Boosting pro-vitamin A content and bioaccessibility in leaves by combining engineered biosynthesis and storage pathways with high-light treatments

Biofortification of green leafy vegetables with pro-vitamin A carotenoids, such as beta-carotene, has remained challenging to date. Here, we combined two strategies to achieve this goal. One of them involves producing beta-carotene in the cytosol of leaf cells to avoid the negative impacts on photosynthesis derived from changing the balance of carotenoids and chlorophylls in chloroplasts. The second approach involves the conversion of chloroplasts into non-photosynthetic, carotenoid-overaccumulating chromoplasts in leaves agroinfiltrated or infected with constructs encoding the bacterial phytoene synthase crtB, leaving other non-engineered leaves of the plant to sustain normal growth. A combination of these two strategies, referred to as strategy C (for cytosolic production) and strategy P (for plastid conversion mediated by crtB), resulted in a 5-fold increase in the amount of beta-carotene in Nicotiana benthamiana leaves. Following several attempts to further improve beta-carotene leaf contents by metabolic engineering, hormone treatments and genetic screenings, it was found that promoting the proliferation of plastoglobules with increased light-intensity treatments not only improved beta-carotene accumulation but it also resulted in a much higher bioaccessibility. The combination of strategies C and P together with a more intense light treatment increased the levels of accessible beta-carotene 30-fold compared to controls. We further demonstrated that stimulating plastoglobule proliferation with strategy P, but also with a higher-light treatment alone, also improved beta-carotene contents and bioaccessibility in edible lettuce (Lactuca sativa) leaves. Biofortification of leaves and other green tissues with pro-vitamin A carotenoids such as beta-carotene still remains challenging mostly due to the negative impact that altering carotenoid contents may have in photosynthesis. Here, we combined extraplastidial production and artificial chromoplast differentiation together with chemical (hormones) and physical (high-light) treatments to boost the beta-carotene contents of leaves while improving bioaccessibility, using Nicotiana benthamiana as the test system and lettuce (Lactuca sativa) as the crop model. image