Abstract
Carotenoids comprise a diverse range of naturally occurring stereoisomers, which differ in their physico-chemical properties. Their biosynthesis begins with phytoene, which is a rarity among carotenoids because it is colourless. Phytoene is sought after as a skin protectant against harmful UV range B (290-320 nm) and C (100-290 nm) light, and as a natural skin-whitening agent and is synthesized from geranylgeranyl diphosphate. Geranylgeranyl diphosphate is catalysed by phytoene synthase and phytoene desaturase to phytoene and phytofluene, respectively. The subsequent steps involve desaturation, isomerisation and cyclisation reactions to form α- and β-carotene stereoisomers, via all-trans lycopene. The marine microalga Dunaliella salina is the richest source of β-carotene, but it can accumulate phytoene and phytofluene as well. In the present study, different analytical tools including High-Performance Liquid Chromatography (HPLC), Ultra-Performance Convergence Chromatography (UPC2-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize and quantify the phytoene isomeric configurations in D. salina in order to explore both the feasibility of D. salina as a cell factory for phytoene production and to gain new insight into the carotenoid synthesis pathway in D. salina. D. salina, similar to tomato, produced predominantly 15-cis phytoene isomer (>98%) and a trace amount of all-trans phytoene (<2%). High light stress, red light stress, or use of a phytoene desaturase inhibitor or a mitotic disrupter herbicide led to the accumulation of 15-cis phytoene but not all-trans phytoene. 9-cis phytoene was not detected in any of the extracts of D. salina biomass. Our main findings suggest that 15-cis phytoene is the most abundant isomer in D. salina and that it is subject to a series of isomerisation and desaturation reactions to form all-trans and 9-cis β-carotene.