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

<i>Nannochloropsis oceanica</i> 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: <i>N. oceanica</i> Δ12 fatty acid desaturase (NoΔ12FAD), <i>N. oceanica</i> Δ6 fatty acid desaturase (NoΔ6FAD), and <i>N. oceanica</i> Δ5 fatty acid desaturase (NoΔ5FAD). Functional validation in yeast confirmed their specific desaturation activities, and localization experiments showed they reside outside the plastid of <i>N. oceanica</i>. Genetic manipulation of individual <i>NoFAD</i> 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 <i>NoFAD</i> 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 <i>N. oceanica</i>, highlighting its production through the ω6 pathway via sequential desaturation and elongation steps outside of the chloroplast.