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Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18232-7. doi: 10.1073/pnas.0809737105. Epub 2008 Nov 14.

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize.

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Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida, Avenue Alcalde Rovira Roure, 191, 25198 Lleida, Spain.


Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between beta-carotene hydroxylase and bacterial beta-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of beta-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes.

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