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Physiochemical and Transgenic Approaches to Increase Artemisinin Production
Prof. M. Z. AbdinCentre for Transgenic Plant Development
Department of BiotechnologyJamia Hamdard
New Delhi110062INDIA
IN COLLABORATION WITH Dr.D.C.JainIpca,Mumbai
ARTEMISININ
Antimalarial (Li et al., 1982)
Anticarcinogenic (Lai and Narendra, 1995; Efferth et al., 2001)
Antihepatitis B (Romero et al., 2005)
Antileishmanial (Sen et al. 2007)
Antischistosomiatic (Borrmann et al. 2001)
Herbicidal (Chen et al., 1987; Duke and Lydon, 1987)
AntiHIV (Kim et al., 1993)
O
O
OO
O
H
HH
Structure of artemisinin
Global Demand of Artemisinin – 180 Metric Tonnes/ year
Global Production of Artemisinin 120 Metric Tonnes/year
Average Artemisinin Yield 2025kg/ha
Average Artemisia annua leaves production – 1.5 ton/ha (India)
Average Artemisinin content in leaves 0.50.9% (India)
Average Artemisinin Yield – 1015 kg/ha
Artemisinin Production in India – 2 metric tonnes/Year
Artemisinin Demand in India – 2530 metric tonnes/year (Ipca)
NATIONAL and INTERNATIONAL
STATUS OF ARTEMISININ
Cytosolic MVA Pathway Plastidial DXP Pathway
3 acetylCoA Glyald 3p + Pyruvate HMGS DXS
HMGCoA DXP HMGR DXR
MVA MEP
IPP DMAPP
GPP FPS
Squalene FPP Amorpha4,11diene SQS ADS
CYP71AV1
Sterol Dihydroartemisinic acid Artemisinic acid Arteannuin B
Dihydroartemisinic acid Artemisinin Dihydroarteannuin B hydroperoxide
Artemisinin biosynthetic pathway
Mevinolin Fosmidomycin
Planta Med (2003) 69: 289299 Plant Cell Rep (2007) 26: 21292136
b
e
a. Rosetteb. Boltingc. Prefloweringd. Floweringe. Postflowering
A. annua L. plants in the experimental field of Jamia Hamdard
a c
d
S.No. Parameters Organs Stages studied
Rosette Bolting Preflowering Flowering Postflowering Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE
Flower NA NA NA 2.24± 0.14 NA1 HMGR activity Leaf 1.02 ± 0.11 1.62 ± 0.13 2.92 ± 0.12 2.15 ± 0.14 0.74± 0.06 (nmol MVA/h/mg protein ) Stem 0.52 ± 0.04 0.83 ± 0.09 1.41 ± 0.14 0.71 ± 0.08 0.35 ± 0.02 Flower NA NA NA 0.75 ± 0.03 NA 2 Artemisinin Leaf 0.15 ± 0.01 0.56 ± 0.03 0.72 ± 0.06 0.52 ± 0.04 0.23± 0.01 (% d w) Stem 0.01 ± 0.001 0.03 ± 0.002 0.05 ± 0.004 0.04 ± 0.003 0.02± 0.001
Flower NA NA NA 0.21 ± 0.02 NA 3. Artemisinic acid Leaf 0.05 ± 0.01 0.29 ± 0.03 0.22 ± 0.06 0.20 ± 0.02 0.13± 0.01 (% d w) Stem 0.001 ± 0.0001 0.005 ± 0.0003 0.004 ± 0.0004 0.003 ± 0.0003 0.001± 0.0001
4. Mevalonate Flower NA NA NA 13.24 ± 0.015 NA (µg/g-1fw) Leaf 7.65 ± 0.006 10.50 ± 0.009 17.57 ± 0.012 12.15 ± 0.010 5..74±0.06 Stem 2.52 ± 0.004 4.83 ±0.007 7.41 ± 0.014 5.71 ± 0.008 1.85± 0.003
Relationship between HMGCoA reductase activity, artemisinin, artemisinic acid andMevalonate content in different organs of A. annua L. plants at various phenological stages
HMGR activity expressed as nmol MVA formed/h/mg protein; artemisinin and artemisinic acid contents expressed as % d w and mevalonate expressed as µg/g1 fw ; Each value represents mean ± SE (n = 3)NA: Not detected
0
10
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60
70
80
90
100
0 25 50 75 100 125 150 175 200
% in
hibi
tion
in th
e in
corp
orat
ion
of la
bel (
14C
) int
o M
VA
and
art
emis
inin
MVA
Artemisinin
Mevinolin concentration (µM)
Effect of mevinolin on the synthesis of MVA and artemisinin in twigs of A. annua L. plants in the presence of 120 µM HMGCoA (314C) .
0
10
20
30
40
50
60
70
80
90
100
120 140 160 180 200 220 240 260 280
MVA
Artemisinin
HMGCoA (314C) concentration (µM)
% in
crea
se in
the
inco
rpor
atio
n of
labe
l (14
C) i
n M
VA
and
art
emis
inin
Effect of increasing concentration of HMGCoA (314C) (120280 µM) in the synthesis ofMVA and Artemisinin in twigs of A. annua L. in presence of 150 µM of mevinolin .
Effect of mevinolin and fosmidomycin on artemisinin accumulation in twigs of A. annua L. plants.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control FOS MEV
Art
emis
inin
acc
umul
atio
n (m
g g1
dw)
Effect of growth hormones on artemisinin content and HMGR activity in the leaves of Artemisia annua L.
The hormone treatments were given to Artemisia annua L. plants as fine spray at vegetative stage. The artemisinin and enzyme activity were determined in the leaves collected at preflowering stage as per the protocols standardized in our laboratory (Abdin et al., 2005)
3.96 (00)
5.00 (26.2)
5.12 (29.2)
5.55 (40.1)
0.065 (00)
0.085 (30.7)
0.087 (33.8)
0.090 (38.4)
CONTROL
IAA
GA3
IAA + GA3
HMG-CoA Reductase Activity (nmol MVA
h-1mg-1 protein)
Artemisinin content (% DW)
Hormones (100 ppm)
pBin AR (~12.5 kb)
HMGCoA Reductase (3 kb)
BamHI SalI
EcoRI BamHI SalI EcoRI/HindIII
CaMV35S (0.8 kb) OCS (0.2 kb)
EcoRI EcoRI/HindIII
HMGCoA Reductase (3 kb)
Cocultivation of leaf explants with Agrobacterium tumefaciens (after 48 hrs)
A. Control B. A. tumefaciens infected
A B
Regeneration on Kanamycin containing selection mediumE and F. Shoot regenerating from Callus G and H. Clonal propagation of Transformed shoots I, J and K. Transgenic shoot on multiplication medium
L. Transgenic shoot on rooting medium
Genetic Manipulation of Genetic Manipulation of Artemisia annua Artemisia annua for higher for higher artemisinin contentartemisinin content
(B) Southern analysis after genomic DNA was digested with Sal1 and hybridized with HMGRspecific gene probe.Lane 1: Plasmid DNA, Lane 2: Negativr control (DNA from wild type plant), Lanes 39: DNA samples from transgenic lines.
(A) PCR analysis in 7 kanamycinresistant plants for the presence of npt IIgene: lane 1: DNA ladder (1 Kb), lane 2: Positve control plasmid DNA, lane 3: Negative control DNA of a wild type plant, lanes 410: Putative transgenic plant DNA.
MOLECULAR ANALYSIS OF TRANSGENIC PLANTSMOLECULAR ANALYSIS OF TRANSGENIC PLANTS
TRANSGENIC PLANTSTRANSGENIC PLANTS
302.1 ± 37.5 (-4.13)11.2 ± 1.5 (-6.67)
TL78
318.0 ± 35.1 (0.92)12.2 ± 1.4 (1.67)TL67
371.1 ± 31.5 (17.77) 33.0 ± 1.2 (175.0)
TL56
369.0 ± 38.3 (17.11)31.1 ± 0.9 (159.17)
TL45
386.0 ± 33.2 (22.50)35.2 ± 1.0 (193.33)
TL34
318.0 ± 34.8 (0.92)13.0 ± 1.2 (8.33)TL23
320.1 ± 30.2 (1.59)13.1± 1.6 (9.17)TL12
315.1 ± 35.6 (0.0)12.0 ± 1.6 (0.0)Micro-propagated wild type
1
**Artemisinin Content (µg/g dw)
*HMGR Activity (nmol MVA mg protein-1 h-1)
Plant TypeS. No.
*Each value represents mean of 3 replicates ± SD Values in paraentheses are %
**Each value represents mean of 5 replicates ± SD increase/decrease over control
TL – Transgenic Lines Planta Medica (2009)75: 16
291 (00)576 (98)
892 (207)
CONTROLSalinity (160 mM)Lead (Pb; 500 ppm)
ARTEMISINIIN CONTENT (mg/g DW)
STRESS
EFFECT OF ABIOTIC STRESSES ON ARTEMISININ CONTENT IN THE LEAVES OF Artemisia annua L.
The Salt (NaCl) and lead stress was given at bolting stage (100 DAS) and artemisinin was determined 10 days after the treatment
Environ. Exp. Bot. (2005) 53: 185-193.
22.70 ± 0.60 (54.38)
0.71 ± 0.01 (27.29)
0.400±0.006
7.95 ± 0.18
(14.98)
3.19 ± 0.10
(21.55)
T4 (N40+40 + P40 +
K40 + S15+15)
19.51 ± 0.73 (32.71)
0.66 ± 0.02 (17.94)
0.393 ±0.006
7.54 ± 0.24
(8.97)
2.95 ± 0.06
(12.47)
T3
(N40+40+P40+K40)
16.21 ± 0.31 (10.24)
0.59 ± 0.01 (5.35)
0.386 ±0.004
7.13 ± 0.19
(3.11)
2.75 ± 0.06
(4.94)
T2 (Organic Manure, 15 ton ha-1)
14.70 ± 0.52 (0.0)
0.56 ± 0.01 (0.0)
0.381 ±0.006
6.92 ± 0.28
(0.0)
2.62 ± 0.08
(0.0)
T1 (Control)
Artemisinin Yield (Kg ha-1)
ArtemisinnContent inLeaves (% dw)
LeafStemratio
StemBiomass(t ha-1)
LeafBiomass(t ha-1)
Treatment
Each value represents mean ± SE (n = 9) Values in parentheses are % increase over control
Effect of fertilizers on artemisinin content and yield in Artemisia annua L.
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