Installing the neurospora carotenoid pathway in plants enables cytosolic formation of provitamin A and its sequestration in lipid droplets

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PACs formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs, including β-carotene. This strategy led to the accumulation of significant amounts of phytoene, γ- and β-carotene, in addition to fungal, health-promoting carotenes with thirteen conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-CoA reductase substantially increased cytosolic carotenes production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs) that represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, β-carotene accumulated in the cytosol of citrus callus cells was more light-stable, compared to plastidial β-carotene. Moreover, engineering cytosolic carotenes formation increased the number of large-sized CLDs and the levels of β-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PACs biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and paves the way for further optimization of carotenoid biofortification in crops.