Compartmentalized metabolic engineering of plant for artemisinin biosynthesis

Transcription

1 Compartmentalized metabolic engineering of plant for artemisinin biosynthesis Shashi Kumar Group Leader, Metabolic Engineering International Centre for Genetic Engineering and Biotechnology, U. N. Organization, New Delhi, India

2 Compartmentalized MBE (chloroplast, mitochondria and nuclear organelles) for artemisinin biosynthesis P. falciparum Artemisinin biosynthesis by sequential metabolic engineering. Six genes AACT, HMGS, HMGRt, MVK, PMK and PMD (in red font) encoding yeast MEV pathway were integrated into chloroplast genome to generate a high IPP pool. Homoplastomic plant s nuclear genome was transformed with six genes (ADS, CYP71AV1, AACPR, DBR2, IDI and FPP) of artemisinin biosynthetic pathway. Subcellular targeting of DBR2, AACPR and CYP71AV1 were done by chloroplast transit peptide. Dihydroartemisinic acid (DHAA) converts itself to artemisinin via self photochemical-oxidation, a non-enzymatic reaction.

3 MEV/Mevalonate pathway IPP MEP/DXP pathway Bacteria, and malaria parasites have MEP while higher plants have both MEP (plastids) and MEV (cytosol) Pathways produces two five-carbon building blocks called isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are used to make a diverse class of over 50,000 known biomolecules such as cholesterol, heme, vitamin K, coenzyme Q10, and all steroid hormones, metabolic drugs, latex, squalene etc.

4 MBE of yeast Mev pathway into chloroplasts to enhance IPP LF P aada AACT HMGS HMGRt MVKPMK PMD RF

5 Functionality of Yeast MEV (IPP) pathway in chloroplast Fosmidomycin containing medium

6 Mevalonate Conc. (nmoles/g DW) Metabolic Engineering Volume 14, Issue 1, January 2012, Pages Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts Shashi Kumar 1, Jay D Keasling 2, Henry Daniell 3, Katrina Cornish 4, Colleen McMahan 5, Maureen Whalen 5 Shashi Kumar a, b, 1, Frederick M. Hahn a, Edward Baidoo c, Talwinder S. Kahlon a, Delilah F. Wood a, Colleen M. McMahan a, Katrina Cornish b, 2, Jay D. Keasling c, Henry Daniell d, Maureen C. Whalen a,, 1 International Centre for Genetic Engineering and Biotechnology, New Delhi, India 2 Joint Show BioEnergy more Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, United States 3 University of Pennsylvania, Philadelphia, PA, USA 4 Choose The Ohio an State option University, to locate/access Wooster, OH, this USA article: 5 WRRC, USDA, Albany CA, USA Check if you have access through your login credentials or your institution Purchase $39.95 Carotenoid Check access Lipid doi: /j.ymben doi: /j.ymben WT EV MEV6 Squalene Get rights and content

7 Use of excess flux of IPP to produce artemisinic acid (precursor to artemisinin) Artemisinin is produced by trichomes on Artemisia annua leaves artemisinin... Antimalarial and Anticancer drug...is produced by trichomes......found on Artemisia annua leaves... Low artemisinin is produced by trichomes on Artemisia annua leaves

8 Artemisinic acid biosynthesis in chloroplast Spec-mf 23S-R trni P-rrn16S PM P1 MVK MDD AACT HMGS HMGRt T P2 IPP FPP ADS CYP71AV1 AACPR aada T K Negative Electrospray Ionization (ESI) Mass spectrometric (MS) m/z spectra of artemisinic acid Mevalonate pathway (8.1 kb) 3 UTR P-psbA-5 UTR SphI Artemisinic acid pathway J Biosci Mar;39(1):33-41 Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and impact on plant growth Saxena B, Subramaniyan M, Malhotra K, Bhavesh NS, Kumar S Southern hybridization (~ 6.1 kb) (6.8 kb) SalI Probe (0.84kb) SphI 3 UTR trna PCR (~ 2.2 kb) 13 C NMR Spectra of artemisi nic acid (a) control

9 Rationalized MBE for Artemisinin via dihydroartemisinic acid (DHAA) pathway BamHI (13123) T-MEV6 with additional IPP + SalI (7) AccI (8 ) St ui (13119) Pst I (17) ClaI (12270) EcoRI (12202) SspI (12091) ApaLI (11985) 35S-PolyA T SpeI (12739) KpnI (2 3) XhoI (13098) XbaI (613) EcoRV (619) 35S-PolyA T MluI DraI (10163) DraI (9879) EcoRV (8726) DBR2 CsVMV= rbcs Sde FPP Nos-polyA St op KDEL EcoRV (7885) EcoRI (11893) ocs-polya P-APX IPP AACPR 0 CsVMV= Cox Mitochondria, Chloroplast pcox-nuc-art e genome targeted 13128bp via nuclear transformation ApaI (7401) AccI (6827) BclI (6651) CTP 5200 PvuII ( 5695) 1600 CsVMV DraI (6183) PacI (5780) =ADS BclI (1823) KDEL AccI (2258) SphI (2533) 3 5Sde CYP7 1AV 1 CTP PvuII (3235) PacI (3320) AccI (3339) st op ApaLI (4 935) EcoRI (4843) ocs-polya XmaI (4859) SmaI (4861) Dual transformation Targeted enzymes to three different subcellular compartments

10 Advantage of expressing dihydroartemisinic acid (DHAA) biosynthesis pathway (Spontaneous Conversion of DHAA to ART) +O 2 DHAA (in transgenics) Artemisinin Conversion of DHAA to artemisinin is a spontaneous photo-oxidative process and does not require additional enzymes and energy The exposure of light after harvesting the plant favored this bioconversion (Farhi et al., 2011). The maximum bioconversion of DHAA to artemisinin was achieved when A. annua plants were sun-dried (Ferreira and Luthria, 2010). To minimize adverse effects and negative impact of artemisinin on growth of transgenic lines (Toxic)

11 WT DT Nuclear genome transformation of homoplastomic plant Hygro+Spec

12 Identification of pathway-metabolites in DT Artemisinin (LC-MS/MS) Dihydroartemisinic acid (LC-MS/MS) Isopentenyl diphosphate (LC-MS/MS) Amorphadiene (GC-MS)

13 Artemisinin biosynthesis in alternative plant Bioconversion of DHAA to artemisinin spontaneously in presence of light and oxygen Maximum in DT4 transgenic line Maximum titer 0.8µg/g DW (~0.08%)

14 DHAA analysis by LC-MS & Amorphadiene by GC-MS of DT plants Intensity, cps XIC of +MRM (2 pairs): / Da fro Std- DHAA Max cps. Std-amorphadiene amorphadiene I n t e n s i t y, c p s Time, min XIC of +MRM (2 pairs): / Da from... Max cps A. Annua DHAA DT XIC of +MRM (2 pairs): / Da 10 fro Max cps. Time, min DT Mass spectra amorphadiene Intensity, cps Time, min

15 Enhanced accumulation of isoprenoid IPP IPP/DMAPP are universal isoprenoid precursors IID gene introduction in nuclear genome for maintaining equilibrium ratio between IPP and DMAPP Nearly ~3-fold enhancement in IPP Sufficient IPP is essential for high artemisinin biosynthesis

16 DT4 DT3 DT2 DT1 WT DMSO Functionality of Artemisinin 24 hours 48 hours Extracts from DT1- DT4 inhibited parasite Growth in 24-48h assay Oral feeding of intact whole plant DT4 in Balb/C mice reduced the parasitemia levels.

17 Mechanism of action of artemisinin Fluorescent Artemisinin Nitrobenzodiazole Figure - Confocal images of parasite infected RBC incubated with Fluorescent Artemisinin Nitrobenzodiazole conjugate without iron chelator DFO before (1a) and after wash (1b), and with DFO (100 µm) before (1c) and after wash (1d) In 1991, Meshnick and collaborators showed that artemisinin interacted with intraparasitic heme, and suggested that intraparasitic heme or iron might function to activate artemisinin inside the parasite into toxic free radicals (Meshnick et al., 1991). The malaria parasite is rich in heme-iron, derived from the proteolysis of host cell hemoglobin (Rosenthal and Meshnick, 1996). This could explain why artemisinin is selectively toxic to parasites.

18 Conclusions ART is frontline treatment for rapid clearance of malarial parasitemia Lengthy biosynthesis period (18 months) and low yield of ART from native plant. Higher cost of chemical synthesis limits the ART supply for malarial treatment. Low supply from the natural sources, and the nonavailability of an antimalarial vaccine, has necessitated producing this drug with an alternative method. Chloroplast and nuclear genome transformation has produced the complete artemisinin, stop the growth of Plasmodium falciparum Higher biomass and rapid growth of tobacco plant may provide sufficient supply of ART for endemic regions

19 Acknowledgments MBE Group Irshad Karan Nitin Amit Kunbhi Charli Prachi Randhir Amita Collaborators Funding DBT International Centre for Genetic Engineering and Biotechnology colleagues, friends and ICGEB Director Prof. Dinakar Salunke