Creating an Improved Diatoxanthin Production Line by Knocking Out CpSRP54 in the zep3 Background in the Marine Diatom Phaeodactylum tricornutum

Diatoxanthin is a photoprotective carotenoid found in a few groups of microalgae displaying in vitro anti-inflammatory and anti-cancer properties, making it a promising candidate for nutraceutical, pharmaceutical, and cosmetic applications. However, large-scale production is currently nonexistent because of two major challenges: Instability during microalgae harvesting, where diatoxanthin is rapidly converted back to its inactive precursor diadinoxanthin under non-stressful light conditions, and dependence on prolonged exposure to high-intensity light, which is costly and technically challenging during indoor high-cell-density cultivation. The first limitation was previously addressed by knocking out zeaxanthin epoxidase 3 (ZEP3) in the marine diatom <i>Phaeodactylum tricornutum</i>, resulting in a mutant that stabilized diatoxanthin under non-stressful light conditions. Here, we report an improved diatoxanthin production line where both of the described challenges have been overcome. This was achieved by creating <i>P. tricornutum</i> mutants where the phenotype of the <i>zep3</i> mutant was combined with the light-sensitive phenotype of the <i>chloroplast signal recognition particle 54</i> (<i>cpsrp54</i>) mutant. Growth rates were maintained at wild-type levels at light intensities ≤ 150 µmol photons m^-2 s^-1 in the <i>zep3cpsrp54</i> mutants, but prolonged medium light exposure resulted in a 1.5- and 7-fold increase in diatoxanthin concentration compared with <i>zep3</i> and wild-type, respectively. When returned to low light, the <i>zep3cpsrp54</i> cultures retained ~80% of their accumulated diatoxanthin. The improved production lines allow for diatoxanthin accumulation without the use of high-intensity light and with limited loss of diatoxanthin when returned to non-stressful light conditions.