Folate Biofortification of Potato by Tuber-Specific Expression of Four Folate Biosynthesis Genes

Insufficient dietary intake of micronutrients, known as "hidden hunger", is a devastating global burden, affecting two billion people. Deficiency of folates (vitamin B9), which are known to play a central role in C1 metabolism, causes birth defects in at least a quarter million people annually. Biofortification to enhance the level of naturally occurring folates in crop plants, proves to be an efficient and cost-effective tool in fighting folate deficiency. Previously, introduction of folate biosynthesis genes GTPCHI and ADCS, proven to be a successful biofortification strategy in rice and tomato, turned out to be insufficient to adequately increase folate levels in potato tubers. Here, we provide a proof of concept that additional introduction of HPPK/DHPS and/or FPGS, downstream genes in mitochondrial folate biosynthesis, enables augmentation of folates to satisfactory levels (12-fold) and ensures folate stability upon long-term storage of tubers. In conclusion, this engineering strategy can serve as a model in the creation of folate-accumulating potato cultivars, readily applicable in potato-consuming populations suffering from folate deficiency. Biosynthesis of folates (vitamin B9) in plants originates in the cytosol and in plastids and is finalized in mitochondria, yielding polyglutamylated folate species. Engineering of the limiting steps in the cytosolic and plastidial branches, a successful strategy for biofortification of rice kernels and tomato fruit, provided unsatisfactory results upon implementation in potato tubers. Here, additional engineering of the first and final steps in the mitochondrial branch of folate biosynthesis, HPPK/DHPS and FPGS respectively, resulted in adequate enhancement of folate levels in potato tubers, yielding a 12-fold increase. This presents a valuable tool in fighting the global burden of folate malnutrition.