Physiochemical and Transgenic Approaches to Increase Artemisinin Production

Transcription

1 Physiochemical and Transgenic Approaches to Increase Artemisinin Production Prof. M. Z. Abdin Centre for Transgenic Plant Development Department of Biotechnology Jamia Hamdard New Delhi INDIA IN COLLABORATION WITH Dr.D.C.Jain Ipca,Mumbai

2 H O ARTEMISININ O O H H O O Structure of artemisinin Antimalarial (Li et al., 1982) Anti carcinogenic (Lai and Narendra, 1995; Efferth et al., 2001) Anti hepatitis B (Romero et al., 2005) Anti leishmanial (Sen et al. 2007) Anti schistosomiatic (Borrmann et al Anti HIV (Kim et al., 1993) Herbicidal (Chen et al., 1987; Duke and Lydon, 1987)

3 TIOL and INTERTIOL STATUS OF ARTEMISININ Global Demand of Artemisinin 180 Metric Tonnes/ year Global Production of Artemisinin 120 Metric Tonnes/year Average Artemisinin Yield 20 25kg/ha Average Artemisia annua leaves production 1.5 ton/ha (India) Average Artemisinin content in leaves % (India) Average Artemisinin Yield kg/ha Artemisinin Production in India 2 metric tonnes/year Artemisinin Demand in India metric tonnes/year (Ipca)

4 Cytosolic MVA Pathway Plastidial DXP Pathway 3 acetyl CoA HMGS Mevinolin Glyald 3p + Pyruvate DXS HMG CoA HMGR DXP DXR Fosmidomycin MVA MEP IPP DMAPP GPP FPS Squalene SQS FPP ADS Amorpha 4,11 diene CYP71AV1 Sterol Dihydroartemisinic acid Dihydroartemisinic acid hydroperoxide Artemisinic acid Artemisinin Arteannuin B Dihydroarteannuin B Artemisinin biosynthetic pathway Planta Med (2003) 69: Plant Cell Rep (2007) 26:

5 a d c b e a. b. c. d. e. Rosette Bolting Pre flowering Flowering Post flowering A. annua L. plants in the experimental field of Jamia Hamdard

6 Relationship between HMG CoA reductase activity, artemisinin, artemisinic acid and Mevalonate content in different organs of A. annua L. plants at various phenological stages S.No. Parameters studied Organs Stages Rosette flowering Mean ± SE Flower 1 HMGR activity Leaf 0.74± 0.06 (nmol MVA/h/mg protein ) Stem 0.35 ± 0.02 Flower 2 Artemisinin 0.23± 0.01 (% d w) 0.02± Bolting Mean ± SE Mean ± SE 2.92 ± ± ± ± ± ± 0.03 Stem 0.01 ± ± ± ± Post Mean ± SE 2.24± ± 0.13 Flowering Mean ± SE 1.02 ± 0.11 Leaf Flower Pre flowering 2.15 ± ± ± ± ± ± Artemisinic acid Leaf 0.05 ± ± ± ± 0.02 HMGR 0.13±activity 0.01 expressed as nmol MVA formed/h/mg protein; artemisinin and artemisinic acid contents expressed as % d w and 1 mevalonate expressed as µg/gstem fw ; Each value represents mean ±0.005 SE (n =±3) (% d w) ± ± ± : Not detected 0.001±

7 % inhibition in the incorporation of label (14C) into MVA and artemisinin 100 MVA 90 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 HMG CoA (3 14C).

8 % increase in the incorporation of label (14C) in MVA and artemisinin MVA 80 Artemisinin HMG CoA (3 14C) concentration (µm) Effect of increasing concentration of HMG CoA (3 14C) ( µm) in the synthesis of MVA and Artemisinin in twigs of A. annua L. in presence of 150 µm of mevinolin. 280

9 Artemisinin accumulation (mg g 1dw) Control FOS MEV Effect of mevinolin and fosmidomycin on artemisinin accumulation in twigs of A. annua L. plants.

10 Effect of growth hormones on artemisinin content and HMGR activity in the leaves of Artemisia annua L. Hormones (100 ppm) Artemisinin content (% DW) HMG-CoA Reductase Activity (nmol MVA h-1mg-1 protein) (00) 3.96 (00) IAA (30.7) 5.00 (26.2) GA (33.8) 5.12 (29.2) IAA + GA (38.4) 5.55 (40.1) CONTROL 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 pre flowering stage as per the protocols standardized in our laboratory (Abdin et al., 2005)

11 Genetic Manipulation of Artemisia annua for higher artemisinin content HMG CoA Reductase (3 kb) BamHI EcoRI EcoRI pbin AR (~12.5 kb) EcoRI/HindIII BamHI CaMV35S (0.8 kb) A SalI SalI EcoRI/HindIII HMG CoA Reductase (3 kb) OCS (0.2 kb) Co cultivation of leaf explants with Agrobacterium tumefaciens (after 48 hrs) A. Control B. A. tumefaciens infected B Regeneration on Kanamycin containing selection medium E 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

12 MOLECULAR ALYSIS OF TRANSGENIC PLANTS (A) PCR analysis in 7 kanamycin resistant plants for the presence of npt II gene: lane 1: D ladder (1 Kb), lane 2: Positve control plasmid D, lane 3: Negative control D of a wild type plant, lanes 4 10: Putative transgenic plant D. (B) Southern analysis after genomic D was digested with Sal1 and hybridized with HMGR specific gene probe. Lane 1: Plasmid D, Lane 2: Negativr control (D from wild type plant), Lanes 3 9: D samples from transgenic lines.

13 TRANSGENIC PLANTS

14 S. No. Plant Type *HMGR Activity (nmol MVA mg protein-1 h-1) **Artemisinin Content (µg/g dw) 1 Micro-propagated wild type 12.0 ± 1.6 (0.0) ± 35.6 (0.0) 2 TL1 13.1± 1.6 (9.17) ± 30.2 (1.59) 3 TL ± 1.2 (8.33) ± 34.8 (0.92) 4 TL ± 33.2 (22.50) 5 TL ± 1.0 (193.33) 31.1 ± 0.9 (159.17) 6 TL ± 31.5 (17.77) 7 TL ± 1.2 (175.0) 12.2 ± 1.4 (1.67) 8 TL ± 1.5 (-6.67) ± 37.5 (-4.13) *Each value represents mean of 3 replicates ± SD **Each value represents mean of 5 replicates ± SD TL Transgenic Lines ± 38.3 (17.11) ± 35.1 (0.92) Values in paraentheses are % increase/decrease over control Planta Medica (2009)75: 1 6

15 EFFECT OF ABIOTIC STRESSES ON ARTEMISININ CONTENT IN THE LEAVES OF Artemisia annua L. STRESS ARTEMISINIIN CONTENT (mg/g DW) CONTROL Salinity (160 mm) Lead (Pb; 500 ppm) 291 (00) 576 (98) 892 (207) 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:

16 Effect of fertilizers on artemisinin content and yield in Artemisia annua L. Treatment Leaf Biomass (t ha-1) Stem Biomass (t ha-1) Leaf Stem ratio Artemisinn Content in Leaves (% dw) Artemisinin Yield (Kg ha-1) T1 (Control) 2.62 ± 0.08 (0.0) 6.92 ± 0.28 (0.0) ± ± 0.01 (0.0) ± 0.52 (0.0) T2 (Organic Manure, 15 ton ha-1) T3 (N40+40+P40+K40) 2.75 ± 0.06 (4.94) 7.13 ± 0.19 (3.11) ± ± 0.01 (5.35) ± 0.31 (10.24) 2.95 ± 0.06 (12.47) 7.54 ± 0.24 (8.97) ± ± 0.02 (17.94) ± 0.73 (32.71) T4 (N P40 + K40 + S15+15) 3.19 ± 0.10 (21.55) 7.95 ± 0.18 (14.98) 0.400± ± 0.01 (27.29) ± 0.60 (54.38) Each value represents mean ± SE (n = 9) Values in parentheses are % increase over control

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