Proof of concept (metabolic engineering) & trojan horse Golden rice Proof of concept (metabolic engineering) & trojan horse
Metabolic engineering F E (4) P C A B Q S Aumentare il contenuto di VitaminaA
Via dei carotenoidi Cunningham & Gant (1998)
GGPP PHS crtI Cunningham & Gant (1998)
Oltre il licopene crtY Oltre il -carotene
VitA deficiency: 124 M children affected/year 250,000 children go blind/y 1-2 M deaths/y
GGPP è presente nell’endosperma di riso
Costrutti Cotrasf.
Southern blot crtB = psy crtI = pds DNA digerito con: * I-sce I * Kpn
Cotrasformazione crtB = psy crtI = pds crtY = lyc In entrambi i casi la maggior parte degli eventi sono singole inserzioni
Il colore del seme Lycopene? -carotene wt I trasformanti singoli pB19hpc segregano il carattere giallo 3:1 pB19hpc pZPsC + pZLcyH
HPLC dei pigmenti pZPsC + pZLcyH pB19hpc Punto atteso di eluizione del licopene pB19hpc HPLC dei pigmenti
Come aumentare il contenuto di β-carotene (proVitA)? β-carotene prodotto in molte le linee (lcy o costitutiva o indotta) Quantità massima 1.6 g/g 100 g retinol equivalents /300 g riso al giorno Come aumentare il contenuto di β-carotene (proVitA)? Precursore GGPP Traducibilità (es. codon usage) altre specie Transcription factor (come identificarli?) Paine, et al., (2005) Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology 23:482-487
Golden Rice II (b) Photograph showing individual maize calli cotransformed with the plasmid containing the maize psy (right, Zm psy) and an empty vector (EV) control (left). (c) Histogram showing the total colored carotenoid content of maize calli transformed with a given psy gene (from Arabidopsis thaliana (At), Daucus carota (Dc), Narcissus pseudonarcissus (Np), Zea mays (Zm), Capsicum annuum (Ca), Oryza sativa (Os) or Lycopersicon esculentum (Le)). Data shown represents the 75th percentile for each population of transgenic calli expressed as a percentage of the median empty vector (EV) control value. The second y-axis (diamonds) shows the percentage of calli from each population with a carotenoid content more than fivefold that of the EV median Paine, et al., (2005) Nat Biotech. 23:482-487 Expression of a psy transgene increases the carotenoid content of maize callus
Narciso (Golden rice I) Carotenoid enhancement of the rice endosperm by transformation with psy orthologues and crtI b) Polished wild-type and transgenic rice grains containing the T-DNA (as above) with the daffodil psy (Np) or maize psy (Zm) showing altered color due to carotenoid accumulation. (c) Histogram showing the total carotenoid content of T1 rice seed containing a T-DNA (as above) with the psy gene from either rice, maize, pepper, tomato or daffodil from the five events with the highest carotenoid content for each T-DNA. Measurement error tended to be proportional to absolute carotenoid content and pooling across all 25 transformants resulted in a measurement standard error of 6.3% approximately. dwt, dry weight. (d) Schematic diagram of the T-DNA in pSYN12424 used to create Golden Rice 2. Narciso (Golden rice I) Paine, et al., (2005)
Carotenoid content of endosperm
Carotenoid content & composition in T2 seed endosperm * large scale (>>100) primary transformants * Different variety * No antibiotic select. Paine, et al., (2005)
Conclusioni Trasformanti a singola inserzioni accumulano 20-30 mg/kg di carotenoidi; il β-carotene rappresenta l’80% Il carattere è stabile ed ereditabile La quantità contenuta in 72 g di riso dovrebbe coprire (assumendo conversione sfavorevole di 12:1) il 50% del fabbisogno giornaliero di un bambino.
Shewmaker et al (1999) Plant J. 20:401-412 Colza 1-2 Cold pressed oil from control and transgenic seeds; 3-4 Complete hexane extraction from control and transgenic seeds Shewmaker et al (1999) Plant J. 20:401-412
mRNA Protein Motivi del grande accumulo? 1) La sovraespressione dell’enzima è veramente a livelli notevoli. Protein
Livello degli altri enzimi nella via? 2) L’accumulo avviene per un periodo molto più lungo (il flusso istantaneo non è così elevato
Il prodotto dove si accumula? Control Il prodotto dove si accumula? Ci sono cambiamenti nella struttura dei plastidi CONTROLLA EFFETTI GERMIN. Prodotti sequestrati negli oil bodies? Transgenic
Pomodoro Roemer et al (2000) Verifica LIVELLO ALTRI ENZIMI Beta Carotene increased 3 fold; endogenous carotenoid genes are upregulated
Banana??? Vedi ppt di Beyer alla PAS Generali: Patata: - G. Diretto, S. Al-Babili, R. Tavazza, V. Papacchioli, P. Beyer, G. Giuliano (2007) Metabolic Engineering of Potato Carotenoid Content through Tuber-Specific Overexpression of a Bacterial Mini-Pathway. PLoS ONE 4:e350 Cassava - Welsch R, Arango J, Bär C, Salazar B, Al-Babili S, Beltrán J, Chavarriaga P, Ceballos H, Tohme J, Beyer P. (2010) Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene. Plant Cell 22:3348-56. Banana??? Vedi ppt di Beyer alla PAS Generali: -Beyer P (2010) Golden Rice and 'Golden' crops for human nutrition. N Biotechnol. 27:478-81. -Giuliano G, Tavazza R, Diretto G, Beyer P, Taylor MA.(2008) Metabolic engineering of carotenoid biosynthesis in plants Trends Biotechnol. 26:139-45. http://www.ask-force.org/web/Vatican1/PAS-08-Beyer-Golden-Rice-Crops-20090515.pdf
Naqvi et al., (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. PNAS 106: 77627767 Vedi lezione sulle vitamine
Iron deficiency Lucca P, Hurrell R, Potrykus I. (2002) Fighting iron deficiency anemia with iron-rich rice. J Am Coll Nutr. 21:184S-190S. OBJECTIVE: Iron deficiency is estimated to affect about 30% of the world population. Iron supplementation in the form of tablets and food fortification has not been successful in developing countries, and iron deficiency is still the most important deficiency related to malnutrition. Here we present experiments that aim to increase the iron content in rice endosperm and to improve its absorption in the human intestine by means of genetic engineering. METHODS: We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to twofold. To increase iron bioavailability, we introduced a thermo-tolerant phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered major enhancers of iron absorption, we over-expressed the endogenous cysteine-rich metallothionein-like protein. RESULTS: The content of cysteine residues increased about sevenfold and the phytase level in the grains about one hundred and thirtyfold, giving a phytase activity sufficient to completely degrade phytic acid in a simulated digestion experiment. CONCLUSIONS: This rice, with higher iron content, rich in phytase and cysteine-peptide has a great potential to substantially improve iron nutrition in those populations where iron deficiency is so widely spread
Bibliografia Ye X. et al.; Engineering the Provitamin A (β-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm (2000) Science 287:303-305. Paine, et al., (2005) Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology 23:482-487 Tang et al., (2009) Golden Rice is an effective source of vitamin. Am J Clin Nutr 89:1776–83. Lucca et al. (2002) Roemer et al. Elevation of the provitamin A content of transgenic tomato plants (2000) Nat. Biotech. 18:666-669 Fraser et al., (2002) PNAS 99:1092-1097 Shewmaker et al., (1999) Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. Plant J. 20:401-412 www.potrykus.org Intervista a Potrykus: “In the wrong movie” http://www.botanischergarten.ch/debate/InTheWrongMovie.pdf Vedi anche: Golden Rice and Beyond -- Potrykus 125:1157 -- PLANT PHYSIOLOGY