ISOLATION AND CHARACTERIZATION OF THE PHYTOENE DESATURASE GENE AS A POTENTIAL SELECTIVE MARKER FOR GENETIC ENGINEERING OF THE ASTAXANTHIN-PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)(1).

Phytoene desaturase (PDS) is a rate-limiting enzyme in carotenoid biosynthesis. Algal PDS is inhibited by some herbicides, leading to the bleaching of the cells due to destruction of chl. Specific point mutations in PDS confer resistance to the herbicide norflurazon, suggesting that mutated PDS could be used as a dominant selectable marker for genetic engineering of algae, for which very few selective markers are available. In this study, we report the isolation and characterization of the PDS gene from the astaxanthin-producing green alga Chlorella zofingiensis Dönz. The open reading frame (ORF) of this PDS gene, interrupted by six introns, encoded a polypeptide of 558 amino acid residues. The deduced protein sequence showed significant homology to phytoene desaturases of algae, cyanobacteria, and higher plants. Expression of the PDS gene in Escherichia coli demonstrated that the enzyme was able to convert phytoene to ζ-carotene. The PDS gene in Chlorella was shown to be up-regulated by high light and glucose treatment. With a single amino acid change (L516R), the mutated PDS-L516R was still active and exhibited ∼36-fold greater resistance to the bleaching herbicide norflurazon than the unaltered enzyme. Thus, the modified PDS gene could be a useful tool for genetic engineering of carotenoid biosynthesis in C. zofingiensis and perhaps also in other algae.