(77f) Cloning and Characterization of the Gene Encoding Phytoene Desaturase From Chlorella Protothecoides

Phytoene desaturase (PDS) is a key rate-limiting enzyme in carotenoid biosynthesis. However, little is known about the enzyme and the molecular basis of the carotenoid biosynthesis processes in the microalga, Chlorella protothecoides. In this study, a 2054 bp cDNA fragment with a 1656 bp open reading frame (ORF) was cloned which encodes for PDS in C. protothecoides. Homology studies showed that the deduced amino acid sequence had a significant overall similarity with those sequences of other green algae and higher plants. Three motifs were found in the PDS sequence, one at the N-terminus for binding dinucleotides; one in the middle of the sequence for carrying substrate; and the third at the C-terminus for binding carotenoid. Phylogenetic analysis showed that the PDS gene in C. protothecoides was more similar to those in algae than to those in bacteria, higher plants, and cyanobacteria. The PDS gene expressed in Escherichia coli produced a protein with the expected molecular weight of 61 kDa. By heterologous complementation in E. coli strain, which accumulates phytoene for containing a plasmid pACCRT-EB, and HPLC analysis of the corresponding pigments, C. protothecoides PDS was shown to catalyze two desaturation steps converting phytoene to ζ-carotene. Taken as a whole, these results demonstrated that the cloned cDNA sequence of the PDS gene in C. protothecoides was actually a PDS-type gene. These findings provide insight into the regulatory mechanism of the PDS in C. protothecoides at the molecular level.