Strategie “intelligenti” per aumentare i flussi

1. Strategie “intelligenti” per aumentare i flussi Quali suggerimenti dalla MCA per i 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

3. (7) S A B C P Interventi a rischio? Espressione di varianti de-sensitizzate di enzimi regolati a feedback (i cosiddetti “enzimi limitanti”). Asp kinasi (Thr, Met) 3-17X  Karchi (1993) P5CS (Prolina)  Hong (2000) Antranilato sintasi (Trp) 3-5X  Cho (2000) AK + DHPS 100X  Falco (1995) …..etc  Chen (2004) Ad esempio, nel caso dell’Asp Kinasi, l’aumento di aa libero è notevole (3-17 volte), ma piccolo (≃6%) considerando gli aa totali (libero + combinato).

4. Zhu, X. and Galili, G. (2003) Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds. Plant Cell, 15, 845-853.

6. Association between average free Lys content and rate of seedgermination …our results imply that the expression of a feedback-insensitive DHPS coupled with a reduction in Lys catabolism may be the preferred choice for increasing Lys accumulation in seeds of crop plants to remarkably high levels. La combinazione ha un effetto deleterio (poor germination).

7. An attempt to translate the research discussed above into commercial application has been recently reported by the Monsanto/Renessen companies who generated a high-Lys maize genotype (LY038) expressing a bacterial feedback-insensitive DHDPS in an embryo-specific manner (Dizigan et al., 2007). This genotype was further used to produce a maize LY038 9 MON 810 hybrid, which also proved to be superior in broiler chicken performance, compared to the same lines that lacked the LY038 trait (Lucas et al., 2007). The LY038 maize has also been approved for commercial use in the livestock feeding industry in a number of countries. However, as indicated on the ‘GMO Compass’ Internet website, the LY038 maize was subsequently withdrawn. Based on the information existing on the internet site http://foodfreedom.wordpress.com/2009/11/11/high-lysine-gmmaize-withdrawn-safety-concerns/, withdrawalwasapparentlydue to concerns made by the EU regulators on the European Food Safety Agency (EFSA) GMO panel about the safety of LY038 maize for human consumption, even though LY038 maize was targeted to the feeding industry.

8. Galili and Amir (2013)

9. A. thaliana Riboswitches B. subtilis N. crassa E. coli Alternative RNA splicing Manipolare l’omeostasi dei metaboliti può essere una strategia efficace?

10. TPP aptameri sono presenti in numerose specie vegetali L’80% dei nucleotidi , escludendo il dominio P3, sono conservati in tutti gli aptameri vegetali considerati Aptamero vegetale Aptamero batterico

11. Model for TPP riboswitch function in plants • DOMANDA: cosa succede se si riduce l’affinità dell’aptamero per TPP?

12. (5) S A B C P P ATP Increasing demand Espressione di pompe ATP-dipedenti che sottraggono il prodotto della via metabolica. In presenza di inibizioni a feedback l’effetto di stimolazione sarà ovviamente anche in altri punti lungo la via metabolica. * Corynebacterium glutamicum A lysine–inducible, energy–dependent lysine exporter (lysE) has been isolated. Sahm et al, FEMS Micro. Rev. 16, 243–252, (1995) Kelle et al, Biotechnol. Bioeng. 51, 40–50 (1996) Broer et al, Appl. Env. Microbiol. 59, 316–321, (1993) Vrljic et al, Mol. Microbiol. 22, 815–826, (1996) * Trovare pompe con specificità desiderata La sottrazione del prodotto si può ottenere non solo con pompe ATP-dipendenti ma anche con altri modi (quali?).

13. (5) S A B C P Incorporazione in polimeri Se incorporo P in polimeri tendo a sottrarre prodotto dall’equilibrio e quindi a stimolare almeno l’ultima reazione. * Altro modo: formazione di cristalli * Incorporazione in fase diversa (es.: trigliceridi in lipid bodies) P-P-P-P-P-P-P-P-P-P-P-P…

14. S A B C P Aumentare il contenuto in aa Esprimendo proteine ricche nell’aa carente (es Met) si aumenta il “demand” e come conseguenza si stimola il “supply” La sottrazione di prodotto è un’alternativa più efficace rispetto a “spingere” il substrato all’imbocco della via metabolica tramite desensitizzazione dell’enzima iniziale. Sintesi proteica

15. Mertz (1964) Science 145:279-280 Maize is particularly poor in Lys and Trp The maize mutant opaque-2 (o2) shows a significant increase in Lys (+70%) & Trp.

16. The mutation mainly affects the synthesis of the 22-kDa a-zein.  The mutation simply increases demand: the reduction in the 22-kDa a-zein, devoid of Lys and poor in Trp is compensated by an increase in other storage proteins (albumins and globulins), leaving supply unchanged;  Supply is not limiting. Essentially the same result, a large increase in Lys, is obtained by transgenesis through selective inhibition of zein isoform synthesis (Huang et al., 2004, 2005, 2006). Lys translocated to developing seeds is in excess and a substantial fraction undergoes degradation in the endosperm (Silva and Arruda, 1979).

17. Galili and Amir (2013)

18. Expression of a potato pollen-specific sb401 protein (containing 16.7% Lys) in maize grains increased both grain protein content by up to 39% as well as grain Lys content by up to 54.8% in the different transgenic lines, compared to the control maize (Onouchi et al., 2005; Yu et al., 2005) Galili and Amir (2013)

19. J. Lai and J. Messing (2002) Increasing maize seed methionine by mRNA stability. Plant J 30:395-402. Metionina - Maize seeds … have variable levels of protein-bound met. This variability is a result of post-transcriptional regulation of the Dzs10 gene, which encodes a seed-specific high met storage protein. -Sales of synthetic methionine to animal producers exceed one billion dollars annually. - it is likely that the methionine level of the human cereal-legume diet is inadequate in many developing countries. Non-transformed hybrid (4) was compared to three different inbred lines: BSSS53 (3), which has high Dzs10 levels, Mo17 (2) with low levels, and A654 (1) with no detected Dzs10.

20. Amino acid composition analysis of hydrolysed ground maize kernels expressed as percentage of the sample with standard deviations in parentheses Wu (2003) Enrichment of cereal protein lysine content by altered tRNAlys during protein synthesis, Plant Biotechnol. J. 1:187–194. Wu (2007) Altered expression of plant lysyl tRNA synthetase promotes tRNA misacylation and translational recoding of lysine, Plant J. 50:627–636.

21. Galili and Amir (2013) The level of Met increased up to 62% in transgenic rice plants expressing the sesame 2S albumin (Lee et al., 2003) and in lupin plants expressing a sunflower 2S albumin (up to 97%) (Molvig et al., 1997). Nevertheless, the Brazil nut protein and, to a lower extent, also the sunflower 2S albumin were subsequently found to be allergenic in some people, questioning their potential biotechnological use (Bartolome et al., 1997; Kelly and Hefle, 2000).

22. Huang T, Tohge T, Lytovchenko A, Fernie AR, Jander G (2010) Pleiotropic physiological consequences of feedback-insensitive phenylalanine biosynthesis in Arabidopsis thaliana. Plant J. 63:823-35 Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY (2004) Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol Bioeng. 86:718-27. Wakasa, K., Hasegawa, H., Nemoto, H., Matsuda, F., Miyazawa, H., Tozawa, Y., Morino, K., Komatsu, A., Yamada, T., Terakawa, T. and Miyagawa, H. (2006) High-level tryptophan accumulation in seeds of transgenic rice and its limited effects on agronomic traits and seed metabolite profile. J. Exp. Bot. 57, 3069–3078. Dubouzet, J.G., Ishihara, A., Matsuda, F., Miyagawa, H., Iwata, H. and Wakasa, K. (2007) Integrated metabolomic and transcriptomic analyses of high tryptophan rice expressing a mutant anthranilate synthase alpha subunit. J. Exp. Bot. 58, 3309–3321. Se ne trovano molte altre su Medline cercando con: feedback-insensitive

23. Increasing aa content Molvig L, Tabe LM, Eggum BO, Moore AE, Craig S, Spencer D, Higgins TJ. (1997) Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene. Proc Natl Acad Sci U S A. 94:8393-8 L.M. Tabe and M. Droux (2002) Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein. Plant Physiol.128:1137–1148. G. Amira, et al., (2005) Soluble methionine enhances accumulation of a 15 kDa zein, a methionine-rich storage protein, in transgenic alfalfa but not in transgenic tobacco plants. J. Exp. Bot., 56: 2443 - 2452. P. Chiaiese, et al., (2004) Sulphur and nitrogen nutrition influence the response of chickpea seeds to an added, transgenic sink for organic sulphur. J. Exp. Bot., 55: 1889 - 1901.0

24. Referenze La bibliografia si trova in buona parte nel file pdf Morandini (2003) P. Morandini and Salamini F. Plant biotechnology and Breeding, allied for years to come (2003) Trends Pl. Sci. 8:70-5. Per l’ingegneria tramite TFs, vedere anche: Gantet P, Memelink J. (2002) Transcription factors: tools to engineer the production of pharmacologically active plant metabolites. Trends Pharmacol Sci. 23:563-9. Broun P. (2004) Transcription factors as tools for metabolic engineering in plants. Curr Opin Plant Biol. 7:202-9. (e relative referenze) Broun P et al., (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci U S A. 101:4706-11 A. Aharoni, et al. (2004) The SHINE Clade of AP2 Domain Transcription Factors Activates Wax Biosynthesis, Alters Cuticle Properties, and Confers Drought Tolerance when Overexpressed in Arabidopsis PLANT CELL 16: 2463 - 2480. http://www.hort.purdue.edu/rhodcv/hort640c/referen/pMCA.htm (lista di referenze sulla Ingegneria Metab. nelle piante)