Plant Physiol. (1991) 95, 152-156 Received for publication July 5, 1990 0032-0889/91/95/01 52/05/$01 .00/0 Accepted September 7, 1990 Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni

Hitoshi Shibata*, Satoru Sonoke, Hideo Ochiai, Hideji Nishihashi, and Masaharu Yamada Applied Biological Science, Faculty of Agriculture, Shimane University, Matsue-Shi, Shimane 690, Japan (H.S., S.S., H.O.); and Central Research Laboratories of Dainippon Ink and Chemicals, Inc., Sakura-Shi, Chiba 285-01, Japan (H.N., M.Y.)

ABSTRACT R2 To evaluate and characterize stevioside biosynthetic pathway I in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19- oic acid) and steviol-glucosides (steviol-13-0-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glu- cose donor, were prepared from the soluble extracts of S. rebau- diana leaves. Enzyme fraction 1, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-0-methylsteviol, but not to iso- steviol and 13-0-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-0-glucopyranoside. Enzyme fraction 11, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed I the glucose transfer from UDP-glucose to steviol-13-O-glucopyr- anoside, steviolbioside and stevioside, but not to rubusoside (13,19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-0-glucopyranoside, ste- R1 violbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indi- cate that in the steviol-glucoside biosynthetic pathway, steviol- 13-0-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside Steviol H H producing rebaudioside A. Steviolmonoside H 13-Glc Rubusoside B-Glc 8-Glc Steviolbioside H O-Glc-o-Glc(2-1) Stevioside 3-Glc O-Glc-O-Gic(2-1) Rebaudioside-A O-Glc O-Glc-O-Glc(c2-1( ) One remarkable, sweet constituent of Stevia rebaudiana IO-Glc(3-1) Bertoni, a wild shrub belonging to Compositae family, has been named stevioside, and proven to some 300 times as sweet as sucrose. Stevioside is a glucoside composed of three Figure 1. Chemical structures of steviol and steviol-glucosides. glucose molecules and an aglycon, steviol (ent-13-hydroxy kaur-16-en-19-oic acid), a diterpenoidic carboxylic alcohol (Fig. 1). In addition to stevioside, the leaves of S. rebaudiana acid (17) and 17-['4C]kaurene (1) are reportedly incorporated contain several kinds of the steviol-related glucosides such as into the steviol skeleton; thus, steviol is believed to be synthe- steviolbioside, rebaudioside A, and others as the minor con- sized from kaurene (18), which is also a precursor of gibber- stituents. Stevioside and rebaudioside A, the latter is steviol- ellins. The later stage of steviol-glucoside biosynthesis, the tetraglucoside, are known to be the main sweet constituents enzymatic glucosylations toward steviol and steviol-gluco- (10). As a consequence of their high content (5-10% of dry sides, remains to be characterized. In the soluble extracts from weight) in sweet glucosides, S. rebaudiana leaves are now the leaves of S. rebaudiana Bertoni, we have detected the becoming important in Japan, Brazil and other countries as activities of glucosylation from UDP-glucose toward steviol, a new noncaloric sweetner. steviol- 13-0-glucopyranoside, steviolbioside and stevioside, Several works on the relationship between sweetness and and report here some properties of these enzymatic glucosy- chemical structures of steviol-glucosides (2, 3, 5, 9, 11, 13) lations and characterization of the reaction products, then and the methods for chemical synthesis of stevioside (15) are discuss on the main biosynthetic pathway of stevioside and already reported. On the biosynthesis of steviol, ['4C]acetic rebaudioside A from steviol in S. rebaudiana Bertoni. 152 GLUCOSYLATION OF STEVIOL AND STEVIOL-GLUCOSIDES 153

MATERIALS AND METHODS protein solution. Once the labeled UDP-glucose was added, the reaction was run at 35°C for 60 min, and stopped by Preparation of Steviol and Steviol-Glucosides adding 200 ,uL 1-butanol saturated previously with water. Stevioside and rebaudioside A were extracted from S. re- After mixing, the organic layer was separated by centrifuga- baudiana leaves according to the methods in (10), then puri- tion. Under the same experimental conditions, no ['4C]glu- fied finally by HPLC under the conditions described below. cose nor UDP-['4C]glucose was found to be transferred into Steviol was prepared by the chemical degradation ofstevioside 1-butanol layer from the aqueous layer. Aliquots of the bu- with NaIO4 and KOH, then crystallized in methanol (15). An tanol extract were taken for radioactivity measurements, and isomeric iso-steviol was obtained by hydrolysis of stevioside the amount of the glucosylated ['4C]glucose was calculated. with 10% aq H2SO4 (20). Methylation of steviol with diazo- Other aliquot was subjected to TLC or HPLC analysis. methane (19) or with CH3I (7) afford 19-O-methylsteviol or 13-O-methyl-steviol, respectively. Steviolbioside was prepared TLC and HPLC by saponification of stevioside (20). A specific ,3-glycosidation which produced rubusoside from stevioside, was performed Steviol-glucosides and the reaction products of glucosyl- with a microbial enzyme (H Nishihashi, unpublished data). transferase containing authentic standards were spotted on Saponification of rubusoside afforded steviol-13-O-glucopyr- precoated silicagel 60 F254 plates (0.25 mm thick; E. Merck, anoside. Steviol and steviol-glucosides thus prepared were Darmstadt, FRG) and developed with chloroform-methanol- checked for their purity by TLC and HPLC, and then were water (10:6:1). After development, chromatograms were air- used as authentic standards or substrate for enzymatic dried, sprayed with 50% sulfuric acid then heated at 100°C glucosylation. for 15 min to visualize the glucosides. Labeled compounds on the TLC plates were localized by autoradiography and Plant Material quantified by Berthold TLC-Multi-Tracemaster LB285. For the HPLC analysis of stevioside and rebaudioside A, Stevia (Stevia rebaudiana Bertoni cv Houten) plants were the liquid chromatograph (Tosoh LC system) was equipped field grown, and the leaves were harvested in late summer with a prepacked TSK Gel-NH2-60 column (25 cm x 4.6 mm from fully grown shoots, frozen in liquid nitrogen, and kept i.d.) and the operating conditions were: ambient temperature; at -300C. flow rate of eluting solvent, acetonitrile-water (79:21), 1 mL/ min; wavelength of UV detector, 210 nm. Radio LC detector Preparation of Enzyme Fractions (Tosoh RS-8000) was also fitted to analyze ['4C]glucose- labeled reaction products. Steviol-13-O-glucopyranoside and Frozen stevia leaves (about 200 g) were homogenized in a steviolbioside were separated on TSK gel ODS-80 Tm (15 cm blender in the presence of 600 mL 0.2 M K-phosphate (pH x 4.6 mm i.d.) using 70% methanol as the solvent. 7.5) containing 5 mM MgCl2, 10 mm EDTA, 20 mm 2- mercaptoethanol, 2 g sodium iso-ascorbate, and 10 g polyclar AT. The homogenate was passed through two layers of Mir- RESULTS acloth and the filtrate was centrifuged for 30 min at 20,000g. Glucosyltransferases Toward Steviol and Steviol- The enzyme fraction was precipitated by (NH4)2SO4 (30-75% Glucosides saturation) and redissolved in a minimal volume of 50 mM K-phosphate (pH 7.5) containing 1 mm EDTA and 10 mm 2- Although numerous studies have been concerned with the mercaptoethanol (buffer A). Excess (NH4)2SO4, and a small sweet steviol-glucosides, surprisingly little is known about the amount of steviol-glucosides still present in the fraction were glucosylation to the steviol skeleton in S. rebaudiana leaves removed by filtration through Sephadex G-25, equilibrated which accumulate sweet glucosides in a yield 5 to 10% per and eluted with buffer A. The protein fraction was designated dry leafbasis. As we could detect the UDP-glucose dependent the crude enzyme, and chromatographed on DEAE-Toyo- glucosylation activities for steviol and steviol-glucosides in the pearl 650M column (20 x 2.5 cm i.d.). The column was crude extracts (Table I), the extract was applied onto DEAE- washed with 200 mL buffer A before the following linear Toyopearl column to obtain partially purified enzyme prep- gradient was applied: 300 mL 0 to 0.5 M KCI in buffer A. arations. Two distinct active fractions, enzyme I and II were Two distinct active fractions, passed through the column and obtained under the chromatographic conditions cited. Sub- eluted with about 0.15 M KCI, were separately concentrated strate specificities of these enzyme fractions were examined by ultrafiltration (UP-20 filter, ADVANTEC, Japan) and (Table I). A background radioactivity found in the assay designated enzyme I and enzyme II. without substrate, was subtracted from the radioactivity de- termined with substrate in all other experiments. Such blank Enzyme Assays activity was assumed to depend on the steviol and its gluco- sides present still in the enzyme preparations, because it The usual standard reaction mixture for the glucosyltrans- increased with the amounts of protein added to the reaction ferase assays contained the following in a total volume of 250 mixture. Crude extract acted on steviol, steviolbioside, and ;tL: 0.1 M Tris-HCI (pH 7.5), 10 to 40 iM steviol or one of stevioside, but not on rebaudioside A. Enzyme I was specific steviol-glucosides, 1 or 2 uM (18.5 or 37 KBq, respectively) for steviol. Enzyme II was free from the activity toward steviol UDP-['4C(U)]glucose (11.5 GBq/mmol) purchased from and could act on steviol- 13-O-glucopyranoside, steviolbioside American Radiolabel Chemicals, Inc., and up to 100 ,L and stevioside, but again not on rebaudioside A (Table I). 154 SHIBATA ET AL. Plant Physiol. Vol. 95, 1991

Table I. Substrate Specificity of Glucosyltransferases to Steviol and Table Ill. Reactivity of Steviol Derivatives with Enzyme I Steviol-Glucosides The reactions were performed with enzyme 1 (1.3 mg protein) for Crude enzyme and enzyme I and 11 were prepared as described in 1 h in the presence of 40 Mm steviol or its derivatives and 13 Mm UDP- the text. To the each reaction mixtue (250 ML), crude enzyme (480 [14C]glucose. MAg protein), enzyme 1(210 Mig protein), or enzyme 11(360 Mg protein) Activity: ['4C]Glucose was added, then the reaction was run for 1 h. Transferred I'4C]Glucose Transferred dpm/h Acceptor Crude Enzyme Enzyme 11 Steviol 11,400 dpm x 10-3/h iso-Steviol 0 1 9--Methylsteviol 17,700 None 2.8 0.3 2.5 1 3--Methylsteviol 200 Steviol 4.1 22.9 2.1 Steviolmonosidea 0.4 12.7 Steviolbioside 5.0 0.5 23.9 Stevioside 3.2 0.8 9.6 Rebaudioside A 2.8 0.5 2.1 mer, iso-steviol could not serve as substrates. These results indicate that the 1 3-hydroxyl group of steviol is glucosylated a Steviol-1 3-0-glucopyranoside. first to produce steviol-1 3-O-glucopyranoside. Putative steviol-['4C]glucosides obtained from the enzy- matic reactions were analyzed by TLC. Authentic standards Among steviol-glucosides, steviolbioside was preferred as the (spot numbers 1-5 in Fig. 2) were separated well from each substrate for enzyme II. other on the TLC plate. The radioactivity on the plate was scanned and quantified by the TLC-Radioscanner. The prod- Effect of Nucleotides and Nucleotide-Glucoses uct from steviol with UDP-['4C]glucose (lane F) was found to be identical in RF to authentic steviol-l 3-O-glucopyranoside. The enzymatic glucosylation using UDP-glucose produces This glucoside (lane E) was converted into compound with the glucoside and UDP. Some UDP-glucose dependent glu- an identical RF to that ofsteviolbioside. The reaction products cosyltransferases are reportedly inhibited by UDP (6, 8, 12). of stevioside (lane B) and steviolbioside (lane C) showed This was the case for glucosylation toward steviol and steviol- identical RFs with those of rebaudioside A and stevioside, glucosides. UDP, in contrast to ADP, strongly inhibited the respectively. On the other hand, rubusoside (lane D) could glucosylation catalyzed by enzyme I and II (Table II). ADP-, not accept ['4C]glucose and rebaudioside A (lane A) appeared CDP-, and GDP-glucose had no effect on the enzyme activity. to give no distinct reaction product. Analysis by HPLC of the reaction mixture of steviol-13-0- Characterization of the Reaction Products glucopyranoside revealed the presence of radioactive com- Steviol have two sites, 1 3-hydroxyl and 1 9-carboxyl groups, pound (45% of total 14C) with a retention time of 11.6 min, for accepting glucose to produce steviol-glucosides (Fig. 1). that coeluted with authentic steviolbioside (Fig. 3A). The To evaluate which group of steviol is glucosylated first, we remaining [14C] was eluted at 2.2 min under the reverse phase used 13-0-methylsteviol and 19-O-methylsteviol as the sub- conditions, suggesting the presence of ['4C]stevioside. About strate for enzyme I (Table III). Steviol and 19-O-methylsteviol 80% ofthe radioactivity in the reaction product ofsteviolbio- accepted ['4C]glucose, but 1 3-O-methylsteviol and a stereoiso- side appeared as the main peak with the retention time corresponding to stevioside (Fig. 3B). The main product (84% of total 14C) of the reaction with stevioside was identified as rebaudioside A (Fig. 3C). Table II. Influence of Nucleotides and XDP-Glucose on Glucosyl- transferases Under the reaction conditions reported here, we used rela- tively low concentrations of UDP-glucose (1-2 FM) as com- Enzymes I and 11 were assayed with steviol and steviolbioside, with to respectively. In the control with no additive, 5100 dpm of the [14C] pared those ofthe glucose acceptor (10-40 uM) obtain labeled glucoside was formed in a 1-h incubation with enzyme 1 (360 the sole reaction product. In the presence of higher concen- Mg protein), and 8300 dpm of the glucoside with enzyme 11(310 ,Ag trations of UDP-glucose (1 mM) with steviol-1 3-0-glucoside protein). and enzyme II, at least three products corresponding to ste- Activity violbioside, stevioside, and rebaudioside A were detected on Addition the TLC plates (data not shown). These results confirm that Enzyme Enzyme II steviol-13-0-glucopyranoside, steviolbioside, and stevioside mM % of control were glucosylated by the enzyme II from UDP-glucose pro- UDP 0.1 48 95 ducing steviolbioside, stevioside, and rebaudioside A, 0.5 20 75 respectively. 1 7 15 5 3 3 ADP 1 96 109 DISCUSSION ADP-glucose 1 104 114 The chemical structure ofstevioside was established in 1955 CDP-glucose 1 102 94 (14, 20). Although biosynthetic route of steviol skeleton has GDP-glucose 1 92 105 been reported (1, 17), little information about the biosynthesis GLUCOSYLATION OF STEVIOL AND STEVIOL-GLUCOSIDES 155

lation of steviol-glucosides, hydrophobic steviol have to be IA converted to any hydrophilic derivative. The glucosylation ". reaction is expected to produce a water soluble reaction B product, thus permitting its segregation to the vacuoles (4). The glucosylation to steviol and steviol-glucosides would play a role, physiologically, to produce the water soluble products. c ] 1,500 dpm However, the reason remains to be solved why stevioside and 1- rebaudioside A have three and four glucose moieties in their D molecules. For the synthesis of rebaudioside A from steviol- 13-0- glucopyranoside, hydroxyl and carboxyl groups have to be glucosylated. It is very interesting to elucidate whether these different groups are glucosylated by one enzyme or by two individual enzymes. Purified UDP-glucose:flavonoid 3-0-glu- I I 1 ft cosyltransferase from Hippeastrum petals can act on the hy- droxyl groups of flavanones, flavones, flavonoids, and antho- Origin 1 2 3 4 5 Front cyanidins (6). Partially purified UDP-glucose:IAA glucosyl- Figure 2. TLC of labeled products synthesized from UDP-[U-14C] glucose by enzyme I or 11. The following substrates were used: A, rebaudioside A; B, stevioside; C, steviolbioside; D, rubusoside; E, steviol-13-0-glucopyranoside; F, steviol. Steviol was reacted with 1 60n enzyme 1, other glucoside with enzyme II. Numbers indicated by A Steviolbioside arrows show the position of authentic standards: 1, rebaudioside A; 400 t 2, stevioside; 3, steviolbioside; 4, steviol-13-O-glucopyranoside; 5, 200 steviol. E- 40- of steviol-glucosides from steviol has been reported since that 8 20 time. This report is concerned with the extraction, separation I and characterization of glucosyltransferases involved in the 10 15 20 steviol-glucoside biosynthesis in S. rebaudiana. Two distinct [TIME] enzymes are identified based on their separation by DEAE- _ Toyopearl chromatography and substrate specificity differ- B ences. All glucosylation steps from steviol to rebaudioside A were catalyzed by these two enzyme fractions.

Steviol having two sites to accept glucose to form steviol- 1- glucosides is glucosylated first at 1 3-hydroxyl group, then the E-4 glucose bound to this position is glucosylated, leaving still 19- carboxyl group and producing steviolbioside which has been u detected as the minor constituent of steviol-glucoside (10) in S. rebaudiana. After the formation of bisglucose moiety bound to steviol at 13-position, steviolbioside, the 1 9-carboxyl [TIME] group is ready to accept glucose. This finding will be supported further by the results that rubusoside, a steviol-bisglucoside 800 _ occupied with two glucose molecule at 13 and 19-positions, could not serve as the substrate for the glucosyltransferase *400 (Fig. 2). Thus, the glucosylation to 19-carboxyl group occurs P- after the formation of bisglucose structure at 13-position of E-4 the steviol skeleton. The glucosylation products of steviolbio- 8 side and stevioside were found to be stevioside and rebaudio- side A, respectively. ent-Kaurene, the first hydrophobic intermediate in gibber- ellin biosynthesis is subsequently oxidized by microsomal or [TIME] chloroplastic mixed function oxidases to yield ent-7-a-hy- droxy kaurenoic acid (16). Similar mixed function oxidase Figure 3. Identification by HPLC of the [14C]glucose labeled reaction may act on ent-kaurene to yield ent-13-hydroxy kaurenoic products synthesized from UDP-[14C]glucose by enzyme 11. The fol- lowing were acid, which is also a substrates used: A, steviol-13-0-glucopyranoside; B, steviol, hydrophobic compound. There steviolbioside; and C, stevioside. The upper parts of the three panels is no indication on the localization of steviol-glucosides in showed the elution profiles of the authentic standards monitored by vacuoles, which is the known organelle to accumulate many an absorbance at 210 nm, the lower panels indicated the distribution plant secondary products (6). If this is true for the accumu- of ['4C] monitored by Radio LC detector. 156 SHIBATA ET AL. Plant Physiol. Vol. 95, 1991 transferase forming an ester of IAA and glucose is possible to 9. Kasai R, Kaneda N, Takano 0, Yamasaki K, Sakamoto I, Mor- imoto K, Okada S, Furukawa H (1981) Sweet diterpene-gly- utilize hydroxy acids such as hydroxycinnamic acids and p- cosides of leaves of Stevia rebaudiana Bertoni. Synthesis and coumaric acid, which inhibit also IAA-ester formation (12), structure-sweetness relationship of rebaudioside-A, D, E and suggesting that the enzyme can act only on the carboxyl their related glycosides. Bull Chem Soc Jpn 1981: 726-735 group. However, it remains to be established whether gluco- 10. Kohda H, Kasai R, Yamasaki K, Murakami K, Tanaka 0 (1976) syltransferases forming ether or ester linkage can act on both New sweet diterpene glucosides from Stevia rebaudiana. Phy- tochemistry 15: 981-983 hydroxyl and carboxyl groups. 11. Kusama S, Kusakabe I, Nakamura Y, Eda S, Murakami K (1986) Transglucosylation into stevioside by enzyme system from LITERATURE CITED Streptomyces sp. Agric Biol Chem 50: 2445-2451 12. Leznicki A, Bandurski RS (1988) Enzymatic synthesis of indole- 1. Bennett RD, Lieber ER, Heftmann E (1967) Biosynthesis of acetyl 1-O-fl-D-glucose. II Metabolic characteristics of the en- steviol from (-)-kaurene. Phytochemistry 6: 1107-1110 zyme. Plant Physiol 88: 148 1-1485 2. Darise M, Mizutani K, Kasai R, Tanaka 0, Kitahata S, Okada 13. 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