Folate Synthesis, Catabolism, and Engineering in Plants

University Of Florida, Gainesville FL

Investigators

Abstract

Folates are essential cofactors for one-carbon transfer reactions in all organisms. Unlike plants and microorganisms, humans cannot synthesize folates and so require them in the diet. A lack of folates is the world's most common nutrient deficiency and has grave consequences that include neural tube defects in infants and vascular disease in adults. Since plant foods are the single largest source of folates in human diets, enhancing plant folate content by metabolic engineering is an appealing way to improve human nutrition worldwide. At present too little is known about folate synthesis, catabolism, and regulation in plants to undertake the rational engineering of folate levels. This research addresses the missing basic biochemical knowledge and will take the first steps towards engineering itself. Of the nine enzymes specific to folate synthesis, only three have been cloned from plants. It is therefore planned to clone the other six enzymes by combining genomic approaches and functional complementation, and to biochemically characterize the recombinant proteins. Genomic sequence data indicate that some of these enzymes differ so strikingly in structure from their counterparts in other organisms that they are likely to have novel properties. The engineering work will explore the extent to which flux in the whole folate pathway is regulated via the committing enzymes of its pterin and p-aminobenzoate branches. These enzymes will be overexpressed singly and together, relying primarily upon non-plant enzymes that are expected to be insensitive to end-product inhibition. A second engineering approach will seek to divert folates towards overaccumulation in a stable form (5-formyltetrahydrofolate) by using antisense RNA to block recycling of this compound. Folates and pathway intermediates in engineered plants will be quantified by HPLC, and pathway flux will be measured using radiolabeled precursors. Folate catabolism will also be investigated using radiolabeled substrates to establish the degradative reactions that occur and the rate at which the folate pool turns over. Tomato will be used for both the cloning and the engineering work, because: (a) Fruits have lower folate contents than leaves, showing that enhancement is in principle possible. (b) Folates are subject to huge losses during cooking, making fruits and fruit juices an efficient vehicle to deliver folates because they are consumed fresh. (c) Tomato is readily transformable and is a major world crop. It is also possible that the tomato fruit will tolerate unphysiologically high folate levels because it is an organ programmed to die.