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

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

“…In the case of strategy C, i.e., the possibility to produce carotenoids in compartments other than plastids, the 2-fold increase in total carotenoid levels and 4-fold increase in β-carotene contents confirms a remarkable capacity of leaf cells to accumulate carotenoids outside chloroplasts. The precise location where extraplastidial β-carotene is synthesized and the form in which it is stored in the cytosol is unknown, but recent results suggest that pro-vitamin A carotenoids produced in the cytosol of N. benthamiana leaf cells accumulate in lipid droplets (Zheng et al, 2023). In any case, our data suggest that factors besides storage might be more limiting to further increase the contents of this pro-vitamin A carotenoid.…”

“…Therefore, a version of the enzyme with GFP fused to its N-terminus (referred to as GFP-crtB or c-crtB) was used to retain the protein in the cytosol of agroinfiltrated leaf cells (Andersen et al, 2021;Llorente et al, 2020). Furthermore, a truncated version of hydroxymethylglutaryl CoA reductase (tHMGR) was used to increase the MVA pathway flux and produce extra IPP and DMAPP for GGPP synthesis (Figure 1B) (Andersen et al, 2021;Zheng et al, 2023). Following agroinfiltration with this gene combination (named HEcBIY), samples were collected at 6 days post-infiltration (dpi) for HPLC analysis of carotenoid levels (Figure 1B).…”

“…The addition of tHMGR to increase the MVA pathway flux was key for the success of strategy C as it allowed to boost the production of extraplastidial carotenoids (Andersen et al 2021;Zheng et al, 2023). We therefore reasoned that increasing the metabolic flux through the MEP pathway might also contribute to improving the contents of plastidial carotenoids produced with strategy P (Figure 1A).…”

“…In the case of β-carotene, its bioaccessibility in lettuce artificial chromoplasts produced by crtB was doubled compared to that in chloroplasts (Morelli and Rodriguez-Concepcion, 2022). Recent results showed that extraplastidial β-carotene produced with a fungal pathway engineered in plant cells accumulates in cytosolic lipid droplets (Zheng et al, 2023), an environment that might be beneficial for bioaccessibility. To test whether the extraplastidial β-carotene produced with our bacterial pathway might be more bioaccessible, we performed an in vitro assay developed in our lab (Morelli and Rodriguez-Concepcion, 2022) to estimate the bioaccessibility of carotenoids in leaf samples.…”

“…MVA-derived IPP and DMAPP are mostly used to produce polyterpenes, sesquiterpenes, and triterpenes such as phytosterols (the major product of the pathway), while very low levels of GGPP are synthesized for the biosynthesis of diterpenes and protein prenylation. Interestingly, cytosolic IPP and DMAPP can be used to produce carotenoids by introducing bacterial or fungal enzymes that produce GGPP and convert downstream carotenoids (Figure 1) (Majer et al, 2017;Andersen et al, 2021;Zheng et al, 2023). This strategy preserves the chloroplast carotenoid profile without disrupting the photosynthetic complexes and allows the accumulation of carotenoid intermediates in the cytosol by separating them from the endogenous (plastidial) enzymes that convert them into downstream carotenoid end-products.…”