Folate engineering in tomato fruit

The B vitamin folate comes mainly from plant sources. Folate deficiency is a worldwide health problem associated with spina bifida and other birth defects, anemia, cardiovascular diseases, and some cancers. The United States and other western countries mandate fortification of grain products with synthetic folic acid to help their populations reach the Recommended Dietary Allowance (RDA), which is 400 mug per day for adults and 600 mug for pregnant women. However, food fortification strategies are hard to implement in poor countries, where folate deficiency causes at least 200,000 severe birth defects every year. Moreover, concerns have arisen about the effects of chronic exposure of western populations to synthetic folic acid.;Folate biofortification of plant foods through metabolic engineering is thus an attractive way to increase the intake of the natural vitamin in rich and poor countries alike. This work consisted in the engineering of folates in plants using tomato fruit as a model. Plants synthesize folates from pteridine, p-aminobenzoate (PABA), and glutamate moieties. In the first engineering step, pteridine synthesis was enhanced by overexpressing the first enzyme in the pteridine pathway, GTP cyclohydrolase I, in ripening fruit. Pteridine levels increased up to 140-fold, but folate enhancement was just two-fold. Folate levels did not increase further due to a depletion of PABA pools in the engineered fruit. To solve this problem, PABA production was engineered by overexpressing the first enzyme of PABA synthesis, aminodeoxychorismate synthase. This caused an increase of 19-fold in total PABA. When transgenic PABA- and pteridine-overproduction traits were combined by crossing, vine-ripened fruit accumulated up to 25-fold more folate than controls. The accumulated folates showed normal proportions of one-carbon forms, with 5-methyltetrahydrofolate the most abundant, but were less extensively polyglutamylated than controls. Pteridine and PABA levels in double transgenic fruit were >20-fold higher than in controls but neither dihydropteroate nor dihydrofolate accumulated, pointing to a flux constraint at the dihydropteroate synthesis step. This work demonstrates the feasibility of developing crops with levels of folate sufficient to supply the entire RDA in a single serving. Furthermore, analyzing folate-overproducing plants gives insight into regulation of folate synthesis, which is not understood.