Biotecnologie ambientali

1. Biotecnologie ambientali Plastiche biodegradabili e altri polimeri da piante

2. PROGRAMMA • Le piante coltivate e la sindrome da domesticazione: shattering e dormienza • Rischi e benefici ambientali delle piante transgeniche in paragone a quelle convenzionali. • Convenzione di Rio, Protocollo di Cartagena e normativa sulle piante create con tramite ingegneria genetica • Piante per una maggiore sostenibilità ambientale (es. plastiche biodegradabili), per il risanamento (fitodepurazione) e come biosensori di contaminazione. • Interazione pianta-microrganismo: le risposte di difesa delle piante e generazione di specie resistenti. Tumori vegetali e trasgenesi naturale. • Interazione simbiotiche pianta-microrganismo: fissazione dell’azoto (batteri azoto fissatori) ed efficienza nella nutrizione minerale (funghi vescicolo arbuscolari)

3. Plastiche • Sono richiesti > 50 anni per la degradazione • La maggior parte finisce in discarica o agli inceneritori • La dispersione impropria (accidentale o volontaria) comporta inquinamento ambientale e danni a diversi organismi*. With a production of 265 million tonnes p.a. (2009), plastics are a necessity in today’s economy. In one year, roughly 80 million metric tons of polyethylene will be manufactured. The average cost of polyethylene is ≈1 €/kg. * http://www.dailymail.co.uk/news/article-519770/Banish-The-Bags-The-Mail-launches-campaign-clean-country---planet.html

4. Bioplastiche non equivale a biodegradabili; nell’accezione comune significa: prodotte a partire da organismi e non da petrolio. Conviene distinguere Biobased e Biodegradable Mater-bi Novamont http://www.acquistiverdi.it/sites/default/files/imagecache/Original_confirma/images/prodotti/shopper_in_mater_bi_novamont.jpg http://en.european-bioplastics.org/technologymaterials/materials/

5. In Europe, the current price for starch plastics ranges from €2.00 to €5.00 per kg depending on the grade. Con una resa (realistica) di 2 t/ha di prodotto finale diventa un target fattibile Qualche numero sulle plastiche http://www.packaging-int.com/upload/image_files/suppliers/images/companies/2606/b-cups-l.jpg ~50 Mt produzione annuale europea di cui solo 0.7 Mt sono Bioplastiche Quelle biodegradabili sono essenzialmente: PLA (Poly Lactic Acid) 150,000 t / y PHA (Poly Hydroxy Alkanoates) 50,000 t / y Starch-based biopolymer 120,000 t / y Produzione globale di amido 70 Mt (2010) prezzo 0.50 - 1.30 € / kg (it excludes starchy plants consumed directly) (in funzione di origine, purezza…) Target realistico: rimpiazzare il 20% della plastica attualmente prodotta con plastica biodegradabile:

6. Starch Protein Oils Cellulose Rubber Lignin Cyanophycin … PHB PLA van Beilen & Poirier (2012) Plants as factories for bioplastics and other novel biomaterials

7. Ojumu et al., 2004 Polyhydroxyalkanoates (PHAs) • Polyesters accumulated inside microbial cells as carbon & energy source storage

8. ~250 different bacteria have been found to produce some form of PHAs http://www.unil.ch/webdav/site/dbmv/shared/Poiriers/Fig1.jpg

9. PHB/PHA sono biodegradabili, ma hanno un costo non (ancora) competitivo con la plastica prodotta da petrolio http://www.unil.ch/webdav/site/dbmv/shared/Poiriers/Fig2.jpg

10. La via biosintetica del PHB parte dal Acetil-CoA ed è composta da 3 enzimi • PHA synthase • β-ketothiolase • NADPH-dependent acetoacetyl-CoA reductase http://www.unil.ch/webdav/site/dbmv/shared/Poiriers/peroxisomal_PHB_and_citrate_synthase.jpeg

11. Metabolic pathways leading to the production of PHB, P(HB-co-HV) and mclPHA in plants

12. Science 256:520-523 (1992) La sintesi di PHB in pianta è stata dimostrata 20 anni fa Transgenic plant producing the biodegradable plastic polyhydroxybutyrate (PHB). PHB is seen by electron microscopy as white granules inside the nucleus of a leaf mesophyll cell of Arabidopsis thaliana.

13. Transgenic plant producing the biodegradable plastic polyhydroxybutyrate (PHB). The PHB granules are visualised by epifluorescence microscopy as foci of red fluorescence. http://www.unil.ch/dbmv/page13444_en.html

14. Numerosi ostacoli che imponevano basse rese sono stati superati (es. localizzazione nel plastidio/perossisoma invece che nel citosol) http://www.unil.ch/webdav/site/dbmv/shared/Poiriers/Fig5.jpg

17. Molti tentativi. In genere le rese sono basse. Più alta è la resa, maggiori sono gli effetti sulla crescita

19. To be commercially viable PHA must be > 15% dry weight Bohmert-Tatarev et al., (2011) Plant Physiology 155:1690–1708. T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.

20. spectinomycinR A=tiolasi B=reduttasi C=sintasi Acinetobacter sp. Thiolase (phaA) and synthase (phaC; Schembri et al., 1995) and the Bacillus megaterium reductase (phaB; McCool and Cannon, 1999) and aadA gene conferring resistance to spectinomycin

21. genetic arrangement of the wild-type psbA locus Expected genetic arrangement of transformants (pCAB) PCR primers Southern blot of PstI-digested genomic DNA from transplastomic and wild-type lines 3.04 kb band Le piante hanno integrato nel DNA plastidiale il costrutto

22. 6 weeks 10 weeks Plants from pCAB transformations were found to possess a slightly paler green phenotype and grew slower than wild-type plants

23. T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed.

24. PHB granules; PHB accumulation in tobacco leaves G, PHB granules; P, plastoglobuli; S, starch granules. Bars = 1 μm. Transmission electron micrographs from samples of leaf 17 (where leaf 1 is defined as the oldest leaf of a plant)

25. PHB production in T1 progeny of CAB lines 2 and 6. T1 Lines have delayed flowering but are fertile  Il carattere è trasmissibile e sembra stabile

26. La crescita è rallentata (ci mettono più tempo a fiorire) e raggiungono una minore altezza

27. La ridotta velocità di crescita alla fine comporta una biomassa minore Screening T1 seeds obtained from self-pollination on medium containing spectinomycin. This procedure allows the detection of residual wild-type copies of the plastome in the parent plant, since these copies should be maternally inherited, producing seeds with spectinomycin-sensitive seedlings.  Range: 0.6% to 11.6% depending on the line analyzed

28. Synthesis of a biopolymer at a level of 10% dry weight in leaves of sugar beet would yield 0.5 tonnes of biopolymer /ha. With 2 Mha of sugar beet currently grown in the EU, this single crop could yield 1 Mt of biopolymers/ year in addition to 24 Mt of sugar/ year. Yields of tobacco leaf biomass can be increased by using different agronomic practices compared to growing tobacco for smoking, and yields of up to 14 tonnes/ ha have been reported, which compares reasonably well with sugar beet at 18 tonnes/ ha In sintesi: le bioplastiche biodegradabili da piante sono a portata di mano, ma solo se il presente clima ostile cambia...

29. Gomma Mercato mondiale della gomma: Naturale 9 Mt/y 1.80 €/kg Sintetica 3 Mt/y 1.30 €/kg Perché la produzione minore se il prezzo è inferiore? La qualità della gomma sintetica è inferiore, specialmente per applicazioni dove sono richieste alte prestazioni. The polymer chain has two trans- and multiple cis-bonds

30. Schematic pathway for rubber biosynthesis from isopentenyl diphosphate (IPP).

31. La gomma naturale è ancora prodotta come ai primi del 1900 Circa il 90% deriva dalle piantagioni del Sud-Est asiatico Altre piante producono lattice che contiene gomma

32. Sources of natural rubber. (a) Tapping of the Para rubber tree (H. brasiliensis); (b) a field of Guayule (P. argentatum) at Ehrenberg, Arizona (kindly provided by Yulex Corporation; http://www.yulex.com/); (c) Russian dandelion (T. koksaghyz) (kindly provided by Dirk Prüfer); (d) Canadian goldenrod (S. canadensis).

33. Biosintesi della gomma a partire dall’isopentenil pirofosfato (IPP) Non è solo la sintesi del polimero che deve essere aumentata, ma anche quella del lattice e delle strutture che lo contengono Meccanismo proposto di polimerizzazione tramite carbocatione

34. Altri materiali interessanti p-hydroxybenzoic acid (pHBA) The global market for Liquid Crystal Polymers is approximately 10,000 metric tons per year, with applications in electrical and optical connectors, integrated circuit boards, vehicle ignition components, and mobile phone components. LCPs are thermotropic polyesters and the major monomer used in the manufacture of these copolymers is pHBA McQualter, R.B., Chong, B.F., Meyer, K., Van Dyk, D.E., O'Shea, M.G., Walton, N.J., Viitanen, P.V. and Brumbley, SM. (2005) Initial evaluation of sugarcane as a production platform for p-hydroxybenzoic acid. Plant Biotechnol. J.3, 29-41.

36. Cyanophycin, or multi-L-arginyl-poly (L-aspartic acid), is a non-protein, non-ribosomally produced amino acid polymer composed of an aspartic acid backbone and arginine side groups (http://en.wikipedia.org/wiki/Cyanophycin )

37. Bibliografia Bohmert-Tatarev et al., (2011)High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate Plant Physiology 155:1690–1708. Tilbrook K, Gebbie L, Schenk PM, Poirier Y, Brumbley SM (2011) Peroxisomal polyhydroxyalkanoate biosynthesis is a promising strategy for bioplastic production in high biomass crops. Plant Biotechnol J. van Beilen and Poirier (2008) Production of renewable polymers from crop plants. Plant J. 54:684–701. Poirier, Y., Dennis, D.E., Klomparens, K. and Somerville, C. (1992) Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants. Science, 256, 520–523. Shen, Haufe, Patel (2009) Product overview and market projection of emerging bio-based plastics. http://www.epnoe.eu/content/download/7670/109501/file/PROBIP2009%20Final%20June%202009.pdf van Beilen and Poirier (2007) Establishment of new crops for the production of natural rubber. Trends Biotechnol. 25:522-9 in Plants as factories for industrial products, pharmaceuticals, biomaterials, and bioenergy). Mooney (2010) The second green revolution? Production of plant-based biodegradable plastics. Biochem. J. 418:219–232.