The ω6 Pathway toward Eicosapentaenoic Acid Biosynthesis outside the Chloroplast in the Industrially Relevant Alga Nannochloropsis oceanica

Nannochloropsis oceanica is an emerging chassis for industrial eicosapentaenoic acid (EPA) production, yet its biosynthesis pathway remains incompletely defined. Here, we delineate the pathway by characterizing three key enzymes: N. oceanica Δ12 fatty acid desaturase (NoΔ12FAD), N. oceanica Δ6 fatty acid desaturase (NoΔ6FAD), and N. oceanica Δ5 fatty acid desaturase (NoΔ5FAD). Functional validation in yeast confirmed their specific desaturation activities, and localization experiments showed they reside outside the plastid of N. oceanica. Genetic manipulation of individual NoFAD genes (knockdown/overexpression) reshaped fatty acid profiles, particularly EPA, while having a minimal effect on the transcription of other EPA biosynthesis genes. Moreover, impairing EPA biosynthesis via NoFAD knockdown diminished the alga's resilience to temperature and light stress, underscoring the role of EPA in stress adaptation. Collectively, our findings offer new insights into EPA biosynthesis in N. oceanica, highlighting its production through the ω6 pathway via sequential desaturation and elongation steps outside of the chloroplast.