A DUF4281 domain-containing protein (homologue of ABA4) of Phaeodactylum tricornutum regulates the biosynthesis of fucoxanthin

Although fucoxanthin is one of the most valuable carotenoids, its biosynthesis pathway is still poorly understood. Neoxanthin has been regarded as the intermediate of fucoxanthin in recent years, however, multiple details still need to be explored. Previous study performed in Arabidopsis thaliana demonstrated a DUF4281-domain containing protein (ABA4) was necessary for the biosynthesis of neoxanthin, and the mutation of ABA4 led to a huge alteration of pigment composition. In Phaeodactylum tricornutum, a function unknown protein (designated as PtABA4 in this study) displayed high homology to ABA4, and also contained DUF4281 domain. To investigate its function on neoxanthin synthesis and fucoxanthin accumulation, we conducted endogenous overexpression and knockdown (RNA interference) of PtABA4 individually, as well as in vitro activity assay using recombinant protein. The overexpression and knockdown of PtABA4 lead to sharp increases and decrease of fucoxanthin, respectively, demonstrating a key role of PtABA4 on fucoxanthin accumulation. Unexpectedly, the knockdown of PtABA4 results in the decreases of upstream carotenoids of neoxanthin, rather than compensatory increases. In terms of in vitro activity assay, neither the recombinant protein produced by Escherichia coli nor the overexpressed transformant displays neoxanthin synthase activity. Taking all results into consideration, we speculate that the PtABA4 may constitute one multienzyme complexes with other membrane associated enzymes and participate in the formation of several carotenoids, rather than neoxanthin only. The ubiquity of DUF4281 domain across all taxa and the absence of xanthophylls in bacteria provide indirect evidence for our assumption. Above all, this study here provides a target gene for enhancing the fucoxanthin content by genetic engineering methods, and deeps the understanding about the function of DUF4281 domain.