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Science Community Lecture. Oregon State University Corvallis, 13 October 2005 Genetic engineering of pro-vitamin A production in rice. Ingo Potrykus Professor Emeritus Plant Sciences ETH Zuerich Vitamin A Deficiency & Rice The problem : Rice as major staple does not contain any provitamin A. The consequences: 400 million rice-eating poor suffer from vitamin A-deficiency. 6‘000 die per day, 500‘000 become blind per year. The transgenic concept: Introduce, under endosperm-specific regulation, all genes necessary to establish the biochemical pathway. Why genetic engineering in addition to the traditional interventions? The genetic basis in the rice gene pool does not offer a basis for a conventional approach. In developing countries 500 000 children per year go blind and up to 6 000 per day die from vitamin A-malnutrition. And this will continue year by year, if we do not complement traditional interventions. ‚Biofortification‘ – improvement of the micro-nutrient content of crops on a genetic basis – can reduce malnutrition in a costeffective and sustained manner. The majority of the vitamin A-deficient depend upon rice which totally lacks provitamin A and is poor in other micronutrients such as iron, zinc,and essential amino acids. Biochemical pathway engineering from GGPP to pro-vitamin A in rice endosperm. Narcissus Phytoene synthase Narcissus Phytoene desaturase Narcissus Carotene desaturase - - Narcissus Provitamin A Lycopene cyclase Biochemical pathway engineering from GGPP to pro-vitamin A in rice endosperm. Phytoene synthase Narcissus Erwinia - Double desaturase - Narcissus Provitamin A Lycopene cyclase LB I-sce I I-sce I Kpn I GGPP pZPsC Gt1 pr psy nos! 35S pr crtI nos! RB kb Phytoene 23.1 9.4 6.6 4.4 phytoene synthase 2.3 Restriction enzymes: a) I-sce I, b) Kpn I. Probe: psy kb Lycopene 23.1 9.4 6.6 4.4 phytoene desaturase 2.3 LB 35S! -Carotene crt I Spe I I-sce I I-sce I Restriction enzymes: a) I-sce I, b) Kpn I. Probe: pZcycH aph 4 35S pr 35S! cyc Gt1 pr RB kb 23.1 9.4 6.6 4.4 lycopene cyclase Restriction enzymes: a) I-sce I, b) Spe I. Probe: cyc Provitamin A producing rice: HPLC analysis Provitamin A Provitamin A Provitamin A Analysis of Regulatory Clean events In Indica rice IR64 and Japonica rice TP309: Provitamin A, -oryzanol, Vitamin E -carotene -carotene -cryptoxanthin zeaxanthin lutein Carotenoid (ng/g dm) 1200 1000 800 600 400 200 0 200 180 160 140 120 100 80 60 40 20 0 -Oryzanol (µg/g dm) 25 Vit E (µg/g dm) 20 15 10 5 0 TP309 WT IR 64 TP309 IR64 IR 64 IR 64 Wildtype Golden Rice Biochemical pathway engineering from GGPP to pro-vitamin A in rice endosperm. Narcissus Erwinia - Phytoene synthase Double desaturase - Provitamin A Experiments in progress to increase and stabilize the pro-vitamin A content in rice. Group Peter Beyer, Freiburg, Germany. 1. Use of codon-optimized crtI 35S term CaMV35S Promoter CrtI-Synthetic CaMV35S PolyA PSY T-Border (r) Gt-1 GluB-1 T-BorderPMI (l) nos 3. To raise expression with appropriate transcription factor. Gt1-Promoter CaMV35S Promoter PMI Rap 2.2 pC1380MPI-Gt1-AtRAP2.2 5. To convert -carotene into retinoic acid (vitamin A) pZPDiox 2. To channel more carbon into the pathway Pyruvate + D-Glyceraldehyde 3-phosphate DXS DXR CMT CMK MECPS 4. To block -carotene degrading and converting enzymes. At least 8 putative carotenoid cleaving enzymes identified in rice genome. One of these leads to decolorization of carotene accumulating E.coli. ß-carotene dioxygenase JA Payne et al., Nature Biotechnology 23: 482-487, 2005. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. The level of expression can be regulated with he choice of phytoene synthase genes. The total amount of pro-vitamin A ranges, so far, from 0.8 to 40 g/g endosperm. Genes from ‚proof-ofconcept experiment, Science 2000 More than 80% of the carotenoids consist of the most effective pro-vitamin A - -carotene. JA Payne et al., Nature Biotechnology 23: 482-487, 2005. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Wildtype Golden Rice „Golden Rice“ contains the genes required to activate the biochemical pathway leading to accumulation of provitamin A. Proof of concept was complete in 1999. And this was the end of public funding. How much GoldenRice has a child to eat to prevent vitamin A-malnutrition? Lines used for subsequent calculation. Wildtype SGR 1 SGR2 Further lines with much higher content of provitamin A in the pipeline. SGR1: 1.6g reguatory clean; jointly developed by public & private sector. SGR2: 16g developed by private sector; donated to the humanitarian project! Amount depends upon the typical diet. Example Bangladesh Calculation from International Food Policy Institute: (1) Share of calorie intake for rural Bangladesh. 79% energy intake from rice but no provitamin A! Provitamin A in vegetables and fruit Vitamin A in fish & animal food Contribution to WHO/FAO recommended nutrient intake of provitamin A from a conventional diet in Bangladesh from vegetables/friut, animal/fish, and ‚Golden Rice‘ GoldenRice 16 g line 32 g line is also availale Golden Rice could minimize vitamin A-malnutrition at no costs, if it could replace ordinary rice. Also populations with 40% energy from rice would be protected because of higher provitamin A background. Exposure evaluation Bioavailability study. Event independent studies Requirements for GoldenRice deregulation. Golden Rice Modelling analysis for intended use. Deregulation Protein production and equivalence Extraction from GMO plant or heterologous source Biochemical characterisation Function/ specificity/ mode of action. Protein evaluation No homology with toxins and allergens. Rapid degredation in gastric /intestinal studies. Heat lability No indication of acute toxicity in rodents. Further allergenicity assessments (Daffodil!) Molecular characterization and genetic stability Single copy effect; marker gene at same locus. Simple integration; Mendelian inheritance over Event dependent studies Requirements for GoldenRice deregulation. Deregulation three generations (minimum). No potential gene disruption. No unknown open reading frames. No DNA transfer beyond borders. Golden Rice No antibiotic resistance gene or origin of replication. Insert limited to the minimum necessary. Insert plus flanking plant genome sequenced. Phenotypic evidence for stability over 3 generations Biochemical evidence for stability. Unique DNA identifier for tracebility/detection. Golden Expression profiling Rice Gene expression levels at key growth stages. Evidence for seed-specific expression. Event dependent studies - continued Requirements for GoldenRice deregulation. Deregulation Phenotype analysis Field performance, typical agronomic traits, yield compared to isogenic lines. Pest and disease status to be same as isogenic background. Compositional analysis Data from 2 seasons x 6 locations x 3 reps. on proximates, macro and micro nutrients, antinutrients, inherent toxins and allergens. Data generated on modified and isogenic background. Environmental risk assessment Minimize potential for gene flow. Evaluate any change in insect preference – by field survey. Data submitted must be of scientific publication quality Genetically Modified Rice Adoption: Impact for Welfare and Poverty Aleviation. K Anderson, LA Jackson, CP Nielsen. World Bank Policy Research Working Paper 3380, August 2004 The paper uses the ‚global economy-wide computable general equilibrium model‘ to analyse the potential economic effects of adopting first and second generation GMO crops in Asia. ‚The results suggest that farm productivity gaines could be dwarfed by the welfare gains resulting from the potential health-enhancing attributes of Golden Rice‘. Projected gaines from Golden Rice adoption by developing Asia would amount to $ 15.2 billion per year globally. Enhanced productivity of Asian unskilled labor in $ billion: China 7.209; India 2.528; Other S+SE Asia 4.140. Iron Deficiency Aneamia The problems : Rice contains very little iron (1), an inhibitor of iron resorption (2), and does not support iron uptake from a vegetative diet (3). The consequences: Rice-eating poor suffer from iron deficiency. Iron deficiency affects nearly 3 billion people. It impairs body growth, mental & motor development, activity, intellect & emotion; it favours infectious diseases. It is the major cause for maternal and child death in pregancy. The transgenic concept: Decrease the inhibitor (1), increase iron content (2), add uptake-promoting substances (3). High Iron Rice: Decrease in inhibitor Phytase activity Phytate content in rice seeds after PU [nmol free P/ g rice/ min] simulated small intestine conditions 10’000 1.00 WT 8’000 4’000 IP4 IP3 0.75 non treated % 6’000 TR after acid treatment 0.50 2’000 0.25 0 0.00 WT TR IP6 IP5 n.t. 1h, 6.5 37°C n.t. 1h, 6.5 37°C High Iron Rice: „Thermostability“ of the fungal phytase residual activity of after 20 min at 100ºC the purified phytase [%] with rice seeds 25 100 80 20 60 15 in the transgenic seeds 10 PU (mol free P / min/ mg) PU (mol free P /g/min) 8 TR 6 WT 50% 40 10 20 5 0 0 0 20 40 60 80 100 Temperature [ºC] 4 2 0 0 5 10 15 Minutes 20 „High-Iron“ Rice has 2-fold iron, 7-fold uptake-promoting cystein, and high inhibitor-degrading activity. However, the inhibitor-degrading enzyme (phytase) does not retain its heat stability when in the apoplast; and when active in the phytate vesicles, is provoking replacment of degraded phytate. Experiments are, therefore, in progress for a) expressing the phytase in a different compartment, b) enhancing iron uptake via excretion from the roots of mucogenic acid, and c) supporting transport of iron via an iron transporter. Introgression of the GoldenRice trait into Vietnamese varieties. High iron/high yield variety Khang Dan and High yielding/good grain quality/ aromatic varieties. IR1490 OM2031 CS2000 DS2001 ASS996 Jasmine 85 MTL 250 OM 3536 Engineered provitamin A IPP/DMAPP-Isomerase pathway in rice endosperm. PP IPP PP DMAPP GGPP-Synthase PP GGPP Phytoene-Synthase Phytoene Phytoene Desaturase -Carotene-Desaturase Phytofluene -Carotene Neurosporene Lycopene isomerase , -Lycopene Cyclase Golden Rice Science is moving towards nutritional optimization, but regulators will not deregulate. Regulation is set to look at risks, not benefits. 24‘000 death per day are irrelevant! other ? Vitamin A Lipids ? Vitamin E -Oryzanol Carotenoids: -carotene,lutein, zeaxanthin Lycopene Iron & zinc bioavailability: Ferritin, Phytase, GoldenRice follow-up projects. „Biofortification“- genetics-based improvement of the nutritional value of crops for sustained reduction of micronutrient malnutrition. Two international programs, using traditional and molecular techniques, are supporting the concept of „biofortification“ with support from The World Bank and the NIH-Gates Foundation. International, multidisciplinary. „Grand Challenges in Global Health.“ Rice Sorghum Cassava Banana „HarvestPlus“. CGIAR others ? Vitamin A Lipids ? Vitamin E Rice -Oryzanol Maize Carotenoids: Wheat -carotene,lutein, zeaxanthin Cassava Sweet Potato Beans Iron- & Zinc Bioavailability: Ferritin, Phytase, Acknowledgement ETH Zürich for support of ‚proof-of-concept‘ work. • Group Peter Beyer & Ingo Potrykus for sciene. • Humanitarian Golden Rice Board for guidance and decisions. • Humanitarian Golden Rice Network for development of varieties. • Syngenta (Adrian Dubock) for key contributions in product development, deregulation, strategic advice. • AgBiotech companies for IPR donation • Syngenta Foundation, Rockefeller Foundation, USAid, HarvestPlus, Gates Foundation, Government of India for financial support. No support from EU or EU national agencies!