Characterization of fucoxanthin-deficient strains in the haptophyte <i>Tisochrysis lutea</i> induced by heavy-ion beam irradiation

Although microalgae such as diatoms and haptophytes have been studied to optimize fucoxanthin production, the complete biosynthetic pathway of fucoxanthin remains unclear. In this study, we subjected the haptophyte Tisochrysis lutea cells to heavy-ion beam irradiation to induce random mutations and obtained two greenish strains, GR1 and GR2, following exposures to 45 Gy and 100 Gy, respectively. The GR1 strain exhibited slow growth, whereas GR2 showed growth comparable to the wild-type strain. Neither GR1 nor GR2 accumulated fucoxanthin; instead, both strains accumulated fucoxanthin biosynthetic intermediates, haptoxanthin and phaneroxanthin, and harbored 74 and 148 mutation sites, respectively. As expected, higher radiation doses resulted in a greater number of mutations. Over 80% of these mutations consisted of short nucleotide insertions, primarily 4 to 8 bp in length. Additionally, mutations were identified in orthologs of the ZEP1 and CRTISO5 genes known in the diatom Phaeodactylum tricornutum to encode enzymes that convert haptoxanthin to phaneroxanthin and phaneroxanthin to fucoxanthin in GR1 and GR2 strains, respectively. The loss of fucoxanthin decreased photosynthetic capacity to some extent. However, the amounts of chlorophyll a and c did not change, suggesting that haptoxanthin and phaneroxanthin functioned as photosynthetic accessory pigments in the light-harvesting antennae. Because the genomic analysis results aligned with those from pigment analysis, our findings demonstrate that ZEP1 and CRTISO5 in T. lutea cells are involved in fucoxanthin biosynthesis and support the broader application of heavy-ion beam irradiation in fundamental microalgal research.