Ingegneria metabolica e caratteri intelligenti

1. Ingegneria metabolicae caratteri intelligenti Un esempio di ingegnosità umana applicata alle piante

2. Tolleranza agli erbicidi Science (2004) 304:1151 - 1154

3. Glyphosate Glycine (N-(phosphonomethyl) glycine is a non-selective systemicherbicide, absorbed through the leaves, used to kill weeds, especially perennials. Some crops having been genetically engineered to be resistant to it. Glyphosate was first sold by Monsanto under the tradename Roundup, but is no longer under patent. (da Wikipedia) da Wikipedia

4. Meccanismo di azione Glyphosate kills plants by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (ESP). ESP is subsequently dephosphorylated to chorismate an essential precursor in plants for the aromaticamino acids: phenylalanine, tyrosine and tryptophan[16], [17]. These amino acids are used as building blocks in peptides and to produce secondary metabolites such as folates, ubiquinones and naphthoquinone. The shikimate pathway is not present in animals, which obtain aromatic amino acids from their diet. da Wikipedia

5. Herbicide tolerance/resistance There are two primary mechanisms of tolerance in herbicide tolerant crops. One is the insertion of a gene that encodes for a modified version of the target enzyme for the herbicide, which allows the enzyme to function in the presence of the herbicide. The other is the insertion of a gene that encodes for a herbicide-detoxifying enzyme. Most glyphosate tolerant crops fall into the first category. Roundup Ready – All have a herbicide resistant target enzyme EPSPS. RR Canola also has a GOX gene that degrades glyphosate. Monsanto glyphosate resistant oilseed has two modes of resistance built in - a transgene where the target enzyme is modified so that it is unaffected by glyphosate, and another gene encoding an enzyme degrading glyphosate. Neither was good enough and together they work well. Dupont is coming out with crops that rapidly degrade glyphosate, made by gene shuffling.

6. Glyphosate Acetyl Transferase Amino acid sequence of B. licheniformis GAT enzymes Enzymatic N-acetylation of glyphosate at pH 7. da Castle et al. 2004

7. Directed evolution Summary of directed evolution iterations for improvement of glyphosphate N-acetyltransferase activity. The complexity of each library is determined by the number of enzyme sequences possible given the diversity encoded by the parental genes (not counting mutations that arise by PCR).

8. Evolution of GAT activity Una diminuzione di Km significa che l’enzima raggiunge la Vmax a concentrazioni di S minore (è più attivo anche se la conc. è bassa). Un aumento di Kcat (K2) determina un aumento nel numero di molecole trasformate nell’unità di tempo. Vmax = K2 x Et Alla 11a iterazione si ottengono enzimi con un rapporto Kcat /Km 10.000 volte più alto rispetto all’enzima iniziale Nelle prime 3 si ottiene un aumento sostanziale di Kcat e nelle successive una riduzione decisa di Km Parental and evolved GAT enzymes were purified and kcat, KM, and kcat/KM measured in 20 mM Hepes, pH 6.8, 10% ethylene glycol, 0.17 mM AcCoA, and ammonium glyphosate at 21°C (11). (A) The lowest KM, (B) the highest kcat, and (C) the highest kcat/KM ratios observed among the variants analyzed in each shuffling iteration. Although plateaus in kcat and KM occurred, the total catalytic efficiency and specificity of new GAT enzymes continued to increase (C).

9. Sequence of 11th-iteration GAT enzyme Residui cambiati: 39/146 (27%) Sequence analysis of the evolved 11th-iteration GAT enzyme with greatest kcat/KM for glyphosate. Blue residues represent the original diversity sites from the parental enzymes. Green residues were introduced in the fifth-iteration synthetic library and magenta residues in the eighth-iteration library. Orange residues arose by random mutagenesis as follows: V114A, 1st iteration; G37R, 2nd iteration; I135V, 2nd iteration: I132T, 3rd iteration; R47G, 5th iteration; Y31F, 6th iteration; I91V, 6th iteration; G38S, 9th iteration; L36T, 9th iteration; L106I, 10th iteration; Q67K, 11th iteration. The DNA sequence of this gat gene is given in GenBank AY597418

10. Transgenic maize plants expressing GAT Piante con gene al 5° ciclo Trattate con 6X la dose normale di glifosate Piante con geni al 10 e 11° ciclo Piante con gene al 7° ciclo (A) Untransformed maize regenerants 10 days after 1x glyphosate spray. (B to D) Plants on the right are shown 10 days after glyphosate spray application. On the left are untreated T0 plants. (B) Transformed maize T0 plants expressing a fifth-iteration gat variant. Although the plants were tolerant to 4 x glyphosate, they showed a setback in growth. (C) Many events expressing a seventh-iteration gat variant showed no symptoms or growth setback at 4x glyphosate compared to unsprayed controls. (D) Maize plants expressing 10th- and 11th-iteration gat genes were tolerant to 6x glyphosate with no visual damage or growth setback. 1x glyphosate = 26 oz/ac Roundup UltraMAX

11. Synthetic DNA shuffling to increase diversity for recombination. (A) The YITI sequence from B. subtilis and the related sequence from B. cereus are 58 and 47% identical, respectively, to the best GAT enzyme from the fourth iteration of shuffling (GAT cycle 4, GenBank AX543353). Synthetic library construction allows for the incorporation of as much of the available diversity as is feasible with degenerate oligonucleotides and that can reasonably be sampled in the selection and screening of the library. (B) At the eighth iteration of shuffling another synthetic library was designed to introduce additional diversity. The best seventh iteration gat gene (GAT cycle 7, Genbank AY597417) was used and new residues came from L. inocua and Z. mobilis putative NAT proteins. Identities shared among sequences are shaded gray. Paragone tra i geni di partenza e quello al 4 o 7° ciclo

12. Tobacco as well

13. Dicamba resistance Behrens et al. (2007) Dicamba resistance: enlarging and preserving biotechnology-based weed management strategies. Science 316:1185-8. Altri esempi intelligenti Chen R, Li H, Zhang L, Zhang J, Xiao J e Ye Z (2007) CaMi , a root-knot nematode resistance gene from hot pepper ( Capsium annuum L.) confers nematode resistance in tomato Plant Cell Reports 26(7): 895-905