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Una visione sistematica per l’ingegneria metabolica

Una visione sistematica per l’ingegneria metabolica. Cosa ha da dire la MCA ai biotecnologi vegetali?. +. +. +. +. TF. Ingegneria metabolica. probabilmente efficace. probab. inefficace. (1). (2). (3). (4). (5). (6). (7). (8). (9). S. S. S. S. S. S. S. S. S. A. A. A.

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Una visione sistematica per l’ingegneria metabolica

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  1. Una visione sistematica per l’ingegneria metabolica Cosa ha da dire la MCA ai biotecnologi vegetali?

  2. + + + + TF Ingegneria metabolica probabilmente efficace probab. inefficace (1) (2) (3) (4) (5) (6) (7) (8) (9) S S S S S S S S S A A A A A A A A A Y E B B B B B B B B B Z W X C C C F C C C C C Q P P P Q P P P P P P ATP Flux change: an asymmetricproblem

  3. (2) S A B Inattivazione di enzimi Spegnimento, parziale o totale, di geni tramite: - Knock out - Antisenso • Cosoppressione • RNA interference Glucosinolati (Cyt. P450) Biosintesi di lignina Ac. grassi poli-insaturi (desaturasi) Morfina (COR) Caffeina Lisina Amido ...... Domesticazione?

  4. Inactivation of Allergens and Toxins Per approfondire: manoscritto con 150 referenze Piero Morandini Dept. of Biology Milan University (Italy)

  5. The dangers of nature and food Gene inactivation strategies Manipulating crops (toxins) Manipulating crops (allergens) Transgenic vs. classical approaches Consequences & conclusions Most of the material presented is the work published by other groups Outline

  6. Nature: a ‘mine field’ Toxic substances abound in nature, both in cultivated and wild plants With time we learned how to avoid some (or at least limit their intake), inactivate others through various processes: Proper storage Cooking (e.g. heat inactivation) Food processing (e.g.: maceration, fermentation) precise mapmetal detector Knowledge handed through culture and technology (to detect, avoid & inactivate) are crucial for survival Technology and knowledge buffer us from the toxic effect of nature

  7. A field trip into the ‘mine field’ Focus mainly on small metabolites (but toxic proteins also abound in nature)

  8. http://www.poppyseedtea.com/ Poppy seed tea can kill you

  9. Solanum tuberosum http://commons.wikimedia.org/wiki/File:Potato_sprouts.jpg

  10. March 27, 1925 340-341 http://depthofprocessing.blogspot.com/2009 /05/are-potato-peels-nutritious.html Poisoning and Toxicology Handbook by Leikin & Paloucek 4th edition, Informa Health Care, 2007 ISBN 1420044796, 9781420044799 Solanine abounds in green parts, sprouts and diseased potatos

  11. LD50=30 mg/kg Solasonine Solasonine accumulates in Solanum sodomeum http://kanaya.naist.jp/knapsack_jsp/image.jsp?word=C00002265

  12. Datura stramoniumangel's trumpet or devil's weed http://www.luciolongo.it/Fotografie%20strane%20che%20ho%20fatto%20in%20giro.htm * Lazzarini, D. et al. (2006) Food poisoning by Datura stramonium: an unusual case report. Intern. Emerg. Med. 1:88-90. * Suk, SH and Kwak, YT (2009) Toxic encephalopathy after taking dried seeds of Datura stramonium in two elderly subjects. Geriatr. Gerontol. Int. 9:326-328. * Spina, SP and Taddei, A (2007) Teenagers with Jimson weed (Datura stramonium) poisoning. Canadian Journal of Emergency Medicine 9:467-468. * Arnett, A.M. (1995) Jimson Weed (Datura stramonium) Poisoning. Clinical Toxicology Review 18. (Available at: http://www.erowid.org/plants/datura/datura_info5.shtml). Capsicum annuumpeperoncino www.floralimages.co.uk/pdaturstram1.htm * Snyman, T., Stewart, M.J. and Steenkamp, V. (2001) A fatal case of pepper poisoning. Forensic Sci Int. 124, 43-46. *http://members.tripod.com/prof_anil_aggrawal/poiso010.html http://www.ubcbotanicalgarden.org/potd/2006/10/capsicum_annuum_cultivars.php

  13. Teenagers with Jimson weed (Datura stramonium) poisoning A teenager brought a Jimson weed plant to a party after watching youths misusing the plant on a popular television show. Eight teenagers opened the seed pods, each chewing and ingesting the seeds from 2 to 3 pods (~100-300 seeds) in combination with alcohol. A 16-year-old white male and a 15-year-old female of Asian descent presented to our tertiary care emergency department (ED) with a severe acute anticholinergic toxidrome after this ingestion, which was 1-2 hours before presentation. The male patient … presented with visual hallucinations, disorientation, incomprehensible and nonsensical speech, and dilated sluggish pupils. The patient was tachycardic (heart rate 120 beats/min), febrile (38°C) and had a blood pressure of 120/60 mm Hg. The female patient was very agitated, disoriented, swearing and spitting at ED staff after ingestion of approximately 100 Jimson weed seeds and vodka. On exam, she had dilated pupils, sinus tachycardia (heart rate 160 beats/min), tachypnea (40 breaths/min), fever (37.8°C)… * 188 human cases over a 6-year period in Texas. 78% of cases were the result of intentional abuse, largely in teenagers. * 23 cases were reported in British Columbia over 3 years (2004-2006). * ingestion was possibly associated with a teen fatality in British Columbia in 2006. * The use of Jimson weed can be prevalent in the teenage party community as a means to achieve an inexpensive euphoria. Jimson weed seeds are easily purchased online. Case report: http://www.cjem-online.ca/v9/n6/p467 http://uvalde.tamu.edu/herbarium/final/dain_fr.jpg

  14. Xanthotoxin or 8-methoxypsoralen Celery contains several natural pesticides http://en.wikipedia.org/wiki/Psoralen Psoralens form adducts with DNA upon exposure to light and therefore cause photodermatitis (= light dependent dermatitis)

  15. Giant hogweed http://nyis.info/plants/Images/GH_burns.jpg http://nyis.info/plants/Images/GH_plant.jpg For giant hogweed to affect a person, sap from a broken stem or crushed leaf, root, flower or seed must come into contact with moist skin (perspiration will suffice) with the skin then being exposed to sunlight. Giant hogweed, like celery, is a member of the carrot and parsley family See also http://www.pathguy.com/sol/12523.jpg

  16. Two days and three days after contact and moderate light exposure Image from the author’s body, as punishment for not recognizing the plant in an orchard while collecting plums (despite all the academic knowledge...)

  17. Colchicine intoxication http://en.wikipedia.org/wiki/File: Colchicum_speciosum000.jpg http://pandasthumb.org/archives/2010/08/08/ Samarasinghe.Gloriosa_superba.JPG Colchicine is a potent poison: it blocks microtubule formation and thereore separation of chromosomes at cell division. http://en.wikipedia.org/wiki/Colchicine

  18. Ide et al. (2010) [Case of colchicine intoxication caused by tubers of Gloriosa superba] [in Japanese] Chudoku Kenkyu. 23:243-5. Gloriosa superba is one of the poisonous plants growing in Japan. It contains potent alkaloid such as colchicine … We report here a case of colchicine intoxication caused by tubers of Gloriosa superba. A 58-year-old male ingested about 25 g of Gloriosa superba tubers by mistake. He believed that it was wild yam. He developed abdominal pain, vomiting and diarrhea 30 minutes after the ingestion of the plant. Forty five hours later he was taken to the emergency department. Unfortunately he died due to progressive multiple organ failure about one hour after the admission. It was two days after Gloriosa superba ingestion. Kupferschmidt and Campbell (2005) Colchicine Poisoning. A 5-Year European Poisons Centres Survey. Clinical Toxicology 43:399 Poisoning from ingestion of meadow saffron leaves or from colchicine tablets is a rare but regular medical emergency. Number of fatal cases, and the respective number of colchicine tablet and C. autumnale poisonings during the years 1999–2003. Reported 355 cases of colchicine poisoning, 15 with a fatal outcome, in the EU. In 119 cases (34%), ingestion of C. autumnale material was involved (only 1report with no information about the source of colchicine, i.e. plant or tablet).  an estimate of 1100 cases with 45 fatalities per 5 years.

  19. A matter of dose (threshold)  reduction below a threshold considered safe

  20. http://www.springerlink.com/content/37827612x62xp348/ The issue of toxic substances in plants is not new nor is gone Cultivated plants have less toxins than wild relatives (e.g. potato) What happened? Mutants selected by human & animal tests Trial and error (or trial and death) Many crops still produce low levels of toxins (capacity is there!) Their content may increase by breeding or spontaneously: e.g. Potato, Celery, Zucchini (courgette)…

  21. Bottle gourd Celery How to reduce toxic substances in plant?  Target the gene(s) Gene  RNA  Protein Toxins are either proteins or are produced through proteins http://nonsense123.files.wordpress.com/2008/06/bottle-gourd.jpg Which approaches? Zucchini Blackjack http://www.people.cornell.edu/pages/kjc34/distribution.html http://www.growfruitandveg.co.uk/grapevine/vegging-out/courgettes-doing-my-head_18095.html

  22. Cucurbitacins in Plant Food  Di Jørn Gry, Inge Søborg and Hans Christer Andersson Recupera formule di struttura... Libro su goggle books

  23. Degradation product Gene inactivation strategies a) inactivate a protein (enzyme) Regulator b) increase toxin degradation c) target a regulator of the synthesis Halkier (2006) Annu. Rev. Plant Biol. 57:303-33

  24. Tools of the tradeto inactivate (plant) genes • ‘Classical’ mutation (base change, insertion, deletion…) • Insertional mutagenesis (transposons or T-DNA) • RNA mediated (antisense, RNAi, miRNA, hpRNA…) collectively known as post-transcriptional gene silencing (PTGS), often involving epigenetic changes Different methods may end up exactly in the same result (inactivation of a gene) and the same change at the DNA level

  25. Each method has pros & cons • None suites all situations • Hard or impossible distiguish natural / non-natural

  26. Transcription Direct gene inactivation • The gene is mutated (becomes non functional) • RNA missing or aberrant • Protein missing or non functional • Usually irreversible Protein toxin ‘good’ phenotype

  27. ‘Classical’ mutation • Mutations arise spontaneously in any organism (endogenous or environment) • Frequency can be enhanced by various treatments: UV, X and γ-rays, chemical mutagens, and mitogens (indirectly) • Crop plants accumulated many mutations Just one base change out of 15,000 Konishi et al., (2006) Science 312:1392-1396

  28. Identification of acyanogenic forage Sorghum by a combination of biochemical screening and TILLING (by Cecilia Blomstedt) ISB News Report, Feb. 2012, p.9-10 One line was totally cyanide deficient (tcd1) in both shoot and root tissue throughout all stages of growth and development. Molecular modeling, based on the solved crystal structure of relevant P450s, indicated that the tcd1 mutation, a proline to leucine amino acid change, is believed to interfere with the structural organization of the CYP79A1 protein and prevent substrate binding and subsequently a loss of catalytic activity. …confirmed the complete loss of CYP79A1 activity and dhurrin production Sorghum mutant lines growing in the field in Queensland, Australia (~9 weeks old) (A) The M5 generation of selected mutant lines of specific interest, including the tcd1 and acdc1-3 mutants, growing in the field for seed collection to be used in future experiments. (B) acdc1 mutant ~0.8m tall. (C) tcd1 mutant ~0.6m tall.

  29. T-DNA STOP Gene = meaningful sentence ATG Insertional mutagenesis Transposons: genetic elements able to jump around in the genome Retroviruses: virus making new copies (through RNA) able to integrate into the genome. T-DNA: bacterial DNA inserted into the plant genome Inactivate genes by: A large bit of DNA ‘breaks’ the gene  sentence meaning is lost or altered

  30. Gene silencing petunia expressing a maize gene The presence of multiple copies of the maize gene causes partial or complete silencing of an endogenous gene

  31. Antisense RNA Transcription Duplex formation Protein toxin Transcription Block of translation Protein Indirect gene inactivation • The gene is intact but its expression inhibited • RNA is either missing, destroyed or non functional • Another gene is responsible for the change • Only knowledge of the sequence required toxin ‘good’ phenotype

  32. Gossipolo, un esempio illuminante • Cotton produces 1.65 kg of seed for 1 kg of fiber • Due to gossypol, a cardio- and hepatotoxic terpenoid  seed unfit for consumption by humans and monogastric animals) • Seed contains 21% oil and 23% high-quality protein Sunilkumar et al. (2006) P.N.A.S. 103:18054–18059

  33. Cottonseed may help feed the world • Used as feed for ruminant animals (whole seeds or meal after oil extraction) • 44 million metric tons (Mt) of cottonseed (9.4 Mt of protein) • Could fulfill protein requirements of half a billion people each year (50 g/day rate) Sunilkumar et al. (2006) PNAS 103:18054-9

  34. RNAi inactivating δ-cadinene synthase in the seed Chlorophyll Carotenoids … Proposed biosynthetic pathway of gossypol Sunilkumar et al. (2006) PNAS 103:18054-9

  35. RNAi construct for δ-cadinene synthase T-DNA region of the vector used to transform cotton Sunilkumar et al. (2006) PNAS 103:18054-9

  36. Gossypol content of seeds in individual T1 lines Levels of gossypol in pooled samples of 30 mature T1 seeds from 26 transgenic lines

  37. Individual transgenic seeds showed up to a 99% reduction in compared with wild type Sunilkumar et al. (2006) PNAS 103:18054–18059

  38.  a strong reduction of gossypol in seed Levels of gossypol (mg/mg seed) for each individual seed Sunilkumar et al. (2006) PNAS 103:18054-9

  39. Gossypol and other protective terpenoids are not reduced in leaves, floral organs, and roots Gossypol (G) hemigossypolone (HGQ) total heliocides (H) Sunilkumar et al. (2006) PNAS 103:18054–18059

  40. Transgenic seed exhibits a large reduction in Gossypol level. A monogenic trait: the reduced gossypol trait cosegregates with the transgene  Much more predictable, stable, specific Sunilkumar et al. (2006) PNAS 103:18054–18059

  41. No systemic reduction of gossypol and other protective terpenoids Spatial and temporal confinement of RNAi-mediated suppression of the gene Sunilkumar et al. (2006) PNAS 103:18054-9

  42. Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination Rathore (2012) Plant Biotechnology Journal 10:174-183. …unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.

  43. Transgenic vs. conventional • A glandless mutant was obtained with conventional strategies. Varieties with this trait were a failure under field conditions (extraordinarily susceptible to a host of insect pests) • Terpenoids protect the plant from both insects and pathogens • The transgenic approach achieved a goal classical breeding was unable to obtain (specific reduction in seed) Targeted gene silencing can be used to modulate biosynthetic pathways in a specific tissue to obtain a desired phenotype. Impossible by traditional breeding  Texas A&M University and U.S. Department of Agriculture Sunilkumar et al. (2006) PNAS 103:18054–18059

  44. Take home message (I) “[this] approach…not only improves food safety but also provides an additional and potentially extraordinary mean to meet the nutritional requirements of the growing world population without having to increase either crop yields or acreage planted” (Sunilkumar et al., 2006) “Our hope is to get through regulatory approval process in the U.S. first. However, it takes $50-100 million to go through the process. At this point, we don't know where the money is going to come from, but we are exploring various possibilities. Getting U.S. approval will make it easier to then get permit in other countries. We will be especially interested in some African countries and some Asian countries. “ (personal communication by Keerti S. Rathore) Gene technology could improve food safety, food security and reduce environmental impact. Regulation is a major obstacle

  45. Lathyrus sativus A hardy tropical/subtropical legume Important source of nutrition but contains a neurotoxin: oxalyldiamino-propionic acid (ODAP) http://www.treknature.com/gallery/Asia/India/photo152618.htm

  46. http://www.gudjons.com/Mittel/Lathyrus-tub.jpg http://www.grainlegumes.com/fckeditor/aepfiles/File/Species/Lathyrus_sativus_pod_(L.delaRosa)_600.jpg • Beans from this so-called “famine crop” (consumed by poor people in Asia and Africa) causes lathyrism. • a paralytic disease (spastic paraparesis) prevalent among adults in Central India who have consumed large quantities of L. sativus seeds for several months • Safe content for ODAP is < 0.2%. Content in varieties range: 0.30-3.3 • Classical breeding approaches are in progress, but what about a transgenic approach targeting the toxin biosynthetic pathway only in the seed?

  47. Biosynthesis Proposed biosynthetic pathway for β-1 in Lathyrus sativus (products in brackets have not been detected) Yan et al., (2006) Phytochemistry 67:107–121

  48. Fonio & pearl millet cause goiter Gressel (2008) Genetic Glass Ceilings

  49. Cyanide in Cassava • 4th most important source of calories in the tropics. • Staple for about 800 million people worldwide. • Average annual per capita consumption of cassava in 2003 was 300 kg in Dem. Rep. Congo. • Processing (and cooking) to reduce cyanide levels, but results in loss of proteins, vitamins, and minerals. • Cyanide-associated health disorders have been attributed to eating poorly processed cassava http://www.food-info.net/uk/products/rt/cassava.htm

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