Functional Identification of Two Types of Carotene Hydroxylases from the Green Alga <i>Dunaliella bardawil</i> Rich in Lutein

Abstract

The salt-tolerant unicellular alga <i>Dunaliella bardawil</i> FACHB-847 can accumulate large amounts of lutein, but the underlying cause of massive accumulation of lutein is still unknown. In this study, genes encoding two types of carotene hydroxylases, <i>i.e.</i>, β-carotene hydroxylase (DbBCH) and cytochrome P450 carotenoid hydroxylase (DbCYP97s; DbCYP97A, DbCYP97B, and DbCYP97C), were cloned from <i>D. bardawil</i>. Their substrate specificities and enzyme activities were tested through functional complementation assays in <i>Escherichia coli</i>. It was showed that DbBCH could catalyze the hydroxylation of the β-rings of both β- and α-carotene, and displayed a low level of ε-hydroxylase. Unlike CYP97A from higher plants, DbCYP97A could not hydroxylate β-carotene. DbCYP97A and DbCYP97C showed high hydroxylase activity toward the β-ring and ε-ring of α-carotene, respectively. DbCYP97B displayed minor activity toward the β-ring of α-carotene. The high accumulation of lutein in <i>D. bardawil</i> may be due to the multiple pathways for lutein biosynthesis generated from α-carotene with zeinoxanthin or α-cryptoxanthin as intermediates by DbBCH and DbCYP97s. Taken together, this study provides insights for understanding the underlying reason for high production of lutein in the halophilic green alga <i>D. bardawil</i> FACHB-847.