Folate biofortification of lettuce by expression of a codon optimized chicken GTP cyclohydrolase I gene

Folates are essential coenzymes involved in one-carbon metabolism. Folate deficiency is associated with a higher risk of newborns with neural tube defects, spina bifida, and anencephaly, and an increased risk of cardiovascular diseases, cancer, and impaired cognitive function in adults. In plants folates are synthesized in mitochondria from pterin precursors, which are synthesized from guanosine-5'-triphosphate (GTP) in the cytosol (pterin branch), and p-aminobenzoate (PABA), derived from chorismate in plastids (PABA branch). We generated transgenic lettuce lines expressing a synthetic codon-optimized GTP-cyclohydrolase I gene (gchI) based on native Gallus gallus gene. Immunoblotting analyses confirmed the presence of the gchI in transgenic lines. Twenty-nine transgenic lines were generated and 19 exhibited significant increase in the folate content, ranging from 2.1 to 8.5-fold higher when compared to non-transgenic lines. The folate content in enriched lettuce would provide 26% of the Dietary Reference Intakes for an adult, in a regular serving. Although the lettuce lines generated here exhibited high folate enhancement over the control, better folate enrichment could be further achieved by engineering simultaneously both PABA and pterin pathways.