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144 Biotechnological Production of Lignans
Linum flavum Suspension, Root
like tissue
6MPTOX, 5-demethoxy-6-methoxy-
PTOX
Linum flavum Hairy roots 6MPTOX
Linum flavum Hairy roots Coniferin
Linum elegans Callus, Suspension 6MPTOX, -peltatin
Linum leonii Callus Justicidin B
Linum leonii Hairy roots Justicidin B
Linum lewisii Cell cultures Justicidin B, Isojusticidin B
Linum linearifolium Callus, Suspension,
Shoots, Hairy roots
PTOX, 6MPTOX, Justicidin B
Linum mucronatum ssp.
armenum
Shoot, Suspension
Linum narbonese Callus Justicidin B
Linum nodiflorum Suspension 6MPTOX
Linum nodiflorum Suspension 6MPTOX, PTOX, DPTOX
Linum persicum Callus, Cell cultures
PTOX, 6MPTOX, B- and -peltatin
Linum tauricum Callus, Suspension,
Shoots, Hairy roots
6MPTOX
4-demethyl-6MPTOX
Linum serbicum Callus, Suspension 6MPTOX, -peltatin
Picea glechni Suspension Pinoresinol, Dihydrodehydrodiconiferil
alcohol
Podophyllum hexandrum Callus, Suspension PTOX
Podophyllum hexandrum Embryogenic callus PTOX
Podophyllum peltatum Callus PTOX
Podophyllum peltatum Embryogenic
suspension
PTOX, DPTOX, 4- DPTOX
Podophyllum sp. Callus PTOX
Sesamum indicum Callus, Suspension,
Hairy roots
Sesamin, Sesamolin
* (The data are cited in Ionkova, 2007; Konuklugil et al., 2007; Ionkova et al., 2006,
2007; Vasilev et al., 2008; Vasilev and Ionkova, 2005).
Development of in vitro cultures
The lignans are a large group of natural substances, which occur in a range of plant species,
but only in low concentrations. Their natural abundance, however, is scarce and their
chemical synthesis is not yet economically feasible. Podophyllotoxin (PTOX) is a lignan,
which is used as adduct for the semisynthesis of etoposide and tenoposide. Both
antineoplastic drugs are of importance for therapy and today and in the future will be a high
demand for the commercial drug and its precursors for the treatment of leukaemia. For the
production of podophyllotoxin different routes are known: extraction and isolation from
Podophyllum hexandrum, Linum and other species, production in plant cell cultures (Van
Uden et al., 1990c) and organic synthesis (Wink et al., 2005). Extraction and isolation of
podophyllotoxin from Indian Podophyllum hexandrum species is not economical and is
characterized by a high price for the final product.
Another problem is cultivation of the plant, which is why mostly the natural product is
extracted from wild collected species. The main reason for this is the limited growth rate
and culture conditions for accelerated growth. As a consequence, P. hexandrum is actually
endangered in the wild in India, especially in the Himalayan region. PTOX and related
Biotechnological Production of Lignans 145
compounds are present in the plant families Juniperaceae, Berberidaceae, Lamiaceae and
Linaceae (Ionkova, 2007).
Podophyllum species
Up to this moment aryltetralin lignans are actually derived exclusively from plant sources.
Due to their restricted natural abundance and important pharmacological application,
identification of new sources or rational in vitro synthesis is very important for the
production of therapeutic candidates for cancer chemotherapy.
Podophyllotoxin was produced by cell culture of Podophyllum hexandrum under in
vitro culture conditions. A maximum of 4.26 mg/l of podophyllotoxin was produced when
P. hexandrum was cultivated in a 3 l stirred tank bioreactor. The compound extracted from
the cell culture was applied to the human breast cancer cell line (MCF-7) and 1 nM
podophyllotoxin was able to inhibit the growth of the cancer cells by 50%. The most
profound inhibitory effect of podophyllotoxin was observed when it was applied in the
beginning of cell growth (Chattopadhyay et al., 2004).
Submerged cultivation of Podophyllum hexandrum for the production of
podophyllotoxin was carried out in a 3 l stirred tank bioreactor fitted with a low-shear
Setric impeller. The specific requirements of the medium, such as carbon source (sugar)
and light, were established for the growth of and podophyllotoxin production by P.
hexandrum in suspension cultures. Substitution of sucrose by glucose resulted in higher
growth and podophyllotoxin production. The biosynthesis of podophyllotoxin was
favoured when plant cells were cultivated in the dark. An agitation speed of 100 rpm was
sufficient to mix the culture broth in the bioreactor without causing any significant cell
damage. Biomass and podophyllotoxin accumulation in the 3 l bioreactor under batch
growth conditions were 6.5 g/l and 4.26 mg/l, respectively, in 22 days. This resulted in an
overall podophyllotoxin productivity of 0.19 mg/l/d), which represented an increase of
27% in comparison to its productivity in a shake flask. Podophyllotoxin production was
found to be a combined growth-associated and non-growth associated process
(Chattopadhyay et al., 2002).
Linum species
Antitumour activity will undoubtedly continue to be the most clinically relevant property of
lignans. The production of anticancer compounds, such as lignan podophyllotoxin, by plant
in vitro cultures from different plant species has been demonstrated. Cell cultures of
different Linum species of section Syllinum are shown to produce considerable amounts of
lignans, mainly MPTOX. Although both PTOX and MPTOX have comparable cytotoxic
activity, due to the different substitution in position 6 MPTOX is not used for the
production of anticancer drugs (Kuhlmann et al., 2002). Results, summarized in Table 9.2,
show that a broad range of experiments have been carried out, resulting in enhancement of
lignan production of in vitro cultures from Linum species. Since PTOX is the preferred
precursor for the semi-synthesis of anticancer drugs like Etoposide and Etopophos®, the
accumulation of predominantly PTOX is especially interesting. L. linearifolium is now
beside L. album and L. persicum the third Linum species of section Syllinum with PTOX
(ca 0.8% DW) as the main lignan (Kuhlmann et al., 2002).
146 Biotechnological Production of Lignans
As a new biotechnological alternative is our success in stereoselective hydroxylation of
deoxypodophyllotoxin by recombinant CYP3A4 in Escherichia coli to epipodophyllotoxin
(Vasilev et al., 2006a), the direct precursor for the semi-synthesis of anti-cancer drugs
etoposide (VP-16) and teniposide (VM-26).
A large number of aryltetralin lignans were identified from in vivo and in vitro species
of the Linum genus in our research group (L. tauricum Willd. ssp. bulgaricum (Podp.)
Petrova; L. tauricum Willd. ssp. tauricum; L. tauricum Willd. ssp. serbicum (Podp.)
Petrova; L. tauricum Willd. ssp. linearifolium (Lindem.) Petrova; L. elegans Sprun. ex
Boiss.; L. flavum L. ssp. sparsiflorum (Stoj.) Petrova; L.capitatum Kit. ex Schult. var.
laxiflorum (Stoj.) Petrova; L. cariense Boiss; L. altaicum Ledeb.; L. austriacum var
euxinum Juz.; L. lewissi Pursh.; L. campanulatum L.; L. setaceum Brot.; L. africanum, L.
strictum L.; L. leonii F. W. Schulz.; L. narbonense L.). Lignans in different samples of
Linum species, mainly occurring in Bulgaria, were analysed by HPLC-ESI/MS and HPLC-
UV/DAD. The ESI/MS fragmentation pathways recently established for aryltetralin lignans
are now extended to ester and glycoside derivatives. In total, ca. 40 different lignans,
mainly of the aryltetralin type, were identified (Vasilev et al., 2008). 6-
Methoxypodophyllotoxin and its glucoside were present as major constituents in all
samples. Differences between the investigated taxa were observed especially with respect
to the accumulation of 6-deoxy-7-hydroxy-aryltetralins such as podophyllotoxin and of 6-
hydroxy-7-deoxy-aryltetralin lignans of the peltatin type. Studies on extracts of in vitro
cultures of L. narbonese and L. leonii have shown cytotoxic activity, due to the presence of
arylnaphthalene lignan Justicidin B (Vasilev et al., 2006b).
Increasing the yields of plant cell cultures for the productions of podophyllotoxin and
related lignans
Different methods and approaches have been applied to in vitro cultures producing PTOX,
its derivatives and other lignans (Fig. 9.2 (Ionkova, 2007)).
Fig. 9.2. Podophyllotoxin (R
1
=OH, R
2
=H, R
3
=OCH
3
), Deoxypodophyllotoxin
(R
1
=H, R
2
=H, R
3
=OCH
3
), -peltatin (R
1
=H, R
2
=OH, R
3
=OCH
3
) – A,
Epipodophyllotoxin– B.
H
3
CO
OCH
3
O
O
O
H
H
R
O
R
R
A
B
C D
1
2
3
H
3
CO
OCH
3
O
O
O
H
H
OCH
3
OH
O
A
B
C D
A B
Biotechnological Production of Lignans 147
Optimization of conditions of culturing, of plant in vitro cultures, for enhancement of
lignan production.
Multiple experiments from different working groups have been done for the enhancement
of lignan production from in vitro cultures with established synthesis (Table 9.2).
Table 9.2. Optimization of lignan in vitro production.
Species,
(in vitro culture)
Lignans
synthesized
Varying factors with
impact on the
culture
Results of optimization
Callitris drummondii
(Suspension)
PTOX-beta-D-
glucoside
Illumination 0.02% in the dark
0.11% in light
Forsythia x intermedia
(Suspension)
Pinoresinol
Matairesinol
Carbon source 2% sucrose:
0.001% lignans
6% sucrose:
0.07% pinoresinol;
0.1% matairesinol
Ipomoea cairica
(Callus)
Arctigenin, trace-
Logenin
Phytohormones,
Carbon source
4 mg/l 2,4-D, 3% maltose,
pH 6.4 - 0.03 % lignans
Juniperus chinensis
(Immobilized cells)
PTOX Various calcium
alginate
concentrations
1.8% Ca-alginate gel:
5-fold increased levels,
compared to free cell
suspension; maximum
excretion of PTOX in the
medium
3% Ca-alginate gel:
(0.21–0.025 mg/g dw)
4–5-fold increase
6% Ca-alginate gel:
small amount
Linum africanum
(Suspension)
PTOX
DPTOX
Source of explant
material, light,
IAA, NAA, 2,4-D,
Cytokinin/Kinetin
ratios
Highest synthesis:
Kinetin 10ml/l, IAA:
0.4ml/l, 2,4-D: 0.2ml/l;
PTOX: increases in dark
conditions (no difference
between callus and
suspension)
DPTOX: increases when
cultivated in the dark and
has higher levels in callus
than suspension
Linum album
(Suspension)
PTOX and
related lignans
Illumination 0.2% - dark
0.5% - light
Linum album
(Suspension, shake-
flasks: 1000ml – 50ml
medium, 300ml
–
50
PTOX Oxygen supply
Increasing of
shaker speed
Enhancement of PTOX
accumulation
148 Biotechnological Production of Lignans
ml medium,
establishment of
bioreactor)
L. altaicum
(Callus, Suspension)
Justicidin B
Isojusticidin B
Dark Justicidin B
between 0.92–0.96%.
L. austriacum ssp.
euxinum
(Callus, suspension)
Justicidin B Dark Justicidin B
between 0.50–0.96%
Linum campanulatum
(Callus, Suspension)
Justicidin B Illumination,
Kinetin, IAA, 2,4-D,
Succrose
Higher lignan content:
dark conditions
1.41 % in the dark
0.40 % in light
L. leonii
Hairy roots
Agrobacterium
strains
L. lewisii
(Callus, Suspension)
Justicidin B Dark Justicidin B
between 0.16–0.30%
Linum nodiflorum
(Suspension)
6MPTOX Illumination Light: 0.6 %
Dark: trace amounts
Linum tauricum ssp.
tauricum
(Suspension, Shoots,
Callus, Hairy roots)
4-Demethyl-6-
MPTOX
6MPTOX
Phytohormones
Methyl jasmonate
Agrobacterium
strains
Suspension: 4 mg/l NAA,
maximal production of 4-
demethyl-6-methoxy-
podophyllotoxin (2.16
mg/g dw)
Callus: 0.1mg/l 2,4-D,
0.2mg/l IAA, 2.0mg/l
Kinetin: maximal
production of 6-methoxy-
podophyllotoxin
(3.99 mg/g dw)
Podophyllum
hexandrum
(Suspension)
PTOX Illumination Light: 0.03%
Dark: 0.09%
Podophyllum
hexandrum
(Callus)
PTOX Phytohormones 2,4-D + Kinetin: 0.077%
Podophyllum
hexandrum
(Suspension, 3l-
stirred-tank
bioreactor)
PTOX Mathematical
model for
developing
nutritient-feeding
strategies, low
shear conditions in
fed-batch modes of
operation,
prolonging the
productive log-
phase of
cultivation.
Improvement to 48.8 mg/l
PTOX, corresponding
volumetric productivity of
0.80 mg/l per day
Podophyllum
hexandrum
(Suspension)
PTOX pH,
Phytohormones,
Carbon source,
Inoculum
pH 6.0
IAA: 1.25 mg/l
Glucose 72 g/l
Inoculum level: 8 g/l
Biotechnological Production of Lignans 149
Podophyllum
hexandrum
(Suspension)
PTOX Sugar, Nitrogen
source, Phosphate
MS medium, NH
4
+
Salts:
Nitrate: 1:2, 60 g/l
Glucose: highest growth
and PTOX accumulation
Podophyllum peltatum
(Callus)
PTOX Red light (660 nm)
Carbon source
Phytohormones
Enhancement of
production in red light
Sucrose: 0.057%
Maltose: 0.023%
2,4-D + Kinetin: 0.57%
Rollinia mucosa
(Jacq.) Bail. (Callus)
Furofuranic
lignans:
Epigambin,
Magnolin,
Epiyangambin
Origin of plant
material, Explant
type, Growth
regulators (2,4-D,
NAA, BA, PIC)
Foliar blade explants:
biomass, synthesis
enhancement;
PIC: best biomass
production;
NAA, 2,4-D:
Epiyangambin, Magnolin
(dependent on explant
source);
PIC: Epiyangambin:
Calli from foliar blade
* (The Data are cited in Vasilev and Ionkova, 2005; Ionkova et al., 2006, 2007; Konuklugil et
al., 2007; Vasilev et al., 2008).
Selection of high producing strains
A cell line of Linum album, accumulating mainly PTOX at a level of 0.2% on dry mass
basis was reported by Empt et al. (2000). With this cell line, about 28 mg PTOX can be
produced in 1l of culture medium in 11 days (Petersen and Alfermann, 2001).
Development of differentiated and transformed cultures
A 14-day old hairy root culture of Linum album Kotschy. (Linaceae) was reported to
produce about 2.5% of 6MPTOX on dry mass basis (Windhövel et al., 2003). A hairy root
culture of L. austriacum L. was reported by Mohagheghzadeh et al. (2002), which
synthesized the arylnaphthalene lignans justicidin B and isojusticidin B. Hairy root cultures
of Linum leonii, obtained by genetic transformation using the agropine-type strain
Agrobacterium rhizogenes 15834, accumulate justicidin B (Vasilev et al., 2006b). The
products encoded by rol A and rol C genes were found to have a synergistic effect on root
induction and to induce increased sensitivity to auxin. The transformation of these genes
from A. rhizogenes into the hairy root was checked by PCR (polymerase chain reaction).
Proof of transformation was given by the PCR products showing that rol A and rol C genes
were present in the hairy roots of L. leonii. Genetically modified hairy roots produced 5-
fold higher yields of justicidin B (10.8 mg/g DW) compared to untreated callus (Vasilev
and Ionkova, 2005) and show that differentiated roots produce higher amounts of
secondary compound. This suggests that this technique may be used to enhance the
accumulation of justicidin B. In addition to the production, we investigated the cytotoxic
effect of justicidin B in three chronic human myeloid leukaemia-derived cell lines (LAMA-
84, K-562 and SKW-3), that show a lower responsiveness to cytotoxic drugs due to a
strong expression of the fusion oncoprotein BCR-ABL (a non-receptor tyrosine kinase).
IC
50
values of justicidin B were 1.1, 6.1 and 1.5 μM for the chronic myeloid leukaemia
150 Biotechnological Production of Lignans
(LAMA-84), pre-B-cell lymphoma (K-562) and chronic lymphoid leukaemia (SKW) cell
lines respectively. These IC
50
were comparable to the anticancer drug etoposide (a semi-
synthetic lignan derivative).
Influence of stress factors
The influence of methyl jasmonate has been reported on pinoresinol and matairesinol
production in Forsythia t intermedia Zab. (Oleaceae) suspension culture (Dewick, 1994).
In a medium, containing 2% sucrose, the amount of pinoresinol was increased eightfold
(0.058±0.015mg/g dry mass), matairesinol about fivefold (0.029±0.026 mg/g dry mass). In
a medium containing 6% sucrose, pinoresinol was enhanced to 0.086±0.19 mg/g dry mass
and matairesinol to 2.24±1.00mg/g dry mass. The accumulation of PTOX and 6MPTOX
was enhanced about twofold, expressed on dry mass basis in a given cell line of suspension
culture of Linum album after the addition of methyl jasmonate, by 7.69±1.45 mg/g dry
mass and 1.11±0.09mg/g dry mass respectively (Van Furden et al., 2005). The PTOX
production of Juniperus chinensis L. (Cupressaceae) callus culture was stimulated to 6.4-
fold by addition of COS (chito-oligosaccharides), a biotic elicitor with most active
component chitopentaose, detected by HPLC (Premjet et al., 2002). The concentration 100
μM of the elicitor methyl jasmonate in the nutrition medium of suspension of Linum
tauricum ssp. tauricum, leads to substantial increase of the levels of lignans 4DM-
6MPTOX and 6MPTOX from traces, reaching a maximum of 0.1180 mg/g dw and 0.1250
mg/g dw respectively. The results (Ionkova et al., 2006) indicate that addition of
extracellular methyl jasmonate can not only increase the biosynthesis of both 4DM-
6MPTOX and 6MPTOX in a L. tauricum ssp. tauricum suspension culture, but also change
the ratio of both compounds, in comparison with the intact plant and callus cultures,
towards the more pharmacologically valuable 4-DM-6MPTOX (Vasilev et al., 2005a).
Feeding of precursors and/or biotransformation
Coniferin feeding to in vitro culture to produce PTOX derivatives has been reported in
literature (Van Uden et al., 1990a; Woerdenbag et al., 1990). Levels of PTOX increased
13- to 56-fold after application of coniferin. The initiation of Linum flavum L. hairy roots
was reported by Han-wei Lin et al. (2003a) as a source of coniferin. Significant variation
was reported for its accumulation between hairy root lines, originating from different L.
flavum seedlings and/or A. rhizogenes strains. After culturing the roots in Linsmaier and
Skoog medium (LS) with 2,4-D (2,4-dichlorophenoxyacetic acid) and NAA
(naphthalenacetic acid) as growth regulators coniferin reached 58 mg/g dry mass. Another
experiment is the cross species co-culture of L. album hairy roots, as a source of coniferin,
and Podophyllum hexandrum cell suspensions (Lin et al., 2003b). Increasing PTOX
concentrations in coculture was observed.
Feeding of coniferin led to fast increase of pinoresinol content, and no influence on
matairesinol synthesis in Forsythia t intermedia suspension culture (Schmidt and Petersen,
2002). Feeding of phenylalanine and coniferyl alcohol in
Juniperus chinensis callus
culture, led to 3.6- and 2.2- fold increase of PTOX, respectively (Premjet et al., 2002).
Certain enzymes of podophyllotoxin biosynthetic pathway have been isolated and
characterized and podophyllotoxin and 6MPTOX have been established to be stored in the
vacuole (Schmidt and Petersen, 2002). Elucidation in details of the biosynthetic pathway of
Biotechnological Production of Lignans 151
PTOX, isolating all enzymes and genes responsible for their encoding, would lead to new
strategies for enhancement of the yields of this valuable substance by means of a
combination of biotechnological and biochemical methods.
A novel system for the production of 2,7'-cyclolignans was recently demonstrated.
Deoxypodophyllotoxin is stereoselectively converted into epipodophyllotoxin by
recombinant human cytochrome P450 3A4 (CYP3A4). CYP3A4 is the main human
metabolizing enzyme. As a new biotechnological alternative, recently we described the
successful stereoselective hydroxylation of deoxypodophyllotoxin by recombinant
CYP3A4 in Escherichia coli. Epipodophyllotoxin (Fig. 9.3) has been detected as the only
metabolite in yields up to 90% (Vasilev et al., 2006a). Therefore, the heterologous
expression of CYP3A4 in E. coli presents an interesting alternative for the large-scale
production of epipodophyllotoxin.
O
O
CH
3
O
OCH
3
O
O
OCH
3
O
O
CH
3
O
OCH
3
OCH
3
O
O
OHH
CYP3A4
Fig. 9.3. Stereoselective hydroxylation of deoxypodophyllotoxin by recombinant
CYP3A4 in Escherichia coli to epipodophyllotoxin.
Conclusion
Results, summarized in Table 9.1 and Table 9.2, show that a broad range of experiments
have been carried out, resulting in enhancement of lignan production in in vitro cultures.
Cell cultures of different plant species are shown to produce considerable amounts of
lignans. Although both PTOX and MPTOX have comparable cytotoxic activity, due to the
different substitution in position 6 MPTOX is not used for the production of anticancer
drugs (Kuhlmann et al., 2002). Since PTOX is the preferred precursor for the semi-
synthesis of anticancer drugs like etoposide and etopophos®, the accumulation of
predominantly PTOX is especially interesting. L. tauricum ssp. linearifolium is now beside
L. album and L. persicum the third Linum species of section Syllinum with PTOX as the
main lignan (Ionkova et al., 2007).
Development of differentiated cultures as a general rule results in higher production of
active substances. This approach however is not economically feasible for scale-up of
production, as it encounters problems of in vitro cultivation and processing of great
biomass and longer growth periods than undifferentiated cultures. For this reason,
differentiated cultures are usually not used for in vitro production of lignans. An efficient
alternative of differentiated cultures are the genetically transformed, hairy roots cultures.
Their advantage is the shorter cycle (10–14 day), combined with the state of differentiation
which makes possible the stable production of active substances. Stress factors as biotic
152 Biotechnological Production of Lignans
(chito-oligosaccharides (COS) in Juniperus chinensis) and abiotic elicitors (methyl
jasmonate in Forsythia t intermedia, Linum tauricum ssp. tauricum) have been
demonstrated to enhance production of lignans. The feeding of a cheaper precursor as
phenylalanine, coniferin and coniferil alcohol in plant cell and tissue cultures of lignin-
producing plants, results in higher levels of production, as plant cells represent a ‘ready’
and organized system for bioprocessing and synthesis of target compounds.
A new biotechnological alternative is the successful stereoselective hydroxylation of
deoxypodophyllotoxin by recombinant CYP3A4 in Escherichia coli to epipodophyllotoxin
(Vasilev et al., 2006a), the direct precursor for the semi-synthesis of anticancer drugs
etoposide (VP-16) and teniposide (VM-26).
Due to the pharmaceutical importance and the low content in the plants the present
review focuses on alternative production systems for podophyllotoxin and related lignans.
Accumulation of lignans in plant tissue and organ cultures has been discussed based on the
work of different authors. A survey of literature data has shown positive results in
experiments with optimization of conditions of culturing, selection of high producing cell
lines, influence of stress factors and feeding of precursors in plant in vitro cultures for the
enhancement of production of lignans.
Recently, the interest of international pharmaceutical industries has been directed more
and more to plant based anticancer compounds. In this case, it has been shown that
production of podophyllotoxin and related lignans in cell cultures is possible. We believe
that cell cultures of different plants as a source of biologically active lignans can play a role
in this respect.
Abbreviations
PTOX – podophyllotoxin; 6MPTOX – 6-methoxypodophyllotoxin; DPTOX –
deoxypodophyllotoxin; 4DM-6MPTOX – 4-demethyl-6-MPTOX; HR – hairy root
cultures.
Acknowledgements
Financial support from Ministry of Education and Science, Sofia, Bulgaria (grant D002-
128/2008 Iliana Ionkova) is acknowledged.
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