Biofortification of plant-based food: enhancing folate levels by metabolic engineering.

H umans require a minimum daily intake of essential micronutrients, vitamins, and minerals to maintain optimal health. Micronutrient malnutrition, the dietary insufficiency of one or more micronutrients, has far-reaching negative health consequences at all stages of life and was a pervasive health issue for all countries at the turn of the 20th century. Micronutrient malnutrition has been significantly alleviated for those populations in developed countries as a result of programs established in the mid-20th century that fortified processed foods with the necessary micronutrients. Similar fortification efforts have had only modest success in developing countries because an industrial level of agriculture, food-processing, and distribution that is limited or lacking in many of the targeted countries is required. Micronutrient malnutrition thus has remained a widespread and persistent global health problem in developing countries where it continues to exact an enormous toll on individuals, populations, and society (1). In the past decade, a complementary approach to fortification of processed foods, termed biofortification, has been undertaken to deliver the necessary daily micronutrients directly in the staple crops being grown and consumed by at-risk populations in developing countries (2). All plants have the biochemical activities necessary to synthesize or accumulate a near full complement of essential dietary micronutrients (the exceptions being vitamins D and B12). Unfortunately, the plant-based foods most abundantly consumed by at-risk populations (e.g., rice, wheat, cassava, and maize) contain levels of several individual micronutrients that are insufficient to meet minimum daily requirements, even when the crop is the most abundant component of the diet. Biofortification seeks to increase the levels of specific, limiting micronutrients directly in crops by combined breeding and geneticengineering approaches. Significant progress has been made in biofortification of vitamin E and provitamin A in plants (3–5), and in this issue of PNAS, the work of Diaz de la Garza et al. (6) extends the approach to include folate, one of the most important of the B vitamins in the human diet. The recommended dietary allowance for folate ranges from 400 g per day for adults to 600 g per day for pregnant women (7). Plant-based foods are the primary source of folate in the human diet. Folate is a complex molecule that is assembled from three different molecular components: pteridine, para-amino benzoic acid (PABA), and glutamate. The pteridine moiety is synthesized in the plant cytosol, PABA is synthesized in the chloroplast, and folates are synthesized from these two precursors in the plant mitochondria (Fig. 1). In addition to biosynthetic enzymes, membrane-bound transporters for folates and various pathway intermediates must be present (8). Previous studies (9, 10) have shown that overexpression of the first committed enzyme of the cytosolic pteridine pathway, GTP cyclohydrolase I (GCHI), resulted in a 100to 1,000-fold increase in pteridine levels but only a 2to 3-fold increase in folates. In tomato fruit engineered for GCHI overexpression, endogenous PABA levels were depleted, suggesting that PABA synthesis might limit folate levels in the transgenic lines. Consistent with this hypothesis, folate levels could be further increased in tomato fruit overexpressing CGHI by exogenous application of PABA (9). Diaz de la Garza et al. (6) report engineering of PABA levels in tomato fruit by overexpression of the nuclearencoded, chloroplast-targeted enzyme aminodeoxychorismate (ADC) synthase (ADCS). The best ADCS transgenic lines accumulated PABA in tomato fruit at levels nearly 20-fold higher than controls, although folate levels were still unchanged relative to controls. When