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Biotecnologie ambientali

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Presentazione sul tema: "Biotecnologie ambientali"— Transcript della presentazione:

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. *

4 Conviene distinguere Biobased e Biodegradable
Mater-bi Novamont Bioplastiche non equivale a biodegradabili; nell’accezione comune significa: prodotte a partire da organismi e non da petrolio. Conviene distinguere Biobased e Biodegradable

5 Qualche numero sulle plastiche
~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 In Europe, the current price for starch plastics ranges from €2.00 to €5.00 per kg depending on the grade. Produzione globale di amido 70 Mt (2010) prezzo € / kg (it excludes starchy plants consumed directly) (in funzione di origine, purezza…) Target realistico: rimpiazzare il 20% della plastica attualmente prodotta con plastica biodegradabile: Con una resa (realistica) di 2 t/ha di prodotto finale diventa un target fattibile

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

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

8 ~250 different bacteria have been found to produce some form of PHAs

9 PHB/PHA sono biodegradabili, ma hanno un costo non (ancora) competitivo con la plastica prodotta da petrolio

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

11 Metabolic pathways leading to the production of PHB, P(HB-co-HV) and mclPHA in plants
The pathway for PHB synthesis from acetyl-CoA (top left) has been implemented in the cytosol, plastid and peroxisome. The pathway for P(HB-co-HV) synthesis in the plastid was created by combining the PHB biosynthesis pathway with a pathway generating 3-propionyl-CoA via a threonine deaminase and pyruvate decarboxylase (top right). Synthesis of P(HB-co-HV) in the cytosol involves an unidentified source of propionyl-CoA or 3-hydroxyvaleryl-CoA. Other PHA co-polymers, such as mclPHA, have been mainly synthesized in the peroxisome using the 3-hydroxyacyl-CoA intermediates generated by the b-oxidation cycle (bottom left). Synthesis of mclPHA from the conversion of R-3-hydroxyacyl-ACP to R-3-hydroxyacyl-CoA is achieved via a bacterial 3-hydroxyacyl-CoA–ACP transacylase (bottom right).

12 Science 256: (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.

14 Numerosi ostacoli che imponevano basse rese sono stati superati
(es. localizzazione nel plastidio/perossisoma invece che nel citosol)

15

16

17 van Beilen and Poirier (2008) Production of renewable polymers from crop plants. Plant J. 54:684–701
Molti tentativi. In genere le rese sono basse. Più alta è la resa, maggiori sono gli effetti sulla crescita

18 Poirier and Brumbley (2010) Metabolic Engineering of Plants for the Synthesis of Polyhydroxyalkanaotes. Microbiology Monographs 14/2010, , In: Plastics from Bacteria - Natural Functions and Applications (Chen G. G-Q. ed), pp Springer Berlin Heidelberg.

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 C=sintasi B=reduttasi 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 Le piante hanno integrato nel DNA plastidiale il costrutto
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 Binding sites of PCR primers used to verify correct insertion of the transgenic DNA behind the psbA coding sequence (KMB 77, KMB 41, KMB 36, and KMB 153) are shown in B along with the size of each predicted PCR product. C, Agarose gel electrophoresis of PCR products demonstrating correct integration of the transgenic DNA. M, Marker; Wt, wild-type plant. Samples 2, 3, 6, and 8 are derived from CAB plant lines 2, 3, 6, and 8 in the R2 regeneration cycle (for explanation of regeneration cycles, see Fig. 3). D, Southern blot of PstI-digested genomic DNA from transplastomic and wild-type lines probed with probe 4, a DNA fragment with homology to the aadA gene. Genomic DNA was isolated from the following lines: Wt, the wild type; PB1 R2, line obtained after plastid transformation of pUCaadA in the R2 regeneration cycle; CA 4 R2, line obtained after plastid transformation of pCA in the R2 regeneration cycle; CAB R1 lines 6 and 11, lines obtained after plastid transformation of plasmid pCAB in the R1 regeneration cycle; CAB R2 lines 2, 3, 4, 6, 8, 10, and 11, lines obtained after plastid transformation of pCAB in the R2 regeneration cycle. The arrow shows the expected 3.04-kb band for the expected integration event for CAB lines. 3.04 kb band Le piante hanno integrato nel DNA plastidiale il costrutto

22 6 weeks 10 weeks Phenotype of transplastomic lines obtained after transformation of pCAB. A, Wild-type (left) and transplastomic CAB 2 R2 (right) plants after growth in tissue culture and subsequent transfer to soil. Both plants were grown in the greenhouse in soil for 19 d. The total ages of the wild-type and CAB 2 R2 plants (including tissue culture growth) are 6 and 10 weeks, respectively. Line CAB 2 R2 was obtained after plastid transformation of pCAB, isolation of a regenerant, and one additional cycle of shoot regeneration from an excised leaf (for R2 regeneration cycle description, see Fig. 3). B, Wild-type (left) and transplastomic CAB 2 R2 T1 (right) plants after 63 and 85 d of growth, respectively, in the greenhouse. Line CAB 2 was grown from T1 seed produced from a line obtained after plastid transformation of plasmid pCAB, isolation of a regenerant, and performance of one additional cycle of shoot regeneration from an excised leaf (R2 regeneration cycle). Growth of plants in A and B was staggered so that comparison of plants at similar developmental stages could be recorded. Mesh bags shown in B were used for seed collection. C, Leaves harvested from wild-type and transplastomic CAB plants of equivalent age. Each plant was grown in tissue culture and then transferred to soil and grown for an additional 20 d in a greenhouse. The total age of each plant at the time of harvest was 2 months. Each harvested leaf is leaf 8, where leaf 1 is defined as the oldest leaf of the plant. Lines are as follows: WT, the wild type; CAB 4 R1, line obtained after plastid transformation and isolation of a regenerant (for R1 regeneration cycle description, see Fig. 3); CAB 11 R1, line in the R1 regeneration cycle. Plants from pCAB transformations were found to possess a slightly paler green phenotype and grew slower than wild-type plants

23 PHB production in leaf samples of greenhouse-grown transplastomic lines over three regeneration cycles. Regeneration cycles at right are as follows: R1, lines obtained after plastid transformation of pCAB and isolation of regenerant; R2, R1 lines that were subjected to one additional cycle of shoot regeneration from an excised leaf; R3, R2 lines that were subjected to another additional cycle of shoot regeneration from an excised leaf. Samples from the R2 regeneration cycle of line 4 and the R1 regeneration cycle of line 10 were not analyzed. Data points represent averages of two mature to senescent leaf samples harvested from the indicated line. One of these samples was harvested from leaf 4 to 7 (where the oldest leaf of a plant is defined as leaf 1), and the other sample was harvested from leaf 8 to 12. Error bars represent sd. dwt, Dry weight. 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 accumulation in tobacco leaves
PHB granules; PHB accumulation in tobacco leaves G, PHB granules; P, plastoglobuli; S, starch granules. Bars = 1 μm. PHB accumulation in tobacco leaves. Transmission electron micrographs were obtained from samples of leaf 17 (where leaf 1 is defined as the oldest leaf of a plant) from wild-type plants and CAB line 2.1, a plant obtained from seeds formed from a plant in the R2 regeneration cycle (for definition of the regeneration cycle, see Fig. 3). Cells from the palisade and spongy mesophyll layers as well as from vascular bundles were analyzed. 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 T1 Lines have delayed flowering but are fertile
PHB production in T1 progeny of CAB lines 2 and 6. PHB production in T1 progeny of CAB lines 2 and 6. Seeds obtained from plants in the R2 regeneration cycle from CAB lines 2 and 6 (Fig. 3) were germinated on tissue culture medium in the presence of spectinomycin as described in “Materials and Methods.” Plantlets were transferred to soil and grown in the greenhouse. Leaf tissue samples were taken after 1 and 2 to 3 months of growth. WT represents the wild-type line. Lines 2.8, 2.7, 2.3, 2.2, 2.5, 2.6, 2.4, and 2.1 are T1 progeny from parental CAB line 2. Lines 6.2, 6.3, 6.5, 6.8, 6.6, 6.4, 6.7, and 6.1 are T1 progeny from parental CAB line 6. Each data point is an average of two different leaf tissue samples, and the error bars represent sd. One of these samples was harvested from leaf 3 to 5 (where the oldest leaf of a plant is defined as leaf 1), and the other sample was harvested from leaf 8 to 13. dwt, Dry weight. 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
Comparison of the average days to flowering (A) and height (B) of wild-type and PHB-producing CAB lines. Plants were grown in the greenhouse, and days to flowering (when first flowers formed) and the height of the plant at seed set were recorded for each line. Each data point is an average of values from eight transplastomic lines or six wild-type plants. Error bars represent sd. Lines are as follows: wt, the wild type; CAB 2 R2 T1 and CAB 6 R2 T1, T1 lines obtained from seed of parental CAB lines 2 and 6 in the R2 regeneration cycle. The PHB value of parental lines 2 and 6 is shown in Figure 3.

27 La ridotta velocità di crescita alla fine comporta una biomassa minore
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. These seedlings are able to germinate in the presence of the antibiotic but are visually distinguishable from spectinomycin-resistant seedlings by their bleached phenotype. 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
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; (c) Russian dandelion (T. koksaghyz) (kindly provided by Dirk Prüfer); (d) Canadian goldenrod (S. canadensis).

33 Meccanismo proposto di polimerizzazione tramite carbocatione
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 Pathway of rubber biosynthesis from isopentenyl diphosphate. (a) Natural rubber is produced from a side branch of the ubiquitous isoprenoid pathway, with 3-hydroxy-methyl-glutaryl-CoA (HMG-CoA) as the key intermediate derived from acetyl-CoA by the general mevalonic-acid pathway. Mevalonate diphosphate decarboxylase (MPP-D) produces isopentenyl diphosphate (IPP), which is isomerized to dimethylallyl diphosphate (DMAPP) by IPP isomerase (IPI). IPP is then condensed in several steps with DMAPP to produce geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) by the action of a trans-prenyltransferase (TPT). The cis-1,4-polymerization that yields natural rubber is catalyzed by cis-prenyltransferase (CPT), which uses the non-allylic IPP as substrate. (b) Structures of IPP, DMAPP, GPP and FPP. In solution, the diphosphate groups will be partly ionized. (c) Proposed ‘living carbocationic polymerization’ mechanism (adapted from [39]). In the first step, the diphosphate group of FPP or a growing rubber chain is removed, leaving a carbocationic intermediate. In the second step, this intermediate reacts with IPP to extend the chain. The resulting extended rubber molecule can leave the active site or undergo a next chain-elongation step. The resulting polymer chain has two trans- and multiple cis-bonds as shown in Figure 1. 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 Liquid crystal polymers (LCPs) have exceptional qualities compared with conventional resins. They are creep and electrically resistant, flame retardant, and perform well at elevated temperatures (Tullo, 1999). The global market for LCPs is approximately metric tons per year (Tullo, 1999), 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 the aromatic hydroxyacid, p-hydroxybenzoic acid (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,

35

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. 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.


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