Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis

Abstract

Plants are the main source of folate in human diets, but many fruits, tubers, and seeds are poor in this vitamin, and folate deficiency is a worldwide problem. Plants synthesize folate from pteridine, p-aminobenzoate (PABA), and glutamate moieties. Pteridine synthesis capacity is known to drop in ripening tomato fruit; therefore, we countered this decline by fruit-specific overexpression of GTP cyclohydrolase I, the first enzyme of pteridine synthesis. We used a synthetic gene based on mammalian GTP cyclohydrolase I, because this enzyme is predicted to escape feedback control in planta. This engineering maneuver raised fruit pteridine content by 3-to 140-fold and fruit folate content by an average of 2-fold among 12 independent transformants, relative to vector-alone controls. Most of the folate increase was contributed by 5-methyltetrahydrofolate polyglutamates and 5,10-methenyltetrahydrofolate polyglutamates, which were also major forms of folate in control fruit. The accumulated pteridines included neopterin, monapterin, and hydroxymethylpterin; their reduced forms, which are folate biosynthesis intermediates; and pteridine glycosides not previously found in plants. Engineered fruit with intermediate levels of pteridine overproduction attained the highest folate levels. PABA pools were severely depleted in engineered fruit that were high in folate, and supplying such fruit with PABA by means of the fruit stalk increased their folate content by up to 10-fold. These results demonstrate that engineering a moderate increase in pteridine production can significantly enhance the folate content in food plants and that boosting the PABA supply can produce further gains.

Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts

“…H 4 BPt does not occur in plants (Kohashi et al, 1980;Díaz de la Garza et al, 2004;Hossain et al, 2004), but it is widely used to assay the activity of AAHs for which it is not the in vivo cofactor (Leiros et al, 2007;Siltberg-Liberles et al, 2008;Pey and Martinez, 2009). Tests with the natural (R) and unnatural (S) forms of H 4 BPt showed that only the R form was active and that the S form had no inhibitory activity (see Supplemental Figure 4 online).…”

“…The main plant pterins are the folate synthesis intermediates dihydroneopterin, dihydromonapterin, and hydroxymethyldihydropterin (Kohashi et al, 1980;Díaz de la Garza et al, 2004; Hossain et al, 2004;Orsomando et al, 2006). These might be reduced to the tetrahydro level by a dihydropterin reductase like those in bacteria or protists (Gourley et al, 2001;Pribat et al, 2010).…”

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways

“…3C), and increased GCH1 activity only affects the cytosolic (pterin) branch. In previous studies, the overexpression of GCH1 has resulted in massive increases in pterin levels but only a doubling of folate levels, presumably because the plastidial p-aminobenzoate (PABA) branch was depleted (20,21). Similar limitations affected plants with the PABA branch enhanced by overexpression of aminodeoxychorismate (ADC) synthase (ADCS), reflecting depletion of the pterin branch (22).…”

Folate Polyglutamylation is Required for Rice Seed Development

“…A significant proportion of plant folates exists in a polyglutamylated form (Storozhenko et al 2007, Orsomando et al 2005, Díaz de la Garza et al 2004). This study has employed LC-MS/MS to reveal that approximately 40% of rice folate forms detected in leaf and 20% of folate in seed exists either as tetra or penta forms of the 5-CH 3 -and 5/ 10-CHO-H 4 Pte ( Table 2).…”