Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures.

Abstract

Hyoscyamine-6beta-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation.

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Journal of Plant Physiology 164 (2007) 521—524
SHORT COMMUNICATION
Biotransformation of hyoscyamine into
scopolamine in transgenic tobacco cell cultures
Elisabeth Moyanoa, Javier Palazo ´nb,?, Mercedes Bonfillb, Lidia Osunac,
Rosa M. Cusido ´b, Kirsi-Marja Oksman-Caldenteyd, M. Teresa Pin ˜olb
aDepartament de Cie `ncies Experimentals i de la Salut, Universitat Pompeu Fabra, Avda. Dr Aiguader 80,
E-08003 Barcelona, Spain
bSeccio ´n de Fisiologı ´a Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal 643,
E-08028 Barcelona, Spain
cCentro de Investigacio ´n Biome ´dica del Sur, Instituto Mexicano del Seguro Social, Argentina 1, Col. Centro, C.P. 62790,
Xochitepec Morelos, Mexico
dVTT Technical Research Centre of Finland, Plant Biotechnology, P.O. Box 1000, FIN-02044 VTT (Espoo), Finland
Received 28 February 2006; accepted 16 June 2006
KEYWORDS
Biotransformation;
Hyoscyamine-6b-hy-
droxylase;
Hyoscyamine;
Scopolamine;
Tobacco cell cultures
Summary
Hyoscyamine-6b-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its
epoxide scopolamine, a compound with a higher added value in the pharmaceutical
market than hyoscyamine. We report the establishment of tobacco cell cultures
carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV
35S. The cell cultures were derived from hairy roots obtained via genetically
modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The
cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine
produced was quantified by HPLC. The transgenic cell suspension cultures showed a
considerable capacity for the bioconversion of hyoscyamine into scopolamine, and
released it to the culture medium. Although the scale-up from shake-flask to
bioreactor culture usually results in reduced productivities, our transgenic cells
grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased
the scopolamine accumulation.
& 2006 Elsevier GmbH. All rights reserved.
Introduction
In the last decade, several investigators have
used metabolic engineering of pharmaceutically
important tropane alkaloids in order to improve the
conversion of hyoscyamine to the more valuable
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?Corresponding author. Tel.: +34934024492.
E-mail address: javierpalazon@ub.edu (J. Palazo ´n).

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scopolamine (Oksman-Caldentey and Arroo, 2000).
In this context, hairy roots have been obtained that
overexpress the genes coding for the two key
enzymes involved in the biosynthesis of these
alkaloids, N-methylputrescine transferase (PMT;
EC2.1.1.53)and hyoscyamine-6b-hydroxylase
(H6H; EC 1.14.11.11). PMT is the enzyme involved
in the removal of putrescine from the polyamine
pool, as it catalyses the N-methylation of the
diamine to form N-methylputrescine, a well-
defined precursor in tropane alkaloid biosynthesis
(Hashimoto et al., 1989). The conversion of
hyoscyamine into its epoxide scopolamine is a
two-step process catalysed by H6H (Hashimoto et
al., 1993a). Several studies have reported that
overexpression of pmt and/or h6h genes in plants
and hairy roots of Solanaceae such as Atropa,
Datura, Hyoscyamus and Duboisia results in higher
scopolamine production (e.g. Yun et al., 1992;
Hashimoto et al., 1993b; Jouhikainen et al., 1999;
Moyano et al., 2002, 2003; Rocha et al., 2002;
Palazo ´n et al., 2003; Rothe et al., 2003; Zhang et
al., 2004).
Metabolic engineering can also provide techni-
ques to transfer a whole metabolic pathway, or
some of its steps, from one plant species to
another. We recently obtained three tobacco hairy
root lines carrying the 35S-h6h gene from Hyoscya-
mus niger. The root cultures did not biosynthesize
scopolamine, but showed an in vitro capacity to
convert exogenous hyoscyamine added to the
culture medium into scopolamine (Ha ¨kkinen et
al., 2005). The aim of the present work was to
investigate the capacity of dedifferentiated cell
cultures derived from these hairy roots to biocon-
vert exogenous hyoscyamine into scopolamine in
order to scale up the process to bioreactor level. To
our knowledge, this is the first time that such a
bioconversion process has been attempted.
Material and methods
The tobacco root line H6H14, carrying the 35S-
h6h transgene from Hyoscyamus, was obtained as
described previously by Ha ¨kkinen et al. (2005). It
was selected because it showed a high transgene
expression and the greatest capacity to convert
hyoscyamine into scopolamine out of all the root
lines obtained. In order to dedifferentiate root
tissues and obtain a cell suspension culture,
fragments of 100mg fresh weight of the H6H14
root line were transferred to MS medium added to
11.5mM IAA and 1mM kinetin as previously de-
scribed by Palazo ´n et al. (1995). After 3–4
subcultures over 4 weeks, the hairy roots were
transformed into friable callus masses from which
cell suspension cultures were established. Callus
pieces (E100mg) were inoculated into a 100mL
flask (Sigma) containing 40mL MS liquid medium
supplemented with the aforementioned hormonal
concentration and capped with a Magenta B-Cap
(Sigma) and then placed in a rotatory shaker at
100rpm in the dark at 251C. In order to obtain a
fine cell suspension, cell cultures were passed
through a stainless steel sieve (0.76?0.76mm)
every 2 weeks for 6 months and maintained in the
same culture conditions. Transgenic tobacco cell
cultures were entrapped in alginate beads as
described by Gillet et al. (2000) and cultivated as
for free cell cultures.
Free and immobilized cell cultures were fed with
hyoscyamine added to the culture medium. About
0.570.02g of transgenic cells were grown in 40mL
liquid MS medium supplemented with 11.5mM IAA
and 1mM kinetin. The hyoscyamine hydrochloride
(Sigma) was dissolved in water and the stock was
added to the final concentration of 100 or 200mg/
L. In control samples, only water was added. The
cells were grown as described above for a culture
period of 28 days. Alkaloid levels were determined
both in the cells and the culture medium at the end
of the culture period.
The growth was measured after 28 days of
culture and expressed as Growth Index (GI) (final
and initial fresh weight ratio). In the case of
immobilized cell cultures, the beads were first
disrupted as described by Bentebibel et al. (2005).
The tropane alkaloids scopolamine and hyoscya-
mine were extracted and analysed from lyophylised
cells and medium as described by Plank and Wagner
(1986).
For bioreactor cultures, a commercially available
5-L turbine stirred tank bioreactor (Applikon
Dependable Instruments, Schiedam, The Nether-
lands) (Cusido ´ et al., 2002) was used. The culture
was aerated through a sintered steel sparger. The
flow was set at 0.8L/min at the beginning of the
experiment and then gradually increased up to
1.5L/min and maintained at this level with a mass
flow control system until the end of the culture
period (Brooks, Veenendaal, The Netherlands). The
working volume was maintained at 3L culture
medium. The inoculum was 80g/L of free cells
fresh weight, and the cell cultures were kept at
251C in the dark for a culture period of 4 weeks.
Results and discussion
As previously reported (Ha ¨kkinen et al., 2005),
we obtained several transgenic tobacco root lines
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carrying the 35S-h6h transgene. Three of these root
lines showed a high capacity to bioconvert the
added hyoscyamine into scopolamine. Depending
on the root line, the scopolamine production
ranged from 5.7 to 64.1mg/L, the best results
being achieved with the root line H6H14. In order
to scale-up the process, this root line was now
dedifferentiated and a fine cell suspension was
obtained. The biomass production, measured as GI
at the end of the culture (GI ¼ 13.6) was very
similar in the hairy root cultures (GI ¼ 14.0)
(Ha ¨kkinen et al., 2005). Adding hyoscyamine to
the medium of transformed cell cultures did not
significantly affect their growth capacity (Table 1).
Goossens et al. (2003) have also reported that the
tobacco cell line BY-2 is able to tolerate hyoscya-
mine even up to 3g/L.
Similarly to the original H6H14 hairy root line
from which our cell suspension culture was derived,
the transgenic cell cultures showed a moderate
capacity to bioconvert hyoscyamine into scopola-
mine, when the cell suspensions were fed with 100
or 200mg/L hyoscyamine (Table 1). The total
amount of scopolamine achieved at the end of
the culture period was 16 and 21.6mg/L, respec-
tively, which is approximately 38% of that in the
transgenic roots. There was no lack of substrate for
bioconversion, and the best ratio of hyoscyamine
bioconversion into scopolamine (16.0%) had already
been obtained by feeding the culture with 100mg/
L hyoscyamine. Nevertheless, the capacity of
transgenic cell cultures to secrete the produced
scopolamine into the culture medium (secretion
rate) was lower (18–20%) than that of the root line
(56%).
Cell suspensions were immobilized in 1.25% of
alginate and 0.8M of CaCl2, which were the
exceptional conditions found to be optimal by
Gillet et al. (2000) for the production and excretion
of scopolamine by immobilized N. tacabum cells.
After a culture period of 28 days, the growth
(measured as GI) was over 10 times lower than in
free cell suspensions, indicating that the immobi-
lized cells remained in a stationary growth state
throughout the culture period. The total scopola-
mine levels (cell-associated+extracellular) were
dramatically reduced by cell immobilization (Table
1). When the culture medium was supplemented
with 100 and 200mg/L hyoscyamine, the scopola-
mine contents were only 2.2 and 2.5mg/L, respec-
tively. That is, they were 7.3- and 8.6-fold lower
than those obtained in the free cell cultures. Such
low scopolamine levels observed in the immobilized
cultures could be due to problems in the diffusion
of hyoscyamine, since the take-up of this precursor
from the medium by immobilized cells was low, the
percentage of absorption being only about 28% of
the alkaloid supplied to the culture medium (Table
1). In contrast, the entrapment conditions were
favourable for scopolamine excretion. The secre-
tion rate from the producer cells to the medium
was 74.0% and 80.7% in the cultures supplemented,
respectively, with 100 and 200mg/L of hyoscya-
mine. The excretion percentages obtained with our
immobilized cell cultures were 4- and 3.9-fold
higher than those obtained in free cell suspensions,
and their capacity to release scopolamine to the
medium was also higher than that of the hairy root
line H6H14.
With the aim of carrying out the envisaged scale-
up of the process, 80g/L fresh weight of the cells
was inoculated in 3L of medium in a 5L turbine
stirred tank bioreactor (Cusido ´ et al., 2002). The
culture medium was supplemented with 200mg/L
ARTICLE IN PRESS
Table 1.
cultures at the end of the culture period (28 days) in liquid medium supplemented with 100mg/L (H100) or 200mg/L
(H200) hyoscyamine.
Growth Index and scopolamine (medium and cell-associated) contents in H6H14 free and immobilized cell
Cell culture ScaleGrowth Index Hyoscyamine?
Scopolamine
Absorption (%) Total (mg/L) Secretion (%) Conversion (%)
Free cells
MS
MS+H100
MS+H200
MS+H200
Shake flask
Shake flask
Shake flask
Bioreactor
13.6
13.5
13.0
4.8
—
38
31.5
45
—
16.070.4
21.671.1
35.570.8
—
18.1
20.8
10.8
—
16.0
10.8
17.7
Immobilized cells
MS
MS+H100
MS+H200
Shake flask
Shake flask
Shake flask
1.1
1.1
1.1
—
28
27
——
74.0
80.7
—
2.270.1
2.570.1
2.2
1.2
Each value is the average of three replicates7SD.
?Bioconversion of hyoscyamine to scopolamine.
Biotransformation in transgenic tobacco cell cultures 523

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hyoscyamine, since in our small-scale assay we had
obtained the highest levels of scopolamine using
this concentration of the precursor. Although the
growth decreased (GI ¼ 4.8) substantially, the
scopolamine level was significantly higher than
that obtained in shake flasks. The total content
(cell-associated+extracellular) of scopolamine was
35.5mg/L, which was 1.6 times higher than that
obtained in small-scale cultures. In this case almost
18% of the hyoscyamine added to the medium was
transformed into scopolamine, which represented
an increase of 65% with respect to the same
alkaloid obtained by bioconversion in shake flasks.
These results suggest that plant cells which do
not produce secondary compounds of interest are
able to do so after bioconversion of a suitable
precursor, if they overexpress the gene coding for
the key enzyme involved in the biosynthesis of such
compounds. We have shown that N. tabacum free
cell suspensions, which normally do not produce
scopolamine, are able to produce this tropane
alkaloid after overexpressing the gene h6h, which
codes for the last part of the scopolamine
biosynthesis, and after being fed with the immedi-
ate precursor hyoscyamine.
Acknowledgements
This research was supported by three grants from
the Spanish MEC (BIO2002-03614, BIO2002-02328
and BIO2005-05583). L. Osuna is grateful for her
research grant (PIV) from the Generalitat de
Catalunya.
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