Plant Cell, Tissue and Organ Culture 69: 177–182, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Glucosylation of exogenous vanillin by plant cell cultures

Yuana, M.J.W. Dignum∗ & R. VerpoorteLeiden/Amsterdam Center for Drug Research, Division of Pharmacognosy, Leiden University, P.O. Box9502, 2300 RA Leiden, The Netherlands (∗requests for offprints; Fax: +31-71-5274511; E-mail:m.dignum@chem.leidenuniv.nl)

Received 30 May 2001; accepted in revised form 8 October 2001

Key words: Catharanthus roseus (L.) G.Don, glucosylation, Ilex dumosa Reissek, Vanilla planifolia Andrews,vanillin

Abstract

After feeding with vanillin,Vanilla planifolia Andrews cell culture was able to produce the highest amount ofglucovanillin compared to Ilex dumosa Reissek and Catharanthus roseus (L.) G.Don cell cultures. The optimumyield of glucovanillin was obtained from V. planifolia cells fed with 6.6 mM vanillin and harvested after 12 h. Theyield was 3.28 mM glucovanillin (49.7%). This glucoside was stored mainly in the cells.

Abbreviations: DW – dry weight; 2,4-D – 2,4-dichlorophenoxyacetic acid; FW – fresh weight; GV – glucovanillin;V – vanillin

Introduction

Glycosylation of exogenously added phenolic com-pounds in plant cell cultures is considered to beone of the major detoxification processes (Winterhal-ter and Skouroumounis, 1997). However, successfulproduction of glucosides requires a strong glucosyl-transferase activity in the recipient cells to introducea glucose molecule to a specific position of the sub-strate without producing any by-products (Tabata etal., 1988). Kometani et al. (1993) found that excess-ive vanillin is toxic to cells and for a Coffea arabicacell culture, 1 mM vanillin seemed to be the criticalconcentration to obtain the maximum efficiency ofglucosylation.

In vanilla beans, all major flavour compounds, in-cluding vanillin, are present as odourless glucosides.During the so-called curing process of vanilla beans,the glucosides are hydrolyzed to their aromatic agly-cons by the action of several enzymes. Since many ofthese vanilla glucosides are present in small amounts,it is not easy to isolate them from a green vanillabean extract. In order to identify the glucosides des-pite the low concentrations, some research has beenperformed to produce this glucoside either chemically

or biotechnologically (Leong et al., 1989; Sommer etal., 1997). Sommer et al. (1997) concluded that themajor limiting step to the production of higher yieldsof glucovanillin in cell cultures is the reduction pro-cess of vanillin to vanillylalcohol, which decreases theamount of available vanillin for further glucosylation.They found that Catharanthus roseus cell cultures ac-cumulated the highest amount of glucovanillin after 16h incubation time with 8.2 mM of vanillin.

Thus, a study of glucovanillin production by usingVanilla planifolia cell cultures would be of interest,because every cell of a vanilla cell culture has all thegenes encoding the enzymes necessary to produce allcomponents and their precursors that constitute naturalvanilla flavor.

In the present study, three different cell lines fromthree different plant families were used to produceglucovanillin, because previous studies showed gluc-osylating capacity for these cell lines. Catharanthusroseus (L.) G.Don (Apocynaceae) has been used forin vivo formation of vanillin glucoside (Sommer et al.,1997). Kraemer et al. (1999) reported glucosylationof ethanol by plant cells of a species of the Aqui-foliaceae family, Ilex paraguariensis. In the presentstudy, I. dumosa was used. Vanilla planifolia Andrews

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(Orchidaceae) itself has the genetic information neces-sary to produce precursors and other constituents ofvanilla flavor. Several treatments such as the variationof fed vanillin concentration and time-course of vanil-lin feeding were studied in a series of experiments toobtain the optimum yield of glucovanillin.

Materials and methods

Cell suspension cultures

Cultures of Vanilla planifolia were cultivated in 250ml conical flasks filled with 100 ml of MS medium(pH 5.9–6), containing per liter: MS salts (Murashigeand Skoog, 1962), 100 mg myo-inositol, 0.1 mg thiam-ine, 0.5 mg pyridoxine, 0.5 mg nicotinic acid, 2 mgglycine, 1 mg 2,4-D and 30 g sucrose. The flasks wereclosed with silicon caps.

Cultures of Catharanthus roseus were grown in 50ml of MS medium (pH 5.8–6), containing per liter:MS salts (Murashige and Skoog, 1962), 100 mg myo-inositol, 10 mg thiamine, 1 mg 2,4-D, 1 mg kinetin,and 20 g sucrose. The flasks were closed with cottonplugs.

Cultures of Ilex dumosa were grown in 50 ml ofB5 medium (pH 5.8–6), containing per liter: B5 salts(Gamborg et al., 1968), 100 mg myo-inositol, 10 mgthiamine, 1 mg pyridoxine, 1 mg nicotinic acid, 1 mg2,4-D, 1 mg kinetin, and 40 g sucrose. The flasks wereclosed with silicon caps.

All suspension cultures were agitated on a gyratoryshaker (110 rpm) under 1950 cd m−2 at 25 (±1) ◦Cand subcultured every 7 days.

Feeding experiments

Effect of cell density on glucoside accumulationThe cells from several flasks of a cell line were pooledand divided by transferring an inoculum of 5, 10 or15 g fresh weight to flasks with fresh medium. Oneml of a stock solution of vanillin in methanol was ad-ded aseptically to 5-days old cells, with a final vanillinconcentration in the media of 6.6 mM. Fed cells wereharvested after 12 and 24 h by separating media fromthe cells over a sintered glass filter. All treatmentswere done in duplicate.

Effect of fed-vanillin concentration on glucosideaccumulationTo study the effect of fed-vanillin concentration toVanilla planifolia cells, the cells were pooled and di-

vided by transferring an inoculum of 10 g fresh weightto flasks with fresh media. After 5 days, vanillin dis-solved in methanol was added aseptically with a finalconcentration of 3.3, 6.6 and 33.0 mM. Fed cells wereharvested after 12 h by separating media from thecells over a glass filter. All treatments were done induplicate.

Effect of growth phase on glucoside accumulationTo determine the time-course of the vanillin biocon-version with 6.6 mM vanillin, vanillin was addedaseptically to the 5-day old V. planifolia cells. Thecells were harvested after 0, 4, 8, 12, 16, 20 and 24 h.To study the accumulation of glucovanillin in relationto the cell cycle of V. planifolia cell culture, vanillinwas added aseptically to 0, 2, 4, 6, 8 and 10-day oldcells with a final concentration of 6.6 mM. The cellswere harvested 12 h after feeding. All treatments weredone in duplicate.

Extraction of biomass

The fresh biomass was frozen in liquid nitrogen anddried using a freeze dryer. The dried biomass wassuspended in acetate buffer (0.03 M, pH 5) and ho-mogenized with an ultra turrax (120 V, 2 min). Theextracts were centrifuged (13 000 rpm, 10 min) andthe supernatant was collected for glucoside analysis.

HPLC method for glucoside analysis andquantification

Quantification of glucovanillin formed in this experi-ment was done with a Waters-HPLC gradient system.As our main interest was to obtain a high yield ofglucovanillin, other glucosidic compounds were onlyidentified and not quantified. Conditions of reversed-phase HPLC were as follows: Phenomenex Hypersil 5C 18 (ODS) column (250 × 4.60 mm) equipped with aRP-18 pre-column. Eluents (1.0 ml min−1) were water(95%): acetonitrile (5%) for eluent A and acetonitrile(95%): water (5%) for eluent B. Both eluents wereacidified with acetic acid 0.75% (v/v). The startingcondition of the gradient system was 100% of eluentA and 0% of eluent B, then the percentage of eluentB was increased to 20% within 30 min. The columnwas rinsed with 100% B for 7.5 min and equilibratedafterwards with 100% A before the next injection. Thedetection was realized by a Waters 991 photodiode-array (PDA) detector recording from 210 to 350 nm.The wavelengths of 254 and 280 nm to detect gluc-osides were chosen according to UV absorption of

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glucosides. The identification of glucosides was doneby comparing retention time and UV spectra withthose of reference compounds.

Concentrations in media are calculated per ml ofmedium; in biomass the amount of V or GV in thetotal biomass after harvesting, is expressed per ml ofmedium.

Results and discussion

The glucosylation capability of each cell line was dif-ferent. Catharanthus roseus was able to glucosylatevanillin. After 12 h, the highest glucovanillin concen-tration was found in the biomass with 10 g inoculumsize (16% conversion) (Figure 1). In the media, how-ever, was still a large amount of vanillin left. Harvest-ing after 24 h showed a decrease of formed glucov-anillin and also a decrease in vanillin (Figure 1). Theresults of vanillin feeding to Vanilla planifolia cellsshowed that the highest glucovanillin concentrationwas found in the biomass upon harvesting 12 h afterfeeding to 10 g of fresh biomass (43.5% conversion)(Figure 1). The amount of vanillin in the media wasmuch lower compared to the results of C. roseus. Har-vesting after 24 h resulted in decreased glucovanillinand vanillin concentrations (Figure 1). In Ilex dumosathere was hardly any glucovanillin formed either in thebiomass or media compared to the two other cell lines.I. dumosa had a larger amount of vanillin transformedafter 24 h, shown by a slightly higher glucovanillin inthe biomass (Figure 1).

Increasing the biomass to 15 g/flask decreased theproduction of secondary metabolites in all cell linestested. In contrast, Sommer et al. (1997) reported a60% conversion using high density cell suspensions(250 g/150 ml) of C. roseus upon feeding 8.2 mM ofvanillin.

The V. planifolia cells produced the highest amountof glucovanillin in the biomass compared with theothers, but little glucoside was found in the media.Most likely in V. planifolia, the glucovanillin is re-cognized as an endogenous compound and is takenup selectively by the vacuole. In the control of V.planifolia (without vanillin addition), no glucovanil-lin could be detected. In the other cell lines, thevanillin is a xenobiotic, which is not as efficientlytaken up in the vacuole and is in part excreted tothe media. Also, Tabata et al. (1988) concluded thatdifferent culture strains may have different glucosyla-tion efficiency for certain compounds. In addition to

this, Hallard et al. (1997) report the accumulationof alkaloids in transgenic cell cultures. Strictosidine,which is an endogenous compound in C. roseus, isstored in the vacuole of C. roseus cells after feed-ing of secologanin and tryptamin. On the other hand,when secologanin and tryptamin are fed to tobaccocells, in which strictosidine synthase is overexpressed,strictosidine is formed and excreted all into the me-dium. Apparently because strictosidine is a xenobioticfor tobacco. Deus–Neumann and Zenk (1986) reportthe selective accumulation of alkaloids by vacuoles.Only endogenous alkaloids were found to be taken upby the vacuoles in the plant. Other alkaloids normallynot found in the plants studied, were not taken up inthe vacuoles.

The vanillin concentration in the media for all thecell lines after 24 h of incubation was lower com-pared with 12 h of incubation. This result did notlead to an increased glucovanillin production (Figure1). Both Kometani et al. (1993) and Sommer et al.(1997) described the highest accumulation after 24 hof incubation in vanillin feeding experiments.

Apparently, vanillin and glucovanillin are re-duced/oxidized to other compounds. Comparing theretention time on HPLC and UV spectra with ref-erence compounds, other compounds were identifiedbesides glucovanillin, such as vanillyl alcohol, vanillylalcohol-phenyl glucoside, vanillic acid, and glucov-anillic acid. There are also several compounds presentin small amounts that could not be identified. The re-duction products were reported as well by Sommer etal. (1997), as identied in C. roseus cultures. However,they did not identify the oxidation products vanillicacid and glucovanillic acid.

In another experiment, the influence of differentvanillin concentrations was investigated in V. planifo-lia cells. Feeding with 3.3 mM of vanillin, as a finalconcentration did not give a comparable yield of gluc-ovanillin as compared with 6.6 mM of vanillin. Com-paring three different concentration treatments (Figure2), the glucovanillin yield was highest in the bio-mass fed with 6.6 mM vanillin concentration (39.9%conversion).

Increasing the concentration to 33.0 mM did notresult in an increased yield of glucovanillin (Figure2). There is still an excessive amount of vanillin inboth the media and the biomass. At high concentration(33.0 mM), half of the vanillin was converted after 12h, but there was hardly any glucovanillin formation.The glucosylation rate of vanillin at 33.0 mM feedingwas apparently less than for lower feeding concen-

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Figure 1. Concentration of vanillin and glucovanillin in biomass (left) and media (right) of C. roseus, V. planifolia and I. dumosa cell culturesafter feeding 6.6 mM vanillin to different cell densities. Cells were harvested after 12 h (top) and 24 hours (bottom). Error bars indicate highestvalue of duplicate samples. Black bars: glucovanillin; grey bars: vanillin.

Figure 2. Concentration of vanillin and glucovanillin in biomass (A)and media (B) of V. planifolia cell cultures (10 g inoculum size)harvested 12 h after feeding different amounts of vanillin. Error barsindicate highest value of duplicate samples.

trations of vanillin, as the glucovanillin yield is low.This might be due to the toxicity of vanillin for thecells, which also can be concluded from the brown-coloration of the cells after 33.0 mM of vanillin wasadded.

In a feeding experiment using Coffea arabica cellcultures, an excessive amount of vanillin was foundto be toxic to cells and 1 mM vanillin seemed to bethe critical concentration at which the cellular capa-city for glucosylation and the toxicity of vanillin arebalanced. It was also concluded that the efficiency ofglucosylation reached more than 80% at 1 mM vanillinand decreased at higher concentrations (Kometani etal., 1993). Apparently, the V. planifolia cell culturescan deal with a considerably higher concentration ofvanillin than Coffea arabica.

The highest accumulation of the glucovanillin byfeeding V. planifolia with 6.6 mM vanillin was ob-served after harvesting the cells after 12 h (39.9%conversion) (Figure 3). The amount of glucovanillinwas higher in the biomass than in the medium. Theamount of vanillin in the biomass and in the mediumdecreased in accordance with the later harvesting timebut the glucovanillin amount was not increased. Therewas possibly further degradation of the glucovanillinor conversion of vanillin to other compounds.

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Figure 3. Concentration of vanillin and glucovanillin in biomass (A) and media (B) of V. planifolia cell cultures (10 g inoculum size) withsingle feeding of 6.6 mM vanillin and harvesting at 4 h intervals. Error bars indicate highest value of duplicate samples.

Figure 4. Concentration of vanillin and glucovanillin in biomass (A) and media (B) of V. planifolia cell cultures (10 g inoculum size) harvested12 h after a single feeding of 6.6 mM vanillin on a certain day in growth cycle. Error bars indicate highest value of duplicate samples.

Feeding with 6.6 mM vanillin in a 2 days intervalshowed that there is no significant difference in gluco-vanillin accumulation. The yield of glucovanillin wasmainly affected by the amount of biomass (Figure 4).The amount of glucovanillin obtained from the bio-mass is constant when expressed as mg g−1 dw of cellsfor each analysis. In absolute amounts, glucovanillinreached the highest concentration at day 10 (49.7%conversion), as there is more biomass formed at day10 compared to day 2. The amount of vanillin left,either in the biomass or in the media, was lower uponfeeding at later time points because the amount of cellsalong the growth cycle increased, and consequentlythe uptake and conversion of vanillin increased as well(Figure 4). However, the decrease of the vanillin con-

centration was higher than the increase of the amountof glucovanillin.

Conclusions

For producing glucovanillin, the use of Vanilla cellcultures gave better results than other plant cell cul-ture lines tested in this study and previous studies byother groups. The data presented here indicate that itis difficult to compare results of feeding experimentsperformed in different places. Even if cell cultures ofone plant genus are used, there are always differencesbetween the individual cell lines.

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The optimum conditions in this study for gluco-vanillin production were, feeding the cells with 6.6mM of vanillin on the tenth day after subculturingat a cell density of 10 g fresh weight per flask. Thehighest yield is obtained in the biomass upon harvest-ing 12 h after feeding. Using these conditions, 49.7%of vanillin was converted to glucovanillin.

Acknowledgements

The authors want to thank Mr. P. Brodelius for supply-ing the Vanilla planifolia Andrews callus culture.

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