Functional identification of carotene hydroxylases from the green alga Chromochloris zofingiensis

Chromochloris zofingiensis is a promising microalgal species for the production of carotenoids, especially astaxanthin. However, the molecular mechanisms underlying carotenoid biosynthesis remain poorly understood, restraining the enhancement of astaxanthin production. Therefore, elucidating the functional roles of enzymes involved in its carotenoid biosynthesis pathway is of significant importance. In this study, the carotene hydroxylases of C. zofingiensis, including a non-heme diiron monooxygenase (CzBCH) and members of the cytochrome P450 superfamily (CzCYP97A/B/C), were functionally characterized. Sequence alignment revealed that CzBCH and CzCYP97A/B/C are conserved compared to related species. Subcellular localization studies demonstrated that these proteins are targeted to chloroplasts. Functional complementation assays in Escherichia coli showed that both CzBCH and CzCYP97A hydroxylate the β-ring of α- and β-carotene, whereas CzCYP97C specifically hydroxylates the ε-ring of α-carotene and zeinoxanthin. Moreover, both CzCYP97A and CzBCH can collaborate with CzCYP97C to catalyze the conversion of α-carotene into lutein. Bioinformatics analyses predicted potential substrate interactions for CzCYP97A and CzBCH. Additionally, protein-protein interactions between CzCYP97A and CzCYP97C, as well as between CzBCH and CzCYP97C, were identified through computational modeling and experimentally validated using bimolecular fluorescence complementation assays. These interactions may be beneficial for lutein biosynthesis, yet detrimental to zeaxanthin biosynthesis and subsequent astaxanthin biosynthesis. The findings elucidate the functional roles of carotene hydroxylases in C. zofingiensis, and provide insights into the regulatory mechanisms governing carotenoid biosynthesis, including those of lutein and astaxanthin.