage reaction for the glucoside esculin (12) were analysed by RP-HPLC at 0, 30, 60 and 90 min incubation time; Figure 7: Removal of different aglycoi.es through adsorbtion by PVPP. The removal of a) frwrø-resveratrol (100%), b) esculetin (70%), c) daidzeϊn (81%), and d) scopoletin (92%) by PVPP was analyzed by RP-HPLC. The efficiency was defined as the ratio of compounds removed by PVPP over that in the untreated samples;

Figure 8t Lack of D-glucose adsorption by PVPP, The BPAEC chromatograph of D-glucose (13) samples treated with and without PVPP axe illustrated demonstrating that no significant loss of D-glucose occurred by filtration through PVPP;

Figure 9: The correlation of the colorϊmetrie detection at JA OSBW . 3 ^d HPLC analysis, HPLC quantifications of glucosides are normalized on the strongest peak and annotated in, percentage: a) daidzein glucosisdes and b) trans-msverstiol glycosides;

Figure 10: 1 H-NMR spectral data for daidzein and &-«^-resveratrol mono-glucosides;

Figure 11: MS analysis of daidzein glycosides, a) 4'-φ-glc*αosϊde (4) (ns/sr.415.11 βvT H]), b) 7-<9-glucosϊde (5) (jnfa 415.10 OT-H]), daϊdzein (3) (m/z: 253.03 PvT-H]), c) daidzein di-glucoside (6) (m/z: 577.10 EMT-H]), other peaks annotated are derived fragments; and

Figure 12: MS analysis of /rsw-resveratcol glueosides. a) 4'-£?~glcuosϊde (S) (m/z:

389.13 [MT-H]), trans-resveratrol (7) (m/z: 227.08 |M -H] b) 3-0-gIucoside (9) (m/z: 389.13 [M~-H]), c) trans-resveratrol di-glucoside (10) (m/z: 551.18 [M -H]), other peaks annotated are derived fragments,

Table 1 shows the coding seqences of 107 Arabidopsis glycosyltransferases; and

Table 2 is a selection of coding sequences ofλrabidopsis glycosyltransferases that show regioselective modification of diadzein or trans-resveratrol.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the cnntext otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise .

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.


All reagents were of analytical grade. Scopoletin , daidzein, esculetin , esculin, trans-resveratroi, dadzein-7-0-β-D- glucσpyranoside (daidzini), glucose oxidase and almond β-glucosidase were obtained from Sϊgma-AIdricJb (U.K.). Horseradish, peroxidase and ABTS™ were purchased from Calbioch.em φ (U.K.). -trans-Resvertarol 3-0-β-D-glucoρyranoside (piceid) was obtained from from Alexis® Biochemicals (UK). MiIIiQ purified water was used for the preparation of all solutions.

Analytical Methods

Reverse-phase. HPLC (RP-HPLC); RP-HPLC (Agilent HOO system with Photodiode Array Detector * Agilent, U,K.) analysis was carried out using a Columbus

5-μ Cl8 column (150x 3.20 mm, Phenomenex, U,K,). Glucosides were separated from their respective aglycones using a linear gradient of acetonitrile/ 0.1% formic acid (v/v) in

HaO: 10-45% trans-resveratrol/glucosides), 10-50% (daidzein/glucosides) at

0.5 -mL/min over 20 min and monitored at 2SO nm and 250 nm Separation of scapoletin/scopolin and esculelin/escuilin was carried out using the conditions described previously. [1 1]

High performance Anion Exchange Chromatography (HPAEC): HPAEC coupled with integrated amperometric detection (IAD) (Dionex, U.K.) was used to detect D-glucose using a CarbαPac™ PAlO column (2 x 250 mm, Dionex). Seven different monosaccharides including L-Fuoose, L-rhamnose, D-galactose, L-arabinose. D-glucose, D-manose and D-xylose were used as references, The D-glucose was separated isocratically at a flow rate 0.35 mL/min with 24 mM NaOH (pH > 12.5) over 18 min . The column was then washed with a linear gradient of NaOH from 24 miM to 200 mM over 5 min. The IAD waveform was set following manufacturer's recommendation.

1 H-NMR: Glucosides, produced in. a large-scale bioeatalysis, were extracted from the culture media into n-butanol, purified using HPLC, re-extracted with n-butanol, dried under vacuum and solubilized in CD 3 O0 for 1 H-NMR. analysis (Bruker AMX 500-MHz 1 H-NMR. spectrometer). The data were processed and analyzed using Bruker

XWIN -NMR. software version 2,6,

ESI- MS: Negative ion electrαspray MS and MS/MS data (Applied Biosystems

QSTAR Pulsar i hybrid quadropole time-of-flight instrument) were collected and processed using ANALYST QS (Applied Biosystems) software. The mass spectrometer was operated in. negative ion mode with, an ion spray voltage of -2500 V at 300 0 C and the nebulisor and turbo gases set at 70 units. Parent ions were fragmented by collision

wrapped in alu foil for light protection and incubated at 25 0 C (250 rpm). After 44 h the cultures were centrifαged (400Og 5 15 mϊn) and the supernatants analyzed.

Stage 2, cleavage: supernatants (100 μl) were transferred to a microtiter plate, 1 μl of β-glucosidase (1 U) was added and the plate incubated for 90 min at 37°C.

Stage 3_ detection: 50 μl of the reaction mix were transferred to a 96-well filtration plate (Abgene, UJC.), mixed with an equal volume of PVPP aqueous suspension (25 g/L), shaken for 1 h at 25°C before ceatriffiigadon. (1 QOOg, 5 mm). To each filtrate, 50 mM acid buffer (MES) (pH 7.0), ABTS™ (0.1 mU), peroxidase (2 U) and glucose oxidase (2 U) were added to a final volume of 125 μl. The formation of the green dye was monitored at 405 πm at 30 min using a plate reader (Bio-Tec Instruments Inc., U 1 S-A).


The method, illustrated in scheme 1, was established and optimized for a 96-weIi plate format using the conversion of the hydrosycoumarin, scopoletitt (1) to scopolin (2) as a model system. In vitro catalysis had already demonstrated that the substrate was recognized by multiple recombinant arabidopsis GTs. cl0] Cells were cultured in standard media before transfer to D-glucose-minus medium in which L-arabinose was the carbon source. Following induction, addition of substrate aad incubation 3 cells were separated and the media from each well were collected and samples either analyzed directly using reverse-phase (RP) HPLC or treated with β-glucosidase, filtered through polyvlnyl- polypyrrolidone (PVPP) to remove remaining aglycone and levels of D-glucose detected in an enzymatic assay. Figure 1 illustrates the GT activities towards scopoletin and demonstrates a linear relationship between the amount of scopolin formed hx each reaction and D-glucose detection. The whole-cell biocatalysis and screen identified 45 GTs with activity towards scopoletin, sci-fmaiing and extending the earlier data from in vitro catalysis. Invariably, a negative in the D-glucose detection assay correlated with a negative result in the RP-HPLC analysis.

The utility of the method to discover novel biocatalysts was investigated using the isoflavone, daidzein (3) and the sulbene, 2?r<m-resveratroi (7)> Both compounds exist as


glucosides, have attracted considerable pharmaceutical ■interest, [3-27] and chemical synthesis of their different glycosides has been attempted but resulted in poor yields and lack of regiosetective d iscrimination , 28-30] Daidzein, as well as other isoflavones, occurs naturally in legumes as the 7- and 4'-β-0-glucosides (4 daidzin, 5)- [31] trans-Resveratrol (7), a naturally occurring hydroxystilbene is found as glucosides 1321 and methoxides. t33] Piceid (3-β-0glucoside) (8) and resveratroloside (4'-β-0-glucoside) (9) are the most abundant conjugates. Bioactivity of these compounds has been reported in relation to cancer prevention.. [34"36]1 coronary heart d isease, [37-38] antioxidant activity [39;40] and estrogenic activity. [41;42] Since neither daidzein nor trans-resveratrol is reported to occur in arabidposis, they represent non-natural substrates for the GT screen.

The utility of the screening method and regiosetective biocatalysis by the GTs are illustrated in Figures 2 and 3. Thirteen GTs recognized daidzein and twenty-five GTs were identified that glucosylated trans-resveratrol. As previously described for scopoletin , RP-HPLC quantification of the glucosides formed in the biocatalysis revealed a linear correlation to D-glucose detection for both substrates (Figures S5, supporting information). The mono- and di-gϊucosides of daidzein (4-6) and trans-resveratrol (S-IO), eluting earlier than the two aglycones under the RP-HPLC conditions used (Figures 2b and 3b), were identified using external standards when available, or by electrospray liquid chromatography-mass spectrometry (LC-MS). 1 H-NMR analysis was used to confirm, the structure of the monoglucoεides (Table I, supporting information,). From the thirteen GTs that recognized daidzeim , three (GTs 84Al , 73B2 and 73Bl) were found to be 100% regioselective for the 7-OH; the remaining enzymes glucosylated the 4'-OH and 7-OH positions to varying degrees, and one GT, 73C4, produced the diglucoside in addition to the monoglucosides (Figure 2b). Similarly, regioselective glucosylation of trans-resveratrol was observed. From the twenty-five enzymes that recognized the substrate,, five GTs were specific for the 3-OH position (GTs 71Dl, 71C2, 8SAl, 72Dl and 71 C4) and one GT 74Bl was specific for the 4'-OH position (Figure 3b). Only trace levels of a diglucoside were observed under the reaction conditions used. As before, for both daidzein and trans-resveratrol biocatalysis, the D-glucose based detection system did not miss any positive enzyme activities; however in these assays, two false positives in screens of each, compound were observed, where an intense absorption was not associated with any product formation,


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