Isolation and Characterization of Phytoene Desaturase cDNA Involved in the β-Carotene Biosynthetic Pathway in Dunaliella salina

Abstract

The green alga Dunaliella salina is one of the best and most important biological sources of β-carotene; however, to date the molecular basis of the β-carotene biosynthesis process in D. salina is still unresolved. The dehydrogenation of phytoene is the second step in the carotenoids biosynthetic pathway, and the phytoene-related desaturases are the key enzymes in the β-carotene biosynthetic pathway. A phytoene desaturase (Pds) cDNA with a 1752 bp open reading frame was cloned by RT-PCR and RACE-PCR methods on the basis of a modified switching mechanism at 5‘ end of the RNA transcript (SMART) technology from D. salina. The predicted protein sequence displays a high identity (up to 65%) with phytoene desaturases of higher plants and cyanobacteria. The highest amino acid sequence identity (91%) is shared with the phytoene desaturase sequence of Dunaliella bardawil, and a dinucleotide-binding motif lies in the N-terminal. The phylogenetic analysis shows that D. salina Pds is closer to higher plants and cyanobacteria than bacterial and fungi. These results together demonstrated the cloned Pds cDNA of D. salina is a Pds-type gene, and it is postulated that in D. salina the first two dehydrogenations, by which phytoene is converted into ζ-carotene, are carried out by this putative phytoene desaturase. Keywords: Dunaliella salina; β-carotene biosynthesis; phytoene desaturase cDNA; SMART technology