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 valueadded products. Despite the well-studied lipid metabolism, camtenoid biosynthesis in this alga remains less understood. Here we first identified the putative carotenogenic genes via bioinformatics analysis and reconstructed the pathways for camtenoid biosynthesis in N. oceanica. Two key carotenogenic genes, phytoene desaturase (NoPDS) and lycopene beta-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 beta-carotene, suggesting that the step mediated by NoLCYB rather than NoPDS is rate-limiting for camtenoid 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.

Automated summary

In this study, the carotenoid biosynthesis pathway in Nannochloropsis oceanica was reconstructed and two key carotenogenic genes, NoPDS and NoLCYB, were functionally validated. Results showed that NoLCYB played a rate-limiting role in carotenoid biosynthesis in N. oceanica. Additionally, a NoPDS variant was developed as a selectable marker gene alternative for genetic manipulation of N. oceanica. This study not only expands our understanding of carotenoid biosynthesis in N. oceanica, but also provides a useful tool for future genetic engineering of this alga.