Functional characterization of carotenogenic genes provides implications into carotenoid biosynthesis and engineering in the marine alga Nannochloropsis oceanica

Nannochloropsis oceanica represents a promising sunlight-driven eukaryotic cell factory for lipids and value-added products. Despite the well-studied lipid metabolism , carotenoid biosynthesis in this alga remains less understood. Here we first identified the putative carotenogenic genes via bioinformatics analysis and reconstructed the pathways for carotenoid biosynthesis in N. oceanica . Two key carotenogenic genes, phytoene desaturase (NoPDS) and lycopene β -cyclase (NoLCYB), were then selected for further investigation. Both were functionally validated in the engineered Escherichia coli strains and demonstrated to reside in the chloroplast of N. oceanica . Interestingly, whilst NoPDS overexpression showed little effect on carotenoid profiles, NoLCYB overexpression promoted the level of carotenoids particularly β -carotene, suggesting that the step mediated by NoLCYB rather than NoPDS is rate-limiting for carotenoid biosynthesis in N. oceanica . Moreover, a NoPDS variant generated by site-directed mutagenesis was developed as a dominant selectable marker alternative to the antibiotic resistance genes of bacterial origin for N. oceanica transformation. Our results not only help understand carotenoid biosynthesis in N. oceanica , but also expand selectable marker of endogenous origin and provide implications into future genetic engineering of this alga for carotenoid manipulation. • The carotenogenic pathways of N. oceanica were reconstructed • Both NoPDS and NoLCYB resides exclusively in the chloroplast of N. oceanica • NoPDS overexpression in N. oceanica had little effect on carotenoid profiles • NoLCYB overexpression in N. oceanica promoted β-carotene synthesis • A NoPDS variant was developed as a dominant selectable marker for N. oceanica