Metabolic engineering of complex pathways on plants by combinatorial genetic transformation

• Autores: Shaista Batool Naqvi
• Directores de la Tesis: Paul Christou (dir. tes.), Changfu Zhu (codir. tes.)
• Lectura: En la Universitat de Lleida ( España ) en 2009
• Idioma: español
• Tribunal Calificador de la Tesis: Vicente Medina Piles (presid.), Conxita Royo Calpe (secret.), Angharad M. Roscoe Gatehouse (voc.), Albert Boronat (voc.), Eva Stoger (voc.)
• Materias:
  • Ciencias de la vida
    • Genética
      • Ingeniería genética
    • Biología molecular
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• Resumen

  • My research program focuses on the development, optimization and proof of concept demonstration of a novel multi-gene transformation system facilitating the creation and analysis of complex metabolic pathways in plants. Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants. I have developed and used this methodology to dissect and modify complex metabolic pathways in plants, specifically cereals such as maize. In the first instance I transferred five carotenogenic genes controlled by different endosperm-specific promoters together with an herbicide-resistance selectable marker gene into a white maize variety deficient for endosperm carotenoid synthesis. I recovered a diverse combinatorial population of transgenic plants expressing different enzyme combinations and exhibiting distinct metabolic phenotypes. I have also generated and analyzed multi-vitamin corn, expressing high levels of not only the key four nutritionally important carotenoids (ß-carotene, lycopene, lutein and zeaxanthin) but also vitamins C and B9 (folate). I was thus able to demonstrate that it is relatively straightforward to utilize this novel technology to simultaneously engineer multiple metabolic pathways in one and the same plant. The combinatorial maize population I generated was used to dissect mechanistic aspects of the carotenoid pathway, thus elucidating and complementing key rate limiting steps. Concurrently, I elucidated mechanistically a competition between ketolase and hydroxylase enzymes in sequential steps in the extended carotenoid pathway responsible for ketocarotenoid formation. An integral component of my research program was an in depth mechanistic investigation on key aspects of multi-gene transfer in plants in the context of metabolic pathway engineering. I discuss my results in this context and also in terms of how transgenic plants I have generated might contribute towards the alleviation of poverty in the context of food security in developing countries. I have further exemplified multigene transformation by engineering the vitamin E biosynthetic pathway in maize in proof of concept experiments. I was thus able to generate corn plants expressing two key enzymes in vitamin E biosynthesis. This strategy enhanced the total vitamin E content in corn seeds up to 3-folds.