Protoplasma (2014) 251:1359–1371

DOI 10.1007/s00709-014-0638-8

ORIGINAL ARTICLE

Improved sanguinarine production via biotic

and abiotic elicitations and precursor feeding in cell suspensions
of latex-less variety of Papaver somniferum with their gene
expression studies and upscaling in bioreactor
Priyanka Verma & Shamshad Ahmad Khan & Ajay K. Mathur &
Sumit Ghosh & Karuna Shanker & Alok Kalra

Received: 20 November 2013 / Accepted: 15 March 2014 / Published online: 28 March 2014
# Springer-Verlag Wien 2014

Abstract Elicitors play an important role in challenging the tetrahydroprotoberberine cis-N-methyltransferase (TNMT),
plant defense system through plant-environment interaction and protopine 6-hydroxylase (P6H) transcripts was observed.
and thus altering the secondary metabolite production. Culture Among abiotic elicitors, while hydrogen peroxide and carbon
filtrates of four endophytic fungi, namely, Chaetomium dioxide registered low level of sanguinarine accumulation,
globosum, Aspergillus niveoglaucus, Paecilomyces lilacinus, maximum sanguinarine content was detected by 250 μM
and Trichoderma harzianum were tested on embryogenic cell salicylic acid (0.058±0.003 % dry wt.; GI=172.75±13.40).
suspensions of latex-less Papaver somniferum in dose- RT (qPCR) also confirms the downregulation of sanguinarine
dependent kinetics. Besides this, abiotic elicitors salicylic pathway on CO2 supplementation. Various parameters rang-
acid, hydrogen peroxide, and carbon dioxide were also ap- ing from agitation speed (70 rpm), impeller type (marine),
plied for improved sanguinarine production. Maximum bio- media volume (2 l), inoculum weight (100 g), and culture
mass accumulation (growth index (GI)=293.50±14.82) and duration (9 days) were optimized during upscaling in 5-l
sanguinarine production (0.090±0.008 % dry wt.) were reg- stirred tank bioreactor to obtain maximum sanguinarine pro-
istered by addition of 3.3 %v/v T. harzanium culture filtrate. duction (GI=434.00; 0.119±0.070 % dry wt.). Addition of
Interestingly, it was further enhanced (GI=323.40±25.30; 3.3 %v/v T. harzanium culture filtrate and 50-μM shikimate
0.105±0.008 % dry wt.) when T. harzanium culture filtrate was done on the 6th day of bioreactor run.
was employed along with 50 μM shikimate. This was also
supported by real-time (RT) (qPCR), where 8–9-fold increase
Keywords Papaver somniferum . Sanguinarine . Elicitation .
in cheilanthifoline synthase (CFS), stylopine synthase (STS),
CO2 . Bioreactor upscaling . Real-time PCR

Handling Editor: Peter Nick

P. Verma (*) : S. A. Khan : A. K. Mathur : S. Ghosh Introduction
Department of Plant Biotechnology, Central Institute of Medicinal
and Aromatic Plants (CIMAP), Council of Scientific and Industrial Sanguinarine, a well-known alkaloid that belongs to
Research, PO CIMAP, Kukrail Picnic Spot Road, Lucknow 226015,
India
benzophenanthridine type of structure and reported to have
e-mail: priyankaurbest@gmail.com strong antimicrobial, antifungal, and anti-inflammatory activ-
ities, is predominantly localized in the roots of Papaver
K. Shanker somniferum. It is also known to act as a deterrent against
Department of Analytical Chemistry, Central Institute of Medicinal
herbivores and pathogens (Park et al. 2003; Trujillo-
and Aromatic Plants (CIMAP), Council of Scientific and Industrial
Research, PO CIMAP, Kukrail Picnic Spot Road, Lucknow 226015, Villanueva et al. 2012) besides being known for its cytotoxic
India and antiviral activities (Capasso et al. 2002). A study has
shown that sanguinarine is a potent inhibitor of the activation
A. Kalra
of nuclear transcription factor NF-kB3 (Ahmad et al. 2000)
Department of Microbiology, Central Institute of Medicinal and
Aromatic Plants (CIMAP), Council of Scientific and Industrial that has been implicated to play a key role in the regulation of
Research, PO CIMAP, Lucknow 226015, India cell growth, cell cycle regulation, and apoptosis. In the last
1360 P. Verma et al.

two decades, plant cell culture technology has been developed induction or accumulation levels varies with different elicitors
as an alternative source for producing secondary metabolites. (Pitta-Alvarez et al. 2000; Vasconsuelo and Boland 2007).
Instead of growing the plants in their natural habitats where Elicitor-induced sanguinarine accumulation in opium poppy
they are subjected to environmental barriers such as climate cell cultures provides a responsive model system to profile
and temperature change, plant cell cultures produce valuable modulations in gene transcripts, proteins, and metabolites
secondary metabolites under controlled and defined condi- related to alkaloid biosynthesis and other defense-related re-
tions (Smetanska 2008). The lack of a viable large-scale sponses. In addition to all-known sanguinarine biosynthetic
production method for many high-value plant-based products gene transcripts, transcripts encoding several upstream prima-
provides a need for research into improving the yield of ry metabolic enzymes were also induced (Zulak et al. 2007).
pharmaceutically important compounds from plant cell cul- The synthesis of sanguinarine and its analogous compounds
ture. However, increases in productivity from plant cell cul- initiates with the shikimate derivative tyrosine which gets
tures will require a greater understanding of metabolic path- converted to tyramine and L-dopamine via tyrosine/dopa de-
ways which limit their production. Proteins and pathways with carboxylase (TYDC) (Fig. 1). Another tyrosine derivative p-
changes in abundance in high-producing cell lines represent hydroxyphenylacetaldehyde produced by the action of tyro-
potential targets for further increases in production sine aminotransferase condenses with dopamine to produce
(Smetanska 2008). Biotic and abiotic elicitations are targeted norcoclaurine in the presence of norcoclaurine synthase
at plant cell to induce their substantial metabolic alterations (NCS). Norcoclaurine is a branch point for papaverine and
directed at establishing plant defense reactions. Elicitation- (S)-reticuline synthesis. The majority of BIAs are synthesized
based strategies are considered as an efficient approach for from a central intermediate (S)-reticuline which is a conden-
the improvement of valuable compounds from the plants and sation product of tyramine and dopamine. In the case of
secondary metabolite enhancement studies (Radman et al. P. somniferum, the bulk of (S)-reticuline isomerizes into (R)-
2003). A single elicitor can stimulate secondary metabolism reticuline for its subsequent flux toward morphine synthesis
in different cell cultures, and on the other hand, certain plant via codeine and thebaine routes. In Eschscholzia californica,
cultures are responsive to diverse elicitors. Treatment of a it is channelized toward sanguinarine biogenesis via activation
particular culture with different elicitors will result in the of berberine bridge enzyme (BBE) that further converts it to
enhanced accumulation of the same compounds, since these (S)-scoulerine as a committal intermediate toward
elicitors are specific for each plant culture. Although the class sanguinarine route. Sanguinarine alkaloid biosynthesis initi-
of metabolite depends on the plant species, the kinetics of ates with the synthesis of (S)-stylopine, catalyzed by

Fig. 1 The basic skeleton of

benzylisoquinoline alkaloid
pathway in P. somniferum derived
from the shikimate pathway
derivative tyrosine. Tyrosine/
dopa decarboxylase (TYDC),
tyrosine aminotransferase (TYAT),
norcoclaurine synthase (NCS),
berberine bridge enzyme (BBE),
cheilanthifoline synthase (CFS),
stylopine synthase (STS),
tetrahydroprotoberberine cis-N-
methyltransferase (TNMT),
protopine 6-hydroxylase (P6H),
thebaine 6-O-demethylase
(T6ODM), codeinone reductase
(COR), codeine O-demethylase
(CODM). Broken red arrows
shows multisteps
Improved sanguinarine production in latex-less Papaver somniferum 1361

cheilanthifoline synthase (CFS) and stylopine synthase (STS). 1,000-lux intensity illumination. Repeated and frequent
The subsequent step for the dihydrosanguinarine synthesis subculturing every 15th day resulted in establishment of gran-
involves tetrahydroprotoberberine cis-N-methyltransferase ular embryogenic cell suspensions.
(TNMT) and protopine 6-hydroxylase (P6H).
Dihydrosanguinarine is finally oxidized to sanguinarine
(Hagel and Facchini 2012; Mishra et al. 2013). The metabolic Elicitation and pecursor feeding
engineering implications aim to improve the efficiency of a
pathway by altering the enzymatic, transportation, and regu- Biotic elicitors of endophytic fungal origin, namely,
latory mechanisms through several experimental consider- Chaetomium globosum, Aspergillus niveoglaucus,
ations (Facchini and De Luca 2008). These may include Paecilomyces lilacinus, and Trichoderma harzianum were
increased availability and flux of precursor molecules from grown on potato dextrose agar (PDA). As an inoculum, 1-
primary metabolic pool in a desired direction, increment in the cm square disk of actively growing fungal isolates on PDA
percentage of metabolite-synthesizing cells, and providing medium was inoculated in 250 ml of potato dextrose medium
greater protection and stability to rate-limiting enzymes and and incubated on rotary shaker for 20 days (T. harzianum) and
intermediates through semisynthetic combinatorial ap- 15 days (C. globosum, A. niveoglaucus, and P. lilacinus) at a
proaches (Kutchan 1995; Verpoorte and Alfermann 2000; speed of 150 rpm. After attaining full growth, mycelium was
Sato et al. 2001; Ziegler and Facchini 2008). Therefore, the filtered through Whatman filter paper followed by filter ster-
utmost requirement is to generate cell types with ideal phys- ilization with 0.22-μ filter unit and added to 20 ml of 15-day-
iological background to test these various assumptions and old embryogenic cell suspensions in a dose of 3.3, 6, and 10 %
strategies of metabolic alterations (Oksman-Caldentey and v/v. The control experiment was set by treating the suspension
Inze 2004; Verma et al. 2012, 2013). Sanguinarine is a with equal amount of PDA medium only. The abiotic elicitors
plant-defense-related compound, so it is logical to check the used were hydrogen peroxide (H2O2), salicylic acid (SA), and
sanguinarine production by challenging the plant system with carbon dioxide (CO2). The H2O2 and SA solutions were
different biotic and abiotic elicitors. The present study covers added to the culture medium at the concentration of 0.5–
the above-mentioned emerging trends in research and aims to 2.0 mM and 50–250 μM, respectively. For continuously
emphasize on higher accumulation of precursor aromatic ami- maintaining a CO2-enriched gaseous phase around the grow-
no acids with greater physiological advantage in diverting the ing tissues, a two-tier culture flask was designed (Fig. 2). The
metabolic flux toward biogenesis of sanguinarine. Elicitation two-tier flask (each of 100 ml) was designed in such a way
strategies have previously been tested with yeast and Botrytis that the top opening of the lower flask travels through the
mycelia in Papaveracae sps. (Alcantara et al. 2005; Zulak et al. broad base of the upper flask and opened in the gaseous zone
2007; Trujillo-Villanueva et al. 2012), but present investiga- above the surface of the culture medium. The lower flask was
tion for the first time reports the improved flux toward aseptically filled with 60 ml of carbonate buffer consisting
sanguinarine production in latex-less variety Sujata of of 0.2 M KHCO3 and 0.2 M K2CO3 in 3:1 (pH=9.2) for
P. somniferum cell suspensions via combined approach of continuous release of CO2 into the gaseous head space in
elicitation-and precursor-based feeding with their feasibility the upper flask (approx. 2 % CO2 level). The cell suspen-
to be upscaled in the bioreactor. sions growing in the two-tier flask were harvested on the
10th, 20th, 30th, and 40th day. Untreated 20-day-old cell
suspension cultures served as controls. All biotic and abi-
Material and methods otic elicitors were added on the 15th day of inoculation and
harvested on the 5th day of addition except for CO2 which
Cell suspension cultures was supplemented from the zero to 40th day. To obtain
more insight into the regulation of sanguinarine biosynthe-
Leaf explants were taken from in-vitro-established shoot cul- sis in latex-less variety “Sujata” of P. somniferum cell
tures of P. somniferum variety “Sujata” (Sharma et al. 1999; suspensions and particularly to identify possible rate-
Chaturvedi et al. 2013) (Fig. 2a–b). Excised leaf explants were limiting steps in terms of the precursor availability, 15-
cultured on callusing medium comprising of Murashige and day-old embryonic cell suspensions were fed with tyrosine
Skoog (MS) (Murashige and Skoog 1962) basal medium and shikimate and analyzed for improved sanguinarine
supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid production. Aqueous solutions of tyrosine and shikimate
(2,4-D), 0.1 mg/l kinetin (Kn), and 40 mg/l ascorbic acid. were filter-sterilized and added in the range of 1–100 μM.
Obtained embryogenic callus was maintained on respective Tyrosine and shikimate were purchased from Sigma-
media for 6–7 subcycles and further transferred to liquid MS Aldrich, USA. The optimized dose was further added in
medium supplemented with 1 mg/l 2,4-D. Cultures were combination with various biotic and abiotic elicitors. All
incubated on a rotary shaker (120 rpm) at 24±20 °C under the experiments were performed twice and in triplicates.
1362 P. Verma et al.

a b c

d e

g h

k j i
Fig. 2 Raising and establishment of embryogenic cell suspensions from showing settled embryogenic cell mass with suspended non-embryogenic
leaf explants of latex-less variety of P. somniferum and upscaling in cells. Microscopic view of embryogenic mass (g) and suspended cells (h)
bioreactors. a In-vitro-grown plant of P. somniferum variety Sujata. b in 15-day-old cell suspensions and i, j bioreactor-grown embryogenic cell
Leaf explants plated on callusing medium. c Embryogenic callus plated suspensions supplemented with 3 %v/v T. harzanium culture filtrate and
on 1.0 mg/l 2,4-D, 0.1 mg/l Kn, and 40 mg/l ascorbic acid. d Freshly 50 μM shikimate. k Bioreactor-harvested embryogenic cell suspension
subcultured embryogenic cell suspension. e Cell suspension grown under biomass (batch 5). Bar=1 cm (a–f, i–k); 1 mm (g–h)
CO2 supplementation in two-tier flask. f 15-day-old cell suspension

HPLC analysis (10 ml), and extracted with ethyl acetate (3×30 ml). The
aqueous extract was cooled (10 °C), basified with NH4OH
Oven-dried tissue (1.0 g) was ground to fine powder and (pH 8.5), and extracted with chloroform (3 × 30 ml).
extracted with methanol (3×30 ml). Pooled methanolic ex- Combined chloroform extract was dried over Na2SO4 and
tract was filtered, concentrated in vacuum at 40 °C to 10 ml, air-dried to a constant weight. Sanguinarine estimation was
diluted with distilled water (10 ml), acidified with 3 % HCl performed by using high-performance liquid
Improved sanguinarine production in latex-less Papaver somniferum 1363

chromatography-photodiode array detector-mass spectrome- under different sets of conditions. Variables examined and
try (HPLC-PDA-MS) system, Shimadzu, Kyoto, Japan optimized for upscaling included the type of impeller (marine
(Mishra et al. 2013). Ten microliters of the extract was loaded vs. Rushton blade), batch cycle duration (9–20 days), inocu-
onto a C18 (4.6×250 mm, 5 μm) HPLC column. The mobile lum density (50–100 g), and medium volume (2–4 l). The
phase used was consisted of 1.0 % AcOH and acetonitrile. A cultures were aerated through a sintered steel sparger at an air
constant flow rate of 1.2 ml/min was used for all the analysis. flow rate of 2 l/min equivalent to 20 % dissolved oxygen (DO)
Detection was done at 327 nm. Standard of sanguinarine was level. Rotation speed was maintained at 70–100 rpm, and the
purchased from Sigma-Aldrich, USA. The data represent the incubation was carried out at 25±3 °C under high illumination
mean value of three independent experiments each performed (3,000 lux) provided by a specially fabricated LCD light
in triplicate. source kept around the glass vessel. Biomass accumulation
was monitored by measuring the packed cell volume and fresh
Real-time PCR (qPCR) analysis and dry weight determinations. All process parameters during
bioreactor cultivation were monitored and recorded using
Real-time PCR was performed on the StepOne real-time PCR BioExpert Ferm software.
system (Applied Biosystems; Foster City, CA, USA) with
SYBR Green PCR Master Mix (Applied Biosystems). Total
RNA was isolated from cell suspensions supplemented with
optimized doses of biotic and abiotic elicitors following LiCl Results
precipitation method. Random primers were used to generate
complementary DNA (cDNA) from the RNA from each tis- Raising of cell suspensions and growth kinetics
sue, using the high-capacity cDNA reverse transcription kit
(Applied Biosystems). cDNA of untreated cell suspensions Leaf explants plated on callusing medium showed emer-
served as controls. All real-time PCR quantifications were gence of embryogenic callus within 30 days of
performed with a non-template control and the endogenous subculturing. The embryogenic callus was found to be
control actin. The gene expression levels were interpolated slow growing, granular in appearance, and yellowish in
from standard curves for relative expression and normalized to color (Fig. 2). This embryonic callus was maintained on
actin in the same tissue. Real-time transcript analysis was callusing media for 6–7 subcycles and then further trans-
performed with four candidate genes specific to sanguinarine ferred to respective liquid medium. Interestingly, after
biosynthesis, i.e., CFS, STS, TNMT, and P6H. Respective their shifting from semisolid medium to liquid medium,
primers used for the amplification were F-5′-ACCAATGG growth was found to be initially hampered and finally
CGATTCCTCACA-3′, R-5′-CCATTACCACCGCACCTT terminated within 5 days, and callus showed blackening
TG-3′ (CFS); F-5′-TTGGAGACCAGTTATTGTCGTTTC- leading to death. The callus was then further shifted to
3′, R-5′-CGAGCCGAATAATCCGAAGA-3′ (STS); F-5′- liquid MS medium supplemented with 1 mg/l 2,4-D,
ATGGAATGCACATGGCTCGT-3′, R-5′-ACTGTTTCGT where cells showed better growth pattern. Established
GGCTTCCTCC-3′ (TNMT); F-5′-GATTGTGAGATCGA embryogenic cell suspensions on fresh subculturing
CGGGTTTC-3′, R-5′-GTTGCAACTTCCACACATTCAC showed granular yellowish cell mass, while fully grown
TAG 3′ (P6H); and F-5′-TCTCAACCCAAAGGCTAATCG- cell suspensions showed two types of cells—first, yellow-
3′, R-5′-CCCCAGAATCCAAGACAATAC-3′ (actin). The ish granular embryogenic cells that usually settle down in
cycling program consisted of an initial hold at 50 °C for shake flask cultures and, second, suspended non-
2 min and 10-min incubation at 95 °C followed by 40 cycles embryogenic fine cells (Fig. 2). The non-embryogenic
of 95 °C for 15 s and 60 °C for 1 min. Fold increase/relative fine cells were very less in number, approximately 5 %
quantification (RQ) was calculated using the formula RQ= of the total cell mass. Growth kinetic studies revealed that
2−ΔΔCt, where ΔCt=average Ct (gene of interest)−average Ct embryogenic cell suspension attained maximum growth
(actin) and ΔΔCt= ΔCt−ΔCt (control of the experiment; index (GI) of 106.11±8.95 on the 15th day followed by
cDNA of untreated cell suspensions). The experiment was 91.11±10.14 on the 20th day and 89.55±12.84 on the
performed with three biological samples and duplicated twice. 25th day (Fig. 3a). Maximum sanguinarine was also
detected on the 15th day (0.025 ± 0.004 % dry wt.)
Bioreactor upscaling followed by the 20th (0.023±0.003 % dry wt.) and 25th
days (0.023±0.002 % dry wt.), successively. From the
The embryogenic cell suspension culture supplemented with 25th day onward, the yellowish cell suspension started to
optimized elicitation and precursor treatments was upscaled in turn brownish in appearance indicating the initiation of
a 5-l stirred tank bioreactor (Model ADI-1010; Applikon their death phase; therefore, embryogenic cell suspen-
Biotechnology, Holland). Six independent batches were run sions were subcultured on every 15th day.
1364 P. Verma et al.

Fig. 3 Growth kinetics and a

elicitation studies in cell

Sanguinarine (% dry wt.)

0.04 300
suspensions of latex-less variety 0.035
of P. somniferum. a Sanguinarine 250
0.03
and biomass accumulation in cell 200
0.025
suspensions on different days.

GI
0.02 150
Growth and sanguinarine content
of the 20-day-old cell suspension 0.015
100
elicited with different b biotic 0.01
50
elicitors T. harzanium [Tricho 0.005
(T)], A. niveoglaucus [Asper (A)], 0 0
P. lilacinus [Paecilo (P)], and E-5D E-10D E-15D E-20D E-25D E-30D
C. globosum [Chaeto (CH)] and c DAYS
abiotic elicitors salicylic acid
[SA], hydrogen peroxide [H2O2], b
and carbon dioxide [CO2].

Sanguinarine (% dry wt.)

0.12 350
Carbon dioxide was supplied at 0.1 300
the concentration of 2 %, and 250
harvesting was done on 10-day 0.08
200
interval until 40 days (40D)

GI
0.06
150
0.04
100
0.02 50
0 0

CH-3.3% v/ v
Cont.

T-3.3% v/v

T-10 % v/v

A-6 %v/v

P-6 %v/v

P-10 % v/v

CH-6 % v/v
T-6 % v/v

A-3.3% v/v

P-3.3% v/v

CH-10 % v/v
A-10 % v/v
Tricho (T) Asper (A) Paecilo (P) Chaeto (CH)
Biotic Treatments

c
Sanguinarine (% dry wt.)

0.12 350
0.1 300
250
0.08
200

GI
0.06
150
0.04
100
0.02 50
0 0
10D

20D

30D

40D
0.5mM

1.0mM

1.5mM

2.0mM
50 µM

150 µM
100 µM

250 µM

Cont. SA H2O2 CO2 (2%)

Abiotic Treatments

Biotic and abiotic elicitation optimizations and chemical culture filtrate (GI=219.00±19.49; 0.064±0.003 % dry wt.).
analysis C. globosum and A. niveoglaucus (10 %v/v) showed more
than 2-fold increase in biomass accumulation (202.00±25.40
Culture filtrates of four endophytic fungi, namely, and 187.50± 2.80, respectively), but they showed lesser
C. globosum, A. niveoglaucus, P. lilacinus, and T. harzianum sanguinarine accumulation. C. globosum registered 2-fold
were tested in a dose-dependent manner of 3.3, 6, and 10 %v/ increase (0.047±0.009 % dry wt.) in sanguinarine production
v. Culture filtrates were added at late exponential growth while A. niveoglaucus showed only traces of sanguinarine
phase, i.e., 15-day-old embryogenic cell suspension. Among content (0.017±0.006 % dry wt.) comparatively (Fig. 3b).
all the tested biotic elicitors, T. harzanium culture filtrate at the Interestingly, in comparison with biotic elicitors, abiotic elic-
dose of 3.3 %v/v was able to induce maximum biomass itors registered a lower biomass accumulation (Fig. 3c).
accumulation (GI=293.50±14.82) which is also accompanied Among all the three elicitors used, namely, H2O2, SA, and
with highest sanguinarine production (0.090±0.008 % dry CO2, only SA was able to divert flux toward sanguinarine
wt.). It was followed by 10 %v/v of P. lilacinus (GI=232.5± production, with maximum yield registered by 250 μM SA
28.6; 0.071±0.002 % dry wt.) and 6.0 %v/v T. harzanium (0.058±0.003 % dry wt.) with better biomass production
Improved sanguinarine production in latex-less Papaver somniferum 1365

(GI=172.75±13.40). The use of H2O2 as an elicitor resulted embryogenic cell suspension, to check whether it can be
in a decrease in sanguinarine content. Maximum of only further enhanced through precursor availability, two precursor
0.009±0.001 % dry wt., sanguinarine was detected on appli- molecules shikimate and tyrosine were tested alone and in
cation of 0.5 mM H2O2. The most interesting aspect of abiotic combination with elicitors. It was observed that tyrosine
elicitation was found to be CO2 supplementation which re- (10 μM) and shikimate (50 μM) alone were able to enhance
sulted in extension of the growth cycle to 40 days in compar- the biomass production up to more than 2-fold, but no major
ison with the control tissue where cell suspension became changes in sanguinarine production (0.019–0.034 % dry wt.)
brownish within the 25th day. Cells treated with CO2 as were observed (Fig. 4a). Interesting results were obtained
elicitors were healthier, and their GI was 2.5-fold better than when optimum doses of all biotic and abiotic elicitors were
the control (GI=222.3±14.1). It is noteworthy that CO2 sup- employed along with 10 μM tyrosine or 50 μM shikimate.
plementation downregulates the sanguinarine pathway, and Details pertaining to biomass accumulation and sanguinarine
very low level of sanguinarine was detected (0.0015 ± content on different treatments are shown in Fig. 4b. Highest
0.001 % dry wt. on the 40th day) on HPLC profiling (Fig. 3c). sanguinarine production has reached to 4-fold increase (0.105
±0.008 % dry wt.) when 3.3 %v/v T. harzanium culture filtrate
Precursor availability improves sanguinarine production was employed along with 50 μM shikimate with maximum
biomass production (323.40±25.30). Further, a decrease in
Although more than 2- to 3-fold increase in sanguinarine both the content and biomass was observed when shikimate
production was achieved via biotic and abiotic elicitations of was replaced by 10 μM tyrosine (0.092±0.004 % dry wt.;

Fig. 4 Effect of precursor a

molecules tyrosine and shikimate 0.12 350
on elicited embryogenic cell
suspensions of latex-less variety 300
0.1
of P. somniferum. a Dose–
Sanguinarine (% dry wt.)

response of embryogenic cell 250

suspensions on different 0.08
concentrations of tyrosine and
200
shikimate. b Combinatorial effect

GI
of optimum doses of various 0.06
elicitors and precursors. 150
[T. harzanium (T), 0.04
A. niveoglaucus (A), P. lilacinus 100
(P), C. globosum (CH), salicylic
acid (SA), hydrogen peroxide 0.02
50
(H2O2), carbon dioxide (CO2)]
0 0
1 µM 10µM 50µM 100µM 1 µM 10µM 50µM 100µM
cont. Tyrosine Shikimate
Treatments

b
0.12 350
Sanguinarine (% dry wt.)

0.1 300

250
0.08
200
GI

0.06
150
0.04
100
0.02 50

0 0
SA-250µM

SA-250µM
T-3.3 % v/v

P-10 % v/v

CH-10 % v/v

T-3.3 % v/v
A-10 % v/v

H2O2-0.5mM

CO2- 40D

A-10 % v/v

P-10 % v/v

CH-10 % v/v

H2O2-0.5mM

CO2- 40D

cont. 10 µM Tyrosine 50 µM Shikimate

Treatments
1366 P. Verma et al.

GI=295.40±22.13). In both the cases, enhanced sanguinarine harzanium and 250 μM SA was also studied. Highest expres-
content was observed in comparison with supplementation of sion of CFS has been showed by 3.3 %v/v T. harzanium
3.3 %v/v T. harzanium culture filtrate alone, indicating the fortified with 50 μM shikimate (RQ=7.26±0.10), followed
importance of the metabolic flux of precursor molecule. In all by 3.3 %v/v T. harzanium culture filtrate (alone) (RQ=3.63±
the treatments, shikimate was found to be better in diverting 0.30) and 10 %v/v C. globosum culture filtrate. All remaining
the flux toward sanguinarine production. treatments showed lesser or equivalent CFS expression to the
control embryogenic cell suspensions (Fig. 5a). The maxi-
Gene expression studies mum STS expression was observed in 3.3 %v/v T. harzanium
culture filtrate (RQ=11.15±0.49), followed by 10 %v/v P.
The accumulation of sanguinarine in cultured embryonic cell lilacinus culture filtrate (9.95±0.31) and 50 μM shikimate+
culture of latex-less opium poppy requires activation of all the 3.3 %v/v T. harzanium culture filtrate (RQ =7.33± 0.29)
enzymes related to divert flux from reticuline to (S)-reticuline (Fig. 5b). TMNT expression was found to be almost equal in
and further its conversion into sanguinarine (Fig. 1). In the 3.3 %v/v T. harzanium alone (RQ=8.80±0.44) and in combi-
above elicited cultures, gene expression studies of four genes nation with 50 μM shikimate (RQ=8.70±0.29). None of the
(CFS, STS, TNMT, and P6H) related to sanguinarine biosyn- treatment from the remaining cultures showed any significant
thesis were assessed. Candidate treatments for gene expres- change in TNMT expression level except 10 %v/v P. lilacinus
sion analysis were embryogenic cell suspension fed with culture filtrate (RQ=3.6±0.3) (Fig. 5c). Although most of the
3.3 %v/v T. harzanium, 10 %v/v C. globosum, 10 %v/v A. elicitor treatments showed better expression of P6H except
niveoglaucus, and 10 %v/v P. lilacinus culture filtrates, CO2 supplementation and H2O2 where very low expression
250 μM SA, 0.5 mM H2O2, and CO2 supplemented 40-day- was registered, but again, the noticeable enhancement was
old cell suspension. Embryogenic cell suspension fortified observed in 3.3 %v/v T. harzanium culture filtrate alone
with 50 μM shikimate in combination with 3.3 %v/v T. (RQ=4.64±0.30) and in combination with 50 μM shikimate

Fig. 5 Quantitative real-time a b

PCR (qPCR) analysis of four 14
sanguinarine specific genes 8
7 12
against all the optimum doses of
6 10
RQ (STS)
biotic and abiotic elicitors applied
RQ (CFS)

alone or in combination with the 5 8

precursor molecule shikimate. 4 6
a Cheilanthifoline synthase 3
4
[CFS], b stylopine synthase 2
[STS], c tetrahydroprotoberberine 1 2
cis-N-methyltransferase [TNMT], 0 0
and d protopine 6-hydroxylase
[P6H]. [T. harzanium (T),
A. niveoglaucus (Asper),
P. lilacinus (Paecilo),
C. globosum (Chaeto), salicylic
acid (SA), hydrogen peroxide
(H2O2), carbon dioxide (CO2),
RQ relative quantification value]
c d
10 12
9
8 10
RQ (TMNT)

RQ (P6H)

7 8
6
5 6
4
3 4
2 2
1
0 0
Improved sanguinarine production in latex-less Papaver somniferum 1367

(RQ=9.58±0.45) (Fig. 5d). It was observed that CO2 supple- 15 days, and elicitor was introduced along with the precursor
mentation throughout the analysis showed very low expres- on the 15th day, and harvesting was done on the 20th day.
sion levels of sanguinarine-specific pathway genes. Overall Packed cell volume (PCV) of batch 1 was 5.30±1.30/100-ml
expression analysis demonstrated that embryogenic cell sus- suspension. Second batch followed reduced medium volume
pensions treated with 3.3 %v/v T. harzanium culture filtrate in (3 l) and decreased culture duration (15 days; addition on 10th
the presence of 50 μM shikimate were found to be best for day), with incorporation of Rushton-type impeller at the speed
in vitro sanguinarine production in latex-less variety of of 100 rpm. The growth and biomass accumulation was highly
P. somniferum where 8–9-fold increase in the expression levels affected (GI=−40; 0.065±0.020 % dry wt.) with PCV of 3.40
of all the four studied genes was observed. ±0.70 ml/100 ml. Third batch was similar to the second batch
except with low speed, i.e., 70 rpm and medium volume (2 l).
Bioreactor upscaling of cell suspension under optimized For the first time in this batch, cells were found to be growing
sanguinarine production phase (GI=26.5; PCV=8.50±1.80), but still, optimum sanguinarine
production was not achieved. Fourth batch was a replica of the
According to the optimizations done in the previous sections third batch with replacement of Rushton type of impeller to
based on the day of addition, elicitor specificity, dose re- marine type and subsequent increase in the inoculum weight
sponse, and precursor availability, conditions for maximum (100 g instead of 50 g). In this batch, sanguinarine production
sanguinarine production in latex-less variety of P. somniferum as well as growth was significantly improved (0.093±0.020 %
were optimized. A maximum of 0.105±0.008 % dry wt. dry wt.; GI=100; PCV=11.20±1.50 ml/100 ml). Optimum
sanguinarine was produced when 15-day-old embryogenic biomass production (GI=434.00) and sanguinarine content
cell suspensions were fed 50 μM shikimate along with (0.119±0.070 % dry wt.) were registered in the last two
3.3 %v/v T. harzanium culture filtrate and harvested after batches (5th and 6th) where culture duration was reduced to
5 days in shake flask culture. This was also supported by the 9 days, and addition was done on the 6th day with medium
gene expression analysis where 8–9-fold increase in volume of 2 l. The maximum biomass accumulation attained
sanguinarine specific genes had been observed. Cell suspen- under these standardized operational conditions was 5-fold
sions treated with the above optimized conditions were also more than the initial inoculum weight with a concurrent
upscaled in 5-l stirred tank bioreactor. Six individual batches increase in the PCV value of 50.40±3.20 ml/100-ml cell
of upscaling were run in a 5-l stirred tank bioreactor fitted with suspension. The rapid biomass increment was also evident
a steel sparger (Fig. 2). All runs were initiated using 50 and through a steep fall in DO level between 80 and 110 h under
100 g of cells from 15-day-old shake flask cultures (Table 1). such culture conditions (Fig. 6a). As in the case of shake flask
Shikimate and T. harzanium culture filtrate were added to each suspension cultures, the HPLC analysis of cells upscaled in
batch according to the concentration and medium volume bioreactor showed sanguinarine production at their optimum
(50 μM shikimate and 3.3 %v/v T. harzanium culture filtrate). level (Fig. 6b).
In batch 1, wherein the medium volume was 4 l and 50 g
inoculum was used without any impeller, the cells did not
grow well, and both biomass accumulation and sanguinarine Discussion
production were low (GI=−10; 0.085±0.030 % dry wt.).
Here, growth cycle follows normal shake flask conditions Plant cell cultures have been viewed as a promising alternative
where embryogenic cell suspension was allowed to grow for to whole plant extraction for obtaining valuable chemicals. At

Table 1 Optimization of agitation speed, impeller type, media volume, filtrate along with 50 μM shikimate in 5-l stirred tank bioreactor for
inoculum weight, and culture duration to upscale P. somniferum embryo- maximum sanguinarine production
genic cell suspension supplemented with 3.3 %v/v T. harzanium culture

Batch Agitation Impeller Media Inoculum Culture Day of addition of Growth Pack cell volume Sanguinarine
no. speed volume (l) weight (g) duration 3.3 %v/v T. harzanium index (ml/100-ml production
(days) culture filtrate+50 μM shikimate suspension) (% dry wt.)

1 0 No impeller 4 50 20 15th day (134 ml)a −10 5.3±1.3 0.085±0.03

2 100 Rushton 3 50 15 10th day (100 ml)a −40 3.4±0.7 0.065±0.02
3 70 Rushton 2 50 15 10th day (67 ml)a 26.5 8.5±1.8 0.075±0.01
4 70 Marine 2 100 15 10th day (67 ml)a 100 11.2±1.5 0.093±0.02
5 70 Marine 2 100 9 6th day (67 ml)a 434 50.4±3.2 0.119±0.07
6 70 Marine 2 100 9 6th day (67 ml)a 428 49.2±2.1 0.118±0.06
a
Addition was done according to the conc. 3.3 % v/v T. harzanium culture filtrate
1368 P. Verma et al.

Fig. 6 Various process a

parameters recorded during 120
bioreactor cultivation of elicited
embryogenic cell suspensions of
P. somniferum in “batch 5” with 100
marine-type impellers and 9-day
culture cycle. a Characteristic pH temp DO (%) Agitation
steep fall in dissolved oxygen 80
(DO) level between 80 and 110 h
and b HPLC chromatogram of
sanguinarine detection in 60
T. harzanium (3.3 %v/v) and
50 μM shikimate-treated
embryogenic cell suspensions of 40
“batch 5”

20

Time

Sanguinarine

present, unfortunately, only a few plant cell culture processes influences the degree of metabolite production (Lee-Parsons
are implemented for commercial production of bioactive com- et al. 2004), and a combination of elicitors can have synergis-
pounds. The major lacuna which holds back the large-scale tic effects (Sudha and Ravishankar 2002; Yamaguchi et al.
cultivation of plant cells includes low productivity, cell line 2002; Yuan et al. 2002). An ability to identify synergistic
instability, and difficulty in the process of scale-up parameter regulators and to combine them optimally to exploit their
control (Pan et al. 2000). Cell suspension culture systems can effects could have great value in systems for production of
be an alternate approach for large-scale, defined, continuous, secondary metabolites. The appropriate point to add the elic-
and reliable source of bioactive natural products (Namdeo itor is during the exponential phase of growth (Vasconsuelo
2007). In the present study, embryogenic cell suspension of et al. 2003) when the enzymatic machinery is in the maximum
the variety “Sujata” of P. somniferum was taken as source operative status, the response to the elicitor being, in conse-
material for sanguinarine production which is a latex-less quence, more efficiently achieved. As in the case, when used
variety; therefore, it is worthwhile to utilize it in vitro for at early log phase showed immediate increase in betalain
sanguinarine production, as there are chances of diversion of content while suppressing the biomass, leading to low overall
(S)-reticuline toward sanguinarine pathway. Growth kinetics productivity (Savitha et al. 2006). In the present study, culture
of raised embryogenic study showed maximum biomass and filtrates of four fungal elicitors viz. C. globosum,
sanguinarine content on the 15th day. A range of biotic and A. niveoglaucus, P. lilacinus, and T. harzianum were tested
abiotic elicitors along with the precursor molecules were at doses of 3.3, 6, and 10 %v/v. Three abiotic elicitors
added to this exponential phase of growth to improve the (salicylic acid, H2O2, and CO2) were also tested at different
content. In the elicitor treatment technique, timing of the dose- and duration-dependent kinetics. Effects of the two
treatment, as well as the concentration of the elicitor, precursor molecules, tyrosine and shikimate, were also
Improved sanguinarine production in latex-less Papaver somniferum 1369

analyzed in combination with the elicitors. It is well docu- of CO2. Although CO2 promoted growth and extended the
mented that each type of elicitor according to its characteris- exponential phase up to 40 days of the embryogenic cell
tics can induce specific responses that depend on the interac- suspension, a very low level of sanguinarine was detected.
tion of elicitor-plant culture. The concentration of elicitor is a This suggests that this flux may be utilized in the upregulation
factor that strongly affects the intensity of the response and the of alternative paths showed in Fig. 1. Growth-promoting
effective dose, which varies according to the plant species, effect of CO2 supplementation has also been reported by
and can only be found empirically (Vasconsuelo and Boland Solarova and Pospisilova (1997) and Tripathi et al. (2001).
2007). It has been demonstrated that elicitor levels, which In the present study, sanguinarine content was enhanced from
exert stimulatory effects in certain plant systems when applied 0.025±0.008 to 0.105±0.008 % dry wt. on elicitation with
to other ones, are devoid of activity, reflecting different sensi- T. harzianum culture filtrate and shikimate supplementation.
bilities of the molecular components involved in elicitation. Previous reports also showed enhanced benzophenanthridine
As in case of Rubia akane Nakai cell cultures, chitosan type of alkaloids on the application of different elicitors, but in
induces maximum anthraquinone production at a concentra- different species of poppy, i.e., E. californica, Farber et al.
tion of 20 mg/l (Jin et al. 1999), while 200 mg/l of this elicitor (2003) reported enhanced benzophenanthridine alkaloids pro-
is the optimum concentration to improve menthol production duction upon addition of either yeast extract or methyl
by cultured Mentha piperita cells (Chang et al. 1998). jasmonate. Yeast elicitation enhanced sanguinarine but did
Commonly, the literature holds the view that elicitors may not affect dihydrosanguinarine production significantly in
affect plant secondary metabolism by modulating the rates of E. californica cultures (Cho et al. 2007). In the same study,
biosynthesis, accumulation and/or vacuolar transit, turnover methyl jasmonate enhanced dihydrosanguinarine production.
and degradation via G-protein activation, increase in cAMP However, methyl jasmonate did not enhance sanguinarine
level, changes in cytoplasmic Ca2+ concentrations, and phos- production. Dihydrosanguinarine production increased ap-
phorylation of MAP kinases (Vasconsuelo and Boland 2007). proximately 100 % on addition of salicylic acid while
In the present investigation, the maximum biomass accumu- sanguinarine production was not affected at the same condi-
lation (GI=323.40±25.30) and highest sanguinarine produc- tions (Cho et al. 2007). Real-time (qPCR) revealed 8–9-fold
tion (0.105±0.008 % dry wt.) were obtained when the 15-day- increase in the expression levels of CFS, STS, TNMT, and
old embryonic cell suspension of P. somniferum (latex-less P6H which have been registered by 3.3 %v/v T. harzianum
variety Sujata) was elicited with 3.3 %v/v T. harzianum cul- culture filtrate and 50 μM shikimate in the present study. In
ture filtrate along with 50 μM shikimate and harvesting was the previous studies, Facchini and Park (2003) found in-
done on the 20th day. Shikimate and their derivative aromatic creased expressions of the TYDC, 6OMT, CNMT,
amino acids (tryptophan, tyrosine, and phenylalanine) hold a CYP80B1, 4′OMT, and BBE genes following elicitation of
very important position as a precursor molecule in overall opium poppy suspension cultures with a fungal elicitor.
plant metabolism, and that is the reason why these precursor Elicitation of E. californica cultures with methyl jasmonate
molecules play a very notable role in blocking the flux of upregulated the expression of six benzophenanthridine alka-
secondary metabolite pathway and, thus far, acting as a limit- loid biosynthetic enzymes: 6OMT, CNMT, CYP80B1, 4′
ing factor (Verma et al. 2012, 2013). While production has OMT, BBE, and DHBO (Cho et al. 2008a, b). BBE (Dittrich
successfully been enhanced in many plant cell cultures, less is and Kutchan 1991) and P6H (Tanahashi and Zenk 1990)
known about which biochemical pathways are critical for activities are inducible by yeast extract elicitors. Zulak et al.
supporting increased production. The shikimate and aromatic (2007) analyzed the transcript profile of fungal-elicited
amino acid biosynthesis pathways represent a major regulato- P. somniferum cell cultures using expressed sequence tags
ry link of primary and secondary metabolism in plants. Thus, (ESTs) and characterized the profile of primary metabolites
our hypothesis is that primary and secondary metabolic path- and alkaloids. An increase in transcripts of both sanguinarine
ways are coordinated to support increased sanguinarine pro- biosynthetic enzymes and enzymes in primary metabolic
duction. In all the treatments, shikimate was found to be better pathways including sugar metabolism, the shikimate pathway,
in diverting the flux toward sanguinarine production. This and amino acid metabolism was observed. Specifically, all of
could be justified on the basis that tyrosine alone will have the steps between sucrose and sanguinarine were upregulated.
direct effect on alkaloid synthesis, but shikimate will lead to The system biology approach by Zulak et al. (2007) revealed a
synthesis of three aromatic amino acids, i.e., tyrosine, trypto- coordination of primary and secondary metabolism during
phan, and phenylalanine. It may be possible that tryptophan increased benzophenanthridine alkaloid biosynthesis in
and phenylalanine that also are the precursors for other im- P. somniferum cell cultures induced with a homogenate from
portant metabolites have composite effect on growth and the fungus Botrytis cinerea and showed upregulation of eight
accumulation capabilities of the cell (Yamamoto et al. 2000). genes (TYDC, NCS, 6OMT, CNMT, CYP80B1, 4′OMT,
Another interesting aspect of the study presented here is the BBE, and TNMT). The present study also deals with success-
downregulation of sanguinarine pathway via supplementation ful optimization of various parameters associated with
1370 P. Verma et al.

sanguinarine yield in 5-l stirred tank bioreactor. It was found help in understanding gene regulation studies in Papaveraceae
that the marine type of impeller was more suitable for em- family in a better way. A thorough study of the pathway with
bryogenic cell suspensions. Influence of appropriate impeller upregulation/downregulation of specific genes with concur-
types and inoculum density in bioreactor-based cultivation of rent use of elicitor will be the forthcoming area of study for
suspension cells has been highlighted in previous studies efficient sanguinarine production.
(Fulzele and Heble 1994; Huang and Mc Donald 2009). The
use of an improper impeller type can exert undesirable hydro- Acknowledgments The work presented here has been supported by
dynamic forces leading to poor cell viability and metabolite DST-FAST TRACK SERC/LS-261/2012. PV is highly thankful to CSIR
production due to shear mechanical shocks (Huang and Mc for awarding CSIR-RA. Help rendered by Dr. Shiv Shanker Pandey,
Donald 2009). During optimizations, lower productivity of Sonal Mishra, Dr. Rakesh Shukla, and Jyotsana Priya, during experimen-
tation, has been highly acknowledged.
sanguinarine was observed in the initial bioreactor upscaling
cycles in comparison with shake flask cultures. This reduction
could be attributed to various factors such as shear stress,
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