Genomica Funzionale 10-14 Febbraio 2014 Topics covered - Metabolic Engineering; - Concept of Metabolomics; - Metabolomic platforms (LC-MS, GC-MS, NMR, ICP- Set up of a metabolomic protocol and database; - Applications in plant-/food science field; - Bioinformatics applied to Metabolomic data. ENEA “CASACCIA” RESEARCH CENTRE Location and group: 1 Senior Research scientist (G. Giuliano) 1 Junior research scientist 7 post-docs 1 PhD students 3 Undergrad. students Facilities: Coming soon… 48 pins DNA analyzer Microarray platform LTQ Orbitrap mass spectrometer - 1000bp/sequence - Custom oligoarrays - High resolution - 24 samples/h processing - GC-MS - 454 - Instron - Simultaneous analysis of - Accurate mass (MS and MSn) up to 4 orders of magnitude 20-46.000 unigenes - Targeted and untargeted analysis Research activities: DNA sequencing: Photobiology: Carotenoid projects: Microalgae projects: - Tomato - Cryptochromes - Metabolic engineering - Inducible systems - Wheat - Transcription factors - Natural mutants - Biofuels INTRODUCTION 1. Nutrition and diet status overview 2. Nutrient deficiencies 3. Approaches to fight malnutrition 4. Biofiortification strategies …getting a healthy diet… Mediterranean Asian Latin-american Vegetarian Nutrition in developing countries and non developing Worldwide zinc deficiency Dietary Energy Supply AVG DES: 1600-2800 cal Xerophtalmia, renal/liver diseases etc Obesity, Diabete etc Different populations = different diet/metabolism The French paradox, by Serge Renaud, 1992 - Resveratrol? - Alcohol? - Underestimation? - Lipid metabolism/kind issue? - Geography and Lifestyle? Vitamin C deficiencies preferebly affect black people (2002) Rice Nutrition facts of worldwide major crops Wheat Maize Potato Nutritional Indexes Recommended Dietary Allowance (RDA) - Based on scientific knowledge - Presented by a committee of the Food and Nutrition Board (FNB) of the National Academy of Sciences (NAS) during World War II (Lydia J. Roberts, Hazel Stiebeling and Helen S. Mitchell) Carbohydrates Vitamins D Hypervitaminosis Hypervitaminosis A Metastatic calcification Yellow skin discoluration Lipids Aminoacids (proteins) Minerals Different approaches to fight malnutrition… …a mutual evolutionary history: unconscious bio-fortification 12.000-11.000 BC 3.000-2.000 BC Middle east South America Sight + Smell + Taste = pollination and seed dispersal Goff and Klee, 06 (Conscious) fortification/supplementation… Mineral-deficient soils Vitamin A deficiency Mineral Fertilization Pill supplementation Endemic Goitre Rickets Beriberi Thiamine-fortified food Iodate salt Vitamin D supplementation CopenhagenWhy Consensus biofortification? 2008 list of priorities Example of biofortification approach: combat vitamin A deficiency through by enriching crops (e.g. potato) highly consumed in developing countries Source: WHO data, 2005 Biofortification by classic breeding Classic breeding + IRON + ZINC Why genetic/metabolic engineering for biofortification? - Extended gene sources - Easier distribution - Cost effectiveness …before starting a metabolic engineering program… - In vivo study (Bioefficacy)? - Right specie/cultivar - Know-how on endogenous/empyric pathway - Right promoter/gene (and transit peptide) …and after getting a bio-GM-fortified food… - Stability - Did i get what i wanted? - Agronomical performances - How to cook, distribute etc? - Bioavailability - Pleiotropic effetcs How do endogenous pathway work? Transcription regulation INPUT Post-transcriptional regulation - G - Keq - Km - Rate-limiting - Positive and negative feedback regulations Post-translational regulation OUTPUT Metabolic flux analysis Fluxomics Trial and error… Metabolic flux analysis Flux balance analysis Wiechert et al., 07 Covert et al., 08 Bio-fortification strategies, via metabolic engineering, for crop improvement REGULATIVE GGPP PUSH OPP" 4 CrtB 1 15-cis-phytoene 5 CrtI SINK licopene LCY-e BLOCK α-carotene CrtY Recycling 2 β-carotene Metabolic engineering of nutrients 1. Vitamin A (carotenoids) 2. B group vitamins 3. Vitamin C 4. Vitamin E 5. Minerals 6. Flavonoids The tough work of an antioxidant…. - DNA - lipids - proteins Ascorbate Dehydroascorbate …a bit of carotenoid history…. H. W. F. Wackenroder, 1831 J. L. W. Thudichum, 1868 A. Lieben, 1862 R. M. Willsatter, 1910 Carotenoids: chemical structure and characteristics The conjugated system 15-cisphytoene lycopene β-carotene OH HO lutein Absorbance spectra of carotenoids/chlorophylls Carotenoid distribution and their functions in plants Bacteria Fungi Algae High Plants ABA Pollinator attraction Photoreception and Photoprotection in Photosynthesis β-apo-13-carotenone (?) β-ionone Hormone precursors Picrocrocin CHO β-ionol Safranal CHO R 1O Biotic stress responses Predator attraction Lateral branching Aroma production Apocarotenoid distribution and functions in plants/animals Saffron Bixa White peach Carlactone β-ionone Picrocrocin CHO β-ionol Safranal CHO R 1O Biotic stress responses Predator attraction Lateral branching Reduction in tumor cell proliferation Aroma production Carotenoids in nutrition α-carotene" OH β-cryptoxanthin" Brain Cancer β-carotene" Macular degeneration Provitamin A carotenoids Vision Erythema Mucosal protection (mouth, nose, throat, lung) Lung Cancer Prevention of heart and vascular diseases Pancreas Tumor Prostate Cancer Bone and teeth formation Worldwide prevalence Lycopene Xanthophylls Pro-Vitamin A carotenoids Xerophthalmia Carotenoids are found in chloroplasts and chromoplasts Amyloplast (starch) Chromoplast (carotenoids, no chlorophyll) Chloroplast (carotenoids + chlorophyll) 27 Biosynthesis ofengineering carotenoids andcarotenoids their derivatives Metabolic of plant GA3P + Pyruvate DXS DOXP pathway DXR GGPS - Creation of “Golden” crops GGPP OPP" Ye et al., 00 Paine et al, 05 Early o PSY1, PSY2; CrtB OH Fraser et al, 00 HO o CHY Rosati et al., 00 Astaxanthin CrtO DharmapuriCrtW et al., 02 Bkt1 15-cis-phytoene PDS, ZDS, CrtISO; CrtI o lycopene LCY-e Echinenone Ducreux et al, 04 Diretto et al., 07b CrtO, CrtW, Bkt1 LCY-b; CrtY Aluru et al, 08 α-carotene Mann et al, 00 ε-β- OH LUT1 HO lutein LUT5, CHY1, CHY2; CrtZ Fujisawa et al, 08 β-carotene HO OH zeaxanthin ZEP HO β-β- VDE OH O antheraxanthin ZEP VDE O HO Shewmaker et al, 99 OH O violaxanthin NXS ??? O OH OH HO neoxanthin Diretto et al., 07a Golden Rice (I and II) bacterial mini-pathway: Wild-type Golden rice I Golden rice II 2 µg/g DW Ye et al 00 35 µg/g DW Paine et al 05 phytoene synthase phytoene desaturase lycopene β-ciclase Metabolic complementation Golden Rice (I and II): what’s next? 2003 2005 2004 Tuber-specific silencing ü AS-e (Diretto, 06) ü Total car: 2-fold ü β-carotene: 14-fold ü 50 Kg/d -> 50% RDA ü AS-h (Diretto, 07c) ü Total car: 4-fold ü β-carotene: 50-fold ü 14 Kg/d -> 50% RDA ü Desirée ü 0.016 µg/g DW β-carotene ü 700 Kg/d -> 50% RDA lycopene X LCY-e LCY-b δ-carotene LCY-b α-carotene HO zeinoxanthin HO lutein HO Lut5 Lut1 Lut5 CHY1 CHY2 β-carotene zeaxanthin OH HOO antheraxanthin HOO violaxanthin X ZEP OH VDE OH ZEP VDE O OH Effect of using different promoters “35S/Pat” constructs “All Pat” constructs Tuber vs leaf carotenoid content PatI PatB+I PatB+I+Y Leaf Leaf 35S:I Pat:B+35S:I PatB+35S:I+35S:Y A Tuber (log) Tuber (log) Wt - pK-I + pK-BI ++ pKYBI The Golden Potato 120 A α-xanthophylls α-carotene β-xanthophylls β-carotene Early 100 80 Carotenoid content Phytoene synthase 60 phytoene desaturase 40 lycopene β-ciclase 20 0 Wild-type pK-I pP-I B 200 α-xanthophyll synthases ε-cyclase β-xanthophyll synthases CrtY β-cyclases CrtB+CrtI Early pK-BI pP-BI pK-YBI pP-YBI Transcript levels 53 µg/g DW Diretto et al 07b 100 0 Golden potato Wild-type pK-I pP-I pK-BI pP-BI pK-YBI pP-YBI Diretto et al, 10 (in press) 50% RDA = 250gm FW Tuber-specific overexpression OPP GGPP CrtB phytoene CrtI lycopene β-carotene CrtY ü Golden Potato (Diretto, 07b) ü 20-fold Total carotenoids ü 60 µg/g DW β-carotene ü 250 g/d -> 50% RDA Golden rice II: Wheat S. phureja: Potato-CrtB: β-car: 25ug/gr DW Wheat,β-car: β-car: 1ug/gr DW β-car: 10ug/gr DW maizeTot Car: 24ug/gr DW Tot Car: 36ug/gr DW Tot Car: 37ug/gr DW Tot Car: 4ug/gr DW Maize-CrtB+CrtI: β-car: 6ug/gr DW Tot Car: 47ug/gr DW Some bonus post-harvest features of “Golden” potatoes 300 d.p.h. Wildtype pK-YBI pP-YBI β-carotene Total carotenoids Tocopherol Firmness ABA Water loss Sprouting Rate Scossa et al, (submitted) Cluster analysis in “golden” tubers • Lipid biosynthesis • Phytosterol biosynthesis • Scossa et al., In preparation Water loss α-carotene • Tuber firmness • PSY1 • metabolism of fatty acids • β-carotene • • Lipoxygenase Lipoxygenase • desaturation of fatty acids • • Lipoxygenase carotenoid binding proteins • total carotenoids • Lipoxygenases Fatty acid and sterol metabolism Fatty acid biosynthesis acp FA acyl-CoA synthetase Acetyl-CoA Acetyl-acp Acetoacetyl-acp FA acyl-CoA FA acyl-CoA synthetase synthetase Acetoacety-CoA Stearyl-CoA HMG-CoA synthase ω desaturase Tetradecanoic Acid HMG-CoA HMG-CoA reductase Hexadecanoic acid Octadecanoic acid Fatty acid metabolism Mevalonate IPP DMAPP GPP tocopherol transferase GGPP Tocopherols Squalene avenasterol β-sitosterol PSY Chlorophylls Gibberellins Plastoquinones Carotenoid biosynthesis Phytoene Lycopene β-carotene Oleoyl-CoA Polyunsatured fatty acid LOX DMAPP IPP Phytosterol biosynthesis Hexadecanoyl CoA α-carotene Xantophylls End-products of Fatty acid metabolism Polyunsatured Fatty acid metabolism Proposed regulatory network ω3-desaturase Fatty acid Acyl-CoA synthetase HMG-CoA-reductase Golden potato phytosterols Lipoxygenase expression and activity Polygalacturonases Xiloglucan hydrolases Polyunsaturated fatty acids Pectinesterase inhibitors Tuber firmness Water loss 25 160 140 20 100 shore Wt pK- YBI 41 80 pK- YBI 42 pP- YBI 17 Water loss (%) 120 15 Wt pK-YBI 41 pK-YBI 42 pP-YBI 17 pP-YBI 30 10 pK- YBI 30 60 5 40 20 0 0 0 0 50 100 150 200 Days post-harvest 250 300 350 50 100 150 200 Days post-harvest 250 300 350 Modification of membrane properties The “Or” mutation Wild type Or muta nt The “Or” mutation (IV) Fresh Harvested Long-term storage Metabolic engineering of ketocarotenoids Astaxanthin content > 18%/50% of total carotenoids in S. tuberosum/S. phureja (0,6/14ug/gm DW) Astaxanthin market: - 200 $/kg - 200 millions $/year (2015) Morris et al., 06 Astaxanthin content >70% of total carotenoids >0.5% on a dry weight basis Hasunuma et al., 09 …the biofortification you did not expected to obtain… Carotenoids (+ Tocopherols) Romer et al., 02 Polyamines (+ carotenoids) Mehta et al., 02 Ethylene(+ Zn and Cu) Grichko et al., 00 Det1(+ flavonoids) Davuluri et al., 05 Future perspectives in metabolic engineering field… Gene pyramiding 1. Additive effects on the same pathway 2. Simultaneous manipulation of several pathways REGULATIVE GGPP PUSH OPP" 4 CrtB 1 15-cis-phytoene CrtI SINK licopene LCY-e 2 CrtY BLOCK α-carotene (Carotenoid + Acorbate+ Folate biosynthesis) β-carotene (Carotenoid biosynthesis: 1+2+3) 2002 Ferritin + Phytase Naqvi et al., 09 Conclusions - Plant Metabolic Engineering - Several strategies of metabolic engineering have been carried out - Enriched biofortified crops are available for most of vitamins (A, B5, B12, C, E), minerals and flavonoids What’s is it going on in these metabolically engineered plants?